InteractiveFly: GeneBrief

nejire: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - nejire

Synonyms - CREB-binding protein - CBP

Cytological map position - 8F--9A

Function - transcriptional coactivator

Keywords - chromatin modification, histone acetyltransferase

Symbol - nej

FlyBase ID: FBgn0261617

Genetic map position -

Classification - CREB-binding protein

Cellular location - nuclear



NCBI links: Precomputed BLAST

Recent literature
Cutler, T., Sarkar, A., Moran, M., Steffensmeier, A., Puli, O. R., Mancini, G., Tare, M., Gogia, N. and Singh, A. (2015). Drosophila eye model to study neuroprotective role of CREB binding protein (CBP) in Alzheimer's disease. PLoS One 10: e0137691. PubMed ID: 26367392
Summary:
This study utilized Gal4/UAS system to develop a transgenic fruit fly model for Aβ42 (see Drosophila Appl) mediated neurodegeneration. Targeted misexpression of human Aβ42 in the differentiating photoreceptor neurons of the developing eye of transgenic fly triggers neurodegeneration. This progressive neurodegenerative phenotype resembles Alzheimer's like neuropathology. A histone acetylase, CREB Binding Protein (CBP), was identified as a genetic modifier of Aβ42 mediated neurodegeneration. Targeted misexpression of CBP along with Aβ42 in the differentiating retina can significantly rescue neurodegeneration. Gain-of-function of CBP rescues Aβ42 mediated neurodegeneration by blocking cell death. Misexpression of Aβ42 affects the targeting of axons from retina to the brain but misexpression of full length CBP along with Aβ42 can restore this defect. The CBP protein has multiple domains and is known to interact with many different proteins. This structure function analysis using truncated constructs lacking one or more domains of CBP protein, in transgenic flies revealed that Bromo, HAT and polyglutamine (BHQ) domains together are required for the neuroprotective function of CBP. This BHQ domain of CBP has not been attributed to promote survival in any other neurodegenerative disorders. This study has identified CBP as a genetic modifier of Aβ42 mediated neurodegeneration. Furthermore, this study had identified BHQ domain of CBP is being responsible for its neuroprotective function. These studies may have significant bearing on understanding of genetic basis of AD.

Philip, P., Boija, A., Vaid, R., Churcher, A. M., Meyers, D. J., Cole, P. A., Mannervik, M. and Stenberg, P. (2015). CBP binding outside of promoters and enhancers in Drosophila melanogaster. Epigenetics Chromatin 8: 48. PubMed ID: 26604986
Summary:
CREB-binding protein (CBP, also known as nejire) is a transcriptional co-activator that is conserved in metazoans. CBP and the related p300 protein have been used to predict enhancers when they occur with monomethylation of histone H3 on lysine 4 (H3K4me1). This study shows that CBP is bound at genomic sites with a wide range of functions. As expected, CBP was bound at active promoters and enhancers. In addition, the strongest CBP sites in the genome were found at Polycomb response elements embedded in histone H3 lysine 27 trimethylated (H3K27me3) chromatin, where they correlate with binding of the Pho repressive complex. CBP also binds to most insulators in the genome. At a subset of these, CBP may regulate insulating activity, measured as the ability to prevent repressive H3K27 methylation from spreading into adjacent chromatin. It is concluded that CBP could be involved Polycomb repression and insulator activity. A CBP at all functional elements may be to regulate interactions between distant chromosomal regions, and it is speculated that CBP is controlling higher order chromatin organization.

Fernandez-Nicolas, A. and Belles, X. (2015). CREB-binding protein contributes to the regulation of endocrine and developmental pathways in insect hemimetabolan pre-metamorphosis. Biochim Biophys Acta 860(3):508-15. PubMed ID: 26706852
Summary:
CREB-binding protein (CBP) is a promiscuous transcriptional co-regulator. In insects, CBP has been studied in the fly Drosophila melanogaster, where it is known as Nejire. Studies in D. melanogaster have revealed that Nejire is involved in the regulation of many pathways during embryo development, especially in anterior/posterior polarity, through Hedgehog and Wingless signaling, and in dorsal/ventral patterning, through TGF-β signaling. Regarding post-embryonic development, Nejire influences histone acetyl transferase activity on the ecdysone signaling pathway. Functional genomics studies using RNAi have shown that CBP contributes to the regulation of feeding and ecdysis during the pre-metamorphic nymphal instar of the cockroach Blattella germanica and is involved in TGF-β, ecdysone, and MEKRE93 pathways, contributing to the activation of Kr-h1 and E93 expression. In D. melanogaster, Nejire's involvement in the ecdysone pathway in pre-metamorphic stages is conserved, whereas the TGF-β pathway has only been described in the embryo. CBP role in ecdysis pathway and in the activation of Kr-h1 and E93 expression is described in this study for the first time. It is concluded that studies in D. melanogaster may have been suggestive that CBP functions in insects are concentrated in the embryo. Results obtained in B. germanica indicate, however, that CBP have diverse and important functions in post-embryonic development and metamorphosis, especially regarding endocrine signaling. Further research into a higher diversity of models will probably reveal that the multiple post-embryonic roles of CBP observed in B. germanica are general in insects.

Tie, F., Banerjee, R., Fu, C., Stratton, C.A., Fang, M. and Harte, P.J. (2016). Polycomb inhibits histone acetylation by CBP by binding directly to its catalytic domain. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 26802126
Summary:
Drosophila Polycomb (PC), a subunit of Polycomb repressive complex 1 (PRC1), is well known for its role in maintaining repression of the homeotic genes and many others and for its binding to trimethylated histone H3 on Lys 27 (H3K27me3) via its chromodomain. This study identifies a novel activity of PC: inhibition of the histone acetylation activity of CREB-binding protein (CBP). It was shown that PC and its mammalian CBX orthologs interact directly with the histone acetyltransferase (HAT) domain of CBP, binding to the previously identified autoregulatory loop, whose autoacetylation greatly enhances HAT activity. A conserved PC motif adjacent to the chromodomain required for CBP binding was identified and it was shown that PC binding inhibits acetylation of histone H3. CBP autoacetylation impairs PC binding in vitro, and PC is preferentially associated with unacetylated CBP in vivo. PC knockdown elevates the acetylated H3K27 (H3K27ac) level globally and at promoter regions of some genes that are bound by both PC and CBP. Conversely, PC overexpression decreases the H3K27ac level in vivo and also suppresses CBP-dependent Polycomb phenotypes caused by overexpression of Trithorax, an antagonist of Polycomb silencing. It was found that PC physically associates with the initiating form of RNA polymerase II (Pol II) and many promoters co-occupied by PC and CBP are associated with paused Pol II, suggesting that PC may play a role in Pol II pausing. These results suggest that PC/PRC1 inhibition of CBP HAT activity plays a role in regulating transcription of both repressed and active PC-regulated genes.

Sharma, S., Poetz, F., Bruer, M., Ly-Hartig, T. B., Schott, J., Seraphin, B. and Stoecklin, G. (2016). Acetylation-dependent control of global poly(A) RNA degradation by CBP/p300 and HDAC1/2. Mol Cell 63: 927-938. PubMed ID: 27635759
Summary:
Acetylation of histones and transcription-related factors is known to exert epigenetic and transcriptional control of gene expression. This study reports that histone acetyltransferases (HATs) and histone deacetylases (HDACs) also regulate gene expression at the posttranscriptional level by controlling poly(A) RNA stability. Inhibition of HDAC1 and HDAC2 induces massive and widespread degradation of normally stable poly(A) RNA in mammalian and Drosophila cells. Acetylation-induced RNA decay depends on the HATs p300 and CBP, which acetylate the exoribonuclease CAF1a, a catalytic subunit of the CCR4-CAF1-NOT deadenlyase complex and thereby contribute to accelerating poly(A) RNA degradation. Taking adipocyte differentiation as a model, global stabilization of poly(A) RNA was observed during differentiation, concomitant with loss of CBP/p300 expression. This study uncovers reversible acetylation as a fundamental switch by which HATs and HDACs control the overall turnover of poly(A) RNA.
Ghezzi, A., Li, X., Lew, L. K., Wijesekera, T. P. and Atkinson, N. S. (2017). Alcohol-induced neuroadaptation is orchestrated by the histone acetyltransferase CBP. Front Mol Neurosci 10: 103 [Epub ahead of print]. PubMed ID: 28442993
Summary:
Homeostatic neural adaptations to alcohol underlie the production of alcohol tolerance and the associated symptoms of withdrawal. These adaptations have been shown to persist for relatively long periods of time and are believed to be of central importance in promoting the addictive state. In Drosophila, a single exposure to alcohol results in long-lasting alcohol tolerance and symptoms of withdrawal following alcohol clearance. These persistent adaptations involve mechanisms such as long-lasting changes in gene expression and perhaps epigenetic restructuring of chromosomal regions. Histone modifications have emerged as important modulators of gene expression and are thought to orchestrate and maintain the expression of multi-gene networks. Previously genes that contribute to tolerance were identified as those that show alcohol-induced changes in histone H4 acetylation following a single alcohol exposure. However, the molecular mediator of the acetylation process that orchestrates their expression remains unknown. This study shows that the Drosophila ortholog of mammalian CBP, nejire, is the histone acetyltransferase involved in regulatory changes producing tolerance-alcohol induces nejire expression, nejire mutations suppress tolerance, and transgenic nejire induction mimics tolerance in alcohol-naive animals. Moreover, a loss-of-function mutation in the alcohol tolerance gene slo epistatically suppresses the effects of CBP induction on alcohol resistance, linking nejire to a well-established alcohol tolerance gene network. It is proposed that CBP is a central regulator of the network of genes underlying an alcohol adaptation.
Luo, L., Siah, C. K. and Cai, Y. (2017). Engrailed acts with Nejire to control decapentaplegic expression in the Drosophila ovarian stem cell niche. Development 144(18): 3224-3231. PubMed ID: 28928281
Summary:
Homeostasis of adult tissues is maintained by a small number of stem cells, which are sustained by their niches. In the Drosophila female germline stem cell (GSC) niche, Decapentaplegic (Dpp) is the primary factor that promotes GSC self-renewal. However, the mechanism regulating dpp expression in the niche is largely unknown. This study identified a 2.0 kb fragment located in a 5' cis-regulatory region of the dpp locus containing enhancer activity that drives its expression in the niche. This region is distinct from a previously characterized 3' cis-regulatory enhancer responsible for dpp expression in imaginal discs. These data demonstrate that Engrailed, a homeodomain-containing transcription factor that serves as a cap cell marker, binds to this region and regulates dpp expression in cap cells. Further data suggest that En forms a complex with Nejire (Nej), the Drosophila ortholog of histone acetyltransferase CBP/p300, and directs Nej to this cis-regulatory region where Nej functions as the co-activator for dpp expression. Therefore, this study defines the molecular pathway controlling dpp expression in the Drosophila ovarian stem cell niche.
Boija, A., Mahat, D. B., Zare, A., Holmqvist, P. H., Philip, P., Meyers, D. J., Cole, P. A., Lis, J. T., Stenberg, P. and Mannervik, M. (2017). CBP regulates recruitment and release of promoter-proximal RNA polymerase II. Mol Cell 68(3): 491-503. PubMed ID: 29056321
Summary:
Transcription activation involves RNA polymerase II (Pol II) recruitment and release from the promoter into productive elongation, but how specific chromatin regulators control these steps is unclear. This study identifies a novel activity of the histone acetyltransferase p300/CREB-binding protein (CBP) in regulating promoter-proximal paused Pol II. Drosophila CBP inhibition results in "dribbling" of Pol II from the pause site to positions further downstream but impedes transcription through the +1 nucleosome genome-wide. Promoters strongly occupied by CBP and GAGA factor have high levels of paused Pol II, a unique chromatin signature, and are highly expressed regardless of cell type. Interestingly, CBP activity is rate limiting for Pol II recruitment to these highly paused promoters through an interaction with TFIIB but for transit into elongation by histone acetylation at other genes. Thus, CBP directly stimulates both Pol II recruitment and the ability to traverse the first nucleosome, thereby promoting transcription of most genes.
Hundertmark, T., Gartner, S. M. K., Rathke, C. and Renkawitz-Pohl, R. (2018). Nejire/dCBP-mediated histone H3 acetylation during spermatogenesis is essential for male fertility in Drosophila melanogaster. PLoS One 13(9): e0203622. PubMed ID: 30192860
Summary:
Spermatogenesis in many species including Drosophila melanogaster is accompanied by major reorganisation of chromatin in post-meiotic stages, involving a nearly genome-wide displacement of histones by protamines, Mst77F and Protamine-like 99C. A proposed prerequisite for the histone-to-protamine transition is massive histone H4 hyper-acetylation prior to the switch. This study investigated the pattern of histone H3 lysine acetylation and general lysine crotonylation in D. melanogaster spermiogenesis to elucidate a possible role of these marks in chromatin reorganisation. Lysine crotonylation was strongest prior to remodelling and the deposition of this mark depended on the acetylation status of the spermatid chromatin. In contrast to H4 acetylation, individual H3 acetylation marks displayed surprisingly distinct patterns during the histone-to-protamine transition. Nejire, a histone acetyl transferase, is expressed during the time of histone-to-protamine transition. Nejire knock down led to strongly reduced fertility, which correlated with misshaped spermatid nuclei and a lack of mature sperm. protA and prtl99C transcript levels were reduced after knocking down Nejire. ProtB-eGFP, Mst77F-eGFP and Prtl99C-eGFP were synthesized at the late canoe stage, while histones were often not detectable. However, in some cysts histones persist in parallel to protamines. Therefore, it was hypothesized that complete histone removal requires multiple histone modifications besides H3K18ac and H3K27ac. In summary, H3K18 and H3K27 acetylation during Drosophila spermatogenesis is dependent on Nejire or a yet uncharacterized acetyl transferase. Nejire is required for male fertility since Nejire contributes to efficient transcription of protA and prtl99C, but not Mst77F, in spermatocytes, and to maturation of sperm.
BIOLOGICAL OVERVIEW

CREB-binding protein or CBP (in Drosophila, the protein termed Nejire) is a transcriptional coactivator that interacts with a large number of developmentally important transcription factors. CBP and p300 are highly related proteins: mammalian CBP was originally identified by its interaction with CREB (cAMP response-element-binding protein) and p300 was originally identified as a target of the adenoviral E1A oncoprotein. CBP is recruited to DNA by several transcription factors, including CREB (Drosophila homolog: CrebB-17A) and cFos (Drosophila homolog: Fos-related antigen/Kayak). Transcriptional coactivators are considered to be accessory proteins that interact with transcription factors and are required for proper transcription factor function.

Since inactivation of only a single copy of CBP causes severe developmental defects in Rubenstein-Taybi syndrome (Petrij, 1995), it has been proposed that CBP is a limiting integrator of multiple signal transduction pathways at the level of gene activation. Recently, CBP/p300 were reported to have intrinsic histone acetyltransferase (HAT) activity (Ogryzko, 1996 and Bannister, 1996). The identification in Tetrahymena of the first transcription-associated HAT has allowed a major advance in understanding the connection between histone acetylation and gene transcription. HAT has been found to share significant homology to the Saccharomyces cerevisiae adapter GCN5 (Brownell, 1996). GCN5 acetylates specific lysines in a pattern associated with transcriptionally active chromatin, and HAT activity is required for the activation of GCN5-responsive genes. These observations establish the causal link between histone acetyltransferase activity and activation of transcription. HAT activity displayed by multiple families of proteins is associated with gene activation through the modification of chromatin structure. PCAF, a mammalian homolog of GCN5, and CBP are found in a complex with a third family of HATs, the hormone receptor coactivators SRC and ACTR (Chen, 1997 and Spencer, 1997). Importantly, the in vitro substrate specificity of each of these HATs is distinct, suggesting that multiple HATs can act in concert at or near single promoters.

Drosophila CBP was isolated by screening Drosophila libraries under low-stringency hybridization conditions with a C. elegans CBP probe. The hemizygous nej mutant embryos die at stage 9 or 10 during embryogenesis, although some embryos survive to hatching. The most severe phenotype in nef hemizygotes is the twisting of the embryo that occurs at germband elongation.

Drosophila CBP is a coactivator of Cubitus interruptus in Hedgehog signaling. Ci is a known target of the Hh signal transduction pathway; in turn, Ci targets wingless. The expression of wingless is strikingly reduced at the posterior margin of each parasegment in CBP mutants. In addition, engrailed expression, which is maintained by Wg protein, is significantly lower in such mutants than in wild type. These observations suggest the Drosophila CBP might contribute to the functioning of some transcription factors involved in the activation of the Wingless-Engrailed signaling pathway. Ci protein physically interacts with Drosophila CBP. A series of deletion mutants of ci indicates that a region of Ci between amino acids 1020 and 1160 is required for a phosphorylation independent interaction with Drosophila CBP. This region is part of the Ci transactivation domain, C-terminal to five putative Protein kinase A (PKA) sites. Drosophila CBP expression augments transactivation by CI up to a maximum of 62 fold. The dominant gain-of-function ciD mutant phenotype, in which the longitudinal vein 4 of the adult wing is shortened, some posterior row hairs are missing, and the posterior wing margin is flattened, can be explained by the inappropriate expression of ci in the posterior compartment of the wing imaginal disc, where it is usually repressed by Engrailed. A subset of the ciD wing defects is suppressed by the haploinsufficiency of Drosophila CBP. Thus Drosophila CBP is required for the activation of Cubitus interruptus target genes such as patched, and CBP is required for the activator function of Ci but not for the repressor function. Drosophila CBP binds to dCREB2, the Drosophila homolog of CREB, in a phosphorylation-dependent manner, whereas the CBP-Ci interaction is phosphorylation-independent (Akimaru, 1997a).

These observations have been used to explain how two pathways can interfere with each other. PKA phosphorylates dCREB2 and this signaling function appears to repress Hh target genes such as dpp. Since Drosophila CBP binds to dCREB2 in a phosphorylation dependent manner, a limited amount of Drosophila CBP might be recruited to PKA-phosphorylated dCREB2, resulting in a decrease in Ci activity due to a diminished availability of Drosophila CBP. Therefore, a limited amount of Drosophila CBP might be recruited to PKA-phosphorylated dCREB2, resulting in a decrease in Ci activity, explaining the antagonistic actions of PKA and Hedgehog (Akimaru, 1997a).

The role of Drosophila CBP in the regulation of Hh target genes fails to explain the primary developmental effect of Drosophila CBP mutation observed at germband elongation: the clockwise or counter-clockwise twisting of the embryo, just behind the cephalic furrow, often with the posterior side down. The ventral and cephalic furrows appear normal, but the mesodermally derived internal tissues and a block of ectodermal cells are often missing. On the basis of this phenotype, the Drosophila CBP mutant was named nejire, which means 'twist' in Japanese. Mesoderm formation is crucial event that takes place during early embryogenesis. To initiate the differentiation of the mesoderm in Drosophila, multiple zygotic genes such as twist (twi) and snail (sna), which encode a basic-helix-loop-helix and a zinc finger transcription factor, respectively, are required. The transcription of these genes is induced by maternal Dorsal protein, a transcription factor involved in establishment of dorsal-ventral polarity, that is homologous to the NF-kappa B family of proteins. Drosophila CBP mutants, devoid of both maternal and zygotic nejire expression, fail to express twi and therefore generate twisted embryos. This is explained by results showing that dCBP is necessary for Dorsal-mediated activation of the twi promoter. In vitro Dorsal has been shown to bind the N-proximal portion of Drosophila CBP in a phosphorylation independent manner. A region of Dorsal lying between amino acids 186 and 356 (a part of the Rel homology domain) is required for interaction with CBP. Further studies indicated that a CBP-Dorsal complex is formed on the twist promoter (Akimaru, 1997b).

A function of CBP as a transcriptional co-activator during Dpp signalling

CBP not only exerts an effect on DNA transcription by acting as a histone transacetylase, but it can also acetylate transcription factors. In addition CBP can exert an inhibitory effect on transcription. The interaction of Drosophila CBP with Pangolin demonstrates both these points. T-cell factor (TCF), a high-mobility-group domain protein, is the transcription factor activated by Wnt/Wingless signaling. When signaling occurs, TCF binds to its coactivator, beta-catenin/Armadillo, and stimulates the transcription of the target genes of Wnt/Wingless by binding to TCF-responsive enhancers. Inappropriate activation of TCF in the colon epithelium and other cells leads to cancer. It is therefore desirable for unstimulated cells to have a negative control mechanism to keep TCF inactive. Drosophila CREB-binding protein binds to Drosophila TCF (Pangolin). dCBP mutants show mild Wingless overactivation phenotypes in various tissues. Consistent with this, Drosophila CBP loss-of-function suppresses the effects of armadillo mutation. Moreover, Drosophila CBP is shown to acetylate a conserved lysine in the Armadillo-binding domain of Pangolin, and this acetylation lowers the affinity of Armadillo binding to Pangolin. Although CBP is a coactivator of other transcription factors, these data show that CBP represses TCF. Indeed, point mutations have been found in the HAT and CBP2 domains of the remaining p300 gene in colon and gastric carcinomas that have lost one copy of this gene, indicating that p300/CBP has a tumor-suppressor function (Muraoka, 1996). This is consistent with data showing the Drosophila CBP represses Pangolin to antagonize Armadillo-mediated activation of Wingless target genes (Waltzer, 1998).

Preferential genome targeting of the CBP co-activator by Rel and Smad proteins in early Drosophila melanogaster embryos

CBP and the related p300 protein are widely used transcriptional co-activators in metazoans that interact with multiple transcription factors. Whether CBP/p300 occupies the genome equally with all factors or preferentially binds together with some factors is not known. Therefore Drosophila CBP (nejire) ChIP-seq peaks were compared with regions bound by 40 different transcription factors in early embryos, and high co-occupancy was found with the Rel-family protein Dorsal. Dorsal is required for CBP occupancy in the embryo, but only at regions where few other factors are present. CBP peaks in mutant embryos lacking nuclear Dorsal are best correlated with TGF-β/Dpp-signaling and Smad-protein binding. Differences in CBP occupancy in mutant embryos reflect gene expression changes genome-wide, but CBP also occupies some non-expressed genes. The presence of CBP at silent genes does not result in histone acetylation. Polycomb-repressed H3K27me3 chromatin does not preclude CBP binding, but restricts histone acetylation at CBP-bound genomic sites. It is concluded that CBP occupancy in Drosophila embryos preferentially overlaps factors controlling dorso-ventral patterning and that CBP binds silent genes without causing histone hyperacetylation (Holmqvist, 2012).

By comparison of CBP-bound regions in 2-4 hour old Drosophila embryos to previously mapped transcription factors, an extensive overlap of CBP peaks was found with the key activator of dorsal-ventral patterning, the Rel-family transcription factor Dorsal. The genome-wide distribution of CBP was determined in embryos where Dorsal cannot enter the nucleus (gd7 mutants), and it was found that CBP peaks that overlap regions where Dorsal, but few other factors bind in wild-type are selectively reduced in gd7 mutant embryos. Instead, strong CBP-bound regions in gd7 mutants overlap best with regions bound by the Smad protein Medea, a mediator of Dpp-signaling. Signaling by the TGF-β molecule Dpp is exceptionally sensitive to a small decline in the level of CBP in Drosophila embryos. The current results are consistent with a function for CBP in the genomic response to Dpp-signaling (Holmqvist, 2012).

Less overlap of the CBP peaks is found with mapped activators of anterior-posterior patterning such as Stat92E, Fushi-tarazu (Ftz), Paired, Caudal, and Bicoid. Previous work has indicated that CBP may function as a Bicoid co-activator. When Bicoid and CBP are expressed in S2 cells, they can interact, and Bicoid-mediated activation of reporter genes in these cells is influenced by CBP levels. 43% of the 300 strongest Bicoid-binding regions overlap a CBP peak in wild-type embryos, indicating that CBP may participate in Bicoid-mediated activation in vivo. However, many of the Bicoid peaks are found in HOT regions that bind several transcription factors. Therefore, it may not be Bicoid that targets CBP to these sites. Furthermore, although the shape of the Bicoid gradient is slightly changed in embryos from the CBP hypomorph nej1, activation of Bicoid-target genes is not compromised by the decrease in CBP levels in nej1 embryos. Consistent with a non-essential function for CBP in Bicoid-mediated activation, there is no co-occupancy of CBP and Bicoid at the known target genes hb, otd, kni, and eve. Thus, although CBP may contribute to Bicoid-mediated activation of some target genes, it seems to make a more widespread contribution to Dorsal-mediated activation. In conclusion, both genetic and genomic evidence points to a particularly important function for CBP in controlling the two key events in dorsal-ventral patterning of Drosophila embryos, the Dorsal gene regulatory network and Dpp-signaling. Perhaps CBP serves to coordinate the Dorsal and Dpp pathways in dorsal-ventral patterning (Holmqvist, 2012).

In embryos where Dorsal cannot enter the nucleus, places were found where CBP occupancy is increased, unchanged, decreased or lost. Regions that are unchanged bind several transcription factors, evident in their high HOTness, indicating that in the absence of Dorsal, other factors maintain CBP binding at these sites. Surprisingly, regions where CBP binding is increased are even HOTer, and therefore associated with even more factors in wild-type embryos. Although many CBP peaks in the genome are found where also GAF binds, the regions where CBP occupancy increases in gd7 embryos are lacking strong GAF binding, despite their high HOTness. Perhaps binding of GAF to these sites is not compatible with proper regulation of the corresponding genes. Instead, many of these regions bind Medea and Dichaete, especially the places where CBP binding is strong already in wild-type. In gd7 embryos, Dpp/Medea-regulated genes are expressed in more cells, resulting in increased CBP signal. These data indicate that also Dichaete-regulated genes are more highly expressed in gd7 mutants, and that CBP-binding therefore increases at these regions (Holmqvist, 2012).

Unexpectedly, median gene expression level of genes associated with gd7 Up regions is high in wild-type embryos. Most genes associated with these regions increase in expression even further in the absence of Dorsal, in most cases probably due to an expansion in the number of cells expressing the gene. It is therefore expected that these CBP-binding sites would be situated in promoter regions, and the increase in CBP binding a consequence of increased gene activity. However, it was found that these sites are mainly found in intronic and intergenic regions associated with H3K4me1, a mark of transcriptional enhancers. This indicates that CBP becomes recruited to these enhancers to mediate gene activation, rather than passively associating with active gene regions (Holmqvist, 2012).

CBP occupancy in gd7 embryos is reduced at regions where only few factors bind. The bigger the reduction in CBP occupancy compared to wild-type, the fewer the factors that are associated with such a region in wild-type, i.e. the lower the HOTness of the region. CBP peaks that are reduced in gd7 embryos are much more common at regions where Dorsal binds in wild-type compared to other factors, consistent with a requirement for Dorsal in targeting CBP to chromatin. Although not all of the gd7 Down CBP peaks overlap the top 300 Dorsal-binding regions, 92% overlap Dorsal when all Dorsal-binding regions are considered. Peaks where CBP is reduced in gd7 embryos are found in several known Dorsal target genes, such as twi, brk, htl, and Mef2. Furthermore, 10 of the 20 strongest Dorsal peaks overlap a region where CBP binding is reduced in gd7 embryos. Together, these data show that in early embryos, chromatin binding of CBP to many sites in the genome is dependent on Dorsal (Holmqvist, 2012).

A number of genomic regions were found where CBP occupancy in gd7 embryos is reduced to a level approaching background, the gd7 Lost regions. These regions are mostly devoid of histone modifications and occupied by very few or none of the 40 transcription factors. The factors found at these regions bind at very low levels, indicating that they may not contribute to regulation of the corresponding genes at this stage of development. Further, most genes associated with the gd7 Lost regions are expressed at very low levels or completely silent. These CBP-binding regions may therefore represent regulatory sequences that are poised for subsequent activation. Consistent with this interpretation, mean expression of the corresponding genes increases at later stages of development. Why is CBP occupancy lost from these regions in gd7 embryos? Perhaps these genes are not, and will not be expressed in the dorsal ectoderm, and are therefore not associated with CBP in gd7 mutants that convert the entire embryo into dorsal ectoderm. Alternatively, CBP binding to these regions is dependent on Dorsal. Although binding is weak, Dorsal occupies many of these regions in wild-type. It is possible that even small amounts of Dorsal is sufficient and necessary for CBP recruitment to these sites, and that CBP binding is consequently lost in the absence of Dorsal (Holmqvist, 2012).

Although CBP occupancy is reduced predominantly at Dorsal-binding regions in gd7 mutant embryos, expression of Dorsal target genes is also altered. The decrease in CBP occupancy in mutant embryos may therefore be a consequence of transcriptional inactivity, rather than a lack of recruitment by Dorsal. Indeed, CBP occupancy is on average reduced at down-regulated genes and increased at up-regulated genes. Therefore, although Dorsal and CBP occupancy often coincide, Dorsal may not directly recruit CBP to regulatory DNA sequences. However, there are also places where CBP occupancy is reduced without a corresponding change in gene expression. One such example is at the promoter of the caudal (cad) gene, which is co-occupied by Dorsal and CBP but where CBP binding is reduced more than two-fold in gd7 embryos, although the gene continues to be expressed. Furthermore, Dorsal and CBP associate in vivo. It is believed, therefore, that Dorsal may directly recruit CBP to many sites in the genome (Holmqvist, 2012).

There are also genomic sites where CBP occupancy is not dependent on either Dorsal or gene expression. Several known Dorsal target genes, including sna, neur, ind and ths, continue to associate with CBP in gd7 embryos. Although in general, HOTness is major determinant of CBP occupancy, there is no big difference in HOTness of the Dorsal target gene regions where CBP-binding is reduced (e.g., twi, htl, brk) compared to Dorsal target gene regions where CBP binding is not changed (e.g., sna, ind, ths). What maintains CBP binding on these genes in the absence of Dorsal is not clear. Presumably, other factors recruit CBP to these sites in the absence of Dorsal, but no common factor was found for the regions where CBP binding is unchanged. It is noted, however, that GAGA-factor (GAF) associates with many of the CBP-binding regions in wild-type embryos, but much less with CBP-binding regions in gd7 embryos. It is possible that GAF contributes to the recruitment of CBP to chromatin (Holmqvist, 2012).

Dorsal is converted to a repressor when it binds in proximity to AT-rich sequences, and thereby prevents expression of dorsal ectoderm target genes in the neuroectoderm and mesoderm. Consequently, these target genes, e.g. dpp, zen, and tld, are activated in all cells of gd7 mutant embryos. As expected, CBP occupancy increases at these target genes in gd7 embryos, since more cells express the genes. The Zelda protein is a maternally contributed activator of these genes. It has been previously shown that in nej1 embryos containing reduced amounts of CBP, tld expression is diminished, whereas dpp and zen expression remains unaffected. It is possible, therefore, that more activators than Zelda contribute to activation of tld, zen, and dpp in the dorsal ectoderm. Until these factors are identified, it may not be possible to explain why tld expression is particularly sensitive to a reduction in CBP amount in early embryos (Holmqvist, 2012).

When Dorsal functions as a repressor, it recruits the Groucho co-repressor. The yeast Tup1 protein, which is related to Groucho, was recently shown to block recruitment of co-activators to target genes. By contrast, it was found that CBP continues to associate with the tld and zen genes in the neuroectoderm although they are being repressed by Dorsal/Groucho. Groucho binds the histone deacetylase Rpd3 (HDAC1), which may be important for repression. Indeed, it was found that when tld and zen are repressed by Dorsal in the neuroectoderm and mesoderm, the genes are hypoacetylated despite the presence of CBP (Holmqvist, 2012).

Contrary to the general trend, some genes recruit CBP even though they are silent. Why are these genes not activated? In the cases examined, histone acetylation is low despite the presence of CBP when the genes are not expressed. Since lysine methylation and acetylation are mutually exclusive, histone methylation was measured at CBP-bound regions and it was found that Polycomb-repressed H3K27me3 chromatin is present at Dorsal-target genes in some tissues where these genes are not expressed. Although H3K27me3-decorated chromatin restricts DNA accessibility, it was found that H3K27me3-chromatin does not preclude CBP binding, but restrains histone acetylation at these CBP-bound genomic sites. Interestingly, all histone acetylations that were measured are blocked by H3K27me3-chromatin, not only the mutually exclusive H3K27ac. This indicates that despite the ability of CBP to bind to genes enclosed in H3K27me3-chromatin, the histones are not accessible for acetylation by CBP and other HATs. The data are consistent with a model for Polycomb silencing that allows access of proteins and pol II to DNA, but that restrains pol II elongation. Perhaps high levels of histone acetylation are necessary for release of pol II from the promoter, for example by recruiting the bromodomain protein Brd4 that brings in the P-TEFb kinase to phosphorylate pol II (Holmqvist, 2012).

In cells depleted of CBP and p300, global levels of H3K18ac and H3K27ac are greatly diminished whereas other histone acetylations remain unaffected, suggesting that these are in vivo targets of CBP acetylation. CBP can also acetylate H3K56, which occurs in response to DNA damage. It was found that H3K18ac and H3K27ac levels do not always correlate with changes in CBP occupancy at Dorsal target genes, although H3K18ac levels are most similar to CBP abundance. In part, this can be explained by the presence of H3K27me3-chromatin, that precludes histone acetylation. However, in the neuroectoderm (Tollrm9/rm10 embryos), the twi promoter contains less histone acetylation than in the dorsal ectoderm (gd7 embryos) although H3K27me3 levels are reduced and CBP binding not decreased compared to dorsal ectoderm. Together, these results show that CBP's HAT activity is regulated by substrate availability, but that it may also be regulated by genomic context or signaling (Holmqvist, 2012).

Genome occupancy of CBP/p300 and H3K4me1 can be used to predict cis-regulatory DNA sequences. However, what fraction of regulatory sequences that can be identified in this way is not known. It was found that CBP binding to many known enhancer sequences that are active in early embryos is below the cut-off for high-confidence peaks, although average CBP occupancy was determined to be 1.73 times the genomic background at 97 previously described early embryonic enhancers. The results also show that CBP binding differs greatly between wild-type and mutant embryos, and that some gene regulatory networks rely on CBP to a much larger extent than others. Together, these results suggest that although CBP/p300 binding can be used to successfully identify transcriptional regulatory sequences, many enhancer sequences will be missed because they are not bound by CBP/p300 or bound at levels below criteria for high-confidence peaks. Even though mapping CBP/p300 binding in different cell-types will increase the number of putative regulatory sequences, it is anticipated that a substantial number of enhancers will require alternative strategies for their identification, e.g. genome occupancy of other HATs (Holmqvist, 2012).

In conclusion, this study shows that association of CBP with the genome is dependent on the number and types of transcription factors that bind the DNA sequence, that CBP preferentially associates with some gene regulatory networks, that CBP binding correlates with gene activity, but that CBP also binds silent genes without causing histone hyperacetylation (Holmqvist, 2012).

Chromatin signatures at Notch-regulated enhancers reveal large-scale changes in H3K56ac upon activation

The conserved Notch pathway functions in diverse developmental and disease-related processes, requiring mechanisms to ensure appropriate target selection and gene activation in each context. To investigate the influence of chromatin organisation and dynamics on the response to Notch signalling, this study partitioned Drosophila chromatin using histone modifications and established the preferred chromatin conditions for binding of Su(H), the Notch pathway transcription factor. Manipulating activity of a co-operating factor, Lozenge/Runx, showed that it can help facilitate these conditions. While many histone modifications were unchanged by Su(H) binding or Notch activation, rapid changes were detected in acetylation of H3K56 at Notch-regulated enhancers. This modification extended over large regions, required the histone acetyl-transferase CBP and was independent of transcription. Such rapid changes in H3K56 acetylation appear to be a conserved indicator of enhancer activation as they also occurred at the mammalian Notch-regulated Hey1 gene and at Drosophila ecdysone-regulated genes. This intriguing example of a core histone modification increasing over short timescales may therefore underpin changes in chromatin accessibility needed to promote transcription following signalling activation (Skalska, 2015).

Signalling pathways such as Notch have diverse functions depending on the context in which they are activated and on the specific subsets of genes that are regulated in each context. This specificity necessitates mechanisms that enable Su(H) to recognise and bind to appropriate enhancers and effect relevant gene expression changes. By utilising the comprehensive collection of chromatin modifications gathered by the modENCODE project, this study has generated maps of chromatin states (see The full list of signal tracks) in two Drosophila cell types and related those to the loci that are bound by Su(H). In doing so, the profile of H3K56ac across the genome was also analysed, and that this core histone modification was found to be present at enhancers, and at transcription start sites, similar to the reported distribution in mammalian ES cells. Significantly, the inclusion of H3K56ac-binding data in the computational model helped to discriminate the active enhancers. Even more striking was the robust increase in this core nucleosome modification in response to Notch activation. Such changes were also detected in mammalian cells and at ecdysone-regulated genes in Drosophila, arguing that H3K56ac is likely to be a widespread modification associated with enhancer activation (Skalska, 2015).

Unlike the modifications to exposed histone tails, which primarily provide docking sites for further chromatin modifying proteins, H3K56ac can directly alter nucleosomal DNA accessibility by increasing DNA breathing and unwrapping rate. As a consequence, this modification can influence transcription factor (TF) occupancy within the nucleosome and it has been argued that H3K56ac drives chromatin towards the disassembled state during transcriptional activation. As the increase in H3K56ac appears to precede transcription elongation, it fits with the latter model. Furthermore, as mammalian CSL has been found to bind preferentially to motifs at the nucleosome exit point\, H3K56ac may enhance recruitment, giving a feed-forward benefit that could potentially explain the increase in occupancy following Notch activation. In addition, H3K56ac facilitates divergent transcription by promoting rapid nucleosome turnover and also promotes small RNA production in neurospora, which is consistent with the detection of intergenic enhancer-templated RNAs in the modified regions following Notch activation (Skalska, 2015).

The increase in H3K56ac appears to require CBP-HAT activity, which is also essential for catalysing this modification on free histones. It is plausible therefore that the increase in H3K56ac could occur through the incorporation of pre-modified nucleosomes. The modification of histone dimers requires interaction with the chaperones CAF1 and ASF1, and while genetic evidence that the chaperone subunit dCAF-1-p105 can help promote Notch signalling favours such a model, the current results suggest this is less likely. First, it was found that CBP is required at the time of activation, making it improbable that the increase in H3K56ac is a consequence of loading pre-modified histones. Second, an inhibitor of the CBP bromodomain, which plays an important role in enabling H3K56ac on histone dimers via its interaction with chaperones, had no effect on the increase in H3K56ac. Thus, it seems more likely that the modification occurs at the time of enhancer activation, although it may nevertheless involve nucleosome exchange. For example, SWI/SNF nucleosome remodellers have been found to act in combination with H3K56ac to promote nucleosome turnover and gene activity in yeasts. At several loci where changes were detected in H3K56ac, the modification extended broadly from the site of Su(H)/NICD binding, correlating with domains that already possessed H3K4me1. Along with data from other studies of enhancer activation, and the observation that levels of H3K56ac are affected by mutation of H3K4, this suggests that H3K4me1 is likely to be one of the earliest modifications, prefiguring sites of active enhancer. It may also facilitate the spread of H3K56ac across the regulated regions (Skalska, 2015).

Analysis of the relationship between chromatin states and regions occupied by Su(H) suggests that the pre-existing chromatin environment is likely to make an important contribution to recruitment. First, Su(H)-occupied motifs were almost exclusively located in highly accessible chromatin, with modifications such as H3K4me1 characteristic of enhancer states. Second, expression of the cooperating transcription factor Lz converted enhancers towards this preferred chromatin state where additional Su(H) was recruited. By having a preference for a particular chromatin signature, the vast majority (>91%) of potential Su(H) binding motifs will be masked by unfavourable chromatin. Indeed, the small fraction of sites that do not fit with this pattern may reflect false positives in the ChIP data or in chromatin assignment. The greater paradox is that only 7%-10% of CSL motifs within the favourable Enh chromatin were bound. Furthermore, many of the positions that were differentially bound in two cell types existed in Enh chromatin in both cell types examined. These observations suggest that additional factors restrict CSL binding to a subset of sites located within favourable chromatin. Such factors might include currently unknown histone modifications, protein-protein interactions, 3D organisation and/or DNA sequence properties around the CSL motif (Skalska, 2015).

Once bound, Su(H) itself also helps to shape the local chromatin environment. Depleting cells of Su(H) resulted in an increase in local histone acetylation (H3K27ac, H3K56ac), suggesting that, in the absence of NICD, Su(H) helps to suppress enhancer activity through its association with co-repressors. Thus, a model emerges in which Su(H) is recruited to regions that have already acquired regulatory competence and that it keeps these in a transitional state with low levels of H3K56ac. As there is considerable variability between enhancers, this suggests that each attains an activity that reflects the balance between the transcription factors promoting enhancer activity and those, such as Su(H), that can antagonise it. In those instances where Su(H)-corepressor complexes win out, then the enhancer is suppressed until the complimentary activity of NICD converts it from a transitional to an active state, a conversion that is associated with a large-scale increase in H3K56ac (Skalska, 2015).

The extent that the principles observed in this study will be of general relevance for other signalling pathways remains to be established, although it seems likely that their target gene specificity will be similarly dependant on the pre-existing chromatin substrate. However, it is possible that the inferred transitional enhancer states may be particularly relevant for those pathways/contexts where there is a fine-scale switch between repression and activation, as occurs for Notch and ecdysone signalling. Nevertheless, the correlation of H3K56ac with H3K4me1 suggests that H3K56ac is likely to be of widespread importance in enhancer activation. Whether this will be mediated through its direct effects on DNA-histone core interactions or through intermediate bromodomain containing proteins that link to the core transcription machinery, such as Brd4, remains to be determined (Skalska, 2015).


REGULATION

Targets of Activity

The dorsal ectoderm of the Drosophila embryo is subdivided into different cell types by an activity gradient of two TGFbeta signaling molecules, Decapentaplegic and Screw. Patterning responses to this gradient depend on a secreted inhibitor, Short gastrulation and a newly identified transcriptional repressor, Brinker, which are expressed in neurogenic regions that abut the dorsal ectoderm. The expression of a number of Dpp target genes has been examined in transgenic embryos that contain ectopic stripes of Dpp, Sog and Brk expression. These studies suggest that the Dpp/Scw activity gradient directly specifies at least three distinct thresholds of gene expression in the dorsal ectoderm of gastrulating embryos. Brk was found to repress two target genes, tailup/islet (tup) and pannier, that exhibit different limits of expression within the dorsal ectoderm. These results suggest that the Sog inhibitor and Brk repressor work in concert to establish sharp dorsolateral limits of gene expression. Evidence is provided that the activation of Dpp/Scw target genes depends on the Drosophila homolog of the CBP histone acetyltransferase (Ashe, 2000).

Previous studies have identified mutations in the Drosophila homolog of the mammalian CBP histone acetyltransferase gene, nejire. nej is maternally expressed so that the detection of early patterning defects depends on the analysis of embryos derived from females containing nej germline clones. The complete loss of nej+ activity results in a failure to make mature eggs. However, it is possible to obtain embryos from a strong hypomorphic allele, nej1. These embryos exhibit dorsoventral patterning defects. Recent studies have shown that CBP interacts with Smad proteins including the Drosophila protein Mad, a transcription factor downstream of Dpp signaling. In nej mutant embryos, there is a loss of the amnioserosa and other derivatives of the dorsal ectoderm. The expression of target genes requiring peak levels of Dpp signaling is essentially abolished. For example, hnt expression is lost in the presumptive amnioserosa, but persists at the posterior pole where it might be separately regulated by the torso signaling pathway (Ashe, 2000).

There is a similar loss of the dorsal rho pattern in mutant embryos. In contrast, the lateral, neurogenic stripes are unaffected, indicating that the nej mutant does not cause defects in the patterning of the neurogenic ectoderm. Moreover, the fact that the rho stripes are excluded from ventral regions, as seen in wild-type embryos, suggests that the patterning of the mesoderm is also normal. Thus, the nej mutation does not appear to cause a general loss of transcriptional activation, but instead results in specific patterning defects in the dorsal ectoderm. Target genes that are activated by lower levels of Dpp signaling such as ush and pnr are also affected by the nej mutation. In the case of ush, there is a loss of staining in central regions of the dorsal ectoderm. Moreover, the residual staining pattern is narrower than the wild-type pattern. This is reminiscent of the ush pattern seen in dpp/+ heterozygotes. However, the nej mutation also causes a narrowing of the pnr pattern, whereas expression is normal in dpp/+ embryos (Ashe, 2000).

dpp expression has been examined in two groups of dorsal ectoderm cells at the posterior end of the embryo, in abdominal segment 8 and the telson. These dpp-expressing cells become tracheal cells in the posterior-most branches of the tracheal system (Dorsal Branch10, Spiracular Branch10, and the Posterior Spiracle). These branches are not identified by reagents typically used in analyses of tracheal development, suggesting that dpp expression confers a distinct identity upon posterior tracheal cells. dpp posterior ectoderm expression begins during germ band extension and continues throughout development. The sequences responsible for these aspects of dpp expression have been isolated in a reporter gene. An unconventional form of Wingless (Wg) signaling, Dpp signaling, and the transcriptional coactivator Nejire (CBP/p300) are required for the initiation and maintenance of dpp expression in the posterior-most branches of the tracheal system. These data suggest a model for the integration of Wg and Dpp signals that may be applicable to branching morphogenesis in other developmental systems (Takaesu, 2002).

During early stages of embryogenesis, wg and dpp are expressed in undifferentiated dorsal ectoderm. wg mRNA expression, in 15 stripes along the entire dorsal-ventral axis of the embryo (including the dorsal ectoderm), begins at stage 8. wg expression persists in this striped pattern through stage 17. dpp mRNA is expressed on the dorsal side of the embryo along the entire anterior-posterior axis, beginning at stage 4. dpp mRNA expression persists in a large portion of the dorsal ectoderm through stage 8 and resolves into leading edge cell-specific expression in stage 12 embryos. At this time, the embryonic expression pattern of nej has not been reported. However, some information can be obtained from nej mutant phenotypes. nej zygotic mutant embryos show visible defects in the tracheal system at stage 12. The tracheal system is derived from the dorsal ectoderm, suggesting that nej is expressed in this tissue prior to stage 12 (Takaesu, 2002).

dpp expression in posterior tracheal branch anlagen appears to be initiated by prior episodes of wg and dpp expression in the undifferentiated dorsal ectoderm. The maintenance of dpp expression in posterior tracheal branches appears to require continuous input from wg and from a dpp feedback loop. The initiation and maintenance of dpp expression in posterior tracheal branches also requires continuous nej activity. Overall, the data are consistent with the following combinatorial signaling model. The transcriptional activator Med (signaling for the Dpp pathway) interacts with the transcriptional activator Arm (signaling for the Wg pathway) via the transcriptional coactivator Nej. This multimeric complex initiates and, with continuous signaling, maintains dpp expression in posterior tracheal branches with the help of Zw3. These data extend previous studies of dpp expression and Dpp signaling in several ways. nej has been reported to participate in Dpp signaling. Expression from Dpp responsive enhancers is reduced in nej zygotic mutant embryos. While they show that nej3 enhances dpp wing phenotypes, this study shows that Med1 enhances nej3 embryonic phenotypes. The Dorsal Trunk Branch forms normally in Mad12 zygotic mutant embryos, and the Dorsal Trunk Branch appears normal in Med1 mutants. nej is involved in mediating combinatorial signaling by the Wg and Dpp pathways and the involvement of nej in morphogenesis of Dorsal Branch, Spiracular Branch, and the Posterior Spiracle is demonstrated. A region of the histone acetyltransferase domain of Nej binds to Mad. Further study is needed to reveal the mechanisms used by Nej to interact with Wg and Dpp signaling. Several questions remain about the regulation of dpp expression by Wg, Dpp, and Nej. Two questions arise about the mechanism of signal integration: how is zw3 involved and how is Nej recruited to bridge the two pathways? It is tempting to speculate that, in response to a Wg or a Dpp signal, Zw3 (a serine-threonine kinase) is involved in Nej recruitment. Numerous studies have shown that p300/CBP transcriptional coactivation functions are stimulated by its phosphorylation, but the site of phosphorylation has never been mapped. Other questions concern the molecular nature of the enhancers that direct dpp expression in the posterior tracheal branches. A 54-nucleotide region has been identified that contains two sets of conserved, overlapping consensus binding sites for dTCF and Mad/Med. Analyses of DNA-protein interactions predicted by the data involving this candidate combinatorial enhancer have begun (Takaesu, 2002).

From a broader perspective, mammalian homologs of Dpp and orthologs of Wg are important in branching morphogenesis in a variety of developing tissues. For example, BMP2 is involved in renal branching and Wnt4 plays a role in mammary gland branching. The widespread use of TGF-ß and Wnt signals in branching suggests that a greater understanding of the regulation of dpp tracheal expression and dpps role in specifying the unique identities of posterior tracheal branches will have wide relevance (Takaesu, 2002).

Rapid induction of the Drosophila melanogaster heat shock gene hsp70 is achieved through the binding of heat shock factor (HSF) to heat shock elements (HSEs) located upstream of the transcription start site. The subsequent recruitment of several other factors, including Spt5, Spt6 and FACT, is believed to facilitate Pol II elongation through nucleosomes downstream of the start site. This study reports a novel mechanism of heat shock gene regulation that involves modifications of nucleosomes by the TAC1 histone modification complex. After heat stress, TAC1 is recruited to several heat shock gene loci, where its components are required for high levels of gene expression. Recruitment of TAC1 to the 5'-coding region of hsp70 seems to involve the elongating Pol II complex. TAC1 has both histone H3 Lys 4-specific (H3-K4) methyltransferase (HMTase) activity and histone acetyltransferase activity through Trithorax (Trx) and CREB-binding protein (CBP), respectively. Consistently, TAC1 is required for methylation and acetylation of nucleosomal histones in the 5'-coding region of hsp70 after induction, suggesting an unexpected role for TAC1 during transcriptional elongation (Smith, 2004).

Improved activities of CREB binding protein, heterogeneous nuclear ribonucleoproteins and proteasome following downregulation of noncoding hsromega transcripts help suppress poly(Q) pathogenesis in fly models

Following earlier reports on modulation of poly(Q) toxicity in Drosophila by the developmentally active and stress-inducible noncoding Hsromega gene (Heat shock RNA ω), possible mediators of this modulation were investigated. RNAi-mediated downregulation of the large nuclear hsromega-n transcript, which organizes the nucleoplasmic omega speckles, suppresses the enhancement of poly(Q) toxicity brought about by reduced availability of the heterogeneous nuclear ribonucleoprotein (hnRNP) Hrb87F and of the transcriptional regulator, cAMP response element binding (CREB) binding protein (CBP). Levels of CBP RNA and protein are reciprocally affected by hsromega transcript levels in eye disc cells. The data suggest that CBP and hnRNPs like Hrb57A and Hrb87F physically interact with each other. In addition, downregulation of hsromega transcripts partially rescues eye damage following compromised proteasome activity, while overexpression of hsromega and/or poly(Q) proteins disrupts the proteasomal activity. Rescue of poly(Q) toxicity by hsromega-RNAi requires normal proteasomal function. It is suggested that hsromega-RNAi suppresses poly(Q) toxicity by elevating cellular levels of CBP, by enhancing proteasome-mediated clearance of the pathogenic poly(Q) aggregates, and by inhibiting induced apoptosis. The direct and indirect interactions of the hsromega transcripts with a variety of regulatory proteins like hnRNPs, CBP, proteasome, Drosophila inhibitor of apoptosis protein 1 (DIAP1), etc., reinforce the view that the noncoding hsromega RNA functions as a 'hub' in cellular networks to maintain homeostasis by coordinating the functional availability of crucial cellular regulatory proteins (Mallik, 2010).

Earlier studies have shown that while overexpression of the noncoding hsrω transcripts enhanced (Sengupta, 2006), reducing the cellular levels of these transcripts through RNAi suppressed the neurodegeneration caused by mutant proteins with expanded poly(Q) stretches (Mallik, 2009a). As shown earlier (Mallik, 2009a), expression of the hsrω-RNAi transgene had no effect on poly(Q) transcription but it diminished/eliminated the source of toxicity by inhibiting formation of the inclusion bodies (IBs) and by enhancing clearance of the mutant poly(Q) proteins. The present study provides useful insights into the possible mechanisms through which these noncoding transcripts modulate cellular toxicity of the mutant poly(Q) proteins (Mallik, 2010).

Studies in a variety of poly(Q) model systems have reported that many essential cellular proteins, e.g., transcription factors like CBP, TBP; chaperone proteins, etc., are sequestered by the expanded poly(Q) proteins. In agreement with earlier reports, the present study shows that the poly(Q) damage is enhanced by functional depletion of hnRNPs, CBP, or proteasome components because of expression of dominant-negative mutants or RNAi or null mutations. hsrω-RNAi substantially rescued the poly(Q) toxicity even when additional damage was caused by the presence of mutant alleles of Hrb87F or CBP. In contrast, compromised proteasome activity affected the rescue of poly(Q) damage by hsrω-RNAi (Mallik, 2010).

It is significant that while complete absence of Hrb87F does not affect normal development of Drosophila melanogaster, ~20% reduction in cellular levels of Hrb87F, as seen in the Df(3R)Hrb87F/+ eye discs, resulted in a significant enhancement in the poly(Q) eye phenotype. As noted earlier, the mutant poly(Q) proteins deplete the functional availability of many essential proteins and thus disrupt cellular homeostasis. Therefore, even a 20% depletion of the otherwise dispensable Hrb87F exaggerates the poly(Q) toxicity. Mallik (2009a) has shown that hsrω-RNAi results in disappearance of the omega speckles so that the various proteins, including Hrb87F, sequestered in them become available in the soluble cellular pool. The increased availability of such essential proteins in the functional pool following hsrω-RNAi compensates not only for the genetic deficiency of Hrb87F but also for the functional depletion of this and other proteins by the poly(Q) IBs. This finds support in the fact that targeted overexpression of hnRNP A2/B1 and its Drosophila homologs, Hrb87F and Hrb98DE, suppresses CGG repeat-induced neurodegeneration in the FXTAS fly model (Sofola, 2007). It has recently been shown (Ji, 2009) that poly(ADP) ribosylation and deglycosylation of hnRNPs modulate their activity and their binding with the hsrω transcripts; it was suggested that only nonribosylated hnRNPs can be sequestered by these transcripts. It is, therefore, likely that the release of hnRNPs from the omega speckles following hsrω-RNAi provides for a greater pool of the hnRNPs being available for ribosylation and thus activity (Mallik, 2010).

CBP is one of the important regulators of chromatin structure and transcription and its sequestration by the mutant poly(Q) proteins is believed to be a major cause for neurodegeneration (Li, 2004; Bae, 2005). It is also reported that overexpressing CBP or enhancing its activity suppresses poly(Q) IB formation and neurodegeneration (Taylor, 2003). The findings that developmental defects in eyes caused by expression of dominant-negative forms of CBP or by its depletion through RNAi are rescued by hsrω-RNAi clearly show that the hsrω transcripts can modulate CBP metabolism in eye disc cells. This possibility is confirmed by the finding that levels of CBP transcripts and that of the CBP protein are elevated following hsrω-RNAi and are lowered by hsrω overexpression (Mallik, 2010).

The observations that cellular distributions of hnRNPs, like Hrb87F and Hrb57A, partially overlap with that of CBP and that these proteins are co-immunoprecipitated suggest that CBP interacts with Hrb57A and Hrb87F. Downregulation of hsrω-n RNA results in disappearance of the omega speckles and redistribution of the hnRNPs (Mallik, 2009a). In view of the physical association of these proteins, it is speculated that the enhanced availability of hnRNPs in the diffuse cellular pool may pull more CBP into the diffuse fraction so that a greater amount of CBP becomes available for activity rather than remaining stored/sequestered. Additionally, caspase-6-mediated cleavage and degradation of CBP followed by a subsequent decrease in histone acetylation, another critical step common to several neuropathologies, may also be inhibited by hsrω-RNAi since other studies showed that hsrω-RNAi inhibits caspase activity through stabilization of DIAP1 via its interaction with Hrb57A (Mallik, 2009b). The levels of hsrω-n transcripts may also affect CBP mRNA levels through the variety of RNA-processing and transcription factors that directly or indirectly associate with the hsrω transcripts. As reported earlier, the net increase in CBP levels, following hsrω-RNAi, would inhibit formation of poly(Q) IBs and restore the histone acetylation homeostasis (Mallik, 2010).

It is remarkable that while hsrω-RNAi suppressed the eye phenotypes resulting from expression of CBP-FL AD or CBP RNAi or CBP DeltaNZK, it failed to rescue the lethality or the eye damage following expression of CBP DeltaQ or CBP DeltaBHQ, respectively. This indicates that the transactivation domain of CBP is required for the suppressive action of hsrω-n RNAi. It is likely that the hnRNPs like Hrb87F, Hrb57A, etc., interact with CBP through its Q domain so that the hnRNPs released by disappearance of the omega speckles following hsrω-RNAi fail to compensate the damage caused by expression of dominant-negative CBP DeltaQ or CBP DeltaBHQ. Further studies are required to understand the mechanism(s) of these interactions (Mallik, 2010).

These studies also reveal interaction of ubiquitin proteasome pathway (UPP) with hsrω transcripts and an important role of this interaction in the modulation of poly(Q) toxicity. Restoration of the eye phenotype following targeted disruption of the normal proteasomal activity by hsrω-RNAi indicates that the proteasome activity improves when levels of these noncoding transcripts are reduced. This is also confirmed by the direct demonstration, through the GFP-reporter expression, that the intrinsic UPP is compromised in cells overexpressing hsrω. The finding that proteasome activity is impaired in 127Q-expressing flies is consistent with earlier reports that poly(Q) toxicity in vivo is enhanced by proteasome mutations or by inhibitors of proteasome activity. It is significant that hsrω-RNAi ameliorated the proteasomal dysfunction due to poly(Q) expression, since the proteasome-GFP reporter expression was very low in cells coexpressing poly(Q) and hsrω-RNAi. Restoration of proteasome function in mutant poly(Q)-expressing cells is thus an additional pathway through which hsrω-RNAi suppresses the neurodegeneration. This finds further support in the observation that when the endogenous UPP function is intrinsically compromised by expression of dominant-negative mutants, hsrω-RNAi is no longer as effective in suppressing the poly(Q) damage as in cells with normal proteasome function. The mechanism(s) through which the hsrω transcripts regulate UPP pathways remain to be understood (Mallik, 2010).

Many of the poly(Q) proteins involved in CAG repeat expansion disorders contain caspase consensus cleavage sites and caspase-mediated cleavage of the mutant protein appears necessary for pathogenesis (Evert, 2000). Inhibition of activity of caspases like caspase-1, caspase-3, or caspase-8 or alteration of the caspase cleavage sites in the mutated protein delays and reduces the expanded poly(Q) protein pathogenicity. Other studies show that in cells in which apoptosis is ectopically induced, hsrω-RNAi stabilizes DIAP1 through enhanced association with Hrb57A (Mallik, 2009b). Elevated levels of DIAP1 inhibit caspase activity and thus apoptosis. Further, expression of expanded poly(Q) proteins brings about hyperactivation of JNK, which contributes to neuronal dysfunction and cell death in neurodegenerative disorders. Significantly, hsrω-RNAi suppresses activation of the JNK pathway also (Mallik, 2009b). Inhibition of caspase and JNK activities thus appear to be other paths through which hsrω-RNAi suppresses the poly(Q) toxicity in the fly models (Mallik, 2010).

In summary, it is suggested that hsrω-RNAi suppresses poly(Q) toxicity by modulating several components involved in the pathogenesis of these debilitating diseases. First, hsrω-RNAi enhances the availability of hnRNPs and CBP in functional pools. This in turn would suppress IB formation and restore histone acetylation and transcriptional regulation in cells expressing the mutant poly(Q) proteins. Second, the proteasomal activity is improved when hsrω RNA levels are reduced and this helps the cells to get rid of toxic proteins. Third, the release of hnRNPs from omega speckles following hsrω-RNAi stabilizes DIAP1 (Mallik, 2009b), resulting in inhibition of apoptosis so that neuronal cells, that otherwise would have died, survive. Additionally, in view of the above noted role of JNK in poly(Q) damage, the suppression of JNK activation in eye disc cells following hsrω-RNAi (Mallik, 2009b) may also contribute to amelioration of the poly(Q) damage. Further, the hsrω transcripts are known to interact with several other proteins, including Hsp90, and therefore, it remains possible that other network effects also contribute to the observed suppression of the poly(Q) damage. The observed pleiotropic effects reflect involvement and, therefore, critical importance of the hsrω noncoding transcripts in cellular homeostasis. Since most of the wild-type poly(Q) proteins, whose mutations result in neurodegeneration, are themselves involved in diverse regulatory processes, alterations in the noncoding hsrω transcript pool can be expected to bring about unpredictable and divergent consequences in cells with genetically compromised regulation. These transcripts apparently function as hubs for coordination of several cellular networks and thus ensure homeostasis. Such multiple networking interactions provide a basis for the context-dependent actions of the same molecule in different cells or in the same cell under different conditions. The multipronged action of these noncoding transcripts also provides a new paradigm for a therapeutic target for the human poly(Q) disorders (Mallik, 2010).

Protein Interactions

T-cell factor (TCF), a high-mobility-group domain protein, is the transcription factor activated by Wnt/Wingless signaling. When signaling occurs, TCF binds to its coactivator, beta-catenin/Armadillo, and stimulates the transcription of the target genes of Wnt/Wingless by binding to TCF-responsive enhancers. Inappropriate activation of TCF in the colon epithelium and other cells leads to cancer. It is therefore desirable for unstimulated cells to have a negative control mechanism to keep TCF inactive. Drosophila CREB-binding protein (dCBP) binds to Drosophila TCF (Pangolin). dCBP mutants show mild Wingless overactivation phenotypes in various tissues. Consistent with this, dCBP loss-of-function suppresses the effects of armadillo mutation. Moreover, dCBP is shown to acetylate a conserved lysine in the Armadillo-binding domain of dTCF, and this acetylation lowers the affinity of Armadillo binding to dTCF. Although CBP is a coactivator of other transcription factors, these data show that CBP represses TCF (Waltzer, 1998).

Attempts to demonstrate trans-activation activity by the Drosophila Myb gene product have been unsuccessful so far. Co-transfection of Schneider cells with a plasmid expressing the Drosophila homolog of transcriptional co-activator CBP (dCBP) results in transactivation by Myb. Using this assay system, the functional domains of Myb have been analyzed. Two domains located in the N-proximal region, one of which is required for DNA binding and the other for dCBP binding, are both necessary and sufficient for trans-activation. In this respect, D-Myb is similar to c-Myb and A-Myb, but different from mammalian B-Myb. These results shed light on how the myb gene diverged during the course of evolution (Hou, 1997).

In the visceral mesoderm, dpp is expressed in parasegment (ps) 7 under the control of the homeotic gene Ultrabithorax (Ubx). In this cell layer, dpp stimulates its own expression and the expression of Ubx. dpp also stimulates the expression of wingless (wg), an extracellular signaling molecule of the Wnt family, in the neighbouring ps8. wg in turn feeds back to stimulate Ubx and dpp expression in ps7. Thus, dpp is part of a parautocrine feedback loop by which Ubx maintains its own expression indirectly through controlling dpp and wg. Dpp also diffuses from its mesodermal source through the endodermal cell layer of the embyonic midgut, where it stimulates the expression of D-Fos and of the homeotic gene labial. These inductive steps ultimately specify the differentiation of distinct cell types in the larval midgut epithelium. In order to understand the mechanism by which dpp stimulates transcription, a short enhancer fragment of Ubx, called Ubx B, has been characterized that contains response sequences for dpp and wg signaling in the embryonic midgut. The dpp response sequence of this enhancer is bipartite, consisting of a tandem repeat of Mad binding sites and a cAMP response element (CRE). The presence of the latter raised the question whether the co-activator CBP (CREB-binding protein, binding to CREs) might participate in Dpp-induced transcriptional activation (Waltzer, 1999).

Drosophila CBP loss-of-function mutants show specific defects that mimic those seen in mutants that lack the extracellular signal Dpp or its effector Mad. CBP loss severely compromises the ability of Dpp target enhancers to respond to endogenous or exogenous Dpp. CBP binds to the C-terminal domain of Mad. These results provide evidence that CBP functions as a co-activator during Dpp signaling, and they suggest that Mad may recruit CBP to effect the transcriptional activation of Dpp-responsive genes during development (Waltzer, 1999).

The embryonic midgut of nejire (nej) mutants (whose CBP function is reduced) show phenotypes related to wg gain-of-function phenotypes: increased labial expression in the endoderm, and derepression of the Ubx B enhancer in the visceral mesoderm. These phenotypes do not resemble those seen in dpp or Mad mutants: in Mad mutants, labial expression is strongly reduced, and so is the beta-galactosidase (lacZ) staining mediated by the Ubx B enhancer in the middle midgut. However, the narrow band of lacZ staining normally visible in the visceral mesoderm of the gastric caeca (in ps3) is absent in nej mutant embryos. Indeed, closer inspection reveals that the gastric caeca frequently fail to elongate in these mutants. A similar phenotype is observed in Mad and in dpp mutants. Thus nej, like dpp, is required for the formation of the gastric caeca, and also for the activity of the Ubx B enhancer in the caecal primordia. The activity of this enhancer in these primordia coincides with Dpp expression and depends on dpp function. The formation of the first midgut constriction is often impeded. While this could reflect overactive Wg signaling, it also mimics loss of glass bottom boat (gbb) signaling: Gbb is a Dpp homolog expressed in the visceral mesoderm and whose function is required for the formation of the first midgut constriction (Waltzer, 1999).

The hypothesis that CBP is a co-activator of dpp-induced transcription was tested by examining the Dpp response of the Ubx enhancer in nej mutants. Because it was expected that the repressive effect of CBP on this enhancer would mask a possible activating effect of CBP in cells in which the enhancer is stimulated by Wg signaling, a mutant version of Ubx B, called B4, was used whose positive response to Wg is abolished. B4 activity in the midgut is reduced compared with the wild-type enhancer; however, B4 still contains a fully functional dpp-response sequence and can be efficiently stimulated by ectopic Dpp. B4 can thus be used to selectively monitor the stimulation of Ubx by Dpp in the visceral mesoderm. The activity of Ubx B4 is significantly reduced in nej mutants. LacZ staining is particularly weak in ps6/7 (near the Dpp source), but also in ps10, and is barely detectable in the gastric caeca. Furthermore, in nej mutant embryos derived from nej mutant germlines (nej), lacZ staining mediated by B4 is even weaker than in the zygotic nej mutants: although these nej GLC embryos are somewhat variable in terms of their phenotypes the most severely mutant embryos show lacZ staining in only a few cells in the ps8 region. Similarly, in Mad12 mutant embryos, lacZ staining is much reduced, with some staining remaining in ps6 and ps8. This implies that CBP, like Mad, is required for the Dpp response of the Ubx B4 enhancer (Waltzer, 1999).

The response of B4 to GAL4-mediated ectopic Dpp was examined in nej mutant embryos. If Dpp is expressed throughout the mesoderm, B4-mediated lacZ staining is increased and detectable throughout the midgut mesoderm. In nej mutants, this response of B4 to ectopic Dpp is strikingly disabled: there is barely any lacZ staining in the anterior midgut, and only a moderate increase of lacZ staining in the ps8/9 region, indicating a residual Dpp response in this region. These results strongly support the conclusion that CBP is required for the transcriptional response of the Ubx enhancer to Dpp signalling. They argue that CBP functions downstream of the Dpp signal (Waltzer, 1999).

In the early blastoderm embryo, dpp mediates the subdivision of the dorsal ectoderm into two embryonic tissues: the amnioserosa and the dorsal epidermis. High Dpp levels in the dorsal-most cells specify amnioserosa while lower Dpp levels in dorsolateral regions specify epidermis. Expression of the gene Race (related to angiotensin converting enzyme; the earliest known marker for the amnioserosa) in the dorsal blastoderm embryo depends on dpp signaling. Thus it was asked whether the activity of the Race enhancer depends on CBP function. This enhancer mediates lacZ staining in the presumptive amnioserosa and in the anterior midgut primodium: the former, but not the latter, staining requires dpp. In nej GLC embryos, there is no detectable lacZ staining in the presumptive amnioserosa, although staining remains, and is even slightly enhanced, in the head and in the anterior midgut primordium. This demonstrates that the Race enhancer depends on an activating function of CBP exclusively in a subset of the blastoderm cells, namely in the dorsal-most cells of the embryonic trunk. It suggests that CBP is required for the response of this enhancer to dpp (Waltzer, 1999).

To see whether CBP may be required in other developmental contexts in which dpp functions, the developing tracheae were examined in nej mutant embryos. The tracheal system develops from segmentally repeated clusters of ectodermal cells, the tracheal placodes. These cells undergo a complex process of migration and fusion to generate the final branched structure of the tracheal system. dpp signaling plays a crucial role in this process, and has been implicated in the dorsoventral migration of certain tracheal branches. For example, in punt or thick veins mutants, the branches that normally migrate dorsally or ventrally (the dorsal and ganglionic branches, respectively) fail to develop, whereas the branches that grow out anteriorly (the dorsal trunk and the visceral branches) are essentially not affected. The tracheae in nej mutant embryos were examined using an antibody that stains the lumina of the tracheal trees (2A12). The dorsal trunk and the visceral branches are essentially normal in these mutants. However, in most nej mutant embryos, branching defects are seen: usually, one or two dorsal branches fail to form at each side, and ganglionic branches fail to fuse. Essentially the same defects are also seen in in nej GLC embryos. These defects resemble those found in punt hypomorphs and in Mad12 mutant embryos, although the most apparent defects in the latter mutants are the fusion defects in their ganglionic branches. Once again, the similarity of the tracheal phenotypes of nej mutants when compared to dpp, punt and Mad mutants suggests that CBP may be required during Dpp signaling (Waltzer, 1999).

dpp promotes vein development during pupal stages, and a subclass of dpp mutant alleles cause loss of veins. In particular, in dppS1 homozygous flies, vein 4 fails to reach the margin. This weak dpp allele was exploited to see whether there would be a genetic interaction between dpp and nej. Indeed, while nej heterozygosity on its own shows no abnormality whatsoever in the wing, this condition clearly enhances the vein phenotype of dppS1 homozygotes: in many of the wings from flies of this genetic constitution, neither vein 4 nor vein 2 reaches the margin. This synergy in the wing between nej and dpp loss-of-function alleles is consistent with the notion that CBP functions during Dpp signaling. To clarify the position of CBP in this Dpp response in the wing, it was asked whether the mild dpp overactivation phenotype due to overexpression of a constitutively active form of Sax (Sax*), a Dpp type I receptor, depends on nej gene dosage. Expression of Sax* under the control of engrailed.GAL4 induces ectopic venation and overgrowth of the posterior part of the wing. Moreover, removal of one copy of genes required for Sax signaling, such as Mad or Medea suppresses this phenotype. Likewise, nej heterozygosity suppresses to a considerable extent the wing phenotypes caused by Sax*. This result is consistent with CBP being required for Sax signaling, and it indicates that CBP functions downstream of this Dpp receptor (Waltzer, 1999).

Since Mad mediates transcriptional activation by dpp and appears to be a transcription factor required for every aspect of dpp signaling, it was asked whether CBP might be recruited by Mad as a transcriptional co-activator. To test whether CBP might bind to Mad, the yeast two-hybrid system was used. When these binding studies were begun, Drosophila CBP had not yet been discovered. So fragments of mouse CBP were used to test whether these might bind to Mad, assuming that a putative interaction between the two proteins would be conserved. Indeed, there is a strong degree of homology between mouse and Drosophila CBP. A set of fragments of mouse CBP were used that cover the whole protein and these were fused to a transcriptional activation domain (the 'prey'). This series of prey was tested in two-hybrid assays in yeast with full-length Mad protein fused to the LexA DNA-binding domain (the 'bait'). The C-terminal domain of mouse CBP (CBP1678 to 2441) interacts specifically with Mad in this assay. This interaction was confirmed using a similar set of prey with fragments from the Drosophila CBP protein, which was tested against a series of baits containing different Mad domains. This reveals that a fragment of Drosophila CBP that spans amino acids 2240-2608 (which overlaps the above mentioned C-terminal domain of mouse CBP) interacts specifically with the MH2 domain of Mad (Mad219-455). These specific interactions in the yeast two-hybrid assay between CBP fragments and Mad almost certainly reflect direct binding since yeast does not encode any proteins homologous to either of these. Interactions between CBP fragments and Mad are significantly stronger if the N-terminal domain of Mad is removed, suggesting that MH1 inhibits the binding of CBP to MH2. Inhibitory interactions between MH1 and MH2 have been described previously (Waltzer, 1999).

To confirm these results, direct binding between Mad and CBP in vitro were tested with pull-down assays. In these assays, [35S]methionine-labelled Mad domains and various fragments from Drosophila CBP expressed as GST fusion proteins and immobilized on GST-Sepharose beads were used. Either full-length Mad or its MH2 domain binds to the same Drosophila CBP fragment that interacts with Mad in the yeast assay while Mad's MH1 domain does not bind CBP. Interestingly, deletion of the C-terminal of Mad's MH2 domain (amino acids 372-455) abolishes CBP interaction, demonstrating that the C-terminal 84 amino acids of Mad are required for binding to CBP. The linker domain (L) between MH1 and MH2 seems to be dispensable for Mad interaction with CBP. Weak binding of full-length Mad to CBP2 (CBP2240-2507), the highly conserved domain of Drosophila CBP that overlaps the Mad-binding fragment of CBP, was observed. However, the significance of this binding is uncertain, as this binding activity could not be detected in the reciprocal assay nor in yeast. Finally, to characterize more precisely the mutual binding domains within Mad and Drosophila CBP, the reciprocal experiment was performed using [35S]methionine-labelled C-terminal fragments of CBP and various GST-Mad fusion proteins. CBP binds to GST-MH2, but not to GST alone, nor to GST-MH1+L fusion proteins, which include extended MH1 fragments (Mad1-241), nor to GST-MH2C fusion protein in which the last 84 amino acids of Mad's MH2 domain are deleted. Deletion mapping of the C-terminal region of CBP reveals a minimal fragment of CBP (CBP2413-2608) that is sufficient for binding to Mad. This domain partially overlaps the highly conserved CBP2 domain but in most binding assays, CBP2 by itself is not sufficient for binding to Mad. Altogether, these experiments demonstrate that CBP and Mad bind to one another, and that the stretch between amino acids 2507 and 2640 within Drosophila CBP is critical for CBP's binding to the MH2 domain of Mad (Waltzer, 1999).

tinman encodes an NK-2 class homeodomain transcription factor that is required for development of the Drosophila dorsal mesoderm, including heart. Genetic evidence suggests its important role in mesoderm subdivision, yet the properties of Tinman as a transcriptional regulator and the mechanism of gene transcription by Tinman are not completely understood. Tinman can activate or repress target genes in cultured cells, based on evaluation of functional domains that are conserved between the tinman genes of Drosophila melanogaster and Drosophila virilis. Using GAL4-tinman fusion constructs, a transcriptional activation domain (amino acids 1-110) and repression domains (amino acids 111-188 and the homeodomain) have been mapped and an inhibitory function for the homeodomain has been found upon transactivation by Tinman (Choi, 1999).

Tinman is regulated by Twist and autoregulates its own promoter. The properties of Tin as a transcription factor were assessed using tinman P1 and P1E2m promoters and truncated forms of the Tin expression vectors (d8 and d6). The P1 reporter contains Tin-responsive elements (the E2 cluster) and is activated by Tin. The P1E2m reporter contains mutated Tin binding sites but otherwise is exactly the same as the wild-type P1 reporter, which also contains several weak Tin binding sites. Tin can activate the P1 reporter (6-fold activation). In contrast, Tin down-regulates the P1E2m reporter gene (3-fold repression). In this case, Tin binds to weak binding sites and represses the P1E2m reporter gene. These results indicate that Tin can act either as a transcriptional activator or repressor, depending on the context of the reporters (P1 or P1E2m). These phenomena are dependent on the functional domains of Tin. For example, deletion of the amino-terminal region of Tin abrogates activation of the P1 reporter, indicating that the amino terminus (aa 1-110) of Tin is required for transcriptional activation. Indeed, this Tin mutant (d8) represses gene expression of both the P1 and the P1E2m reporter. Further deletion of Tin (construct d6) relieves this repression, irrespective of the reporter gene used. These results suggest that the region following the amino terminus of Tin (aa 111-188) is required for the repressor activity of Tin. Taken together, these results indicate that, depending on the context of the target genes (for example P1 or P1E2m), Tin can act as either a transcriptional activator or repressor and that these different transcriptional activities are dependent on functional domains of Tin (Choi, 1999).

Tinman-dependent transactivation is augmented by the p300 coactivator; Tinman physically interacts with p300 via the activation domain. In addition, cotransfection experiments indicate that the repressor activity of Tinman is strongly enhanced by the Groucho corepressor. Using immunoprecipitation and in vitro pull-down assays, Tinman is shown to directly interact with the Groucho corepressor, for which the homeodomain is required. Together, these results indicate that Tinman can act as either a transcriptional activator or repressor. The first evidence of Tinman interactions with the p300 coactivator and the Groucho corepressor is provided (Choi, 1999).

CREB-binding protein (CBP) is a coactivator for multiple transcription factors that transduce a variety of signaling pathways. Current models propose that CBP enhances gene expression by bridging the signal-responsive transcription factors with components of the basal transcriptional machinery and by augmenting the access of transcription factors to DNA through the acetylation of histones. To define the pathways and proteins that require CBP function in a living organism, a genetic analysis of CBP has been carried out in flies. Drosophila CBP (dCBP) was overproduced in a variety of cell types and distinct adult phenotypes were obtained. An uninflated-wing phenotype, caused by the overexpression of dCBP in specific central nervous system cells, was used to screen for suppressors of dCBP overactivity. Two genes with mutant versions that act as dominant suppressors of the wing phenotype were identified: the PKA-C1/DCO gene, encoding the catalytic subunit of cyclic AMP protein kinase, and ash1, a member of the trithorax group (trxG) of chromatin modifiers. Using immunocolocalization, it has been shown that the Ash1 protein is specifically expressed in the majority of the dCBP-overexpressing cells, suggesting that these proteins have the potential to interact biochemically. This model was confirmed by the findings that the proteins interact strongly in vitro and colocalize at specific sites on polytene chromosomes. The trxG proteins are thought to maintain gene expression during development by creating domains of open chromatin structure. One model for the function of CBP and p300 is bridging DNA binding transcription factors to components of the basal transcriptional machinery. These results thus suggest a second model for dCBP function, namely interaction with trxG proteins, and imply that dCBP might be involved in the regulation of higher-order chromatin structure (Bantignies, 2000).

Screens for enhancers and suppressors of overexpression phenotypes have been useful in identifying components of regulatory pathways. Nevertheless, overexpression systems have drawbacks and can potentially identify secondary effectors of a nonspecific phenotype. However, it is thought that this screen has identified genes that affect dCBP function for several reasons. (1) The number of deficiencies that suppress the uninflated-wing phenotype is small. A large number of suppressors might suggest that the overexpression of dCBP is not eliciting a specific cell phenotype. (2) Two of the deletions suppress both the wing and the eye overexpression phenotypes, suggesting that the overexpression of dCBP in the two tissues has some common effects. One of the deletions demonstrates that the dosage of PKA can affect the dCBP overexpression phenotype. CBP and dCBP are known to play a role in PKA signaling, so the fact that PKA was identified in this screen is consistent with the idea that dCBP overexpression reflects an overactivation of the PKA pathway. Trivial explanations for the suppression of dCBP overexpression by ASH1 have been ruled out; dCBP overexpression does not cause the death of ASH1-expressing cells, nor do ash1 mutations affect the overexpression of dCBP. A characterization of dCBP loss of function in these cells both in wild-type and ash1 mutant backgrounds is necessary to complete this analysis. A clonal analysis of dCBP mutant cells is not feasible because dCBP is required for cell viability and only small clones can be generated. This analysis will have to await reagents that allow dCBP function to be knocked out in the GAL4-386 cells in the ash1 mutant background. In addition, it will be important to identify the targets of dCBP and ASH1 in these cells as well as the pathways that activate them. Although the genetic analysis is not complete, it is likely that the genetic suppression of dCBP overexpression by ash1 mutations reflects a functional association between ASH1 and dCBP because these two proteins have specific interactions in vitro (Bantignies, 2000).

Overexpression of dCBP in specific CNS cells causes wing inflation defects. In many tissues, overexpression of dCBP causes lethality, suggesting that the dose of this effector is important for its function. The overproduction of dCBP in specific cells of the CNS with two different GAL4 lines produces defects in wing inflation with various degrees of penetrance. However, overexpression of dCBP in wing tissues throughout development does not interfere with wing inflation (Bantignies, 2000).

Previous studies have implicated specific CNS cells in the regulation of wing inflation. In Drosophila, the death of specific cells is triggered after eclosion and is strongly correlated with wing inflation behavior. In addition, two specific neurons in the fly brain are responsible for the production of the neuropeptide eclosion hormone (EH). The specific knockout of EH-producing cells (EH cells) during early development results in eclosion delays and a disruption of eclosion behaviors, such as wing inflation. In the moth Manduca sexta, EH triggers a neuroendocrine cascade that regulates both ecdysis and postecdysis processes such as wing inflation. It was suggested that the frequent failure of EH cell knockout flies to inflate their wings successfully is due to a lack of excitability of neuroendocrine-responsive EH cells that release important signals for proper eclosion behaviors. In Manduca, different neuropeptides, such as bursicon and the cardioacceleratory peptides, are usually released after eclosion to aid in wing expansion. It may be that the neurons that overexpress dCBP are the neurosecretory cells that are targeted by the EH cascade and that produce the peptides that signal the wing inflation process. In this case, the overexpression of dCBP interferes with normal cell function. Of course the wing inflation defect could be due to the death of the neurons caused by the overexpression of dCBP. However, the pattern of cells that overexpress LacZ and dCBP in the GAL4-386 background remains the same throughout development, and cells that overexpress dCBP and express ASH1 are viable at least 24 h posteclosion, so the overexpression of dCBP does not appear to affect the viability of these cells. Two additional GAL4 lines, GAL4-c929 and GAL4-c191, also drive specific expression in the CNS, specifically in most of the peptidergic neurons of the brain and ventral ganglion. At 25°C, escapers were obtained only with the GAL4-c191 line. Approximately 30% of these flies have uninflated or partially inflated wings (Bantignies, 2000).

It is proposed that the overexpression of dCBP in specific CNS cells affects the regulation of signaling pathways that involve dCBP and that are important for proper eclosion behaviors. Preliminary data suggest that at least some of the cells that overexpress dCBP are neuropeptidergic neurons and colocalize with the neuropeptides FMRFamide and PHM. However, antibody incompatibility does not allow for a determination of whether these cells also express ASH1. Clearly, more characterization will be required to determine the exact pathways affected by dCBP. The dominant wing phenotype obtained by overexpressing dCBP with GAL4-386 is a good model to elucidate some of the cells and signaling pathways involved in wing inflation (Bantignies, 2000).

Biochemical experiments show that coactivator dCBP binds strongly to trxG protein ASH1. This observation supports the idea that ASH1 and dCBP interact in vivo and implicates a novel class of chromatin binding proteins in mediating dCBP function. The ASH1 protein contains three motifs that are characteristic of some proteins that regulate transcription and/or are bound to chromosomes: there are two AT hook motifs in the N-terminal region, a SET domain, and a PHD finger in the C-terminal domain. The AT hook motif is important for the binding of some proteins to DNA. PHD fingers are Cys-rich Zn finger-like motifs implicated in protein-protein interactions and are found in other trxG proteins. The SET domain is an approximately 130-aa region found in a number of other chromatin-associated proteins, including the TRX factor, PcG protein Enhancer of Zeste [E(Z)], and the modifier of position effect variegation Suppressor of variegation 3-9. The TRX SET domains have been proposed to mediate association with components of chromatin-remodeling complexes, and ASH1 and TRX interact directly through their SET domains. Binding assays indicate that two N-terminal regions and the SET domain of ASH1 interact strongly with dCBP. However, no interaction with the PHD domain was observed. Thus, the SET and the PHD domains of ASH1 might function for the recruitment of other chromatin-associated proteins, such as TRX, and the N-terminal region could serve to interact with the DNA, possibly through the AT motifs, to direct the targeting of HATs to the promoter. Further biochemical characterization will be necessary to confirm this model, but the interaction between dCBP and ASH1 provides new insights on the possible function of ASH1 in gene regulation (Bantignies, 2000).

The binding of ASH1 to dCBP requires the C-terminal C/H3 domain. In mammalian CBP and p300, this region mediates interactions with numerous sequence-specific transcription factors, the adenovirus E1A protein, TFIIB, RNA helicase A, and P/CAF, a GCN5-like histone acetylase. In dCBP, the C/H3 domain mediates the interaction with transcription factor dTCF and Mad, demonstrating an important role for this domain in dCBP function. This domain contributes to the interaction with chromatin-associated protein ASH1, suggesting that dCBP may function in epigenetic regulatory complexes. The C/H3 domain is adjacent to HAT and might contribute to the regulation of the histone acetylation activity of CBP and p300 or might recruit targets of acetylation close to the enzymatic domain. Thus, it will be interesting to determine whether ASH1 has any effect on dCBP HAT functions or if it is a target of dCBP acetyltransferase activity (Bantignies, 2000).

The bromodomain of P/CAF has been shown to bind histone peptides in an acetylation-dependent manner. The bromodomain of GCN5, a member of the SAGA complex, is required for SWI/SNF remodeling of the nucleosome and stabilizing the SWI/SNF complex on the promoter. Thus, it appears that the bromodomain interacts with acetylated proteins and may form a link between different regulatory complexes. Although the full-length ASH1 does not interact with the bromodomain of dCBP, both the ASH1-458-853 polypeptide and the SET domain do interact with this domain. It may be that full-length ASH1 undergoes a modification, upon binding with the dCBP C/H3 domain, that allows other regions of ASH1 to interact with the dCBP bromodomain. In this case, it would appear that the interaction is not dependent on acetylation (Bantignies, 2000).

These results also show that dCBP and ASH1 colocalized to a number of specific sites on polytene chromosomes, suggesting that they might serve as coregulators of a specific set of genes including the homeotic selector genes. The mapping of the specific sites where dCBP and ASH1 colocalize will help identify target genes that are regulated by ASH1 and dCBP. An analysis of these genes, their promoters, and their regulation by dCBP and ASH1 will further define the functional role of the dCBP-ASH1 interaction (Bantignies, 2000).

The development of Drosophila requires the function of the CREB-binding protein, dCBP. In flies, dCBP serves as a coactivator for the transcription factors Cubitus interruptus, Dorsal, and Mad, and as a cosuppressor of Drosophila T cell factor. Current models propose that CBP, through its intrinsic and associated histone acetyltransferase activities, affects transient chromatin changes that allow the preinitiation complex to access the promoter. Evidence is provided that dCBP may regulate the formation of chromatin states through interactions with the modulo (mod) gene product, a protein that is thought to be involved in chromatin packaging. dCBP and Modulo bind in vitro and in vivo, mutations in mod enhance the embryonic phenotype of a dCBP mutation, and dCBP mutations enhance the melanotic tumor phenotype characteristic of mod homozygous mutants. These results imply that, in addition to its histone acetyltransferase activity, dCBP may affect higher-order chromatin structure (Bantignies, 2002).

Aberrant histone acetylation, altered transcription, and retinal degeneration in a Drosophila model of polyglutamine disease are rescued by CREB-binding protein

Sequestration of the transcriptional coactivator CREB-binding protein (CBP), a histone acetyltransferase, has been implicated in the pathogenesis of polyglutamine expansion neurodegenerative disease. A Drosophila model was used to demonstrate that polyglutamine-induced neurodegeneration is accompanied by a defect in histone acetylation and a substantial alteration in the transcription profile. Furthermore, complete functional and morphological rescue by up-regulation of endogenous Drosophila CBP (dCBP) is demonstrated. Rescue of the degenerative phenotype is associated with eradication of polyglutamine aggregates, recovery of histone acetylation, and normalization of the transcription profile. These findings suggest that histone acetylation is an early target of polyglutamine toxicity and indicate that transcriptional dysregulation is an important part of the pathogenesis of polyglutamine-induced neurodegeneration (Taylor, 2003).

Huntington's disease, spinobulbar muscular atrophy, dentatorubro-pallidoluysian atrophy, and six forms of spinocerebellar ataxia are caused by expansion of CAG trinucleotide repeats, resulting in pathological polyglutamine expansion in the disease proteins. These diseases likely share a common mechanism involving a toxic gain of function by the expanded polyglutamine tract. Evidence indicates that the nucleus is an important site of polyglutamine toxicity and that transcriptional dysregulation may be a primary pathogenic process in polyglutamine disease. Mutant proteins with expanded polyglutamine have been shown to bind and sequester the transcriptional coactivator CREB-binding protein (CBP) in cell culture, animal models, and tissue derived from patients with these diseases. Physical interaction between CBP and mutant protein has been found to depend on the acetyltransferase domain of the former and the polyglutamine tract of the latter. Sequestration of CBP results in reduced acetyltransferase activity and loss of CBP-mediated coactivation in cell culture models of polyglutamine disease. Live-cell dynamic imaging shows that coexpression of GFP-CBP with various proteins with polyglutamine expansions in cell cultures results in not merely colocalization, but functional sequestration of CBP in polyglutamine inclusions. CBP is an important cofactor in transcriptional activation mediated by CREB. A recent report demonstrates that disruption of CREB function leads to progressive neurodegeneration in a pattern similar to that observed in transgenic mouse models of polyglutamine disease. CBP also serves as a cofactor for other transcription factors in addition to CREB, but this finding indicates that disruption of the CREB pathway alone is sufficient to lead to neurodegeneration and further implicates CBP as a target of polyglutamine toxicity (Taylor, 2003 and references therein).

The results presented in this study demonstrate that CBP is a potent modifier of polyglutamine-induced neurodegeneration in vivo and support the hypothesis that histone acetylation may be a target of polyglutamine toxicity. Alterations in CBP activity by sequestration, increased degradation, or decreased expression as seen in this study may contribute to altered acetylation. The loss of polyglutamine aggregation seen with rescue by CBP suggests that CBP itself, as a polyglutamine protein, may directly block accumulation of polyglutamine monomers into aggregates, similar to a blocking peptide, making it more susceptible to degradation. Alternatively, genes regulated by CBP may influence aggregation or accelerate degradation of polyglutamine. The findings presented in this study add to the accumulating evidence that pharmacologics capable of influencing histone acetylation may be of benefit in polyglutamine disease (Taylor, 2003).

CBP-mediated acetylation of histone H3 lysine 27 antagonizes Drosophila Polycomb silencing

Trimethylation of histone H3 lysine 27 (H3K27me3) by Polycomb repressive complex 2 (PRC2) is essential for transcriptional silencing of Polycomb target genes, whereas acetylation of H3K27 (H3K27ac) has recently been shown to be associated with many active mammalian genes. The Trithorax protein (TRX), which associates with the histone acetyltransferase CBP, is required for maintenance of transcriptionally active states and antagonizes Polycomb silencing, although the mechanism underlying this antagonism is unknown. This study shows that H3K27 is specifically acetylated by Drosophila CBP and its deacetylation involves RPD3. H3K27ac is present at high levels in early embryos and declines after 4 hours as H3K27me3 increases. Knockdown of E(Z) decreases H3K27me3 and increases H3K27ac in bulk histones and at the promoter of the repressed Polycomb target gene abd-A, suggesting that these indeed constitute alternative modifications at some H3K27 sites. Moderate overexpression of CBP in vivo causes a global increase in H3K27ac and a decrease in H3K27me3, and strongly enhances Polycomb mutant phenotypes. TRX is required for H3K27 acetylation. TRX overexpression also causes an increase in H3K27ac and a concomitant decrease in H3K27me3 and leads to defects in Polycomb silencing. Chromatin immunoprecipitation coupled with DNA microarray (ChIP-chip) analysis reveals that H3K27ac and H3K27me3 are mutually exclusive and that H3K27ac and H3K4me3 signals coincide at most sites. It is proposed that TRX-dependent acetylation of H3K27 by CBP prevents H3K27me3 at Polycomb target genes and constitutes a key part of the molecular mechanism by which TRX antagonizes or prevents Polycomb silencing (Tie, 2009).

The major findings of this work are: (1) that Drosophila CBP acetylates H3K27; (2) that this acetylation requires TRX; and (3) that it prevents H3K27 trimethylation by E(Z) at Polycomb target genes and antagonizes Polycomb silencing. The remarkably complementary developmental profiles of H3K27ac and H3K27me3 (but not H3K27me2) during embryogenesis suggest that the deposition of H3K27me3, which increases steadily after ~4 hours with the onset of Polycomb silencing, occurs at the expense of a substantial fraction of the H3K27ac already present. This suggests that the establishment of Polycomb silencing might require active deacetylation of this pre-existing H3K27ac. The reciprocal effects of knockdown and overexpression of CBP and E(Z) on H3K27 trimethylation and acetylation in bulk chromatin further suggest that the two modifications constitute alternative chromatin states associated with active and inactive genes. Consistent with this, ChIP-chip experiments revealed that H3K27me3 and H3K27ac are mutually exclusive genome wide. Moreover, in S2 cells, the inactive abd-A gene does not have the H3K27ac modification in its promoter region, but acquires it upon RNAi knockdown of E(Z). It will be important to determine whether such a modification switch occurs genome wide after loss of E(Z) (Tie, 2009).

The ability of E(Z) overexpression to suppress the small rough eye phenotype of CBP overexpressers further supports the conclusion that H3K27 trimethylation by E(Z) antagonizes H3K27 acetylation by CBP and suggests that deacetylation of H3K27 by RPD3, and possibly other deacetylases, might be a prerequisite for subsequent methylation by E(Z) and therefore important for reversal of an active state. Conversely, the ability of CBP and TRX overexpression to increase the global H3K27ac level at the expense of H3K27me3 suggests that either active demethylation of H3K27me3 by the H3K27-specific demethylase UTX (Agge, 2007; Lee, 2007; Smith, 2008), or histone replacement (Ahmad, 2002), might be a prerequisite to acetylation by CBP. Indeed, depletion of Drosophila UTX in vivo using a GAL4-inducible UTX RNAi transgene line results in an increase in H3K27me3, as previously reported (Smith, 2008), and in a marked decrease in H3K27ac. These data, together with the evidence of developmentally programmed reversal of Polycomb silencing, now suggest that the widely accepted stability of Polycomb silencing during development might be more dynamically regulated than previously appreciated (Tie, 2009).

This is the first report that CBP/p300 acetylates H3K27. Recombinant Drosophila CBP acetylates H3K27 and K18 in vivo and in vitro. The greatly reduced H3K27ac levels in CBP-depleted S2 cells also strongly suggest that CBP is the major H3K27 acetylase in Drosophila. The conservation of H3K27 acetylation by human p300, together with the reported association of CBP with the TRX homolog MLL in humans (Ernst, 2001), suggest that it is likely to play a similar role in antagonizing Polycomb silencing in mammals (Tie, 2009).

The genome-wide distribution of H3K27ac, as estimated from human ChIP-chip experiments, appears very similar to that of H3K4me3. This suggests that H3K27ac is much more widely distributed than just at Polycomb target genes, which are estimated to number several thousand in mammalian cells and hundreds in Drosophila. Although these numbers could grow with the identification of additional Polycomb-silenced genes in additional cell types, the recently reported strong correlation of H3K27ac with active genes suggests that it plays an additional role(s) in promoting the transcription of active genes, including those that are never targets of Polycomb silencing. (Note that the H3K27ac at non-Polycomb target genes will not be directly affected by global changes in H3K27me3.) Interestingly, like H3K27me3, H3K27ac appears on the transcribed regions of Polycomb target genes, which might reflect a role for H3K27ac in facilitating transcriptional elongation, and, conversely, a role for H3K27me3 in inhibiting elongation. In addition to its anti-silencing role in preventing H3K27 trimethylation, H3K27ac may also serve as a signal for recruitment of other proteins with additional enzyme activities that alter local chromatin structure further to facilitate or promote transcription. Prime candidates are those containing a bromodomain, a conserved acetyl-lysine-binding module present in several dozen chromatin-associated proteins, including a number of TrxG proteins that also antagonize Polycomb silencing (Tie, 2009).

The results presented in this study provide new insight into how TRX and CBP function together to antagonize Polycomb silencing. Robust H3K27 acetylation by CBP is dependent on TRX, suggesting that H3K27ac plays a crucial role in the anti-silencing activity of TRX. Consistent with this, preliminary genetic evidence suggests that the Polycomb phenotypes caused by TRX overexpression are dependent on CBP, as they are suppressed by RNAi knockdown of CBP. The nature of this dependence is currently unknown, but could involve targeting of CBP by TRX or regulation of the H3K27 acetylation activity of CBP by TRX (Tie, 2009).

The physical association of TRX and CBP and the widespread coincidence of H3K27ac and H3K4me3 sites in the human ChIP-chip data further suggest that the two modifications might be coordinately executed by TRX and CBP. However, the results also raise the possibility that H3K4 trimethylation by TRX itself might be less important for antagonizing Polycomb silencing than H3K27 acetylation. This possibility is also suggested by the discovery of Polycomb-silenced genes in ES and human T cells that contain 'bivalent' marks (both H3K4me3 and H3K27me3) in their promoter regions (although the H3K4me3 levels at these inactive genes are typically lower, on average, than they are at active genes, hinting at the possible importance of quantitative effects of the two marks) (Tie, 2009).

A speculative model is proposed for the regulation of Polycomb silencing that incorporates the activities of TRX, CBP, E(Z), RPD3 and UTX. Repressed genes are marked with H3K27me3. H3K27 trimethylation by PRC2 (which can also control DNA methylation in mammals) requires RPD3 (and possibly other histone deacetylases) to deacetylate any pre-existing H3K27ac. H3K27me3 promotes binding of PC-containing PRC1 complexes, which may inhibit H3K27 acetylation and maintain silencing through 'downstream' events, including those promoted by the H2AK119 mono-ubiquitylation mediated by its RING subunit. Conversely, active genes are marked with H3K4me3 and H3K27ac. H3K27 acetylation by CBP is dependent on TRX and possibly other TrxG proteins, as suggested by the observation that H3K27me3 levels are significantly increased on salivary gland polytene chromosomes from trx, ash1 and kis mutants. The current results predict that this increase will be accompanied by a decrease in H3K27ac. Interestingly, ash1 encodes another HMTase that also interacts with CBP and antagonizes Polycomb silencing. Acetylation of H3K27 is likely to also require the K27-specific demethylase UTX when removal of pre-existing H3K27me3 is a prerequisite for acetylation, e.g. for developmentally regulated reversal of Polycomb silencing at the onset of differentiation. H3K27ac prevents H3K27 trimethylation and might also serve as a signal for recruitment of other TrxG proteins with additional chromatin-modifying activities that may protect the H3K27ac modification and also alter local chromatin structure to promote transcription and further inhibit Polycomb silencing (Tie, 2009).

Gcm/Glide-dependent conversion into glia depends on neural stem cell age, but not on division, triggering a chromatin signature that is conserved in vertebrate glia

Neurons and glia differentiate from multipotent precursors called neural stem cells (NSCs), upon the activation of specific transcription factors. In vitro, it has been shown that NSCs display very plastic features; however, one of the major challenges is to understand the bases of lineage restriction and NSC plasticity in vivo, at the cellular level. This study shows that overexpression of the Gcm transcription factor, which controls the glial versus neuronal fate choice, fully and efficiently converts Drosophila NSCs towards the glial fate via an intermediate state. Gcm acts in a dose-dependent and autonomous manner by concomitantly repressing the endogenous program and inducing the glial program in the NSC. Most NSCs divide several times to build the embryonic nervous system and eventually enter quiescence: strikingly, the gliogenic potential of Gcm decreases with time and quiescent NSCs are resistant to fate conversion. Together with the fact that Gcm is able to convert mutant NSCs that cannot divide, this indicates that plasticity depends on temporal cues rather than on the mitotic potential. Finally, NSC plasticity involves specific chromatin modifications. The endogenous glial cells, as well as those induced by Gcm overexpression display low levels of histone 3 lysine 9 acetylation (H3K9ac) and Drosophila CREB-binding protein (dCBP) Histone Acetyl-Transferase (HAT). Moreover, dCBP targets the H3K9 residue and high levels of dCBP HAT disrupt gliogenesis. Thus, glial differentiation needs low levels of histone acetylation, a feature shared by vertebrate glia, calling for an epigenetic pathway conserved in evolution (Flici, 2011).

Understanding the biology and the potential of stem cells of specific origins is a key issue in basic science and in regenerative medicine. This study shows that NSCs can be fully and stably redirected towards the glial fate in vivo, via a transient, intermediate, state, upon the expression of a single transcription factor. NSC plasticity is temporally controlled and quiescent NSCs cannot be converted; however, plasticity is independent of cell division. Finally, the acquisition of the glial fate involves low histone acetylation, a chromatin modification that is conserved throughout evolution, emphasizing the importance of this mark in glial cells (Flici, 2011).

NSCs produce the different types of neurons and glia that form the nervous system. These precursors can be converted into induced pluripotent cells and even into monocytes, a differentiated fate of an unrelated somatic lineage; however, the in vitro behavior may differ markedly from the in vivo situation. For example, the Achaete-Scute Complex homolog-like 1 transcription factor promotes the expression of oligodendrocyte features upon retroviral injection in the dentate gyrus, but promotes neuronal differentiation from the same progenitors in vitro. The use of NB-specific drivers, markers and conditional overexpression protocols, has led to the demonstration that a single transcription factor can fully convert NSCs into glia in a dose-dependent manner. High Gcm levels probably enable this transcription factor to counteract the endogenous transcriptional program and/or to compensate for the absence of cell-specific co-factors. Quantitative regulation is also required in physiological conditions; for example, the nuclear protein Huckebein enhances the gliogenic potential of Gcm upon triggering its positive autoregulation in a specific lineage. The present study therefore shows for the first time that NSCs can be completely and efficiently redirected in vivo towards a specific fate, also highlighting the importance of quantitative regulation in fate choices (Flici, 2011).

It is widely accepted that NSCs are multipotent precursors; however, their plastic features have not been investigated throughout their life at the cellular level. For example, the existence of a tri-potent NSC with the capacity to generate neurons, astrocytes and oligodendrocytes in the adult brain remains to be demonstrated in vivo. This study demonstrates that NSCs are more plastic at early embryonic stages than at the end of embryogenesis. Furthermore, the intrinsically defined program of quiescence is not compatible with fate conversion, even though quiescent cells are subsequently reactivated. As Drosophila glia are generated at different stages, it is unlikely that a general glial repressor arises late in development and specifically restricts the potential of Gcm. The data rather imply that temporal cues progressively limit NSC plasticity, a feature that may have important consequences in therapeutic applications (Flici, 2011).

It will be of great interest to determine whether such irreversible temporal restriction relies on external cues or whether it reflects an internal clock, as it has been shown for the acquisition of temporal identity, the process by which specific progenies are produced at different developmental stages (Flici, 2011).

Finally, the data show that Gcm does not reprogram neurons. Thus, although other somatic and even germ line cells can be reprogrammed into neurons, these post-mitotic cells seem endowed with an efficient brake to fate conversion. Interestingly, dorsal root ganglia neurons can transdifferentiate from one subtype into another in zebrafish, suggesting that, under some conditions, neurons can adopt a different, but closely related, phenotype. In addition, it cannot be formally excluded that a low percentage of immature neurons adopt a glial or a multipotent phenotype upon Gcm overexpression. Nevertheless, the data indicate that neurons are intrinsically different from other cell types, which may reflect a specific chromatin organization and/or expression of an efficient tumor suppressor molecular network. Transcriptome analyses will help characterizing the molecular signature responsible for the neuronal behavior (Flici, 2011).

Dedifferentiation and transdifferentiation of somatic cells can occur in the absence of mitosis, whereas NSCs plasticity has generally been associated to cell division, as a means to erase transcriptional programs and implement new ones. This study shows that, like terminally differentiated cells, NSCs can be efficiently redirected in the absence of cell division. The concomitant extinction of the endogenous program and activation of the glial program indicate that conversion occurs via an intermediate state, as has been described in B cell to macrophage experimental transdifferentiation. The acquisition of an intermediate state (partial reprogramming) has also been proposed for somatic cell reprogramming. The current findings raise a more general question as to whether intermediate states are common and unstable features of many plastic process including development. These states may reveal competing molecular pathways that in physiological conditions are alternatively consolidated or switched off in response to cell-specific signals. The development of tools enabling tracing these dynamic states will improve understanding of cell plasticity under physiological and experimental conditions (Flici, 2011).

Interestingly, altered tumor suppressor gene expression, which alters the proliferation pathway, leads to ambiguous cell identities, which may reflect the stabilization of intermediate fates. Similarly, Drosophila metastatic cells from brain tumors and several non-dividing NSC cells challenged with Gcm co-express the neuronal and the glial programs. It is proposed that the appropriate activation of the mitotic pathway is necessary for efficient consolidation/extinction of specific fates (Flici, 2011).

The interplay of extrinsic signals, transcription factors and chromatin modifications shape the identity of different cell types. The low and high levels of dCBP as well as H3K9ac truly represent a glial and neuronal signature, respectively. They both depend on gcm, which controls the fate choice, but not on genes downstream to Gcm, which are not sufficient to implement such choice. Thus, full fate conversion is accompanied by a cell-specific chromatin modification (Flici, 2011).

Interestingly, whereas dCBP accumulates at different levels in glia versus neurons and its overexpression or loss affects the levels of H3K9ac, the levels of dGCN5, another HAT that is able to acetylate the H3K9 residue in vivo, are similar in glia and neurons. Moreover, dGCN5 overexpression does not enhance H3K9ac levels nor does it affect the expression of glial genes. These data strongly suggest that the dCBP HAT specifically participates in setting up the H3K9ac signature. It should be noted that dGCN5 is a member of multiprotein complexes, which may explain why its overexpression cannot produce high HAT activity on its own. The balance between HATs and histone deacetylases (HDACs), enzymes with counteracting activities, is thought to be important in the regulation of histone acetylation levels. Although the investigation of histone deacetylation is not in the focus of this paper, the relevance of HDACs in the control of the glia-neuron histone acetylation signature cannot be excluded (Flici, 2011).

The tight regulation of histone acetylation in the nervous system seems to be evolutionarily conserved. Human neuronal disorders are frequently connected to downregulation of histone acetylation and HDAC inhibitors are good candidates as therapeutic tools. Histone acetylation is instrumental for mammalian memory formation and CBP plays an important role in long-term memory processes. Altogether, these data indicate that normal neuronal function requires high levels of histone acetylation (Flici, 2011).

This study shows that low HAT activity is necessary for glial differentiation. The increased levels of histone acetylation by overexpression of dCBP cause downregulation of the majority (but not all) of the tested glial genes, whereas the levels of general nuclear factors remain unchanged. The glial cells do not undergo apoptosis, indicating that high dCBP and histone acetylation levels influence specific pathways rather than generally affecting cell viability. The exact molecular mechanisms are not known, yet the behavior is similar in the mammalian CNS. Oligodendrocyte differentiation requires low levels of histone acetylation, resulting from high amounts of HDACs and low amounts of HATs (CBP and P300). The role of HDACs was further investigated, showing that such enzymes directly repress genes that prevent oligodendrocyte differentiation. Most probably an appropriate balance between HATs and HDACs is the key factor, which produces low levels of histone acetylation and regulates mammalian as well as Drosophila glial differentiation (Flici, 2011).

The broadly accepted model is that histone acetylation weakens the interaction between positively charged histone tails and negatively charged DNA, thereby contributing to transcriptional activation. The current data contradict this simple model. First, the levels of H3K4me3, a histone mark that is connected to actively transcribed genes, are similar in glia and neurons. Second, the total mRNA levels are not different in the two cell populations. Third, and most importantly, dCBP overexpression in glia specifically causes downregulation of a set of glial genes. It seems that the H3K9ac levels reflect specific functional differences between neurons and glia, rather than simply revealing general gene activation. Maybe neurons require more plastic and dynamic regulation of transcription than other cell types and this process requires higher capacity of histone acetylation. Supporting this theory is the finding that a large number of activity-regulated enhancers bind CBP in cortical neuronal cultures. The technological breakthrough will be to analyze the transcriptome and the chromatin landscape of a few cells, which will help understanding the mode of action of dCBP and HDACs in the control of Drosophila glial and neuronal differentiation (Flici, 2011).

Dynamic acetylation of all lysine-4 trimethylated histone H3 is evolutionarily conserved and mediated by p300/CBP

Histone modifications are reported to show different behaviors, associations, and functions in different genomic niches and organisms. This study shows that rapid, continuous turnover of acetylation specifically targeted to all K4-trimethylated H3 tails (H3K4me3), but not to bulk histone H3 or H3 carrying other methylated lysines, is a common uniform characteristic of chromatin biology in higher eukaryotes, being precisely conserved in human, mouse, and Drosophila. Furthermore, dynamic acetylation targeted to H3K4me3 is mediated by the same lysine acetyltransferase, p300/cAMP response element binding (CREB)-binding protein (CBP), in both mouse and fly cells. RNA interference or chemical inhibition of p300/CBP using a newly discovered small molecule inhibitor, C646, blocks dynamic acetylation of H3K4me3 globally in mouse and fly cells, and locally across the promoter and start-site of inducible genes in the mouse, thereby disrupting RNA polymerase II association and the activation of these genes. Thus, rapid dynamic acetylation of all H3K4me3 mediated by p300/CBP is a general, evolutionarily conserved phenomenon playing an essential role in the induction of immediate-early (IE) genes. These studies indicate a more global function of p300/CBP in mediating rapid turnover of acetylation of all H3K4me3 across the nuclei of higher eukaryotes, rather than a tight promoter-restricted function targeted by complex formation with specific transcription factors (Crump, 2011).

Dynamic acetylation of all H3K4me3 mediated by CBP is evolutionarily conserved, observed before the divergence of the single Drosophila enzyme dCBP into the paralogs p300 and CBP in mammals. CBP was discovered as a transcriptional coactivator that binds to CREB and p300 complements this activity. Whereas p300 and CBP are often considered functionally redundant, some studies support unique roles. The majority of genes bound by one also show high levels of the other, suggesting common targeting. They interact with many transcription factors and coactivators, initially suggesting a structural role in promoter complexes; p300 and CBP have been localized to c-fos and c-jun by ChIP and through interactions with other proteins. A purely structural role was challenged following discovery of their acetyltransferase activity with the catalytic domain required for transcription from chromatinized promoter constructs in vitro and in vivo. Recent in vitro studies with reconstituted nucleosome arrays show a requirement for p300 KAT activity to allow decompaction of 30 nm chromatin, nucleosome remodelling, and transcription factor binding (Crump, 2011).

Consistent with previous mass spectrometry and biochemical work, this study shows that H3K4me3 tails are dynamically modified up to the pentaacetylated state, including at lysines 9, 14, and 18, This suggests that the enzyme responsible, p300/CBP, targets specific H3 tails but no specific lysine. In p300/CBP double-knockout mouse fibroblasts, forskolin-induced acetylation of lysine 5, 8, 12, and 16 of histone H4 at c-fos is inhibited, further suggesting no specific targeting of residues. A high level of acetylation is insufficient for efficient gene expression in vivo; treatment of cells with TSA enhances acetylation but interferes with c-fos and c-jun induction. Further, loss of gene expression and Pol II localization caused by p300/CBP inhibition cannot be relieved by preacetylating nucleosomes before inhibition. This indicates a more dynamic role for acetylation in gene expression, suggesting that turnover may be the important factor. Analyses of quiescent cells in which c-fos and c-jun are poised but inactive and inhibition of transcription with DRB both indicate that transcription is not required for dynamic acetylation (Crump, 2011).

The finding that dynamic acetylation and H3K4me3 colocalize on the same nucleosomes across the promoter and start site of c-fos and c-jun raises the question of their cotargeting. Even when the KAT-HDAC enzyme balance is drastically forced in favor of acetylation by HDAC inhibitors, strict targeting to H3K4me3 does not break down. Numerous ChIP studies have established the presence of H3K4me3, H3K9ac, p300/CBP, and HDACs at the promoter and 5' end of many genes , suggesting widespread colocalization (Crump, 2011).

There are two classes of model by which cotargeting of H3K4me3 and rapid dynamic acetylation may occur. The first involves independent targeting to the same loci and H3 tails, as previously shown for serine-10 phosphorylation and lysine-9 acetylation. This suggests that the relevant enzymes may be part of a common process, and cotargeting may arise from independent DNA sequence recognition or unique interactions with the machinery of signal transduction and transcriptional regulation; p300 and CBP have been isolated in complexes containing TATA-binding protein (TBP) and RNA polymerase II (Crump, 2011).

A second class of model is based on dependence of one modification on the other. For example, p300/CBP may mediate dynamic acetylation through direct or indirect recognition of trimethylated lysine 4, which may provide a binding platform or enhance KAT activity. In support of this mechanism WDR5 knockdown, which depletes H3K4 methylation, attenuates the TSA-induced increase in H3K9ac levels at promoters. Other KATs are known to be recruited to H3K4me3 to induce histone acetylation; yeast Yng1 and Yng2, which recognize H3K4me3 via their PHD fingers, form part of the NuA3 and NuA4 KAT complexes, respectively, and mammalian ING4 links HBO1 acetyltransferase activity to H3 lysine-4 trimethylated nucleosomes (Crump, 2011).

A sequential targeting mechanism is conceivable. Unmethylated CpG dinucleotides within CpG islands may primarily recruit CXXC motif-containing proteins, including the H3K4 methyltransferase MLL1 (56) and CGBP/Cfp1, which associates with H3K4 methyltransferases. DNA binding by Cfp1 has recently been shown to restrict Setd1A and H3K4me3 to euchromatic nonmethylated CpG regions. Similarly, ChIP-seq analysis has shown a tight association between Cfp1 and H3K4me3 at CpG islands, and Cfp1 knockdown depletes H3K4me3 levels at nonmethylated CpGs. This provides a plausible mechanism to target H3K4me3 to these regions, which could then recruit p300/CBP for dynamic histone acetylation (Crump, 2011).

Genome-wide phosphoacetylation of histone H3 at Drosophila enhancers and promoters

Transcription regulation is mediated by enhancers that bind sequence-specific transcription factors, which in turn interact with the promoters of the genes they control. This study shows that the JIL-1 kinase is present at both enhancers and promoters of ecdysone-induced Drosophila genes, where it phosphorylates the Ser10 and Ser28 residues of histone H3. JIL-1 is also required for CREB binding protein (CBP)-mediated acetylation of Lys27, a well-characterized mark of active enhancers. The presence of these proteins at enhancers and promoters of ecdysone-induced genes results in the establishment of the H3K9acS10ph and H3K27acS28ph marks at both regulatory sequences. These modifications are necessary for the recruitment of 14-3-3, a scaffolding protein capable of facilitating interactions between two simultaneously bound proteins. Chromatin conformation capture assays indicate that interaction between the enhancer and the promoter is dependent on the presence of JIL-1, 14-3-3, and CBP. Genome-wide analyses extend these conclusions to most Drosophila genes, showing that the presence of JIL-1, H3K9acS10ph, and H3K27acS28ph is a general feature of enhancers and promoters in this organism (Kellner, 2012).

Activation of transcription in higher eukaryotes requires the interaction between transcription factors bound to distal enhancers and proteins present at the promoter. Recent findings indicate that enhancers contain a variety of histone modifications that change during the establishment of specific cell lineages suggesting that these sequences may play a more complex role in transcription than previously thought. Given the presence of common as well as specific histone marks at enhancers and promoters, it is tempting to speculate that epigenetic modifications at these sequences serve to integrate various cellular signals required to converge in order to activate gene expression. Results described in this study support this hypothesis, demonstrating that the proteins that carry out these histone modifications are necessary to establish enhancer-promoter contacts and activate transcription of ecdysone-inducible genes (Kellner, 2012).

The execution of this process in Drosophila requires the recruitment of JIL-1 by mechanisms that are not well understood. Although the direct involvement of JIL-1 in the transcription process has been brought into question due to the failure to observe recruitment of JIL-1 to heat shock genes in polytene chromosomes, results presented in this study clearly indicate that JIL-1 affects transcription at different steps in the transcription cycle. At the promoter region, phosphorylation of H3S10 by JIL-1 results in the recruitment of 14-3-3 and, subsequently, histone acetyltransferases Elp3 and MOF (Karam, 2010). This study found that JIL-1 is also able to phosphorylate H3S28 at both promoters the enhancers. The establishment of the H3K9acS10ph and H3K27acS28ph modifications correlates with the recruitment of 14-3-3 to enhancers and promoters of ecdysone-induced genes. 14-3-3 has been implicated in numerous cellular processes, where it functions as a scaffold protein). 14-3-3 is found as dimers and multimers; each monomer is capable of binding two targets and can mediate and stabilize interactions between two phosphoproteins. Additionally, acetylation facilitates the dimerization of 14-3-3 molecules and their ability to bind certain substrates. Binding assays have demonstrated that 14-3-3 interacts weakly with H3 tail peptides phosphorylated at S10 and S28, but strong binding is detected if the peptide is both phosphorylated and acetylated on the neighboring lysine residues. Given the ability of 14-3-3 to serve as a scaffold for large protein complexes, its demonstrated interactions with H3K9acS10ph and H3K27acS28ph and the presence of these two modifications at enhancers and promoters, it is possible that contacts between these two sequences are stabilized by 14-3-3. This hypothesis is supported by 3C experiments indicating that induction of transcription of the Eip75B gene is accompanied by strong enhancer-promoter interactions. These interactions are lost in JIL-1, CBP, and 14-3-3 knockdown cells. Since these proteins act several steps downstream from transcription factor binding in the pathway leading to enhancer-promoter contacts, and loss of these proteins results in the abolishment of these contacts, it appears that these proteins, rather than specific transcription factors, may be responsible for enhancer promoter interactions at the ecdysone-inducible genes (Kellner, 2012).

Genome-wide studies using ChIP-seq clearly show the presence of JIL-1, H3K9acS10ph, and H3K27acS28ph at enhancers and promoters of most Drosophila genes. There is a clear correlation between the amount of JIL-1, H3K9acS10ph, and H3K27acS28ph at promoters and the level of transcripts associated with the gene. These three marks are also present at enhancers defined by the occurrence of H3K4me1 and H3K27ac, suggesting that the JIL-1 kinase is a regulator of histone dynamics at enhancers and promoters genome-wide. JIL-1, H3K9acS10ph, and H3K27acS28ph are found at low levels at enhancers before activation, which then increase in intensity and drop in baseline when found in combination with H3K27ac, a mark of active enhancers. These conclusions are different from those previously published examining the role of JIL-1 in transcription and dosage compensation (Regnard, 2011). This study concluded that JIL-1 binds active genes along their entire length and that the levels of JIL-1 are not associated with levels of transcription. The differences in the conclusions may be due to the different cell lines used -- male S2 cells versus female Kc cells -- and the emphasis of the analysis by Regnard on the expression of dosage-compensated genes in the male X-chromosome, which may contain JIL-1 throughout the genes as a consequence of their regulation at the elongation step. In addition, the study by Regnard used ChIP-chip on custom tiling arrays of the X chromosome plus cDNA arrays containing the whole genome. This strategy may bias the conclusions and suggest the presence of JIL-1 in the coding region of genes rather than at enhancers and promoters (Kellner, 2012).

Results presented in this study extend the previous list of histone modifications characteristic of active enhancers to include H3K9acS10ph and H3K27acS28ph. Enhancers tend to be cell type-specific and are determined during differentiation with the characteristic H3K4me1 modification. It is unclear how these regions are designated before activation and what keeps them in a poised state ready for activation upon receiving the proper signal from the cell. It is tempting to speculate that the presence of JIL-1 at enhancers prior to activation might play a role in maintaining the enhancer in this poised state. An important question for future studies is the mechanistic significance of the looping between enhancers and promoters in order to achieve transcription activation. One interesting possibility is that various signaling pathways in the cell contribute to building epigenetic signatures at enhancers and promoters in the form of histone acetylation and/or phosphorylation of various Lys/Ser/Thr residues. Acetylation marks at enhancers and promoters may then cooperate to recruit BRD4 (FS(1)H in Drosophila), which contains two bromodomains each able to recognize two different acetylated Lys residues. The requirement for acetylation of histones at enhancers and promoters in order to recruit Brd4 would ensure that several different signaling events have taken place before recruitment of P-TEFb by BRD4 can release RNAPII into productive elongation (Kellner, 2012).

Histone demethylase Utx and chromatin remodeler Brm bind directly to CBP and modulate acetylation of histone H3 lysine 27

Trithorax group (TrxG) proteins antagonize Polycomb silencing and are required for maintenance of transcriptionally active states. Previous studies have shown that the Drosophila acetyltransferase CREB-binding protein (CBP; Nejire) acetylates histone H3 lysine 27 (H3K27ac), thereby directly blocking its trimethylation (H3K27me3) by Polycomb repressive complex 2 (PRC2) in Polycomb target genes. This study shows that H3K27ac levels also depend on other TrxG proteins, including the histone H3K27-specific demethylase Utx and the chromatin-remodeling ATPase Brahma (Brm). Utx and Brm are physically associated with CBP in vivo, and Utx, Brm, and CBP colocalize genome-wide on Polycomb response elements (PREs) and on many active Polycomb target genes marked by H3K27ac. Utx and Brm bind directly to conserved zinc fingers of CBP, suggesting that their individual activities are functionally coupled in vivo. The bromodomain-containing C terminus of Brm binds to the CBP PHD finger, enhances PHD binding to histone H3, and enhances in vitro acetylation of H3K27 by recombinant CBP. brm mutations and knockdown of Utx by RNA interference (RNAi) reduce H3K27ac levels and increase H3K27me3 levels. It is proposed that direct binding of Utx and Brm to CBP and their modulation of H3K27ac play an important role in antagonizing Polycomb silencing (Tie, 2012).

Acetylation of histone H3K27, a strong predictor of active genes, has emerged as one of the central mechanisms for antagonizing/reversing Polycomb silencing since it directly prevents trimethylation of H3K27 by PRC2. Interestingly, H3K27ac is present in animals, plants, and fungi, but H3K27me3 and PRC2 homologs are present only in animals and plants, clearly indicating that H3K27ac has functions other than preventing H3K27 methylation. This study provides evidence that the Drosophila TrxG proteins Utx and Brm modulate H3K27 acetylation by CBP. Utx and Brm are physically associated with CBP in vivo and bind directly to ZF1 and the PHD finger of CBP. Genome-wide ChIP-chip analysis revealed that the chromatin binding sites of Utx, Brm, and CBP coincide on many genes and that strong peaks of all three are highly correlated with the presence of high levels of H3K27ac. Importantly, brm mutants and RNAi knockdown of Brm in vivo result in a decrease of H3K27ac and a concomitant increase of H3K27me3. Similarly, knockdown and overexpression of Utx with no change in the CBP level are sufficient to promote, respectively, a decrease and increase in the bulk H3K27ac level. This suggests that coupled H3K27 deacetylation/trimethylation and demethylation/acetylation are dynamically antagonistic. It further suggests that regulating the balance of these opposing activities is likely to play an important role in determining whether active and silent chromatin states will be maintained or switched (Tie, 2012).

This is the first report that Utx is physically associated with CBP. While functional collaboration between Utx and CBP is required to execute the sequential reactions required to switch Polycomb target genes from silent to active states, it was not obvious that this should require that they be physically associated. The fact that they are suggests that their two reactions are more efficiently coupled. It also suggests that despite the many histone and nonhistone substrates of CBP, H3K27 acetylation on Polycomb target genes to prevent their silencing is sufficiently critical to have evolved a Utx-CBP methyl-to-acetyl switching module, perhaps to counter the complementary coupling effect of the physical association of the H3K27 deacetylase RPD3 with PRC2 to create the antagonistic acetyl-to-methyl switch. Coupling of the Utx and CBP activities may increase the fidelity of maintenance of active chromatin states of Polycomb target genes by ensuring rapid reversal of H3K27ac deacetylation and methylation by RPD3 and PRC2 that may occur either adventitiously or as part of an ongoing dynamic balance between these antagonistic activities. Such coupling could also increase the efficiency of switching Polycomb target genes from a transcriptionally silent to an active state in response to developmentally programmed signals or other cellular signals, ensuring definitive establishment of the new active transcriptional state (Tie, 2012).

While Brm is well known for the chromatin remodeling activity associated with its highly conserved ATPase domain, the current findings identify another activity of Brm associated with its highly conserved BrD-containing C terminus [Brm(1417-1634)], which binds histone H3, enhances H3 binding to the CBP PHD finger, and enhances acetylation of H3K27 by CBP in vitro. The latter effect is most likely due to the simultaneous binding of Brm to H3 and the CBP PHD finger, thereby stabilizing the H3 interaction with the CBP HAT domain. However, it cannot be ruled out that it may also reflect a direct stimulatory effect of Brm on the intrinsic activity of the CBP HAT domain. In any case, this suggests that the physical association and genome-wide colocalization of CBP and Brm do not simply reflect a spatial and temporal coordination of their separate acetylation and chromatin remodeling activities but also reflect regulatory interactions. The reduced H3K27ac level in brm2 mutants and after RNAi knockdown is consistent with the observed enhancement of CBP HAT activity in vitro. However, at this time the possibility cannot be ruled out that the loss of Brm chromatin-remodeling activity in brm2 mutants may also contribute to their reduced H3K27ac level (Tie, 2012).

The physical association of Brm with CBP and with Utx reported in this study is consistent with recent reports that BRG1 can be coimmunoprecipitated with human CBP and p300 from tumor cells and with Utx/Jmjd3 in murine EL4 T cells. However, the region of human CBP reported to bind BRG1 differs from the current findings. It was reported that BRG1 interacts directly with the human CBP fragment containing ZF3, and the proline-rich region of BRG1 is required for this interaction. The current results indicate that only ZF1 and the PHD finger of Drosophila CBP bind directly to the BrD-containing C terminus of Brm. Since the proline-rich region of Drosophila Brm was not assayed due to its insolubility, the possibility cannot be ruled out that it mediates additional contact(s) with CBP (Tie, 2012).

CBP was not found in the previously purified Drosophila Brm-containing complexes, suggesting that only a portion of Brm is physically associated with CBP or that this association may be stabilized only on chromatin and/or may be regulated by other cellular signals. Consistent with this, there are some sites detected by ChIP-chip that are enriched for Brm and Utx without CBP and there are some column fractions containing Brm and Utx without CBP. It is possible that the CBP-Brm association may also serve to recruit Brm to some sites, e.g., recruitment of human Brm or BRG1 to the beta interferon (IFN-β) promoter depends on the prior presence of CBP and leads to subsequent nucleosome remodelin (Tie, 2012).

This study found that Utx, Brm, and CBP colocalize not only with H3K27ac at many regions, including promoters, transcribed regions, PREs, and other presumed cis-regulatory elements. They are also present, albeit at lower levels, on repressed genes marked by strong H3K27me3 domains (e.g., the ANT-C and BX-C). At such sites, the H3K27me3 may be protected from Utx-mediated demethylation by the binding of PC/PRC1. Alternatively, their lower levels at repressed genes may simply result in a dynamic balance of deacetylation/methylation and demethylation/acetylation that overwhelmingly favors the former. It is also possible that Utx and CBP may also be involved in Brm-dependent transcriptional repression at some sites. Recent evidence suggests that maintenance of steady-state levels of histone acetylation is highly dynamic, and the reciprocal changes in H3K27me3 and H3K27ac levels that occur upon altering Utx or E(Z) levels suggest that maintenance of histone methylation levels may also be highly dynamic. Much remains to be discovered about the factors that regulate the demethylase and acetyltransferase activities of Utx and CBP in different chromatin environments (Tie, 2012).

The bromodomains of yeast SWI2/SNF2 and human BRG1 bind specifically to H3K14ac, indicating that this binding specificity has been highly conserved during evolution. Brm(1417-1634) also binds specifically to H3K14ac, and the BrD of Brm is required for CBP binding and histone H3 binding. The BrD-containing C termini of human and plant Brm have been reported to be functionally important in vivo. Surprisingly, the BrD of Drosophila Brm has been reported to be dispensable for viability. A brm transgene containing a deletion of the central 72 residues of the BrD can rescue the late embryonic lethality of brm2 mutants, allowing them to develop into adults. Whether the reduced H3K27ac level of these brm2 mutants is also rescued has not been determined. This rescue could indicate that the Brm BrD is functionally redundant or at least not critical for achieving adequate expression of the genes responsible for the inviability of brm2 mutants. The brm2 mutation behaves genetically as a strong hypomorphic or null allele, but the sequence alteration responsible for its phenotype has not been determined and so the precise nature of its functional deficit is unknown (Tie, 2012).

In summary, this study has shown that Utx and Brm interact directly with CBP and modulate H3K27 acetylation. Utx presumably does so indirectly, at Polycomb target genes, by providing demethylated H3K27 substrate for acetylation by CBP. Their direct physical coupling could provide obvious gains in the efficiency of their two sequential reactions and their consequent H3K27ac yield. The interaction between Brm and CBP may similarly couple their activities, but this study also presents initial evidence to suggest that the binding of the Brm BrD-containing C terminus to H3 and the CBP PHD finger may also directly enhance H3K27 acetylation through its effect on H3 binding by the CBP HAT domain. It is expected that additional TrxG proteins will modulate H3K27 acetylation, including KIS and ASH1, which have recently been shown to affect H3K27me3 levels. The broad distributions of H3K27me3 over many Polycomb target genes is mirrored by similar broad distributions of H3K27ac when those genes are active, suggesting that in addition to its general genome-wide association with active genes, it may play a more specialized dual role at Polycomb target genes, where it also dynamically antagonizes the encroachment of Polycomb silencing (Tie, 2012).

Constitutive scaffolding of multiple Wnt enhanceosome components by Legless/BCL9

Wnt/β-catenin signaling elicits context-dependent transcription switches that determine normal development and oncogenesis. These are mediated by the Wnt enhanceosome, a multiprotein complex binding to the Pygo chromatin reader and acting through TCF/LEF-responsive enhancers. Pygo renders this complex Wnt-responsive, by capturing β-catenin via the Legless/BCL9 adaptor. This study used CRISPR/Cas9 genome engineering of Drosophila legless (lgs) and human BCL9 and B9L to show that the C-terminus downstream of their adaptor elements is crucial for Wnt responses. BioID proximity labeling revealed that BCL9 and B9L, like PYGO2, are constitutive components of the Wnt enhanceosome. Wnt-dependent docking of β-catenin to the enhanceosome apparently causes a rearrangement that apposes the BCL9/B9L C-terminus to TCF. This C-terminus binds to the Groucho/TLE co-repressor, and also to the Chip/LDB1-SSDP enhanceosome core complex via an evolutionary conserved element. An unexpected link between BCL9/B9L, PYGO2 and nuclear co-receptor complexes suggests that these β-catenin co-factors may coordinate Wnt and nuclear hormone responses (van Tienen, 2017).

The Wnt/β-catenin signaling cascade is an ancient cell communication pathway that operates context-dependent transcriptional switches to control animal development and tissue homeostasis. Deregulation of the pathway in adult tissues can lead to many different cancers, most notably colorectal cancer. Wnt-induced transcription is mediated by T cell factors (TCF1/3/4, LEF1) bound to Wnt-responsive enhancers, but their activity depends on the co-activator β-catenin (Armadillo in Drosophila), which is rapidly degraded in unstimulated cells. Absence of β-catenin thus defines the OFF state of these enhancers, which are silenced by Groucho/TLE co-repressors bound to TCF via their Q domain. This domain tetramerizes to promote transcriptional repression (Chodaparambil, 2014), which leads to chromatin compaction apparently assisted by the interaction between Groucho/TLE and histone deacetylases (HDACs) (van Tienen, 2017).

Wnt signaling relieves this repression by blocking the degradation of β-catenin, which thus accumulates and binds to TCF, converting the Wnt-responsive enhancers into the ON state. This involves the binding of β-catenin to various transcriptional co-activators via its C-terminus, most notably to the CREB-binding protein (CBP) histone acetyltransferase or its p300 paralog, resulting in the transcription of the linked Wnt target genes. Subsequent reversion to the OFF state (for example, by negative feedback from high Wnt signaling levels near Wnt-producing cells, or upon cessation of signaling) involves Groucho/TLE-dependent silencing, but also requires the Osa/ARID1 subunit of the BAF (also known as SWI/SNF) chromatin remodeling complex which binds to β-catenin through its BRG/BRM subunit. Cancer genome sequencing has uncovered a widespread tumor suppressor role of the BAF complex, which guards against numerous cancers including colorectal cancer, with >20% of all cancers exhibiting at least one inactivating mutation in one of its subunits, most notably in ARID1A. Thus, it appears that failure of Wnt-inducible enhancers to respond to negative feedback imposed by the BAF complex strongly predisposes to cancer (van Tienen, 2017).

How β-catenin overcomes Groucho/TLE-dependent repression remains unclear, especially since β-catenin and TLE bind to TCF simultaneously (Chodaparambil, 2014). Therefore, the simplest model envisaging competition between β-catenin and TLE cannot explain this switch, which implies that co-factors are required. One of these is Pygo, a chromatin reader binding to histone H3 tail methylated at lysine 4 (H3K4m) via its C-terminal PHD finger (Fiedler, 2008). In Drosophila where Pygo was discovered as an essential co-factor for activated Armadillo, its main function appears to be to assist Armadillo in overcoming Groucho-dependent repression. It has been discovered recently that Pygo associates with TCF enhancers via its highly conserved N-terminal NPF motif that binds directly to the ChiLS complex, composed of a dimer of Chip/LDB (LIM domain-binding protein) and a tetramer of SSDP (single-stranded DNA-binding protein, also known as SSBP). Notably, ChiLS also binds to other enhancer-bound NPF factors such as Osa/ARID1 and RUNX, and to the C-terminal WD40 domain of Groucho/TLE, and thus forms the core module of a multiprotein complex termed 'Wnt enhanceosome' (Fiedler, 2015). This study proposed that Pygo renders this complex Wnt-responsive by capturing Armadillo/β-catenin through the Legless adaptor (whose orthologs in humans are BCL9 and B9L, also known as BCL9-2). The salient feature of this model is that the Wnt enhanceosome keeps TCF target genes repressed prior to Wnt signaling while at the same time priming them for subsequent Wnt induction, and for timely shut-down via negative feedback depending on Osa/ARID1 (Fiedler, 2015; van Tienen, 2017 and references therein).

This study assessed the function of Legless and BCL9/B9L within the Wnt enhanceosome. Using a proximity-labeling proteomics approach (called BioID) in human embryonic kidney (HEK293) cells, a compelling association was uncovered between BCL9/B9L and the core Wnt enhanceosome components, regardless of Wnt signaling. Co-immunoprecipitation (coIP) and in vitro binding assays based on Nuclear Magnetic Resonance (NMR) revealed that BCL9 and B9L associate with TLE3 through their C-termini, and that they bind directly to Chip/LDB-SSDP via their evolutionary conserved homology domain 3 (HD3). These elements are outside the sequences mediating the adaptor function between Pygo and Armadillo/β-catenin, but they are similarly important for Wnt responses during Drosophila development and in human cells, as is shown by CRISPR/Cas9-based genome editing. The results consolidate and refine the Wnt enhanceosome model, indicating a constitutive scaffolding function of BCL9/B9L within this complex. The evidence further suggests that BCL9/B9L but not Pygo undergoes a β-catenin-dependent rearrangement within the enhanceosome upon Wnt signaling (see Model of the Wnt enhanceosome), gaining proximity to TCF, which might trigger enhanceosome switching (van Tienen, 2017).

This study has uncovered genetic and physical interactions between two constitutive core components of the Wnt enhanceosome and the C-terminus of Legless/BCL9. The first of these is ChiLS, the core module of the Wnt enhanceosome (Fiedler, 2015): ChiLS is a direct and specific ligand of the α-helical HD3 element of B9L and, likely, of other Legless/BCL9 orthologs, given the strong sequence conservation of this α-helix. The physiological relevance of this interaction with ChiLS is underscored by genetic analysis in flies. The evidence thus implicates HD3 as an evolutionary conserved contact point between Legless/BCL9 and ChiLS, although the primary link between these two proteins appears to be provided by Pygo (van Tienen, 2017).

A second link between the Legless/BCL9 C-terminus and the Wnt enhanceosome is mediated by the WD40 domain of TLE/Groucho. Given evidence from RIME, this link is also likely to be direct although, for technical reasons, it has not been possible to prove this. The function of the C-terminus of Legless/BCL9 for transducing Wnt signals was revealed by the wg-like phenotypes in Drosophila larvae and flies and by their defective transcriptional Wg responses, and by the loss of transcriptional Wnt responses in BCL9/B9L-deleted human cells. The evidence indicates that Legless/BCL9 undergoes three separate functionally relevant interactions with distinct components of the Wnt enhanceosomewith Pygo, ChiLS and Groucho/TLE. Importantly, BioID revealed that these interactions are constitutive, preceding Wnt signaling, and that they hardly change upon Wnt stimulation. Taken together with its multivalent interactions with the Wnt enhanceosome, this is consistent with Legless/BCL9 being a core component of this complex, providing a scaffolding function that facilitates its assembly and/or maintains its cohesion (van Tienen, 2017).

Following Wnt stimulation, Legless/BCL9 undergoes an additional physiologically relevant interaction, by binding to (stabilized) Armadillo/β-catenin via HD2. Legless/BCL9 thus confers Wnt-responsiveness on the Wnt enhanceosome through its ability to capture Armadillo/β-catenin. In other words, in addition to scaffolding the enhanceosome, Legless/BCL9 also earmarks this complex for Wnt responses. Intriguingly, the BioID data indicated that the capture of β-catenin by Legless/BCL9 triggers its rearrangement within the complex, apposing its C-terminus to TCF. This apparent β-catenin-dependent apposition is consistent with structural data showing that BCL9/B9L HD2 is closely apposed to TCF when in a ternary complex with β-catenin. The evidence supports the notion of Legless/BCL9 acting as an Armadillo loading factor, facilitating access of Armadillo/β-catenin to TCF, but argues against the original co-activator hypothesis which posited that Legless/BCL9 is recruited to TCF by Armadillo/β-catenin exclusively in Wnt-stimulated cells. Whatever the case, the β-catenin-dependent apposition of the Legless/BCL9 C-terminus to TCF is likely to trigger Wnt enhanceosome switching from OFF to ON, resulting in the relief of Groucho/TLE-dependent repression and culminating in the Wnt-dependent transcriptional activation of linked target genes (van Tienen, 2017).

This transition of the Wnt enhanceosome from OFF to ON is accompanied by a proximity gain between Legless/BCL9 and CBP/p300, likely to reflect at least in part its de novo binding to Armadillo/β-catenin. However, the evidence indicates that CBP/p300 is associated with the Wnt enhanceosome prior to Wnt signaling, possibly via direct binding to B9L as suggested by RIME, and that the docking of Armadillo/β-catenin to the Wnt enhanceosome strengthens its association with CBP/p300, and/or directs the histone acetyltransferase activity of CBP/p300 towards its substrates, primarily the histone tails. By acetylating these tails, CBP/p300 appears to promote Wnt-dependent transcription in flies and human cells. Indeed, CBP-dependent histone acetylation has been observed at Wg target enhancers in Drosophila although, interestingly, this preceded transcriptional activation. This is consistent with BioID data, indicating constitutive association of CBP/p300 with the Wnt enhanceosome (van Tienen, 2017).

It seems plausible that histone acetylation at Wnt target enhancers is instrumental in antagonizing the compaction of their chromatin imposed by Groucho/TLE, which depends on its tetramerization via its Q domain as well as its binding to HDACs. Indeed, HDACs were found near the bottom of the BioID lists, and one of the top hits identified by B9L was GSE1, a subunit of the BRAF-HDAC complex. However, CBP/p300 also has non-histone substrates within the Wnt enhanceosome, including dTCF in Drosophila whose Armadillo-binding site can be acetylated by dCBP, which thus blocks the binding between the two proteins and antagonizes Wg responses. It thus regulates Wnt-dependent transcription positively as well as negatively, similarly to Groucho/TLE which not only silences Wnt target genes but also earmarks them for Wnt inducibility, as a core component of the Wnt enhanceosome. It is intriguing that both bimodal regulators are associated constitutively with this complex. A corollary is that the docking of Armadillo/β-catenin to the Wnt enhanceosome changes their substrate specificities and/or activities (van Tienen, 2017).

An important refinement of the initial enhanceosome model is with regard to the BAF complex, which appears to be a constitutive component of the Wnt enhanceosome, as indicated by BioID data. This complex is highly conserved from yeast to humans, and it contains 15 subunits in human cells (Kadoch, 2015), including the DNA-binding Osa/ARID1 subunit. A wealth of evidence from studies in flies and mammals indicates that this complex primarily antagonizes Polycomb-mediated silencing of genes, most notably of the INK4A locus which encodes an anti-proliferative factor, which could explain why the BAF complex functions as a tumor suppressor in many tissues. However, recall that this complex also specifically antagonizes Armadillo/β-catenin-mediated transcription, likely via its BRG/BRM subunit which directly binds to β-catenin. Evidence from studies in Drosophila of Wg-responsive enhancers suggests that this complex mediates a negative feedback from high Wg signaling levels near Wg-producing cells which results in re-repression, imposed by the Brinker homeodomain repressor and its Armadillo-binding Teashirt co-repressor. The same factors may also install silencing on Wnt-responsive enhancers upon cessation of Wnt signaling. Notably, mammals do not have a Brinker ortholog, which could explain some of the apparent functional differences between flies and mammals with regard to the BAF complex (Kadoch, 2015). However, the closest mammalian relatives of Teashirt are the Homothorax/MEIS proteins, a family of homeodomain proteins whose expression can be Wnt-inducible. They are thus candidates for Wnt-induced repressors that confer BAF-dependent feedback inhibition (van Tienen, 2017).

Notably, none of BioID lists contained RUNX proteins. Based on functional evidence from Drosophila midgut enhancers, it is proposed that these proteins (which bind to both enhancers and Groucho/TLE) are pivotal for initial assembly of the Wnt enhanceosome at Wnt-responsive enhancers during early embryonic development, or in uncommitted progenitor cells of specific cell lineages (Fiedler, 2015). However, HEK293 cells are epithelial cells and may thus not express any RUNX factors. In any case, the negative BioID results suggest that RUNX factors function in a hit-and-run fashion. Evidently, the Wnt enhanceosome complex, once assembled at Wnt-responsive enhancers, can switch between ON and OFF states without RUNX (van Tienen, 2017).

In summary, this study has uncovered a fundamental role to Legless/BCL9 as a scaffold of the Wnt enhanceosome, far beyond its role in linking Armadillo/β-catenin to Pygo. Indeed, the function of Legless/BCL9 may extend beyond transcriptional Wnt responses, as indicated by the unexpected discovery of its strong association with nuclear co-receptor complexes. Potentially, these associations underlie the observed cross-talk between Wnt/β-catenin and nuclear hormone receptor signaling, documented extensively in the literature, including evidence for direct activation of the androgen receptor by β-catenin. Furthermore, a strong association between TLE1 and the estrogen receptor has been discovered in breast cancer cells, where TLE1 assists the estrogen receptor in its interaction with chromatin and its proliferation-promoting function. This is reminiscent of the role of Groucho/TLE as a cornerstone of the Wnt enhanceosome, proposed to earmark TCF enhancers for subsequent β-catenin docking and transcriptional Wnt responses (Fiedler, 2015). It will be interesting to test experimentally the putative roles of BCL9/B9L and Pygo in enabling cross-talk between β-catenin and nuclear hormone receptor signaling, both during normal development and in cancer (van Tienen, 2017).

Different enhancer classes in Drosophila bind distinct architectural proteins and mediate unique chromatin interactions and 3D architecture

Genome-wide studies has identified two enhancer classes in Drosophila that interact with different core promoters: housekeeping enhancers (hkCP) and developmental enhancers (dCP). It is hypothesized that the two enhancer classes are occupied by distinct architectural proteins, affecting their enhancer-promoter contacts. It was determined that both enhancer classes are enriched for RNA Polymerase II, CBP, and architectural proteins but there are also distinctions. hkCP enhancers contain H3K4me3 and exclusively bind Cap-H2, Chromator, DREF and Z4, whereas dCP enhancers contain H3K4me1 and are more enriched for Rad21 and Fs(1)h-L. Additionally, the interactions of each enhancer class were mapped utilizing a Hi-C dataset with <1 kb resolution. Results suggest that hkCP enhancers are more likely to form multi-TSS interaction networks and be associated with topologically associating domain (TAD) borders, while dCP enhancers are more often bound to one or two TSSs and are enriched at chromatin loop anchors. The data support a model suggesting that the unique architectural protein occupancy within enhancers is one contributor to enhancer-promoter interaction specificity (Cubenas-Potts, 2017).

This study characterize the protein occupancy, chromatin interactions and architecture profiles for the two enhancer classes found in Drosophila. Each enhancer class has distinct H3K4 methylation states, is bound by both common and distinct architectural proteins, and is involved in distinct types of chromatin interactions. First, it was established that hkCP enhancers exclusively bind CAP-H2, Chromator, DREF and Z4, while dCP enhancers do not and are preferentially enriched for but not exclusively bound by Fs(1)h-L and Rad21. In addition, hkCP enhancers are more likely than dCP enhancers to associate with multiple TSSs, which promotes a higher transcriptional output. Finally, hkCP enhancers preferentially associate with topologically associating domain (TAD) borders, whereas dCP enhancers are enriched at chromatin loop anchors present inside TADs. Interestingly, enhancers activated by both core promoters exhibit more hkCP enhancer like characteristics, indicating that the both CP enhancers may represent an intermediate among the distinctive hkCP and dCP enhancers. Altogether, these results provide strong correlative evidence, supporting a model suggesting that architectural proteins are critical regulators of enhancer-promoter interaction specificity and that the interactions between enhancers and promoters significantly contribute to the generation of 3D chromatin architecture (Cubenas-Potts, 2017).

The importance of architectural proteins in regulating enhancer-promoter interactions in Drosophila is supported by the observation that the vast majority of architectural protein sites present in the genome correspond to enhancers and promoters. Historically, architectural proteins were identified as insulators, which were functionally demonstrated to block enhancer-promoter interactions. The insulator function of architectural proteins correlates with their enrichment at TAD borders. However, several lines of evidence, including ChIA-PET analysis of CTCF- and cohesin-mediated interactions in mammals, suggest that these architectural proteins help mediate long range contacts among regulatory sequences. In Drosophila this study observed that nearly all of the Group 1 and Group 2 architectural protein sites are associated with enhancers or promoters defined by STARR-seq, TSSs or CBP peaks, suggesting that architectural proteins help mediate enhancer-promoter interactions. Notably, Group 3 architectural proteins include the classic insulator proteins CTCF, CP190, Mod(mdg4) and SuHw, and at least 25% of their peaks cannot be explained by enhancers or promoters. It is interesting to speculate that the non-enhancer-promoter sites may be involved in more classical insulator functions or contributing to the chromatin architecture of inactive regions of the genome (Cubenas-Potts, 2017).

The conclusion that architectural proteins are critical regulators of the specificity between enhancers and promoters is supported by two main lines of evidence. First, the current results demonstrate a strong correlation between each enhancer class and distinct architectural protein subcomplexes. Functional evidence supporting this conclusion comes from mutational analyses of the DRE motif in the distinct enhancer classes, which likely recruits DREF and the other hkCP enhancer associated architectural proteins. Zabidi (2015) demonstrated that the tandem DRE motif alone was sufficient to enhance expression of the housekeeping core promoter and that mutation of DRE motifs within an hkCP enhancer reduced its promoter interactions in a luciferase assay. Furthermore, addition of a DRE motif to a dCP enhancer changed its promoter specificity. Because DREF and potentially BEAF-32 bind to the DRE motif, these results strongly support a model suggesting that the differential occupancy of Cap-H2, Chromator, DREF and Z4 in the two enhancer classes is a critical regulator of their specific interactions with the core promoter types. However, the data cannot discount the notion that unique transcription factor binding at proximal TSSs also contribute to the specificity of enhancer-promoter interactions. Although hkCP enhancer identity is most highly correlated with CAP-H2, Chromator, DREF and Z4 localization, these four architectural proteins are not found in isolation within hkCP enhancers. BEAF-32 and CP190 are also strongly enriched in hkCP enhancers, which are also associated with high occupancy APBSs and TAD borders. Thus, the full architectural protein complement at hkCP enhancers is far more complex than the four hkCP-specific architectural proteins. In addition, architectural proteins that are truly unique to dCP enhancers were not detected. Because dCP enhancers exhibit higher cell type specificity, it cannot be discounted that there are additional dCP enhancers present in the Drosophila genome that were not identified by STARR-seq and thus, excluded from this analysis. From these studies, it is unclear if the enrichment of Fs(1)h-L and Rad21, particularly because Fs(1)h-L and Rad21 are present in hkCP enhancers at lower levels, or the absence of BEAF-32, CAP-H2, Chromator, CP190, DREF and Z4 truly distinguishes the architectural protein complexes found at dCP enhancers. In the future, careful biochemical analyses will be required to gain a comprehensive understanding of the complete organization of architectural protein subcomplexes associated with each enhancer class (Cubenas-Potts, 2017).

hkCP enhancers are associated with multi-TSS chromatin interactions and TAD borders. The promoter-clustering by hkCP enhancers results in a dose-dependent increase in transcriptional output for the interacting genes. Thus, one likely molecular mechanism by which hkCP enhancers promote robust transcriptional activation is by increasing the local concentration of RNA Polymerase II and general transcription factors (GTFs) by bringing multiple TSSs into close proximity. It is interesting that the hkCP enhancers, which form promoter clusters, are associated with TAD borders. It is speculated that the hkCP enhancer interactions involve inter-TAD contacts within the A-type compartment, indicative of the formation of transcription factories (70). From this analysis, it is unclear if the hkCP enhancers alone are sufficient for the formation of the 3D interactions or the neighboring TSSs and their associated transcription factors are also contributing to these contacts. It is hypothesized that the genes recruited to the factories contain the housekeeping promoter motifs (DRE, Ohler 1, Ohler 6 and TCT) and that the hkCP enhancer residents Cap-H2, Chromator, DREF and Z4, are critical to the formation of these 3D contacts (Cubenas-Potts, 2017).

dCP enhancers are more likely to be present within TADs and are enriched on the subTAD-like chromatin loop anchors. dCP enhancers do not form promoter clusters, but are more likely to interact with individual TSSs. One possible explanation for this observation is that the genes interacting with dCP enhancers require the binding of sequence-specific transcription factors, and increasing the concentration of GTFs and RNA polymerase II is not an effective mechanism to promote transcriptional output. The chromatin loop association is consistent with dCP enhancers forming a strong contact with a single TSS. However, it is acknowledged that dCP enhancers are likely one of multiple molecular mechanisms contributing to chromatin loop formation. Surprisingly, the chromatin loops that were observed in Drosophila are distinct from the chromatin loops described in humans. A recent study reported approximately 10,000 chromatin loops in the genome of GM12878 lymphoblastoid cells, but this study detected only 458 chromatin loops in Drosophila utilizing a similar method. The reason why there are so few chromatin loops in Drosophila compared to humans is unclear. It is possible that chromatin loops represent a more precise level of architecture within TADs between specific enhancers and promoters in mammals, but because TADs are significantly smaller in flies (median size 32.5 kb compared to 880 kb in mice, the chromatin loops are not as prominent or easily detected in the Drosophila genome. Notably, it appears that the chromatin loops are generated by different architectural proteins in the two species. The chromatin loops in humans are anchored by convergent CTCF motifs, while the results presented in this study demonstrate that the chromatin loop anchors in Drosophila are depleted of CTCF. Because the chromatin loops in Drosophila show a strong enrichment for Fs(1)h-L, a Brd4 homolog, and the architectural proteins Rad21, Nup98, TFIIIC and Mod(mdg4), it is possible that a combination of transcription and architectural proteins is required for chromatin loop formation in flies, which may be different from mammals . Altogether, it is clear that dCP enhancers are involved in individual contacts with TSSs and are likely one mechanism by which chromatin loops form in Drosophila (Cubenas-Potts, 2017).

Surprisingly, only ~20% and ~12.5% of all hkCP enhancer and ~7.5% and ~8.5% of dCP enhancer interactions involve a TSS or enhancer on the opposite anchor, respectively. The biological significance of the enhancer to non-TSS association is unclear. One possible explanation is that current methods for identifying statistically significant interactions are not sufficiently robust and that many of the enhancer to non-TSS interactions are not representative of biologically significant contacts. However, it cannot be discounted that the non-TSS interactions mediated by enhancers are real and the biological significance of these contacts remains to be determined. Throughout this analysis, the patterns of TSS interactions were compared with each enhancer class instead of drawing conclusions about the absolute number of TSSs bound per enhancer, minimizing the impact of any non-specific interactions within the data. Additional molecular studies for the various type of enhancer interactions (enhancer to promoter, enhancer to non-TSS, etc.) will be required to evaluate the various biological contributions of each (Cubenas-Potts, 2017).

This study found that the functional differences between enhancers that activate housekeeping versus developmental genes are reflected in their chromatin and architectural protein composition, and in the type of interactions they mediate. hkCP enhancers are marked by H3K4me3, associate with TAD borders, and mediate large TSS-clustered interactions to promote robust transcription. This class of enhancers contain the architectural proteins CAP-H2, Chromator, DREF and Z4. In contrast, dCP enhancers are marked by H3K4me1, associate with chromatin loop anchors and are more commonly associated with single TSS-contacts. dCP enhancers are depleted of the hkCP-specific architectural proteins and show an enrichment for Fs(1)h-L and Rad21. The results support a model suggesting that the unique occupancy of architectural proteins in the distinct enhancer classes are key contributors to the types of interactions that enhancers can mediate genome-wide, ultimately affecting enhancer-promoter specificity and 3D chromatin organization. In the future, further characterization of the broadly defined housekeeping and developmental enhancers into smaller subclasses may yield additional levels of regulation and formation of unique architectural protein and transcription factor protein complexes as key mediators of long range chromatin contacts (Cubenas-Potts, 2017).

Golgi outpost synthesis impaired by toxic polyglutamine proteins contributes to dendritic pathology in neurons

Dendrite aberration is a common feature of neurodegenerative diseases caused by protein toxicity, but the underlying mechanisms remain largely elusive. This study shows that nuclear polyglutamine (polyQ) toxicity resulted in defective terminal dendrite elongation accompanied by a loss of Golgi outposts (GOPs) and a decreased supply of plasma membrane (PM) in Drosophila class IV dendritic arborization (da) (C4 da) neurons. mRNA sequencing revealed that genes downregulated by polyQ proteins included many secretory pathway-related genes, including COPII genes regulating GOP synthesis. Transcription factor enrichment analysis identified CREB3L1/CrebA, which regulates COPII gene expression. CrebA overexpression in C4 da neurons restores the dysregulation of COPII genes, GOP synthesis, and PM supply. Chromatin immunoprecipitation (ChIP)-PCR revealed that CrebA expression is regulated by CREB-binding protein (CBP), which is sequestered by polyQ proteins. Furthermore, co-overexpression of CrebA and Rac1 synergistically restores the polyQ-induced dendrite pathology. Collectively, these results suggest that GOPs impaired by polyQ proteins contribute to dendrite pathology through the CBP-CrebA-COPII pathway (Chung, 2017).

Neurons have tremendously higher amounts of plasma membrane (PM) than other cell types, due to their highly elongated morphology. Recently, dendritic satellite organelles have been reported to be involved in the maintenance of integrity and dynamics of the PM in distal dendrites out of reach from somatic perinuclear organelles. The dendritic organelles have functional overlaps with the canonical perinuclear organelles. However, the restricted space appears to require the miniaturization of the satellite organelle systems, such as the endoplasmic reticulum (ER) and Golgi outposts (GOPs), compared to those localized around the nucleus, suggesting that the dendritic organelles may show different functional capacity from their perinuclear counterparts. Additionally, the distal areas of the dendrites may have quite different cellular environments, such as variations in the concentration of ions and proteins, compared to the perinuclear region. These differences may account for the vulnerability of dendrites over other neuronal domains to neuropathological insults, including protein toxicity conferred by the accumulation of mutant or misfolded proteins in neurons (Chung, 2017).

In Drosophila dendritic arborization (da) sensory neurons, GOPs were found to be located in dendrites, but not in axons, suggesting that GOPs may act as local stations to supply membranes to the PM in nearby dendrites (Ye, 2007). The synthesis of GOPs was reported to be regulated by secretory pathway-related genes, such as Sec31 and the transcription factor Cut (Iyer, 2013). Additionally, GOPs were shown to be transported toward or away from the soma by machinery consisting of a lava lamp protein (LVA), a golgin coiled-coil adaptor protein, and a motor dynein-dynactin protein complex, and their transport was found to be closely associated with the dynamics of dendrite growth. Moreover, GOPs have been implicated in the growth and maintenance of the dendrite arbor. For example, laser ablation of local GOPs decreased branch dynamics in da neurons (Ye, 2007), and the disruption of GOP trafficking blocked dendrite growth in developing hippocampal neurons, leading to the decreased length of dendrites (Chung, 2017).

Despite such functional roles of the GOPs in the growth and maintenance of the dendrite arbor under non-pathological conditions, it still remains unclear whether the GOPs can also contribute to dendrite aberration observed under pathological conditions induced by protein toxicity. Previously, nuclear proteotoxicity has been reported to cause severe dendrite pathology, but the mechanisms underlying dendrite pathology caused by protein toxicity remain largely elusive. Therefore this study investigated a mechanistic link of the GOPs to dendrite pathology caused by toxic nuclear polyQ proteins using Drosophila da sensory neurons that have been extensively utilized by various researchers as an in vivo cellular model for the study of the mammalian neuronal dendrite system with high fidelity dendrite pathology (Chung, 2017).

Toxic nuclear polyQ proteins cause terminal dendrite defects in C4 da neurons. This study examined morphological characteristics of dendrites of class IV (C4) da neurons expressing pathogenic Machado-Joseph's Disease (MJD) protein, MJDtr-78Q (78Q), known to induce nuclear protein toxicity. For this analysis, dendrite images of C4 da neurons expressing 78Q (78Q OE) and wild-type C4 da neuron controls were compared. A mechanistic model is proposed that links toxic nuclear polyQ proteins to the impaired elongation of terminal dendrites in 78Q OE through the CBP-CrebA-COPII-GOP pathway. Toxic nuclear polyQ proteins decreased the number of GOPs in the dendrites of C4 da neurons. mRNA sequencing revealed that the secretory pathway, including the COPII pathway, was a major pathway that was downregulated by toxic polyQ proteins. The disruption of the COPII pathway decreased GOP synthesis. TF enrichment analysis identified CrebA as a key regulator of the genes involved in the COPII pathway. CrebA overexpression restored the downregulation of the COPII pathway and the loss of GOPs caused by toxic polyQ proteins. It was further found that CBP regulated CrebA expression cooperatively with Cut and that toxic polyQ proteins interfered with CBP upstream of CrebA in the regulation of GOP synthesis. Finally, the co-overexpression of CrebA and Rac1 synergistically restored polyQ-induced defects in dendrite branching and elongation (Chung, 2017).

According to a mechanistic model, however, how the overexpression of Cut, unlike CBP, can potentially contribute to the restoration of GOPs is unclear, although it cooperatively regulates CrebA expression with CBP and interacts with CBP at the protein level. This can be explained by alterations in the stoichiometry between CBP and Cut for their complex formation under polyQ-expressed conditions. The results showed that polyQ proteins appeared to sequester CBP, but not Cut. The sequestration of CBP by polyQ proteins leads to a decreased amount of CBP available for the CBP-Cut complex formation. Accordingly, CBP overexpression can increase the amount of non-sequestered CBP under polyQ-expressed conditions, thereby restoring the CBP-Cut complex formation. By contrast, Cut overexpression does not contribute to an increase in complex formation because it has no effect on the depletion of CBP by polyQ proteins. Moreover, Rac1 was included as a component in the mechanistic model. Rac1 was connected to the polyQ-CBP-CrebA pathway based on the following results. First whether polyQ expression affected the expression of Rac1 was tested and a significant decrease in Rac1 expression was found in polyQ-expressing fly heads compared to w1118. Then TFs were sought that could be affected by polyQ expression and could regulate Rac1 expression. Previously, Cut was shown to regulate Rac1 expression. Thus, to examine the effect of polyQ proteins on Cut, whether polyQ proteins altered the intracellular distribution of Cut proteins was checked in polyQ-expressing da neurons, and no noticeable alteration was found in its distribution. As previously mentioned, Cut interacted with CBP in fly heads. Furthermore, Rac1 was shown to be epigenetically suppressed by H3K27me3, which can be antagonized by CBP-dependent H3K27ac. These data together provide a mechanistic connection of the CBP-Cut-Rac1 pathway to the polyQ-CBP-CrebA pathway through CBP (Chung, 2017).

This study proposed a polyQ-CBP-CrebA pathway for polyQ-induced terminal dendrite defects. However, an alternative possibility that dendrite perturbation may precede through CBP-CrebA-independent pathways, which then can result in altered gene expression, cannot be excluded. Consistent with such a possibility, nuclear polyQ proteins were shown to sequester nuclear pore complex proteins, potentially impairing the transport of proteins and RNA, independently of CBP or CrebA (Grima, 2017). Moreover, nuclear polyQ proteotoxicity induces the mis-localization of several TFs (e.g., TBP, Sp1, and p53) other than CBP, leading to the altered expression of their target genes. The perturbed functions of nuclear pore complexes and TFs may result in dendrite defects via CBP-CrebA-independent mechanisms and consequently alterations in gene expression. However, the restoration of GOP synthesis and PM protein supply via the overexpression of CBP or CrebA and the restoration of dendritic arborization defects via the co-overexpression of CrebA and Rac1 in polyQ-expressing neurons demonstrates that the CBP-CrebA pathway should be one of the important pathways that functionally regulates the polyQ-induced dendrite defects. Nevertheless, the relative importance of the CBP-CrebA pathway to the pathways that include other TFs affected by polyQ proteins can be questioned. However, this study found that the overexpression of the three key TFs (HLH106, gt, and Dref) did not restore the number of GOPs in polyQ-expressing neurons. Moreover, the effect of Sp1, which is known to be downregulated by polyQ proteins, on GOP synthesis was examined, and Sp1 knockdown did not significantly decrease the number of GOPs compared to the controls. Furthermore, it was also found that the overexpression of Cut and Scr, known upstream TFs of CrebA, failed to restore the loss of GOPs in polyQ-expressing neurons comparable to CrebA overexpression. Therefore, the data suggest that the mechanism by which polyQ toxicity induces dendrite pathology can be attributed to the polyQ-CBP-CrebA pathway proposed in this study (Chung, 2017).

The loss of GOPs was one of the most apparent dendrite defects caused by polyQ proteins. Thus, among many cellular processes downregulated by the polyQ proteins, this study focused on three cellular processes, (1) vesicle-mediated transport (VT) and (2) membrane organization (MO) and lipid biosynthetic process (LP), that were considered to be related to GOPs. However, these processes can also be related to other secretory-pathway-related processes in addition to GOP synthesis. For example, neuropeptide maturation and trafficking were previously shown to be modulated by the secretory pathway, suggesting the potential association of neuropeptide-containing vesicle trafficking with the three processes. Thus the effect of polyQ proteins on neuropeptide-containing vesicle trafficking was examined by measuring the amount of neuropeptide-containing vesicles in w1118 and polyQ-expressing neurons using a reporter. PolyQ expression was found to decrease the amount of ANF-EMD vesicles in dendrites and soma compared to controls, suggesting that polyQ perturbs the function of the secretory pathway related to neuropeptide-containing vesicle trafficking in addition to GOP synthesis. Additionally, the three processes can be associated with the synthesis of somatic Golgi. However, the data showed a larger loss of ManII-eGFP-positive puncta in the dendrites of C4 da neurons compared to the soma, suggesting that the three processes can be considered to be related more to GOP synthesis than to somatic Golgi synthesis. Thus, although the polyQ-induced downregulation of genes associated with the abovementioned three processes may impact the secretory pathway in general, the results appear to indicate that the synthesis of GOPs responds more to the downregulation of those genes compared to the synthesis of somatic Golgi (Chung, 2017).

In mRNA-sequencing analysis, fly heads were used to identify genes affected by toxic polyQ proteins. Considering that fly heads contain no C4 da neurons, alterations in gene expression obtained from mRNA sequencing in polyQ-expressing fly heads, compared to w1118 fly heads, should be defined by non-C4 da cells. Due to a small number (~50) of C4 da neurons per larva, however, it is challenging to isolate a sufficient number (>105 cells) of C4 da neurons for mRNA sequencing. Instead of C4 da neurons, fly heads were used for mRNA sequencing to obtain a clue for alteration of gene expression by polyQ proteins. Moreover, mRNA sequencing identified 5,385 differentially expressed genes between polyQ-expressed and control samples, of which 5,325 (98.9%) were downregulated. Such a large number and high percentage of downregulated genes suggests that there might be widespread cell death in polyQ-expressing fly brains. Thus, the extent of cell loss was checked in the polyQ-expressing adult fly brains on which mRNA sequencing was performed by measuring the number of cells that displayed cleaved caspase-3 through immunohistochemistry analysis. PolyQ-expressing brains showed minimal cell loss compared to controls in the condition where mRNA sequencing was performed. Additionally, no observable decrease was found in rRNA in polyQ-expressed adult fly heads compared to w1118 heads. These data suggest that the downregulation of the large number of genes is not likely to be caused by cell death. One alternative explanation can be the previously reported dysregulation of the following histone modifications caused by polyQ proteins: impaired acetylation of H3K27 and dysregulated tri-methylation of H3K9 and H3K27. These dysregulated histone modifications may result in the downregulation of 98.9% of DEGs through a global epigenetic silencing (Chung, 2017).

The normal length of the polyQ tract in humans has been reported to be less than 36 for the MJD protein. In flies, numerous studies have used the truncated MJD protein with only 27 polyQ repeats (MJDtr-27Q) as a control. However, in this study, w1118 rather than 27Q OE was used as the control for 78Q OE in most of the experiments because 27Q OE exhibited strong dendrite pruning defects compared to 78Q OE. A previous study also showed that Httex1p-Q93 caused strong pruning defects in C4 da neurons, whereas Httex1p-Q20 caused no such defects. Taken together, these data suggest that the pruning defects are likely not due to the context of the host proteins but to polyQ toxicity. However, there appears to be a threshold length (between 20Q and 27Q) of the polyQ tract to confer such polyQ toxicity. Nevertheless, further detailed studies should be performed to understand the mechanism underlying the protein toxicity of MJDtr-27Q to induce pruning defects (Chung, 2017).

Finally, this study identified three different nuclear polyQ toxicity models that exhibited decreased GOP synthesis and PM protein supply, indicating a potentially shared mode of nuclear proteotoxicity. Of note, the accumulation of misfolded proteins in the nucleus has been observed in other neuronal diseases or during aging. Thus, the proposed mechanistic model underlying terminal dendrite defects caused by the accumulation of nuclear toxic polyQ proteins can provide significant insights into other neuronal maladies linked to nuclear proteotoxicity and aging that can be further examined in detailed functional studies (Chung, 2017).


DEVELOPMENTAL BIOLOGY

A genetic screen identifies putative targets and binding partners of CREB Binding Protein (CBP) in the developing Drosophila eye

Drosophila CREB Binding Protein (dCBP) is a very large multi-domain protein, which belongs to the CBP/p300 family of proteins, which were first identified by their ability to bind the CREB transcription factor and the adenoviral protein E1. Since then CBP has been shown to bind to over 100 additional proteins and functions in a multitude of different developmental contexts. Among other activities, CBP is known to influence development by remodeling chromatin, by serving as a transcriptional co-activator and by interacting with terminal members of signaling transduction pathways. Reductions in CBP activity are the underlying cause of Rubinstein-Taybi syndrome, which is, in part, characterized by several eye defects including strabismus, cataracts, juvenile glaucoma and coloboma of the eyelid, iris, and lens. Development of the Drosophila compound eye is also inhibited in flies that are mutant for CBP. However, the vast array of putative protein interactions and the wide-ranging roles played by CBP within a single tissue such as the retina can often complicate the analysis of CBP loss-of-function mutants. Through a series of genetic screens several genes have been identified that could either serve as downstream transcriptional targets or encode for potential CBP binding partners and whose association with eye development has hitherto been unknown. The identification of these new components may provide new insight into the roles that CBP plays in retinal development. Of particular interest is the identification that the CREB transcription factor appears to function with CBP at multiple stages of retinal development (Anderson, 2005).

Through the use of several genetic screens attempts have been made to identify genes that function cooperatively with CBP to influence eye development. Either wild type or one of three variants of CBP was expressed within the developing eye of Drosophila. The expression of each CBP protein resulted in a disruption of eye development that is easily visualized by an examination of the external surface of adult eyes. The degree of structural alteration and the underlying cause of said disruptions are unique to the expression of each individual CBP protein. The retinal phenotypes that are generated by the expression of CBP variant proteins reveals considerable information on the role that CBP plays in normal eye development. Each variant protein retains a unique combination of protein domains and is expected to act as a protein sink by binding and soaking up signaling pathway components and transcription factors. Thus the changes in eye specification and ommatidial cell fate are due to a 'loss' of factors that normally function in these processes. Since CBP appears to be normally expressed in every cell within the developing retina it is likely that the putative transcriptional targets and binding partners of CBP that were identified in these screens are biologically relevant for eye development (Anderson, 2005).

Advantage was taken of the Bloomington Stock Center Deficiency Kit to rapidly identify regions of the genome that potentially harbor interacting genes. Through this method, 71 such regions were initially identified. Extant single gene mutants were then screened and 35 complementation groups were identified that potentially interact with CBP. Interacting genes for the remaining 36 genomic intervals could not be identified. This is likely due to the lack of single gene mutations for all predicted genes. Alternatively it is possible that these regions contain two or more genes that must be removed simultaneously in order to modify the rough eye phenotypes associated with CBP expression (Anderson, 2005).

Among the collection of potential interacting genes, the screens identified members of the eye specification cascade, two Hox genes (Ultrabithorax and Sex combs reduced), the CREB transcription factor and several members of the Epidermal Growth Factor Receptor (Egfr) and TGFbeta signaling cascades. Recovery of these factors in the course of an 'eye screen' is supported by known interactions between CBP and many of these factors in other developmental contexts. For instance, CBP is known to interact with the TGFbeta signaling pathway during several stages of embryonic development. Likewise, CBP is known to participate in the regulation of several Hox genes. As another example, CBP is known to interact with the retinal determination genes in both the fly retina and mouse muscle. Furthermore, interactions between CBP and CREB have been well documented in a number of contexts including learning and memory and synapse formation. And finally, the CREB-CBP complex is known to be phosphorylated by RSK, a known component of receptor tyrosine kinase signaling cascades. Thus, it is now possible to link CBP to the Egfr pathway during eye development. These results are further supported by the fact that loss-of-function mutations in either CBP or members of the Egfr cascade lead to near identical phenotypes in the eye, wing, leg and embryo of Drosophila. The screens connect CBP directly to this signaling system (Anderson, 2005).

It should be noted that the approach used in this report to find genes that potentially interact with CBP revealed interactions that would not have been found if the study were limited to a single screen for modifiers of the GMR-CBP FL (GMR driven full length CBP) rough eye phenotype. In that screen only two deficiency stocks were uncovered that could modify the rough eye. Since CBP interacts with well over 100 different proteins the reduction in any one is unlikely to alter significantly the GMR-CBP FL rough eye phenotype. The empirical results bear this out. By expressing CBP protein variants, each containing a unique combination of functional domains, it was possible to induce retinal phenotypes that could be modified by reductions in single gene dosages. Another novel aspect of these genetic screens is that have not only been able to find factors that interact with or are regulated by CBP, but some information is now available about how they modulate the activity of this co-activator. For instance, CBP deltaBHQ (lacking the Histone acetyltransferase domain and the Poly glutamine stretch) retains the N-terminal half of protein, which includes a zinc finger domain, a nuclear hormone receptor binding domain and the CREB binding domain. It is predicted that the genes identified as suppressors of the GMR-CBP deltaBHQ rough eye phenotype may be bound by the zinc finger domain of CBP or the CREB transcription factor. Alternatively, these genes may encode proteins that interact with the N-terminal portion of CBP. For example, the CBP deltaBHQ protein can still interact with the CREB transcription factor through the KIX domain. Previous reports have demonstrated that CREB is phosphorylated by RSK, a member of receptor tyrosine kinase signaling. The screen for modifiers of the GMR-CBP deltaBHQ phenotype yielded several members of the EGFR signaling cascade. The results from these screens will provide insight into how the modular activity of CBP can be integrated with the remaining interacting genes and proteins (Anderson, 2005).

In a significant fraction of cases it remains to be determined which of the newly identified factors will turn out to be binding partners, transcriptional targets or upstream regulators of CBP. This is especially true of many genes that have had no previously ascribed role in eye development. In addition to standard genetic practices of examining phenotypes of loss-of-function mutants, several high-throughput methods including yeast two hybrid assays and genomic microarrays will certainly be useful in sorting out the regulatory relationship between CBP and the interacting genes that are described in this report. Since CBP is thought to function as a biochemical scaffold, the results support a role for CBP in integrating instructions from multiple signaling pathways and regulatory networks. One of the most interesting results from this screen is the identification of CREB as a potential regulator of eye development. The interactions between CREB and CBP are well known and have been described for several developmental contexts. However, this is the first report of a potential role for CREB in Drosophila eye development. It has been further demonstrated that CREB-A is expressed in cells ahead of the morphogenetic furrow and in all developing photoreceptor cells. Loss-of-function retinal mosaic clones indicate that the CREB-A transcription factor is required in all photoreceptor cells with the exception of R8. This phenotype is consistent with that seen in CBP loss-of-function retinal mosaic clones. Interestingly, ectopic expression of CrebA is sufficient to induce ectopic eye formation on the ventral surface of the fly head, a phenotype seen when the Pax protein Eyg is itself ectopically expressed. This might place the CBP-CREB complex within the retinal determination network. While it has been reported that activation of CREB in the vertebrate retina blocks degeneration of retinal ganglion cells (RGCs), it is exciting to speculate on potential roles for CREB in the determination of the vertebrate eye and for the specification of vertebrate cell types. It would also be interesting to determine if mutations within human CREB might also be associated with the same retinal phenotypes observed in patients harboring lesions with CBP (Anderson, 2005).

Of the 36 genomic regions for which the genetic loci responsible for the modification of the rough eye phenotypes has not been identified, three regions are of particular interest because these results suggest the interacting gene or the encoded protein interacts specifically with the N-terminal half of CBP. Deletion of the 76B4-77B1 interval suppresses the rough eye phenotypes that are associated with expression of CBP FL, CBP deltaBHQ and CBP deltaQ proteins while enhancing the CBP deltaNZK (lacking the Nuclear hormone receptor binding domain, the Zinc finger domains and the KIX or CREB binding domain) retinal phenotype. These results suggest that at least one interacting gene resides within this interval and that it is either a target of or the encoded protein is a binding partner of the N-terminal region of CBP. Deletions of the 96F1-97B1 interval enhance the rough eye phenotypes that result from expression of CBP deltaBHQ and CBP deltaQ proteins. Likewise, deletions of the 5C3-6C12 interval suppress the retinal phenotypes of CBP deltaBHQ and CBP deltaQ expression. In both situations it is likely that the interacting gene or its encoded protein functions specifically with the N-terminal domain of CBP. The remaining 33 regions modified the expression of specific constructs. It will be interesting to determine if they encode for members of a common pathway or do they represent a set of factors within differing activities (Anderson, 2005).

Fasting launches CRTC to facilitate long-term memory formation in Drosophila

Canonical aversive long-term memory (LTM) formation in Drosophila requires multiple spaced trainings, whereas appetitive LTM can be formed after a single training. Appetitive LTM requires fasting prior to training, which increases motivation for food intake. However, this study found that fasting facilitates LTM formation in general; aversive LTM formation also occurred after single-cycle training when mild fasting was applied before training. Both fasting-dependent LTM (fLTM) and spaced training-dependent LTM (spLTM) requires protein synthesis and cyclic adenosine monophosphate response element-binding protein (CREB) activity. However, spLTM requires CREB activity in two neural populations--mushroom body and dorsal-anterior-lateral (DAL) neurons--whereas fLTM required CREB activity only in mushroom body neurons. fLTM uses the CREB coactivator CREB-regulated transcription coactivator (CRTC), whereas spLTM uses the coactivator CBP. Thus, flies use distinct LTM machinery depending on their hunger state (Hirano, 2013).

In Drosophila, canonical aversive long-term memory (LTM), which is dependent on de novo gene expression and protein synthesis, is generated after multiple rounds of spaced training. In contrast, appetitive LTM can be formed by single-cycle training. Because both aversive and appetitive LTM require protein synthesis and activation of CREB, it is likely that both types of LTM are formed by similar mechanisms. Appetitive and aversive LTM are known to differ (i.e., octopamine is specifically involved in appetitive but not aversive memory formation). However, it remains unclear why single-cycle training is sufficient for appetitive but not aversive LTM formation. Appetitive LTM cannot form unless fasting precedes training. Although fasting increases motivation for food intake (a requirement for appetitive memory) it was suspected that fasting may activate a second, motivation-independent, memory mechanism that facilitates LTM formation after single-cycle training (Hirano, 2013).

Flies were deprived of food for various periods of time and then subjected to aversive single-cycle training. Fasting prior to training significantly enhanced 1-day memory, with a peak at 16 hours of fasting and a return to nonfasting levels at 20 to 24 hours of fasting. In contrast, 16 hours of fasting did not increase short-term memory (STM, measured 1 hour after training). In this protocol, flies were returned to food vials after training, raising a possibility that the perception of food as a reward after training may enhance the previous aversive memory. This possibility was tested by inserting refeeding periods between food deprivation and training. Although fasting followed by a 4-hour refeeding period failed to induce appetitive LTM, it significantly enhanced aversive 1-day memory; this finding suggests that enhancement of aversive memory occurs through a mechanism unrelated to increased motivation or perception of food as a reward. A 6-hour refeeding period was sufficient to prevent aversive memory enhancement. Continuous food deprivation after training suppressed aversive memory enhancement, which indicates that both fasting before training and feeding after training are required to enhance aversive memory (Hirano, 2013).

Administration of the protein synthesis inhibitor cycloheximide (CHX) abolished 1-day memory enhancement but had no effect on 1-hour memory, supporting the idea that memory enhancement consists of an increase of LTM. Memory remaining after CHX treatment is likely to be protein synthesis-independent, anesthesia-resistant memory (ARM). Fasting for 16 hours neither enhanced protein synthesis-independent memory nor canonical aversive LTM generated by spaced training (spLTM). Furthermore, fasting-dependent memory decayed within 4 days, and food deprivation did not enhance 4-day spLTM, indicating that fasting-dependent memory is physiologically different from spLTM (Hirano, 2013).

Fasting-dependent memory was blocked by acute, dose-dependent, expression of CREB2-b, a repressor isoform of CREB, in the mushroom bodies (MBs). Expression of the repressor from two copies of UAS-CREB2-b under control of the MB247-Switch (MBsw) GAL4 driver, which induces UAS transgene expression upon RU486 feeding, significantly suppressed fasting-dependent memory upon RU486 feeding, whereas expression from one copy of UAS-CREB2-b did not. Defects in LTM formation are highly correlated with CREB2-b amounts. Significantly higher MBsw-dependent expression of CREB proteins was found in flies carrying two copies of UAS-CREB2-b relative to flies carrying one copy. MBsw-dependent CREB2-b expression did not affect STM in either fed or food-deprived conditions. Because the aversive memory enhanced by fasting is mediated by protein synthesis and CREB, this memory is referred to as fasting-dependent LTM (fLTM). Similar to the results in aversive fLTM, MBsw-dependent CREB2-b expression also decreased appetitive LTM but not appetitive STM (Hirano, 2013).

A recent study (Chen, 2012) concluded that CREB activity in MB neurons is not required for spLTM. In that study, CREB2-b was expressed using the OK107 MB driver and GAL80ts was used to restrict CREB2-b expression to 30°C. However, this study found that the GAL80ts construct still inhibited expression of CREB considerably at 30°C. When higher amounts of CREB2-b were acutely expressed in MBs using MBsw, a significant decrease was observed in 1-day spLTM, indicating that CREB activity in the MBs is likely to be required for spLTM (Hirano, 2013).

Consistent with the results of Chen (2012) expression of CREB2-b in two dorsal-anterior-lateral (DAL) neurons impaired aversive spLTM. In contrast, expression of CREB2-b in DAL neurons did not affect aversive fLTM. Moreover, appetitive LTM was also not affected by expression of CREB2-b in DAL neurons. MBsw did not express GAL4 in DAL neurons (Hirano, 2013).

CREB requires coactivators, including CBP (CREB-binding protein), to activate transcription needed for LTM formation. Acute expression of an inverted repeat of CBP (CBP-IR) in MBs significantly impaired spLTM without affecting either STM or 1-day memory after multiple massed trainings, which do not lead to LTM formation. However, neither aversive fLTM nor appetitive LTM was impaired by CBP-IR expression, indicating that an alternative coactivator may be required for fasting-dependent memory (Hirano, 2013).

Recent studies demonstrate the involvement of a cAMP-regulated transcriptional coactivator (CRTC) in hippocampal plasticity (Kovacs, 2007; Zhou, 2006). In metabolic tissues, phosphorylated CRTC is sequestered in the cytoplasm while dephosphorylated CRTC translocates to the nucleus to promote CREB-dependent gene expression. Fasting causes CRTC dephosphorylation and activation. In line with this, significant accumulation of hemagglutinin (HA)-tagged CRTC (CRTC-HA) was found within MB nuclei after 16 hours of food deprivation. Subcellular fractionation indicated that food deprivation causes CRTC-HA nuclear translocation without affecting total CRTC-HA amounts (Hirano, 2013).

To examine the role of CRTC in fLTM and spLTM, a CRTC inverted repeat (CRTC-IR) was acutely expressed using MBsw, and suppression of aversive fLTM was observed but no effect was seen on STM. CHX treatment did not further decrease 1-day aversive memory, and CRTC-IR expression from a second MB driver, OK107, also impaired fLTM formation. CRTC-IR expression from MBsw also impaired appetitive LTM without affecting appetitive STM. In contrast, CRTC-IR expression from MBsw did not impair spLTM . CRTC-IR expression in DAL neurons had no effect on either aversive fLTM or appetitive LTM. Consistent with these results showing lack of fLTM after 24-hour fasting, 1-day aversive memory after 24-hour fasting did not decrease upon CRTC-IR expression in MBs (Hirano, 2013).

To examine the effects of spaced training on fLTM and the effects of fasting on spLTM, fed or fasted flies expressing either CBP-IR or CRTC-IR were space-trained. When CBP-IR was expressed to impair spLTM, 1-day memory after spaced training was impaired in fed conditions but not in fasting conditions, which suggested that spaced training protocols do not block fLTM. When CRTC-IR was expressed to impair fLTM formation, 1-day memory after spaced training was not affected by fasting, which suggested that mild fasting does not impair spLTM formation (Hirano, 2013).

Is activation of CRTC sufficient to generate fLTM in the absence of fasting? HA-tagged constitutively active CRTC (CRTC-SA-HA) was expressed from MBsw, and its nuclear accumulation was observed in the absence of fasting. Acute expression of CRTC-SA-HA from MBsw increased 1-day aversive memory after single-cycle training in fed flies, and this increase was not further enhanced by fasting. In contrast, expression of control CRTC-HA did not alter the fasting requirement for memory enhancement. CRTC-SA-HA expression did not affect feeding itself, which suggested that the memory enhancement is not due to impaired feeding. Taken together, CRTC activity in MBs is necessary and sufficient to form fLTM. Similar to the effects of fasting, CRTC-SA-HA expression did not affect STM or 4-day spLTM (Hirano, 2013).

In mammalian metabolic tissues, CRTC is phosphorylated by insulin signaling, which is suppressed by fasting (see Wang, 2008). CRTC phosphorylation is also regulated by insulin signaling in flies (Wang, 2008). To determine whether reduced insulin signaling activates CRTC and promotes fLTM formation, heterozygous mutants for chico, which encodes an adaptor protein required for insulin signaling, were tested. Although chico1 null mutants are semilethal and defective for olfactory learning, heterozygous chico1/+ mutants are viable and display normal learning (Hirano, 2013).

CRTC accumulated in MB nuclei in chico1/+ mutants in the absence of food deprivation. Under conditions where flies were fed, chico1/+ flies had significantly greater 1-day memory after single-cycle training relative to control flies, whereas 1-hour memory was unaffected. Enhanced 1-day memory in chico1/+ flies was not further enhanced by fasting. Because the chico1/+ mutation does not affect feeding itself, the memory enhancement would not seem to be attributable to impaired feeding. The increased 1-day memory in chico1/+ mutants was suppressed by CHX treatment and CRTC-IR expression using MBsw, which suggests that reduced insulin signaling mimics fLTM through activation of CRTC in MBs (Hirano, 2013).

Single-cycle training after mild fasting generates both appetitive and aversive LTM, and CRTC in the MBs plays a key role in both types of LTM. A CRTC-dependent LTM pathway is unlikely to be involved in increasing motivation required to form appetitive memory, because CRTC knockdown did not affect appetitive STM and because CRTC-SA expression was not sufficient to form appetitive LTM without prior fasting. Although mild 16-hour fasting induced aversive fLTM, longer 24-hour fasting impaired aversive fLTM but not appetitive LTM. Thus, although aversive and appetitive fLTM share mechanistic similarities, they may be regulated by different inputs controlling motivation and fasting time courses. Because nuclear translocation of CRTC was sustained even after 24 hours of food deprivation, prolonged fasting may suppress a CRTC-independent step in aversive fLTM formation. spLTM was not affected by 24-hour fasting prior to training, which suggests that the unknown inhibitory effect of 24-hour fasting does not occur after spaced training. Continuous food deprivation after training suppressed aversive fLTM. In another study, Placais (2013) reports that continuous food-deprivation after spaced training suppresses spLTM as well (Hirano, 2013).

Suppression of aversive LTM by prolonged fasting may ensure that starving flies pursue available food, with less concern for safety. Although the biological importance of aversive fLTM in natural environments is currently unclear, the current results indicate that different physiological states may induce different types of LTM in flies (Hirano, 2013).


EFFECTS OF MUTATION

At germband elongation, a mutation in Drosophila CBP results in a clockwise or counter-clockwise twisting of the embryo, just behind the cephalic furrow, often with the posterior side down. The ventral and cephalic furrows appear normal, but the mesodermally derived internal tissues and a block of ectodermal cells are often missing. On the basis of this phenotype, the Drosophila CBP mutant was named nejire, which means 'twist' in Japanese. Among the four Drosophila CBP mutants, the nej3 mutant exhibits the most severe phenotypes. In addition to the twisting of the embryo, cuticles of the late-lethal embryos exhibit several types of defects: disruption and twisting of the embryonic head skeleton; atrophy of the cephalo-pharyngeal skeleton and the thoracic region; sporadic deletion of naked cuticle between adjacent denticle belts; a decrease in the number of setae found in the denticle belts of the abdominal segments, and occasional reduction of the Filzkorper (Akimaru, 1997b)

The Drosophila X chromosome has been screened for genes whose dosage affects the function of the homeotic gene Deformed. One of these genes, extradenticle, encodes a homeodomain transcription factor that heterodimerizes with Deformed and other homeotic Hox proteins. Mutations in the nejire gene, which encodes a transcriptional adaptor protein belonging to the CBP/p300 family, also interact with Deformed. The other previously characterized gene identified as a Deformed interactor is Notch, which encodes a transmembrane receptor. These three genes underscore the importance of transcriptional regulation and cell-cell signaling in Hox function. Four novel genes were also identified in the screen. One of these, rancor, is required for appropriate embryonic expression of Deformed and another homeotic gene, labial. Both Notch and nejire affect the function of another Hox gene, Ultrabithorax, indicating they may be required for homeotic activity in general (Florence, 1998).

The extant nejire3 allele has been molecularly characterized as a null. However, in the Dfd interaction test, the viability of Dfd hypomorphs is not affected by heterozygous nej3. The failure of a nej null to interact with Dfd suggests that the Dfd-interacting nej alleles are not amorphic. Consistent with this interpretation, the lethal phases of the nej alleles in the current study differ from that of the nej3. Only ~15% of nej3 males die as embryos, demonstrating that the nej maternal component is sufficient for embryogenesis. However, 100% of both nejQ7 males and nejTA57j/nejQ7 females, as well as ~35% of nejS342/nejQ7 females, die as embryos. The premature lethality of the Dfd-interacting alleles indicates that they provide a less functional maternal component, perhaps because of an antimorphic Nej protein. In nejTA57/nejQ7 or nejQ7/Y cuticles, the maxillary and antennal sense organs often show a slight disruption in patterning; the mouth hooks and median tooth are reduced, and the proventriculus is sclerotized. No other phenotypes are consistently observed in cuticles of any nej genotype (Florence, 1998).

At the larval NMJ, an experimental decrease in postsynaptic excitation causes a compensatory enhancement of presynaptic release (see Glutamate receptor IIA and Glutamate receptor IIB). Thus there is a homeostatic regulatory system that maintains postsynaptic excitation through a retrograde signal(s) from muscle to nerve. A homeostatic regulatory system will likely include mechanisms that can monitor postsynaptic excitation and transduce this information through a retrograde signal to modulate presynaptic transmitter release. In principle, homeostatic regulation will require both positive and negative regulation of synaptic function (Marek, 2000).

Nuclear dCBP (Drosophila homolog of the CREB binding protein) located in the postsynaptic cell is required for presynaptic functional development. Viable, hypomorphic dCBP mutations have a ~50% reduction in presynaptic transmitter release without altering the Ca2+ cooperativity of release or synaptic ultrastructure (total bouton number is increased by 25%-30%). Exogenous expression of dCBP postsynaptically, in muscle, rescues impaired presynaptic release in the dCBP mutant background, while presynaptic dCBP expression does not. In addition, overexpression experiments indicate that elevated dCBP can also inhibit presynaptic functional development in a manner distinct from the effects of dCBP loss of function. Pre- or post-synaptic overexpression of dCBP (in wild type) reduces presynaptic release. However, no increase in bouton number is observed, and presynaptic overexpression impairs short-term facilitation. These data suggest that dCBP participates in a postsynaptic regulatory system that controls functional synaptic development (Marek, 2000).

This study used novel insertions in the dCBP sequence to regulate the expression of the gene. Four P(EP) elements have been identified that are located in a region ~3 kb upstream of the dCBP open reading frame. Two of these elements -- —P(EP)1179 and P(EP)1149— -- are oriented to overexpress dCBP, and two are oriented in the opposite orientation. P(EP) elements are mobile genetic elements (P elements) containing upstream transcription activation sequences (UAS sequence) that exploit the tendency for P elements to insert in 5' regulatory regions of a gene. Insertion of such an element in the proper 5'-3' orientation places random genes under the control of the UAS element, allowing them to be expressed in specific tissues under the control of an appropriate GAL4 driver. P(EP)1179 and P(EP)1149 can initiate overexpression of dCBP. Crossing P(EP)1179 or P(EP)1149 to GAL4 drivers that promote expression in nerve or muscle causes tissue-specific overexpression of dCBP, as detected by in situ hybridization using probes specific to the dCBP gene. Overexpression of dCBP has also been demonstrated by RT-PCR, using primers from the P(EP) elements and primers within the dCBP open reading frame. Each PCR product isolated by RT-PCR was sequenced to ensure that the correct open reading frame was driven by the P(EP) element. To ensure that these elements do not regulate overexpression of an additional message located between the P(EP) elements and the start of the dCBP open reading frame, the entire 3 kb genomic region between P(EP)1179 (the furthest insertion upstream of dCBP) and the start of dCBP was subcloned and sequenced. This region does not contain an additional transcript. Furthermore, the overexpression phenotype is phenocopied by overexpression of the dCBP cDNA under UAS control. Based on this sequence data and data obtained from RT-PCR from the P(EP)1179 and P(EP)1149 elements, a more complete characterization of the 5' untranslated region of dCBP is presented that extends to, and most likely beyond, these P(EP) elements. These results also predict that these P(EP) elements will generate a hypomorphic loss of dCBP function. Genetic, histological, and electrophysiological evidence demonstrates that the P(EP) elements P(EP)1149 and P(EP)1179 are hypomorphic mutations in dCBP. dCBP expression in muscle nuclei, as detected by an anti-dCBP antibody, is significantly reduced in the P(EP)1149 hypomorphic mutant (expression being decreased by ~75%–80%, based on reduced fluorescence intensity. Genetic experiments demonstrate that null or strong hypomorphic mutations in dCBP die as late embryos or first instar larvae. P(EP)1179 and P(EP)1149 fail to complement previously characterized hypomorphic alleles of dCBP (including dCBPTA57 and dCBPS342) for synaptic transmission defects. P(EP)1179 or P(EP)1149 trans-heterozygous with dCBPTA57 or dCBPS342 are semiviable as third instar larvae. These trans-heterozygous larvae are developmentally delayed by ~1 day and emerge as sluggish third instar larvae. It was therefore possible to proceed with anatomical and electrophysiological characterization of these dCBP loss-of-function mutations at the third instar larval synapse (Marek, 2000).

It is hypothesized that dCBP is centrally involved in the mechanisms that monitor postsynaptic activity. dCBP P(EP) element mutations are described, detected in a screen for mutations in genes that participate in the homeostatic regulation of presynaptic transmitter release. Perturbations in postsynaptic dCBP can affect presynaptic transmitter release. Furthermore, postsynaptic dCBP can act as both a positive and negative regulator of synaptic function. Finally, CBP in Drosophila and other systems is well suited to participate in a system that monitors postsynaptic activity. CBP function can be regulated by Ca2+ influx through voltage-gated channels and ionotropic receptors (Hardingham, 1999; Hu, 1999). Furthermore, CBP can act as a transcriptional coactivator with CREB and other transcription factors, as well as function as an intracellular signaling molecule via acetylase activity. In conclusion, it is proposed that dCBP is an essential component of the postsynaptic homeostatic mechanism that monitors activity and regulates presynaptic transmitter release (Marek, 2000).

It has been proposed that the homeostatic retrograde increase in presynaptic release is due to a signal that can enhance presynaptic transmission, similar to that proposed for the presynaptic expression of long-term potentiation. The current data suggest an additional model, one whereby a homeostatic increase in presynaptic transmission could also be achieved by relieving an inhibitory signal derived from postsynaptic dCBP function. Homeostatic control of presynaptic function at the Drosophila NMJ ensures that presynaptic release is precisely coupled to the growth of the postsynaptic muscle throughout development. To achieve constant muscle depolarization during development, homeostatic signaling must achieve a progressive and gradual increase in synaptic function. A progressive and gradual increase in synaptic function could be achieved through dCBP-dependent mechanisms since it can both promote and inhibit synaptic development. Activation of dCBP could promote synaptic strengthening, while sustained dCBP activation could inhibit further synaptic development, preventing runaway excitation. This is consistent with the demonstration that postsynaptic dCBP is necessary for normal synaptic development but that sustained overexpression of dCBP can inhibit presynaptic functional development (Marek, 2000).

Newly eclosed flies have wings that are highly folded and compact. Within an hour, each wing has expanded, the dorsal and ventral cuticular surfaces bonding to one another to form the mature wing. To initiate a dissection of this process, two mutant phenotypes were examined. (1) The batone (bae) mutant blocks wing expansion, a behavior that is shown to have a mutant focus anterior to the wing in the embryonic fate map. (2) Ectopic expression of protein kinase A catalytic subunit (PKAc) using certain GAL4 enhancer detector strains mimics the batone wing phenotype and also induces melanotic 'tumors'. Surprisingly, these GAL4 strains express GAL4 in cells, which seem to be hemocytes, found between the dorsal and ventral surfaces of newly opened wings. Ectopic expression of Ricin A in these cells reduces their number and prevents bonding of the wing surfaces without preventing wing expansion. It is proposed that hemocytes are present in the wing to phagocytose apoptotic epithelial cells and to synthesize an extracellular matrix that bonds the two wing surfaces together. Hemocytes are known to form melanotic tumors either as part of an innate immune response or under other abnormal conditions, including evidently ectopic PKAc expression. Ectopic expression of PKAc in the presence of the batone mutant causes dominant lethality, suggesting a functional relationship. It is proposed that batone is required for the release of a hormone necessary for wing expansion and tissue remodeling by hemocytes in the wing (Kiger, 2001).

Comparison of the effects of Ricin A and of PKAc on wing maturation indicates that ectopic PKAc does not simply inactivate hemocytes. Instead, it appears to substitute one normal function of hemocytes for another. Rather than carry out phagocytosis and ECM synthesis, hemocytes enter into an innate immune response in which lamellocytes are differentiated and crystal cells melanize target cells. Evidently, aggregation of lamellocytes within the wing blade interferes with wing expansion, and loss of normal hemocyte function interferes with bonding of dorsal and ventral surfaces. The observation that the effect of ectopic PKAc on the wing is suppressed by overexpression of Pan, the Drosophila homolog of mammalian blood cell transcription factors (lymphocyte enhancer-binding factor 1 and T cell factor), suggests that ectopic PKAc inhibits (or represses synthesis of) Pan, which in turn inhibits Wingless target gene expression. This conclusion is strengthened by the observation that ectopic expression of UAS-dCBP(nej+) using GAL4-30A produces phenotypes similar to those caused by ectopic PKAc. Pan is bound and its transcriptional activity inhibited by dCBP. Expression of PanDeltaN, a dominant-negative inhibitor of Wingless target gene expression, elicits what seems to be a massive induction of the cellular innate immune response. Thus, the Wingless signal transduction pathway may be involved in regulating a choice between the innate immune response and the apoptotic/ECM response (Kiger, 2001).

The dominant-lethal interaction between ectopic PKAc and bae is intriguing. When and how death occurs needs closer examination, as does the cellular focus of bae activity. What role PKAc normally plays in regulating hemocyte behavior remains to be investigated. The association of a wing phenotype with altered hemocyte behavior should provide a means of identifying additional genes involved in hemocyte function during wing maturation (Kiger, 2001).

A genetic system has been developed based upon the hobo transposable element in Drosophila melanogaster. hobo, like the better-known P element, is capable of local transposition. A hobo enhancer trap vector has been mobilized and two unique alleles of decapentaplegic (dpp) have been generated . A detailed study of one of those alleles (dppF11) is reported. This is the first application of the hobo genetic system to understanding developmental processes. LacZ expression from the dppF11 enhancer trap accurately reflects dpp mRNA accumulation in leading edge cells of the dorsal ectoderm. Combinatorial signaling by the Wingless (Wg) pathway, the Dpp pathway, and the transcriptional coactivator Nejire (CBP/p300) regulates dppF11 expression in these cells. This analysis of dppF11 suggests a model for the integration of Wg and Dpp signals that may be applicable to other developmental systems. This analysis also illustrates several new features of the hobo genetic system and highlights the value of hobo, as an alternative to P, in addressing developmental questions (Newfeld, 2002).

During early stages of embryogenesis, wg and dpp are expressed in undifferentiated dorsal ectoderm. wg mRNA expression, in 15 stripes along the entire dorsal-ventral axis of the embryo (including the dorsal ectoderm), begins at stage 8. wg expression persists in this striped pattern through stage 17. dpp mRNA is expressed on the dorsal side of the embryo along the entire anterior-posterior axis, beginning at stage 4. dpp mRNA expression persists in a large portion of the dorsal ectoderm through stage 8 and resolves into leading edge cell-specific expression in stage 12 embryos. The embryonic expression pattern of nej has not been reported. However, some information can be inferred from nej mutant phenotypes. nej zygotic mutant embryos show visible defects in the tracheal system at stage 12. The tracheal system is derived from the dorsal ectoderm, suggesting that nej is expressed in this tissue prior to stage 12 (Newfeld, 2002).

Analysis of dppF11 suggests that dpp expression in leading edge cells is initiated by prior episodes of wg and dpp expression in the undifferentiated dorsal ectoderm. The maintenance of dpp expression in leading edge cells appears to require continuous input from wg and from a dpp feedback loop. The initiation and maintenance of dpp expression in leading edge cells also require continuous nej activity. Overall, these data are consistent with the following combinatorial signaling model: Med (signaling for the Dpp pathway) interacts with Arm (signaling for the Wg pathway) via the transcriptional coactivator Nej. This multimeric complex initiates and, with continuous signaling, maintains dpp expression in leading edge cells (Newfeld, 2002).

These data extend previous studies of dpp expression in leading edge cells and Dpp signaling in several ways. A role for Wg signaling in the regulation of dpp expression in the leading edge has been suggested. dpp leading edge expression is not maintained in arm2 zygotic mutants and does not initiate in arm2 germline clones. nej and Med are involved in the regulation of dpp expression in leading edge cells. While nej3 enhances dpp wing phenotypes, Med1 enhances nej3 embryonic phenotypes. This study suggests a role for nej in mediating combinatorial signaling by the Wg and Dpp pathways (Newfeld, 2002).

Several questions remain about the combinatorial regulation of dpp expression by Wg, Dpp, and Nej. One question is, how is Nej recruited to bridge the two pathways? Numerous studies have shown that p300/CBP transcriptional coactivation functions are stimulated by its phosphorylation but the site of phosphorylation has never been mapped. Perhaps Zeste white3 (a serine-threonine kinase in the Wg pathway) or Thickveins (a serine-threonine kinase in the Dpp pathway) are involved in recruiting Nej to participate in combinatorial signaling (Newfeld, 2002).

Control of chromosome structure is important in the regulation of gene expression, recombination, DNA repair, and chromosome stability. In a two-hybrid screen for proteins that interact with the Drosophila CREB-binding protein (dCBP), a known histone acetyltransferase and transcriptional coactivator, the Drosophila homolog of a yeast chromatin regulator, Sir2, was identified. In yeast, Sir2 silences genes via an intrinsic NAD+-dependent histone deacetylase activity. In addition, Sir2 promotes longevity in yeast and in Caenorhabditis elegans. In this report, the Drosophila Sir2 gene and its product were characterized and the generation of Sir2 amorphic alleles is described. It was found that Sir2 expression is developmentally regulated and that Sir2 has an intrinsic NAD+-dependent histone deacetylase activity. The Sir2 mutants are viable, fertile, and recessive suppressors of position-effect variegation (PEV), indicating that, as in yeast, Sir2 is not an essential function for viability and is a regulator of heterochromatin formation and/or function. However, mutations in Sir2 do not shorten life span as predicted from studies in yeast and worms (Newman, 2002).

Sir2 mutations are recessive suppressors of PEV, consistent with the model that Sir2 is involved in heterochromatin regulation across phylogenetic lines. Furthermore, CBP mutations dominantly suppress PEV, suggesting that Sir2 and CBP may act together to control the pattern of heterochromatin histone acetylation. For example, CBP may be inactivated by acetylation and Sir2 may be required to deacetylate CBP for proper heterochromatin function and/or formation. Alternatively, because CBP is known to acetylate proteins other than histones, Drosophila CBP may facilitate heterochromatin formation through modification of other proteins in the complex. The fact that Sir2 mutations are recessive suppressors of PEV while CBP mutations are dominant modifiers of PEV suggests that CBP's role in maintaining heterochromatin formation or function is more dosage sensitive. It is possible that Sir2 helps to stabilize heterochromatin but is not absolutely required for its formation. Sir2 antibodies can co-immunoprecipitate CBP from Drosophila Kc cells, demonstrating that Sir2 and CBP interact in vivo. Dosage studies of Sir2 and CBP on PEV will clarify the nature of the Sir2-CBP interaction. It will also be important to determine whether the deacetylase activity of Sir2 and the acetyltransferase activity of CBP are important for their functions in heterochromatin formation and/or activity (Newman, 2002).

Myb and CBP/Nejire work together to promote mitosis

Drosophila melanogaster possesses a single gene, Myb, that is closely related to the vertebrate family of Myb genes, which encodes transcription factors involved in regulatory decisions affecting cell proliferation, differentiation and apoptosis. In proliferating cells, Myb promotes both S-phase and M-phase, and acts to preserve diploidy by suppressing endoreduplication. The CBP and p300 proteins are transcriptional co-activators that interact with a multitude of transcription factors, including Myb. In transient transfection assays, transcriptional activation by Myb is enhanced by co-expression of the Drosophila CBP protein, dCBP/Nejire. Genetic interaction analysis reveals that these genes work together to promote mitosis, thereby demonstrating the physiological relevance of the biochemical interaction between the Myb and CBP proteins within a developing organism (Fung, 2003).

The cellular basis of the cuticular defects is observed in the wings and abdomens of myb mutants. In the wings, the lower hair density reflects a reduction in cell number, a consequence of the majority of mutant myb cells being arrested in the G2 phase of their final cell cycle. A fraction of the arrested cells also lose the ability to suppress endoreduplication, resulting in DNA contents of higher than 4C. In the abdomens, epidermal cells that are mutant for Myb proliferate much more slowly than wild type cells and display a variety of mitotic defects, including abnormal numbers of centrosomes, resulting in aneuploidy and polyploidy. Therefore, Myb function appears to be required for multiple aspects of the cell cycle (Fung, 2003).

Do the enhanced cuticular phenotypes observed in myb mutants with reduced CBP levels reflect qualitative or quantitative defects at the cellular level? To address this question, pupal wing and abdominal tissue samples were prepared from females that were: wild type for Myb but heterozygous for a mutation in CBP (w,nej3/w,+); homozygous for a mutation in Myb but wild type for CBP (w,myb1/w,myb1), and simultaneously homozygous for a mutation in Myb and heterozygous for a mutation in CBP (w,nej3,myb1/w,+,myb1). During embryonic and larval development, the animals were incubated at 18°C, but to maximize differences in cellular morphology between the various genotypes, they were shifted to 24°C at puparium formation, thereby reducing Myb function during pupal development (Fung, 2003).

Postmitotic wing tissues were double stained with DAPI to visualize nuclei and rhodamine-labeled phalloidin, an F-actin specific stain, to visualize either cell boundaries (28-30 h after puparium formation, APF) or 'prehairs' (developing hairs at 34-36 h APF). In control samples (heterozygous for a mutation in CBP), each cell boundary encircled a single nucleus of relatively uniform size. After initiation of hair formation, the pattern and morphology of nuclei and prehairs was highly ordered and uniform, with each cell producing a single distally protruding hair. In myb1 mutants, cell and nuclear sizes and shapes were more variable and generally larger than in controls. A few cells with bi-lobed nuclei (appearing like two fused nuclei) were also observed, an abnormality not detected in previous experiments, presumably because of its rarity. The size, shape and orientation of prehairs were also less uniform, and two prehairs extending from a single cell were occasionally observed. The variability in nuclear and cellular sizes and shapes became more pronounced when CBP levels were reduced within the context of a myb1 mutant, with enlarged, misshapen or multi-lobed nuclei and/or or multiple separated nuclei within a single cell being commonly observed. The enhanced cellular defects associated with decreased CBP levels generated more extreme variability in the number, size and orientation of prehairs. Most notably, single cells producing two or more prehairs were common in these samples, correlating with the adult phenotype (Fung, 2003).

To quantitate the visual observations the areas of pupal wing cells and nuclei from the relevant genotypes were measured at their largest photographic cross-section. These results confirm that on average, myb1 nuclei and cells are larger than controls, but also more variable. Both properties are substantially enhanced by decreased levels of CBP as shown by increased averages, ranges, and standard deviations. Six microscopic fields of wing cells (measuring 0.055 mm×0.07 mm) for each genotype were also examined for the presence of cells with either multi-lobed or more than one nucleus, and for cells with more than one protruding hair. No examples of these abnormalities were detected in control samples. For myb1, a total of six cells with bi-lobed nuclei and 16 cells with multiple wing hairs were observed. These defects were greatly enhanced in nej3,myb1/myb1 samples, where a total of 43 cells with multi-lobed or 2 or more separated nuclei and 83 cells with multiple wing hairs were observed (Fung, 2003).

The presence of multi-lobed or multiple separated nuclei in cells that were homozygous for myb1 and heterozygous for nej3, indicates that the cells enter, but do not complete mitosis or cytokinesis, a phenotype that is qualitatively different from most of the myb1 mutant wing cells, which appear to have been arrested before entering into their final mitosis. On the surface, this result is counter-intuitive since it indicates that the myb1 mutant cells with reduced CBP levels appear to be progressing further in the cell cycle than the myb1 mutant cells with normal levels of CBP. However, the presence of fewer, larger wing cells when CBP levels are reduced, indicates that at least a portion of these cells are failing to complete cell division in the previous (second to last) cell cycle, thereby accounting for the enhanced phenotype (Fung, 2003).

The cells that form the wings and other adult thoracic and head structures proliferate throughout larval development, completing their final one or two cell divisions during early pupation. In contrast, the cells that form the adult abdominal epidermis do not divide during larval development, but undergo rapid proliferation after puparium formation. The first detectable defect in abdominal cells that are mutant for Myb is that they proliferate considerably more slowly than wild type cells. Therefore, the rate of abdominal epidermal development was compared between the same three genotypes used for the wing analysis. By 27 h APF, replacement of the larval abdominal epidermal cells by adult cells is already well underway in controls. This process is clearly retarded in myb1 mutants, but the delay is much more dramatic when CBP levels are reduced within the context of a myb1 mutant. In controls, the majority of polyploid larval cells have already been replaced with adult cells by 32 h APF, even though cell proliferation continues until about 40 h APF. In contrast, small regions of larval cells at the segment boundaries (which are the last to be replaced) were still present in myb1 mutants at 42 h APF in, and much larger regions of larval cells remain when CBP levels are reduced, even at 45 h APF. Since the animals were shifted to a non-permissive temperature at puparium formation (24°C), neither the myb1/myb1 nor the nej3,myb1/+,myb1 females survived to adulthood. Therefore, the cellular defects observed in these experiments are expected to be more extreme than those represented by the cuticular defects in adults that were raised entirely at permissive temperature. However, the dramatic delay in replacement of larval cells by adult epidermal cells is likely to account for the undifferentiated cuticle observed between segments and along the dorsal midline in nej3,myb1/+, myb1 adults (Fung, 2003).

Although mutant myb cells proliferate slowly, the mitotic index is actually higher in the mutant cells than in controls throughout pupal development, indicating a specific delay in progression through mitosis. Using an antibody for a mitotic-specific phospho-epitope on histone H3 (PH3) to identify mitotic abdominal histoblasts, it was found that at 32 h APF, the average mitotic index was 4.5±1.1% for w,nej3/w,+; 10.3±0.9% for w,myb1/w,myb1; and 17.4±1.1% for w,nej3,myb1/w,+,myb1. Similar results were also observed at other timepoints. This data demonstrate that delayed progression through mitosis is dramatically enhanced when CBP levels are reduced within the context of a myb1 mutant. The sluggish mitotic progression could account for most, if not all, of the associated reduction in the rate of cell proliferation (Fung, 2003).

In the later cell cycles of abdominal epidermal cells, abnormal mitoses associated with multiple functional centrosomes, unequal chromosome segregation, formation of micronuclei, and/or failure to complete cell division are common in cells that are mutant for Myb. It seemed likely that the mitotic abnormalities and slowed rates of cellular proliferation in myb mutants are directly related to each other, and it was therefore anticipated that the occurrence of mitotic abnormalities would also be enhanced by reduced levels of CBP. However, the data do not support this expectation. No changes were detected in the timing or rate of centrosomal and chromosomal abnormalities between w,myb1/w,myb1 and w,nej3,myb1/w,+,myb1 samples, suggesting that these defects may be at least partially independent of the reduced rate of proliferation. The size and morphology of the cells and nuclei from the two genotypes were also very similar, an observation that is consistent with the rate of mitotic defects not being increased in these samples. These findings are also consistent with the observation in adults that in regions where differentiated cuticle has formed, the phenotype is not appreciably different between myb1 mutants that are wild type or heterozygous for nej3 (Fung, 2003).

Although there are some discrepancies, these results confirm the conclusions of (Hou, 1997) that co-expression of CBP with Myb enhances the ability of Myb to activate transcription of a reporter construct in transient transfection assays. Taken together with their demonstration of direct binding between CBP and Myb in vitro, it is concluded that like their vertebrate counterparts, the Drosophila CBP and Myb proteins physically interact and that CBP acts as a transcriptional co-activator of Myb (Fung, 2003).

CBP-related protein, p300, can acetylate lysines within a highly conserved region of the human c-Myb protein, and the acetylation enhances the DNA-binding and transactivation capabilities of c-Myb. Of the three lysine residues within the conserved region (region III) that may be acetylated (K471, K480 and K485), the first two are conserved at equivalent positions in the Drosophila Myb protein (K450 and K459). The evolutionary conservation of these lysine residues suggests that they may be targets for acetylation by Drosophila CBP and that the mechanism of activating the Drosophila Myb protein via acetylation may also be conserved (Fung, 2003).

Reducing the levels of CBP in animals mutated for Drosophila Myb enhances virtually all aspects of the mutant phenotype: viability is reduced and cuticular and cellular defects are increased. The genetic interaction between Myb and CBP provides direct evidence that the biochemical interaction between CBP and Myb proteins (demonstrated in mammalian and Drosophila systems) is physiologically relevant within the context of a developing animal. Previous studies have shown that Drosophila CBP functions during multiple stages of development and that mutations in CBP/nej produce pleitropic phenotypes, indicating that CBP may be required for multiple developmental processes. Indeed, CBP/nej has been implicated in several signal transduction pathways that regulate developmental patterning, including the Decapentaplegic, Hedgehog, and Wingless pathways. However, the analysis presented here provides the first explicit evidence that Drosophila CBP is directly involved in regulating cell proliferation (Fung, 2003).

A paradox of CBP/p300 function is that these proteins appear to be capable of having opposing effects on cell proliferation. Mice or humans with mutations that led to reduced levels or activity of CBP display markedly increased susceptibility to tumorigenesis, indicating that they function as tumor suppressors. However, a plethora of biochemical and cell culture studies have shown that CBP/p300 physically interacts with, and activates a number of transcription factors known to promote cellular proliferation, including E2F1 and oncoproteins such as JUN, FOS and MYB. Still, direct evidence for CBP/p300 being able to cooperate with any of these transcription factors to drive proliferation within an animal has been lacking. Therefore, the finding that Drosophila CBP is required in concert with Myb for positive regulation of the cell cycle during Drosophila development validates a physiological role for CBP/p300 in promoting cell proliferation in vivo, and supports the proposal that the pro-or anti-proliferative effects of CBP/p300 are dependent on cellular context (Fung, 2003).

The CBP coactivator functions both upstream and downstream of Dpp/Screw signaling in the early Drosophila embryo

The CBP histone acetyltransferase plays important roles in development and disease by acting as a transcriptional coregulator. A small reduction in the amount of Drosophila CBP (dCBP) leads to a specific loss of signaling by the TGF-ß molecules Dpp and Screw in the early embryo. The expression of Screw itself, and that of two regulators of Dpp/Screw activity, Twisted-gastrulation and the Tolloid protease, is compromised in dCBP mutant embryos. This prevents Dpp/Screw from initiating a signal transduction event in the receiving cell. Smad proteins, the intracellular transducers of the signal, fail to become activated by phosphorylation in dCBP mutants, leading to diminished Dpp/Screw-target gene expression. At a slightly later stage of development, Dpp/Screw-signaling recovers in dCBP mutants, but without a restoration of Dpp/Screw-target gene expression. In this situation, dCBP acts downstream of Smad protein phosphorylation, presumably via direct interactions with the Drosophila Smad protein Mad. It appears that a major function of dCBP in the embryo is to regulate upstream components of the Dpp/Screw pathway by Smad-independent mechanisms, as well as acting as a Smad coactivator on downstream target genes. These results highlight the exceptional sensitivity of components in the TGF-ß signaling pathway to a decline in CBP concentration (Lilja, 2003).

These results suggest that several transcription factors that regulate expression of Dpp/Screw signaling components require the dCBP coactivator for their function in Drosophila embryos, and implicate dCBP in regulation of the Dpp/Screw pathway independently of an interaction with Smad proteins. An additional role of dCBP is to regulate Dpp-target genes, acting at a step downstream of Smad protein phosphorylation. It is likely that direct interactions between dCBP and Mad/Medea contribute to regulation of Dpp target genes). Such interactions have been observed in vitro, both in mammalian systems and using Drosophila proteins. However, a major cause of impaired Dpp/Screw signaling in dCBP mutant embryos is due to reduced tolloid expression. This prevents Dpp/Screw from initiating a signaling event in cells that would normally receive the Dpp/Screw signal, presumably by a failure to cleave the Dpp-Sog and/or Screw-Sog complexes. In fact, a majority of embryos that do not express the Dpp/Screw-target gene rhomboid in dorsal cells, also do not contain phosphorylated Smad proteins. Furthermore, the pattern of phosphorylated Smad proteins correlates closely with that of tolloid expression. For example, in many early, cellularizing dCBP mutant embryos, an anterior patch of both tolloid expression and phosphorylated Smad staining remains. At later stages, tolloid expression recovers in dCBP mutant embryos, as does Dpp/Screw signaling as revealed by Smad protein phosphorylation. This recovery of tolloid expression at later stages of development might explain the recovery of phosphorylated Smad proteins in dCBP mutant embryos, by allowing Dpp/Screw to signal. For these reasons, regulation of tolloid expression appears to be a major means of controlling Dpp/Screw signaling by dCBP (Lilja, 2003).

It is likely that reduced screw expression also contributes to the reduction of phosphorylated Smad proteins observed in dCBP mutant embryos. In both screw and tolloid mutants, phosphorylation of Mad is eliminated. Furthermore, progressive reduction in Screw activity leads to a corresponding progressive deletion of dorsal-most cell fate, the amnioserosa. Tsg is required together with Sog to generate peak Dpp activity in dorsal midline cells. Reduced tsg expression in dCBP mutants may therefore contribute to the lack of Dpp/Screw-target gene expression. However, it is not believe that this lack can explain the defects in dCBP mutants, because in tsg mutants, low levels of Dpp signaling persist in a broad dorsal domain, leading to expanded rhomboid expression in dorsal cells. By contrast, in dCBP mutant embryos, expression of genes in response to a low threshold of Dpp activity, such as U-shaped and the dorsal rhomboid pattern, is eliminated (Lilja, 2003).

These experiments do not address whether dCBP regulation of tolloid, screw, and tsg expression is direct or indirect. However, since expression of these genes begins at about the time when zygotic transcription initiates in the embryo, and the effect of dCBP is evident from the onset of expression of tolloid and screw, the notion is favored that dCBP is acting directly on the enhancers of these genes. It is not yet understood whether HATs such as CBP primarily act to acetylate large chromosomal domains, or are directed to specific genes. In the case of the tolloid gene, the results indicate that dCBP is being recruited to the enhancer by a DNA-binding protein, since the isolated enhancer removed from its normal chromosomal location requires dCBP for its activity (Lilja, 2003).

Given its central position in gene regulation and the great number of mammalian transcription factors shown to interact with CBP, relatively few genes are affected by the dCBP mutation. For example, activation and repression mediated by the Dorsal protein are unaffected in the dCBP mutant embryos, as demonstrated by the expression patterns of Dorsal target genes. Also, no defects in early segmentation gene expression could be observed in germline clone mutants. However, the nej1 mutation used in this study to create dCBP mutant germline clone embryos is a weak mutation that results in a very modest reduction in dCBP levels. Since other means of reducing the dCBP amount by approximately two-fold results in similar gene expression defects, Smad proteins and the unidentified activators of tolloid, tsg, and screw expression are particularly sensitive to a decline in dCBP concentration. It may not be a coincidence that screw, tsg, tolloid, and Dpp-target gene expression are all specifically affected by a small dCBP reduction. Perhaps components of the Dpp/Screw signal transduction pathway have evolved to be coordinately regulated by a common coactivator. Given the phylogenetic conservation of the CBP protein and the TGF-alpha signal transduction pathway, as well as the ability of CBP and Smad proteins to interact in vitro, CBP is likely to play an equally important role in TGF-ß signaling in other metazoans (Lilja, 2003).

CREB binding protein functions during successive stages of eye development in Drosophila

During the development of the compound eye of Drosophila several signaling pathways exert both positive and inhibitory influences upon an array of nuclear transcription factors to produce a near-perfect lattice of unit eyes or ommatidia. Individual cells within the eye are exposed to many extracellular signals, express multiple surface receptors, and make use of a large complement of cell-subtype-specific DNA-binding transcription factors. Despite this enormous complexity, each cell will make the correct developmental choice and adopt the appropriate cell fate. How this process is managed remains a poorly understood paradigm. Members of the CREB binding protein (CBP)/p300 family have been shown to influence development by (1) acting as bridging molecules between the basal transcriptional machinery and specific DNA-binding transcription factors, (2) physically interacting with terminal members of signaling cascades, (3) acting as transcriptional coactivators of downstream target genes, and (4) playing a key role in chromatin remodeling. In a screen for new genes involved in eye development the Drosophila homolog of CBP has been identified as a key player in both eye specification and cell fate determination. A variety of approaches was used to define the role of CBP in eye development on a cell-by-cell basis (Kumar, 2004).

The early development of the compound eye is regulated in part by a regulatory network of genes that include the Pax genes twin of eyeless (toy), eyeless (ey), twin of eyegone (toe), and eyegone (eyg); the founding members of the Dach and Eya gene families dachshund (dac) and eyes absent (eya), and the Six class genes optix and sine oculis. Extracellular instructions from the Hh, Dpp, Egfr, Notch, and Wg signaling cascades are integrated into this network at several levels creating additional layers of complexity. A dominant allele of sine oculis, soD, was used as the starting material for a genetic screen to isolate new genes involved in eye specification. The soD allele appears to function as a dominant negative mutant: (1) soD heterozygotes lack compound eyes while heterozygotes of the so3 null allele have wild-type eyes; thus soD is a dominant mutant; (2) embryonic lethality results if the soD mutation is placed in trans to the so3 allele (soD/so3); (3) compound eye development is restored in soD mutants by the addition of wild-type SO protein via UAS-so transgenes -- thus soD has an inhibitory function. The open reading frame of the soD mutant was sequenced and a single valine-to-aspartic acid substitution was found at amino acid 200 (V200D). This mutation occurs within the Six domain, which is implicated in both DNA-binding and protein-protein interactions with EYA. Mutations within this domain of So could negatively affect eye development by either altering its interactions with potential binding partners or causing inappropriate transcriptional regulation of downstream target genes (Kumar, 2004).

The retinal phenotypes of the eye-specific so1 loss-of-function mutant and the soD dominant negative allele differ slightly from one another. SO protein levels are below detection in so1 mutant eye discs while remaining at wild-type levels in soD discs. Similarly, the expression of several other genes that are required for eye development, such as dpp and dac, are not reduced in soD mutants while being disrupted in so1 mutants. Furthermore, in so1 adults the region normally occupied by the compound eyes is replaced by surrounding head tissue. In contrast, soD flies have a large nonpigmented and nondifferentiated field. The lack of retinal tissue in soD adults can be traced back to a complete lack of photoreceptor differentiation during larval eye imaginal disc development as assayed by the absence of ELAV, a pan-neural protein. The presence of this nondifferentiated field in soD adults allows for the isolation of both suppressor and enhancer mutations. Six complementation groups were discovered that suppress, and one complementation group that enhances the soD no-eye phenotype. The enhancing locus is nej, the gene that encodes CBP in Drosophila (Kumar, 2004).

Removal of one copy of nej in a soD background results in an eye phenotype that is now indistinguishable from so1 loss-of-function mutants. Similar to so1 mutants, eye imaginal discs from nej3/+; soD/+ heterozygotes (nej3 is a null allele) are small and undergo increased levels of cell death, while adults lack the nondifferentiated field and instead contain only head tissue. Conversely, expression of CBP throughout the soD retinal field suppresses the no-eye phenotype. Eye imaginal discs are near normal in size and contain large numbers of photoreceptor cell clusters, and adult eyes are fully pigmented although not normally patterned (Kumar, 2004).

CBP is expressed in all cells within the developing eye imaginal disc. Loss-of-function CBP mutations affect the expression of several eye specification genes within the embryonic visual system, protocerebrum, mesoderm, and the developing eye imaginal disc. Using viable loss-of-function allelic combinations, loss-of-function retinal clones, and RNAi interference, this study has demonstrated that each cell type in the developing eye, with the exception of the founder R8 photoreceptor, requires CBP for its specification. Using a 'pathway interference' approach it has been shown that CBP likely functions in the R3/R4 cell fate choice and in the specification of the R7 photoreceptor (Kumar, 2004).

The results presented here indicate a role for CBP in a myriad of developmental decisions within the developing fly retina. It remains to be determined if these effects are through repeated interactions with a small set of master regulatory proteins or with a larger set of signaling molecules and cell-subtype-specific transcription factors. It is more likely that the latter scenario will be correct. This is based on the large body of biochemical data that suggest CBP interacts with more than 100 proteins that are members of many diverse signaling cascades. Furthermore, no single gene has been shown to affect all of the processes that require the activity of CBP. Thus it is hypothesized that CBP functions as a connecting point for signaling, transcription, and chromatin remodeling during all phases of fly eye development (Kumar, 2004).

The sheer number of potential interactions mediated by CBP makes an analysis of this protein inherently difficult. To circumvent this potential problem, a pathway interference approach was used to dissect CBP function by expressing a series of truncated CBPs within the developing eye. The underlying idea behind this approach is that each protein variant will act as a protein sink and soak up a unique set of endogenous factors, thus providing insight into the processes that are affected by CBP. It also provides a first step toward understanding the role that each domain of CBP plays in the developmental process and lays the groundwork for identifying critical components using more biochemical methods. The target proteins are likely to interact with CBP at stoichiometric levels during normal development. However, by increasing the dosage of CBP, the amount of these proteins within a cell becomes limiting and loss-of-function phenotypes can be observed. This approach successfully revealed roles for CBP in the R3/R4 cell fate choice and in R7 fate specification (Kumar, 2004).

How CBP functions in any of these processes is still an unanswered question. Attempts to identify additional components of the regulatory network disrupted by expression of variant CBPs through the restoration of putative interacting and downstream factors were unsuccessful. The addition of any one single factor was insufficient to rescue the effects of any of the CBP variants. Although it is possible that none of the correct factors were tested, it is more likely that the observed phenotypes result from the loss of several proteins and adding just one is insufficient to restore normal eye development (Kumar, 2004).

How are so many developmental decisions in the developing eye regulated by CBP? On the basis of reported roles for CBP/p300 in mammalian development, CBP would appear to be the perfect candidate to act as a 'network manager' during eye development. A scenario can be envisioned in which every cell within the eye disc expresses CBP and a specific combination of transcription factors; some are present in restricted expression patterns while other are more promiscuously expressed. As signals are interpreted at the cell surface and transmitted into the nucleus, the CBP-transcription factor scaffold would interact with terminal members of signaling cascades and execute these instructions by modulating transcription of downstream target genes. Late in development this would translate into the differentiation of specific cell types -- photoreceptors, cone cells, pigment cells, and mechanosensory bristles. This is an attractive model for several reasons. (1) The uncommonly high number of described biochemical interactions suggests that CBP may act as a link between signaling pathways, specific DNA-binding proteins, and the basal transcriptional machinery. These qualities have been shown to be true in vitro. (2) It allows for individual cells to receive several common-use signals but then personalize the output. (3) The ability to interact with members of signaling pathways as well as remodel chromatin allows for very efficient transduction of extracellular instructions. This may be important for the recruitment of photoreceptors into the ommatidial cluster, a process that occurs over a relatively short period of time. This model can be extended to early events in eye specification. CBP is expressed in all cells of the eye and antennal tissues during early development, while expression of selector genes is restricted to the individual tissues. Signaling pathways that include Notch, Egfr, Hh, Dpp, and Wg are known to influence both eye and antennal development. CBP may mediate the interactions between signaling pathways and these selector genes, thereby participating in the process of subdividing the eye-antennal disc into the eye and antenna proper (Kumar, 2004).

Previous reports of CBP in the eye have focused on the role of CBP in the modulation of polyglutamine diseases and retinal degeneration. The work presented here extends these results and points to a role for CBP both in early eye determination and later in cell fate specification. The results that pertain to early eye determination are supported by the synergistic interactions between CBP and SIX, EYA, and DACH proteins observed in mammals. Furthermore, this study has demonstrated a role for CBP in the development of several photoreceptor cell subtypes including the R7 neuron. In recent years it has become increasingly clear that the molecules and mechanisms that control eye development have been preserved in both mammalian and invertebrate retinal systems. It will be interesting to elucidate the molecular and biochemical mechanisms by which CBP influences early eye specification and later photoreceptor cell fate decisions in both invertebrate and mammalian retinal systems (Kumar, 2004).

The cis-regulatory code of Hox function in Drosophila

Precise gene expression is a fundamental aspect of organismal function and depends on the combinatorial interplay of transcription factors (TFs) with cis-regulatory DNA elements. While much is known about TF function in general, understanding of their cell type-specific activities is still poor. To address how widely expressed transcriptional regulators modulate downstream gene activity with high cellular specificity, binding regions were identified for the Hox TF Deformed (Dfd) in the Drosophila genome. This analysis of architectural features within Hox cis-regulatory response elements (HREs) shows that HRE structure is essential for cell type-specific gene expression. It was also found that Dfd and Ultrabithorax (Ubx), another Hox TF specifying different morphological traits, interact with non-overlapping regions in vivo, despite their similar DNA binding preferences. While Dfd and Ubx HREs exhibit comparable design principles, their motif compositions and motif-pair associations are distinct, explaining the highly selective interaction of these Hox proteins with the regulatory environment. Thus, these results uncover the regulatory code imprinted in Hox enhancers and elucidate the mechanisms underlying functional specificity of TFs in vivo (Sorge, 2012).

In order to quantitatively identify genomic regions bound by the Hox TF Dfd in Drosophila, two complementing approaches were employed: ChIP-seq, which has been successfully applied previously to identify stage- and tissue-specific enhancer activities, and computational detection of clusters of TF binding sequences, which allows the identification of cis-regulatory modules irrespective of temporal and spatial context. To generate genome-wide maps of Dfd binding in vivo, ChIP was performed using stage 10-12 Drosophila embryos and a Dfd-specific antibod. Stage-independent in silico Dfd-specific Hox response elements (HREs) were identified by searching for clusters of conserved Dfd binding motifs, as defined by a position weight matrix (PWM), in the non-coding regions of the genomes of 12 distinct Drosophila species. By applying both approaches, 4526 genomic regions containing clusters of Dfd binding sites and 1079 Dfd ChIP-seq enrichment peaks were identified, including two out of the three well-characterized Dfd-HREs, namely rpr-4S3 and Dfd-EAE. To study the regulatory capacity of novel in silico and ChIP-seq detected HREs, cell culture-based enhancer assays were performed for 11 randomly selected HREs, and it was found that reporter expression driven by the identified genomic regions was in all cases dependent on Dfd binding. In vivo activity was tested of 21 arbitrarily selected enhancers in transgenic reporter lines, revealing that 7 out of 11 ChIP-identified and 5 out of 10 in silico-predicted Dfd-HREs recapitulate the spatio-temporal expression of adjacent genes). Most importantly, it was possible to demonstrate Dfd-dependent regulation of both transgenic reporter expression and endogenous gene expression, suggesting that they are bona fide direct Dfd target genes. Thus, the identified Dfd-HREs represent a data set of biologically relevant regulatory regions and an excellent resource to unravel sequence features within Hox responsive enhancers that might be essential for the highly selective Hox target gene regulation (Sorge, 2012).

Transcriptional regulation in many cases relies on the assembly of regulatory protein complexes mediated by closely spaced TF binding sites within a cis-regulatory module and previous studies have shown that Hox proteins employ this mechanism to control target gene activity in small subsets of cells. The novel HREs were systematically scanned for TF binding motifs appearing in close proximity to Dfd binding sites. Using a statistical test for pair-wise distance distributions, w11 overrepresented DNA motifs for known TFs were found adjoining to Dfd binding sites with 5 of the motifs occurring in both the ChIP-seq and in silico-identified Dfd-HREs. When the expression patterns of six of these transcriptional regulators known to bind to the 11 motifs that were identified were examined, colocalization with Dfd was found in different sub-populations of cells in all cases. Colocalization was already known for two TFs, whose binding sites were coupled to Dfd motifs, including Extradenticle (Exd) , which is known to cooperatively bind with Hox proteins to DNA and thereby increase Hox DNA-binding selectivity. It was next asked whether the short-distance arrangements in Dfd-HREs are of biological relevance and translated into the regulation of similar classes of target genes. To this end, the overrepresentation was statistically tested of expression and biological terms of genes associated with HREs harbouring specific combinations of Dfd and close-by motifs. This analysis revealed that only those Dfd-HREs with short distance intervals between the Dfd and adjacent motifs were coupled to similar gene classes, while random distance intervals did not show any correlation. Strikingly, genes associated with specific short-distance HREs had similar expression and functional annotations as the TFs interacting with the Hox adjoining motifs, suggesting that time and place of Hox action is dictated by spatio-temporally restricted co-regulators. Support for this hypothesis stems from the observation that one of the close-distance partners, Optix, regulates similar processes as Dfd, since Dfd and Optix mutants displayed comparable morphological defects in the head region, such as the absence of mouth hooks, a maxillary segment-derived structure known to be specified by Dfd. In addition, one of the genes associated with a Dfd-Optix HRE, the known Dfd target gene reaper (rpr), is expressed in the ventral epidermis primordium as predicted by its HRE architecture, and regulated by Dfd and Optix in ventral-maxillary cells, which also express these factors. A cell-culture assay using the well-established Dfd responsive module responsible for rpr expression in a few anterior-maxillary cells, the rpr-4S3 Dfd-HRE, with wild-type or mutated Dfd binding sites or reduction of Dfd levels by RNAi confirmed the requirement for simultaneous activity of Dfd and Optix on the rpr-4S3 Dfd-HRE for strong reporter gene induction. Optix binding to the rpr-4S3 Dfd-HRE was additionally confirmed by electrophoretic mobility shift assay (EMSA) experiments. Furthermore, transgenic reporter expression induced by the rpr-4S3 Dfd-HRE was lost in Optix mutant embryos or when the Optix binding sites were mutated. These results demonstrate that Optix, one of the newly identified factors, is a Dfd co-regulator required for proper regulation of the important Hox target gene rpr (Sorge, 2012).

Whether The precise spacing between Hox and adjacent binding sites plays a role for enhancer activity was explored. The rpr-4S3 Dfd HRE, which induces gene expression in a few anterior-maxillary cells, has previously been shown to be under the control of Dfd and Glial cells missing (Gcm), a Dfd co-regulator also identified in this study. Dfd and Gcm as well as Optix binding sites within the rpr-4S3 HRE are directly adjacent to each other, thus a 5- and 10-bp spacer was introduced to interfere with potential interactions of the proteins on the enhancer. In all cases, reporter gene expression was strongly reduced or completely abolished, showing that the close-distance arrangements between Dfd and Gcm as well as Dfd and Optix are required for the in vivo activity of the rpr-4S3 enhancer (Sorge, 2012).

While the results regarding the close-distance arrangement of Dfd and Gcm binding sites suggested the formation of a Dfd-Gcm protein complex, like in the case of Dfd and Exd, only independent binding of the two proteins to the rpr-4S3 enhancer was observed in EMSA experiments , supporting the idea of Hox proteins collaborating with other TFs on target HREs in the absence of physical contact. It has been shown before that Hox proteins together with other TFs that bind in the immediate vicinity recruit non-DNA binding cofactors to HREs. To test if such factors could interact with Dfd and the newly identified short distance binding TFs, the modENCODE data set was scanned and it was found that dCBP/Nej, a member of the CBP/p300 family of transcriptional co-activators bearing acetyltransferase activity, binds to the rpr-4S3 enhancer in vivo. As nej has been previously reported to genetically interact with Dfd, its function was examined in Dfd/Gcm-mediated transcriptional activation. Both factors, Dfd and Gcm, are required for transcriptional activation, since expression of Gcm in Drosophila D.Mel-2 cells, which have basal levels of Dfd activity, resulted in strong induction of reporter gene expression, while abolishing Dfd binding to the rpr-4S3 HRE by mutating all Dfd binding sites or by reducing Dfd protein levels in D.Mel-2 cells using RNAi, strongly reduced reporter gene expression in the presence of Gcm. Strikingly, Dfd- and Gcm-mediated reporter gene expression was strongly reduced in nej dsRNA-treated cells, whereas inhibition of protein deacetylation by Trichostatin A (TSA0) restored reporter gene expression. Consistently, rpr expression was abolished in nej mutant embryos. These results demonstrate that dCBP/Nej-mediated protein acetylation/histone modification is important for the combined activity of Dfd and Gcm on the rpr-4S3 HRE. While it was not possible to demonstrate that nej physically interacts with Dfd protein using various assays, EMSA experiments show that nej interacts with Gcm. Furthermore, acetylation of transiently transfected Gcm was detected in cultured Drosophila cells. Acetylation of Gcm is dependent on Nej, as it was reduced upon RNAi-mediated downregulation of nej. These results are consistent with published work demonstrating that in human cells CBP interacts with Gcma, resulting in its acetylation and stimulation of its transcriptional activity. Since about 10% of all Dfd and nej in vivo genomic binding events during embryonic stages 10-12 overlap, the functional interaction of Dfd and nej observed at the rpr locus does not seem an exception. This finding suggests that the interaction of co-activators (and co-repressors) with Hox proteins and close distance binding TFs on enhancer modules could be a commonly used mechanism to achieve highly specific spatio-temporal control of target gene activity. In this scenario, Hox proteins would control downstream genes by direct transcriptional and/or epigenetic regulation depending on HRE composition and thus cofactor identity and recruitment (Sorge, 2012)

Despite very similar DNA binding behaviour in vitro, Hox proteins regulate distinct morphological features along the anterior-posterior body axis in animal systems. To elucidate the mechanistic basis for the differences in their regulatory properties, Dfd-HREs identified in this study were compared to genomic regions bound by the Hox TF Ultrabithorax (Ubx) at identical developmental stages, as identified by the modENCODE consortium. Searching for overrepresented DNA motifs in both enriched ChIP regions, it was found that Dfd and Ubx bind to identical DNA sequences in vivo, reminiscent to in vitro systems. However, individual binding motifs seem to play only a minor role for Hox binding site selection in vivo, since this analysis revealed that Dfd and Ubx exclusively interact with non-overlapping genomic regions in embryonic stages 9-12. Consequently, Dfd- and Ubx-HREs were found to be associated with distinct classes of genes, revealing that genes with roles in the epidermis are primarily under the control of Dfd at the analysed embryonic stages while genes with mesoderm-related functions are predominantly regulated by Ubx. Consistently, it was found that the expression of tartan (trn), one of the genes associated with a Dfd-HRE, is regulated exclusively by Dfd, but not by Ubx, in epidermal cells, while parcas (pcs), one of the genes linked to a Ubx-HRE is under the selective control of Ubx in mesodermal cells. Furthermore, only Ubx-HREs were found to substantially overlap with cis-regulatory elements stage specifically bound by the mesoderm-specifying TFs Myocyte enhancer factor 2 (Mef2), Twist and Tinman. In contrast, the common ability of both Dfd and Ubx to regulate genes involved in nervous system development was underlined by comparable representations of binding motifs for the neuronal-specifying TFs Asense, Deadpan and Snail in Dfd- and Ubx-HREs (Sorge, 2012).

Strikingly, the basic design principles of Dfd- and Ubx-HREs were found to be similar: like in Dfd-HREs, six binding motifs for known TFs were located adjacent to Ubx binding sites and colocalization studies showed that they are expressed in subsets of Ubx-positive cells. Again, Ubx binding sites and motifs for potential co-regulators occurred most frequently in specific short intervals and only those Ubx-HREs with the preferred distance were associated with specific gene classes. This analysis also revealed that four of the six short-distance motifs were specific for Ubx-HREs, which is consistent with the data showing that Hox proteins interact with different and spatially restricted co-regulators to control target gene expression in selected cells. Importantly, in the cases of the close-distance motifs detected in both HREs, namely the binding sites for the TFs Ladybird early (Lbe) and Cut (Ct), the associated target genes were also expressed in non-overlapping tissues. This raised the question of how different Hox proteins can act on distinct target genes, even when their target HREs exhibit similar binding site compositions including short-distance arrangements. Since Lbe is active in both mesodermal and epidermal cells, one Dfd-Lbe and one Ubx-Lbe HRE was exemplarily analysed, and binding of Lbe protein was confirmed to both HREs by EMSAs. As predicted by the presence of Lbe binding sequences. Complex formation between the Hox protein and Lbe was observed in the case of Ubx and Lbe while Dfd and Lbe interact independently with the Dfd-Lbe HRE, indicating that the two Hox proteins employ different mechanisms for binding to the selected HREs. Lbe interaction with the Dfd-Lbe and Ubx-Lbe HREs is essential for in vivo activity, since in both cases ectopic reporter gene expression was observed when Lbe binding sites were mutated. Even more important, reporter gene expression was specifically changed only in segments in which either Dfd or Ubx is active, meaning in the case of the Dfd-Lbe HRE in maxillary cells and in the case of the Ubx-Lbe HRE in abdominal segments A1-A7. Taken together, these results demonstrate that the combined activity of Lbe and the Hox proteins Dfd or Ubx on selected HREs is critical for the precise spatiotemporal and segment-specific control of HRE activity. It was next asked whether additional (DNA- and non-DNA-binding) factors contribute to the predicted cell type-specific expression of the Dfd-Lbe and Ubx-Lbe HREs. Using the Drosophila Interactions Database (DroID; Murali, 2011) and published genome-wide DNA binding studies a search was carried out for unique Dfd-lbe and Ubx-lbe interactors. It was discovered that almost 20% of all Ubx-Lbe HREs but none of the Dfd-Lbe HREs were found to interact with the mesoderm-specifying factor Mef2 in vivo, while H3K9me3 histone marks, which are mediated by one of the unique Dfd-lbe interactors, Enhancer of zeste E(z), are enriched only within Dfd-Lbe HREs. Interestingly, E(z) modifies chromatin also by trimethylating H3K27 residues, a histone mark highly enriched at the genomic region spanning the ChIP-detected Dfd-Lbe HRE. Consistent with the repressive function of this histone modification, loss of Lbe binding to the Dfd-Lbe HRE results in ectopic reporter gene expression, suggesting that Lbe (and Dfd) recruits E(z) to the Dfd-Lbe HRE for cell type-specific target gene repression (Sorge, 2012).

Taken together, these results demonstrate that Hox proteins interact with different regulatory proteins on HREs, which allows them to differentially regulate their target genes despite their similar DNA binding properties. The fact that these interactions occur only in a few cells for a short period of time is very likely one of the major reasons why the identification of factors conferring regulatory precision and specificity to Hox function has met with little success so far (Sorge, 2012).

This study, has identified crucial features of HREs, which are essential for cell type-specific regulation of Hox target genes in vivo. In addition to motif composition the exact spatial arrangement of TF binding elements is critical to translate Dfd function into transcriptional regulation in vivo. These architectural features of Dfd-HREs alone accurately predict target gene function and expression patterns. Furthermore, it was found that epigenetic regulators bind to HREs on a genome-wide scale, suggesting that they generally collaborate with Hox proteins to achieve stable target gene regulation. This is in line with recent findings showing that chromatin modifications at enhancers strongly correlate with functional enhancer activity and tissue specificity. By comparing HREs regulated by Dfd and Ubx, two different Hox proteins with different embryonic regulatory specificities, this study shows that while similar design principles apply, specificity is encoded by distinct sets of co-occurring DNA motifs. Due to the highly dynamic regulatory output of Hox TFs in space and time, cell type-specific approaches are required in future to elucidate all relevant aspects of Hox-chromatin and Hox-cofactor interactions (Sorge, 2012).


EVOLUTIONARY HOMOLOGS

Other Drosophila histone acetyltransferases

PCAF and hGCN5 are distinct human genes that encode proteins related to the yeast histone acetyltransferase and transcriptional adapter GCN5. The PCAF protein shares extensive similarity with the 439 amino acids of yGCN5, but it has an approximately 350 amino acid N-terminal extension that interacts with the transcriptional co-activator p300/CBP. Adenoviral protein E1a can disrupt PCAF-CBP interactions and prevent PCAF-dependent cellular differentiation. A Drosophila homolog of yGCN5 has been cloned and characterized. In addition to the homology to yGCN5, the Drosophila protein shares sequence similarity with the N-terminal portion of human PCAF that is involved in binding to CBP. In the course of characterizing dGCN5, it has been discovered that hGCN5 also contains an N-terminal extension with significant similarity to PCAF. Interestingly, in the case of the hGCN5 gene, alternative splicing may regulate the production of full-length hGCN5. The presence of the N-terminal domain in a Drosophila GCN5 homolog and both human homologs suggests that it was part of the ancestral form of metazoan GCN5 (Smith, 1998).

Comparison of CBP and p300

The cellular protein p300 is a target of the adenoviral E1A oncoprotein and is thought to participate in preventing the G0/G1 transition in the cell cycle, activating certain enhancers and stimulating differentiation pathways. CBP is a protein that is associated with and coactivates the transcription factor CREB, mediating the induction by cyclic AMP of certain responsive promoters. The sequences of p300 and CBP are highly related. p300, like CBP, can stimulate transcription. This activity is directly and specifically inhibited by E1A. CBP exists in a DNA-bound complex containing a member of the CREB family and E1A and CBP interact with one another in vivo. In keeping with the idea that E1A functionally targets CBP, cAMP-dependent transcription is repressed by E1A. Thus, p300 and CBP define a family of transcriptional adaptor proteins that are specifically targeted by the E1A oncoprotein (Arany, 1995).

The 265K nuclear protein CBP was initially identified as a co-activator for the protein kinase A (PKA)-phosphorylated form of the transcription factor CREB. The domains in CBP that are involved in CREB binding and transcriptional activation are highly related to the adenoviral E1A-associated cellular protein p300, and to two hypothetical proteins from Caenorhabditis elegans, R10E11.1 and K03H1.10, whose functions are unknown. CBP and p300 have similar binding affinity for the PKA-phosphorylated form of CREB, and p300 can substitute for CBP in potentiating CREB-activated gene expression. E1A binds to CBP through a domain conserved with p300 and represses the CREB-dependent co-activator functions of both CBP and p300. These results indicate that the gene repression and cell immortalization functions associated with E1A involve the inactivation of a family of related proteins that normally participate in second-messenger-regulated gene expression (Lundblad, 1995).

Mutation of CREB-binding protein (CBP)

Colorectal tumors frequently have loss of heterozygosity on chromosome 22q, suggesting that inactivation of tumor suppressor gene(s) on 22q participates in the tumor development. Neurofibromatosis 2 (NF2) gene and E1A binding protein p300 gene, recently identified on 22q, are thought to be candidates for tumor suppressor genes. In this study, mutation of the NF2 gene in 59 colorectal carcinomas, and mutation of the p300 gene in 27 colorectal and two gastric carcinomas, were analysed using PCR-SSCP, RT-PCR-SSCP and direct sequencing methods. Missense mutations of p300 gene are detected in a colorectal carcinoma, and in a gastric carcinoma, though no mutation of NF2 gene is detected. Both p300 mutations are somatic and coupled to deletion of the second allele of the gene, which suggests inactivation of the p300 gene, in these carcinomas. The mutations are located within the Cys/His-rich regions, which are assumed to play important roles in the function of p300. These are the first cases in which p300 gene has been found to be altered in both alleles, suggesting that inactivation of the p300 gene may be involved in the development of carcinomas, and that this gene may be the target of loss of 22q in carcinomas of the digestive tract (Muraoka, 1996).

The Rubinstein-Taybi syndrome (RTS) is a well-defined syndrome with facial abnormalities, broad thumbs, broad big toes and mental retardation as the main clinical features. Many patients with RTS have been shown to have breakpoints in, and microdeletions of, chromosome 16p13.3. All these breakpoints are restricted to a region that contains the gene for the human CREB binding protein (CBP), a nuclear protein participating as a co-activator in cyclic-AMP-regulated gene expression. RTS results not only from gross chromosomal rearrangements of chromosome 16p, but also from point mutations in the CBP gene itself. Because the patients are heterozygous for the mutations, it is proposed that the loss of one functional copy of the CBP gene underlies the developmental abnormalities in RTS and possibly the propensity for malignancy (Petrij, 1995).

The transcriptional coactivator and integrator p300 and its closely related family member CBP mediate multiple, signal-dependent transcriptional events. Mice lacking a functional p300 gene die between days 9 and 11.5 of gestation, exhibiting defects in neurulation, cell proliferation, and heart development. Cells derived from p300-deficient embryos display specific transcriptional defects and proliferated poorly. Surprisingly, p300 heterozygotes also manifested considerable embryonic lethality. Moreover, double heterozygosity for p300 and cbp is invariably associated with embryonic death. Thus, mouse development is exquisitely sensitive to the overall gene dosage of p300 and cbp. These results provide genetic evidence that a coactivator endowed with histone acetyltransferase activity is essential for mammalian cell proliferation and development (Yao, 1998).

CREB-binding protein (CBP) is a transcriptional co-activator that is required by many transcription factors. Rubinstein-Taybi syndrome (RTS), which is an autosomal dominant syndrome characterized by abnormal pattern formation, is associated with mutations in the human CBP gene. Various abnormalities occur at high frequency in the skeletal system of heterozygous Cbp-deficient mice, but some features of RTS such as cardiac anomalies do not, suggesting that some symptoms of RTS are caused by a dominant-negative mechanism. The characterization of homozygous Cbp-deficient mice is reported in this study. Homozygous mutants die around E10.5-E12.5, apparently as a result of massive hemorrhage caused by defective blood vessel formation in the central nervous system, and exhibit apparent developmental retardation as well as delays in both primitive and definitive hematopoiesis. Cbp-deficient embryos exhibit defective neural tube closure. This is similar to what is observed in twist-deficient embryos. However, a decrease in the level of twist expression is not observed in Cbp-deficient embryos. Anomalous heart formation, a feature of RTS patients and mice mutated in the CBP-related molecule, p300, is not observed in Cbp-deficient embryos. Since both Cbp and p300 are ubiquitously expressed in embryonic tissues including the developing heart, these results suggest that cardiac anomalies observed in RTS patients may be caused by a dominant negative effect of mutant CBP (Tanaka, 2000).

A mouse model is presented of the haploinsufficiency form of Rubinstein-Taybi syndrome (RTS), an inheritable disorder caused by mutations in the gene encoding the CREB binding protein (CBP) and characterized by mental retardation and skeletal abnormalities. In these mice, chromatin acetylation, some forms of long-term memory, and the late phase of hippocampal long-term potentiation (L-LTP) were impaired. The L-LTP deficit is ameliorated in two ways: (1) by enhancing the expression of CREB-dependent genes, and (2) by inhibiting histone deacetyltransferase activity (HDAC), the molecular counterpart of the histone acetylation function of CBP. Inhibition of HDAC also reverses the memory defect observed in fear conditioning. These findings suggest that some of the cognitive and physiological deficits observed on RTS are not simply due to the reduction of CBP during development but may also result from the continued requirement throughout life for both the CREB co-activation and the histone acetylation function of CBP (Alarcon, 2004).

The affinity between DNA and histones in eukaryotic nucleosomes is modulated by phosphorylation, ubiquitination, methylation, and acetylation of the amino termini of histones. The conformational states of these histone tails determine the transcriptional activity of different chromatin domains and their accessibility to transcription factors and other DNA-associated proteins. Rubinstein-Taybi syndrome is only one of several neurological diseases that arise as a consequence of disordered chromatin remodeling. Other congenital syndromes that cause mental retardation in humans share a similar type of defect. Thus, Coffin-Lowry syndrome (CLS), X-linked alpha-thalassemia (ETRX), and Rett syndrome (RT) are conditions that are also caused by mutations in genes encoding enzymes that mediate chromatin remodeling and affect the acetylation state of chromatin indirectly. Coffin-Lowry syndrome results from mutations in the gene encoding RSK2 (see Drosophila RSK), an enzyme that interacts with CBP and phosphorylates histone H3 favoring its acetylation. Rett syndrome is caused by mutations of MECP2, a methyl-CpG binding protein that is thought to recruit HDACs to methylated DNA and mediate chromatin deacetylation. These mutations likely result in deregulation of the expression of a very large number of genes and yet they lead, surprisingly, to a well-defined phenotype. It is therefore likely that specific features of these syndromes are the consequence of dysregulation of perhaps a very few specific target genes. The overlap in the clinical features of these syndromes suggests the possibility that these conditions may share common molecular mediators. A comparison of phenotypic features of different mouse models for these syndromes combined with their molecular characterization using expression and acetylation arrays may reveal some of the common target genes involved in the cognitive disorders and thereby provide valuable information about the etiology of these diseases (Alarcon, 2004).

Experiments with SAHA, a broad HDAC inhibitor, indicate that therapeutical approaches for the treatment of diseases of epigenetic etiology might be possible. It is encouraging that a family of drugs that is currently being tested both in the treatment of cancer and neurodegenerative diseases may enhance L-LTP significantly both in cbp-deficient mutants and control littermates. Moreover, HDAC inhibitors reversed the deficit in fear conditioning of cbp+/- mutants. Further studies should reveal if they also improve the performance of wild-type mice. A number of biotech companies are working to improve the specificity and to reduce the side effects of HDAC inhibitors. If these goals are finally accomplished, it would be worthwhile to test the effectiveness of the new drugs in the treatment of Rubinstein-Taybi patients. However, changes in CBP function have been associated with diverse neurodegenerative conditions, including Huntington's disease (HD) and familial Alzheimer's disease (FAD). While HD has been associated with a reduction in CBP activity, FAD mutations may cause a gain of transcriptional function. Both conditions lead to neurodegeneration, suggesting that caution needs to be exercised in clinical studies involving pharmacological manipulation of CBP activity (Alarcon, 2004).

In conclusion, cbp+/- mice show a severe defect in hippocampal synaptic plasticity paralleling their deficits in some forms of long-term memory. These data suggest that some deficits observed in cbp mutants can be ameliorated using inhibitors of enzymes that compensate for a reduction in CBP function as CREB co-activator (such as rolipram). But, in addition, the data indicate a second deficit in histone acetylation. As a result, it is also possible to compensate for the reduction in CBP by inhibiting histone deacetylases, the enzymes that counteract a CBP role in chromatin remodeling. These findings may open the possibility of dual pharmacological treatment for the neurological deficits observed in RTS patients that reestablish normal CBP function and alleviate some of their symptoms (Alarcon, 2004).

CBP interaction with CREB

Two closely related proteins, p300 and CBP, are functional homologs and global transcriptional coactivators that are involved in the regulation of various DNA-binding transcription factors. p300/CBP interacts with nuclear receptors, CREB, c-Jun, C-Myb (see Drosophila Myb oncogene-like), c-Fos, and MyoD. DNA-binding factors recruit p300/CBP by not only direct but also indirect interactions through cofactors. p300/CBP is not only a transcriptional adaptor but also a histone acetyltransferase. The p300/CBP-histone acetyltransferase domain has no obvious sequence similarity to GCN5, another protein with known histone acetyltransferase activity, or to other previously described acetyltransferases. P300 acetylates all core histones in mononucleosomes and the four lysines in the Histone H4 N-terminal tail. These observations suggest that p300/CBP is not a simple adaptor between DNA binding factors and cellular p300/CBP associated factor (PCAF) or transcription factors; rather, p300/CBP per se may contribute directly to transcriptional regulation via targeted acetylation of chromatin (Ogryzko, 1996 and references).

A phosphoserine binding domain has been characterized in the coactivator CREB-binding protein (CBP) which interacts with the protein kinase A-phosphorylated (and hence activated) form of the cyclic AMP-responsive factor CREB. The CREB binding domain, referred to as KIX, is alpha helical and binds to an unstructured kinase-inducible domain in CREB following phosphorylation of CREB at Ser-133. Phospho-Ser-133 forms direct contacts with residues in KIX, and these contacts are further stabilized by hydrophobic residues in the kinase-inducible domain, which flanks phospho-Ser-133. Like the src homology 2 (SH2) domains which bind phosphotyrosine-containing peptides, phosphoserine 133 appears to coordinate with a single arginine residue (Arg-600) in KIX, which is conserved in the CBP-related protein P300. Since mutagenesis of Arg-600 to Gln severely reduces CREB-CBP complex formation, these results demonstrate that, as in the case of tyrosine kinase pathways, signal transduction through serine/threonine kinase pathways may also require protein interaction motifs that are capable of recognizing phosphorylated amino acids (Parker, 1996).

Human activating transcription factor 4 (hATF4), a member of the cAMP-responsive element-binding protein (CREB) family of transcription factors, is a potent transcriptional activator in both mammalian cells and yeast. The N-terminal 113 amino acids of hATF4 activate transcription efficiently; unexpectedly, the C-terminal bZip DNA binding domain of hATF4 also activates transcription, albeit weakly. These results indicate that hATF4 interacts with several general transcription factors: TATA-binding protein, TFIIB, and the RAP30 subunit of TFIIF. In addition, hATF4 interacts with the coactivator CREB-binding protein (CBP) at four regions: (1) the KIX domain, (2) a region that contains the third zinc finger and the E1A-interacting domain, (3) a C-terminal region that contains the p160/SRC-1-interacting domain, and (4) the recently identified histone acetyltransferase domain. Interestingly, both the N-terminal and C-terminal regions of hATF4 interact with the above general transcription factors and CBP, providing a mechanistic explanation for their ability to activate transcription. Consistent with its role as a coactivator, CBP potentiates the ability of hATF4 to activate transcription (Liang, 1997).

Transcriptional activation of the c-jun gene is a critical event in the differentiation of F9 cells. An element [differentiation response element (DRE)] in the c-jun promoter is both necessary and sufficient to confer the capacity for differentiation-dependent up-regulation. This element binds the differentiation regulatory factor (DRF) complex, of which one component is the adenovirus E1A-associated protein p300, whose sequence is closely related to Creb binding protein. Activation transcription factor-2 (ATF-2) is identified as a DNA-binding subunit of the DRF complex. ATF-2 is a member of the ATF/CREB family of basic region-leucine zipper (bZip domain) transcription factors. p300 and ATF-2 interact with each other in vivo and in vitro. The bromodomain and the C/H2 domain of p300 mediate the binding to ATF-2, which in turn requires a proline-rich region between amino acids 112 and 350 for its interaction with p300. The phosphorylation of the serine residue at position 121 of ATF-2 appears to be induced by protein kinase Calpha (PKCalpha) after treatment of cells with retinoic acid (RA) or induction with E1A. In cotransfection assays, wild-type ATF-2 enhances the transcription of an E2/tk-luciferase construct, in conjunction with p300-E2. However, this is not the case for a mutant form of ATF-2, with a mutation at position 121 (pCMVATF-2Ser121-Ala). These results suggest that ATF-2 and p300 cooperate in the control of transcription by forming a protein complex that is responsive to differentiation-inducing signals, such as RA or E1A, and moreover, that the phosphorylation of ATF-2 by PKC is probably a signaling event in the pathway that leads to the transactivation of the c-jun gene in F9 cells (Kawasaki, 1998).

The efficient replication of human T-cell leukemia virus type 1 (HTLV-1) and viral gene expression are both dependent on the virally encoded oncoprotein, Tax. To activate HTLV-1 transcription, Tax interacts with the cellular DNA binding protein cyclic AMP-responsive element binding protein (CREB) and recruits the coactivator CREB binding protein (CBP), forming a nucleoprotein complex on the three viral cyclic AMP-responsive elements (CREs) in the HTLV-1 promoter. Short stretches of dG-dC-rich (GC-rich) DNA, immediately flanking each of the viral CREs, are essential for Tax recruitment of CBP in vitro and Tax transactivation in vivo. Although the importance of the viral CRE-flanking sequences has been well established, several studies have failed to identify an interaction between Tax and the DNA. The mechanistic role of the viral CRE-flanking sequences has therefore remained enigmatic. In this study, high resolution footprinting was used to show that Tax extends the CREB footprint into the GC-rich DNA flanking sequences of the viral CRE. The Tax-CREB footprint is enhanced but not extended by the KIX domain of CBP, suggesting that the coactivator increases the stability of the nucleoprotein complex. Conversely, the footprint pattern of CREB on a cellular CRE lacking GC-rich flanking sequences does not change in the presence of Tax or Tax plus KIX. The minor-groove DNA binding drug chromomycin A3 binds to the GC-rich flanking sequences and inhibits the association of Tax and the Tax-CBP complex without affecting CREB binding. Tax specifically cross-links to the viral CRE in the 5'-flanking sequence, and this cross-link is blocked by chromomycin A3. Together, these data support a model where Tax interacts directly with both CREB and the minor-groove viral CRE-flanking sequences to form a high-affinity binding site for the recruitment of CBP to the HTLV-1 promoter (Lenzmeier, 1998).

The nuclear factor CREB activates transcription of target genes in part through direct interactions with the KIX domain of the coactivator CBP, in a phosphorylation-dependent manner. The CREB transactivation domain is bipartite, consisting of kinase-inducible and constitutive domains termed KID and Q2, respectively, which function synergistically in response to hormonal stimulation. The Z2 domain has been shown to stimulate transcription via its association with the hTAFII130 subunit of TFIID. The KID region has been found to regulate target gene expression by interaction with CBP and P300 in a phosphoSer-133 dependent manner. The solution structure of the complex formed by the phosphorylated kinase-inducible domain (pKID) of CREB with KIX reveals that pKID undergoes a coil-->helix folding transition upon binding to KIX, forming two alpha helices. The amphipathic helix alphaB of pKID interacts with a hydrophobic groove defined by helices alpha1 and alpha3 of KIX. The other pKID helix, alphaA, contacts a different face of the alpha3 helix. The phosphate group of the critical phosphoserine residue of pKID forms a hydrogen bond to the side chain of Tyr-658 of KIX. The structure provides a model for interactions between other transactivation domains and their targets (Radhakrishnan, 1997).

A fragment of the mixed-lineage leukemia (MLL) gene (Mll, HRX, ALL-1) was identified in a yeast genetic screen designed to isolate proteins that interact with the CREB-CREB-binding protein (CBP) complex. When tested for binding to CREB or CBP individually, this MLL fragment interacts directly with CBP, but not with CREB. In vitro binding experiments refined the identification of the minimal region of interaction to amino acids 2829 to 2883 of MLL, a potent transcriptional activation domain, and amino acids 581 to 687 of CBP (the CREB-binding or KIX domain). The transactivation activity of MLL is dependent on CBP, since either adenovirus E1A expression, which inhibits CBP activity, or alteration of MLL residues important for CBP interaction prove effective at inhibiting MLL-mediated transactivation. Single amino acid substitutions within the MLL activation domain reveal that five hydrophobic residues, potentially forming a hydrophobic face of an amphipathic helix, are critical for the interaction of MLL with CBP. Using purified components, it has been found that the MLL activation domain facilitates the binding of CBP to phosphorylated CREB. In contrast with paradigms in which factors compete for limiting quantities of CBP, these results reveal that two distinct transcription factor activation domains can cooperatively target the same motif on CBP (Ernst, 2001).

One prediction of these studies is that an overlap in MLL- and CREB-dependent target genes exists such that the cooperative interaction of MLL and CREB with CBP would play a role in regulating these genes. Murine knockout models for both CREB and MLL may be instructive in identifying potential shared target genes for these two transcriptional activators. One possible function of the MLL protein at such predicted target genes might be to convert the acute, signal-induced activation of CREB into a sustained, developmentally maintained response analogous to the maintenance role of Trithorax-group proteins during Drosophila embryogenesis (Ernst, 2001).

Fusion of the MLL gene to either CBP or p300 has been reported in several cases of myeloid leukemia or myeloid dysplasia\. The data presented here suggest that MLL utilizes CBP to activate transcription. This finding raises intriguing issues regarding the mechanism of MLL-CBP or MLL-p300 fusions in leukemogenesis. One hypothesis for the oncogenic mechanism of the MLL-fusion gene products is that the acquisition of a new C terminus would impart an enhanced or new activity to the MLL molecule (gain-of-function or neomorphic activity). The MLL-CBP physical interaction presented in this study may be regulated during normal hematopoietic development to modulate the maintenance of endogenous MLL target genes. One testable prediction is that fusion of MLL to CBP would result in a form of MLL that could not be uncoupled from CBP, leading to the temporally inappropriate maintenance of MLL target gene expression. Constitutive expression of certain MLL target genes would consequently contribute to leukemogenesis. Attractive candidates for such target genes are HOX genes, which have been shown to play a role in normal or aberrant hematopoiesis through either loss- or gain-of-function experiments (Ernst, 2001).

The calcium-binding protein DREAM (Drosophila homolog: CG5890) binds specifically to DRE sites (refering to downstream regulatory element of the prodynorphin gene where the DRE sequence is GAGTCAAGG) in the DNA and represses transcription of target genes. Derepression at DRE sites following PKA activation depends on a specific interaction between CREM and DREAM. Two leucine-charged residue-rich domains (LCD) located in the kinase-inducible domain (KID) and in the leucine zipper of CREM and two LCDs in DREAM participate in a two-site interaction that results in the loss of DREAM binding to DRE sites and derepression. Since the LCD motif located within the KID in CREM is also present in CREB, and maps in a region critical for the recruitment of CBP, whether DREAM may affect CRE-dependent transcription was investigated. In the absence of Ca2+ DREAM binds to the LCD in the KID of CREB. As a result, DREAM impairs recruitment of CBP by phospho CREB and blocks CBP-mediated transactivation at CRE sites in a Ca2+-dependent manner. Thus, Ca2+-dependent interactions between DREAM and CREB represent a novel point of cross-talk between cAMP and Ca2+ signaling pathways in the nucleus (Ledo, 2002).

Cyclic AMP-dependent gene expression is controlled at the transcriptional level by several bZIP transcription factors, including CREB, CREM and ATF proteins. They bind to CRE sites in target genes as homo- or heterodimers. Dimerization is achieved by the LZ located next to the basic DNA-binding domain in the C-terminal of the protein. Transcriptional activity by these dimers follows after phosphorylation in their KIDs and the recruitment of the transcriptional cofactor CREB-binding protein, CBP. Importantly, the LCD within the KID domain in alphaCREM, common to all CREM isoforms and almost identical in CREB, is located within a region important for the interaction with the CREB-interacting domain of CBP (amino acids 455-679) known as the KIX domain. In this study, it has been shown that there exists a Ca2+-dependent protein-protein interaction between DREAM and CREB through the LCD located in the KID domain of CREB. As a result of this interaction, DREAM prevents the recruitment of CBP and represses CRE-dependent transcription (Ledo, 2002).

Calcium regulation of CBP function

Recruitment of the coactivator, CREB binding protein (CBP), by signal-regulated transcription factors, such as CREB [adenosine 3',5'-monophosphate (cAMP) response element binding protein], is critical for stimulation of gene expression. The mouse pituitary cell line AtT20 was used to show that the CBP recruitment step (CREB phosphorylation on serine-133) can be uncoupled from CREB/CBP-activated transcription. CBP contains a signal-regulated transcriptional activation domain that is controlled by nuclear calcium and calcium/calmodulin-dependent (CaM) protein kinase IV and by cAMP. Cytoplasmic calcium signals that stimulate the Ras mitogen-activated protein kinase signaling cascade or expression of the activated form of Ras provide the CBP recruitment signal but does not increase CBP activity and fails to activate CREB- and CBP-mediated transcription. These results identify CBP as a signal-regulated transcriptional coactivator and define a regulatory role for nuclear calcium and cAMP in CBP-dependent gene expression (Chawla, 1998).

The transcription factor CREB is involved in mediating many of the long-term effects of activity-dependent plasticity at glutamatergic synapses. Activation of NMDA receptors and voltage-sensitive calcium channels leads to CREB-mediated transcription in cortical neurons via a mechanism regulated by CREB-binding protein (CBP). Recruitment of CBP to the promoter is not sufficient for transactivation, but calcium influx can induce CBP-mediated transcription via two distinct transactivation domains. CBP-mediated transcription is stimulus strength-dependent and can be induced by activation of CaM kinase II, CaM kinase IV, and protein kinase A, but not by activation of the Ras-MAP kinase pathway. These observations indicate that CBP can function as a calcium-sensitive transcriptional coactivator that may act as a regulatory switch for glutamate-induced CREB-mediated transcription (Hu, 1999).

Recruitment of the coactivator CBP by signal-regulated transcription factors and stimulation of CBP activity are key regulatory events in the induction of gene transcription following Ca2+ flux through ligand- and/or voltage-gated ion channels in hippocampal neurons. The mode of Ca2+ entry (L-type Ca2+ channels versus NMDA receptors) differentially controls the CBP recruitment step to CREB, providing a molecular basis for the observed Ca2+ channel type-dependent differences in gene expression. In contrast, activation of CBP is triggered irrespective of the route of Ca2+ entry, as is activation of c-Jun, which recruits CBP independent of phosphorylation at major regulatory c-Jun phosphorylation sites, serines 63 and 73. This control of CBP recruitment and activation is apt to be relevant to other CBP-interacting transcription factors and represents a general mechanism through which Ca2+ signals associated with electrical activity may regulate the expression of many genes (Hardingham, 1999).

CBP as a histone acetyltransferase

The CBP co-activator protein possesses an intrinsic acetyltransferase (AT) activity capable of acetylating nucleosomal histones, as well as other proteins, such as the transcription factors TFIIE and TFIIF. CBP associates with two other histone acetyltransferases: P/CAF and SRC1. Does the intrinsic AT activity of CBP contribute to transcriptional activation? A region of CBP, encompassing the previously defined histone AT (HAT) domain, can stimulate transcription when tethered to a promoter. The stimulatory effect of this activation domain shows some promoter preference and is dependent on AT activity. Analysis of 14 point mutations reveals a direct correlation between CBP's ability to acetylate histones in vitro and to activate transcription in vivo. The HAT domains of CBP and P/CAF share sequence similarity. Examination of the AT domains of recently identified ATs has not revealed any overt sequence similarity between them. Indeed, a BLAST search using CBP does not identify the other ATs, P/CAF, SRC1 or TAFII250. However, careful scrutiny of the relevant HAT domains of CBP and P/CAF reveals considerable sequence similarity over a 100 residue stretch. Over this region of CBP there is 16% identity and 32% similarity with the P/CAF sequence. Although this falls below the detection level for a computer-based search, at least two aspects of the similarity suggest that the similiarity is functionally relevant: (1) there are no major gaps introduced in the sequence, and (2) the conserved residues coincide with motifs found conserved between N-acetyltransferases and the P/CAF-related HAT, GCN5. Aligning the AT domains derived from N-acetyltransferases has allowed the identification of four conserved motifs : A, B, C and D, three of which are analogous to motifs A, B and D found in other N-acetyltransferases. The fourth motif, termed E, is unique to CBP and P/CAF. Mutagenesis shows that all four motifs in CBP contribute to its HAT activity in vitro and its ability to activate transcription in vivo. These results demonstrate that the AT activity of CBP is directly involved in stimulating gene transcription. The identification of specific HAT domain motifs, conserved between CBP and P/CAF, should facilitate the identification of other members of this AT family (Martínez-Balbás, 1998).

The acetylation of histones increases the accessibility of nucleosomal DNA to transcription factors, relieving transcriptional repression and correlating with the potential for transcriptional activity in vivo. The characterization of several novel histone acetyltransferases - including the human GCN5 homolog PCAF (p300/CBP-associated factor), the transcription coactivator p300/CBP, and TAFII250 - has provided a potential explanation for the relationship between histone acetylation and transcriptional activation. In addition to histones, however, other components of the basal transcription machinery might be acetylated by these enzymes and directly affect transcription. The acetylation of the basal transcriptional machinery for RNA polymerase II by PCAF, p300 and TAFII250 was examined. All three acetyltransferases can direct the acetylation of TFIIEbeta and TFIIF; a preferred site of acetylation in TFIIEbeta was identified. Human TFIIE consists of two subunits, alpha(p56) and beta(p34), which form a heterotetramer (alpha2 beta2) in solution. TFIIE enters the preinitiation complex after RNA polymerase II and TFIIF, suggesting that TFIIE may interact directly with RNA polymerase II and/or TFIIF. In addition, TFIIE can facilitate promoter melting either in the presence or absence of TFIIH and can stimulate TFIIH-dependent phosphorylation of the carboxy-terminal domain of RNA polymerase II. TFIIF has an essential role in both transcription initiation and elongation (Imhof, 1997).

PCAF (p300/CBP-associated factor) and human GCN5, two related type A histone acetyltransferases, are unstable enzymes that under the commonly used assay conditions are rapidly and irreversibly inactivated. In addition, free histone H1, although not acetylated in vivo, is a preferred and convenient in vitro substrate for the study of PCAF, human GCN5, and possibly other type A histone acetyltransferases. Using either histone H1 or histone H3 as substrates, it is found that preincubation with either acetyl-CoA or CoA stabilizes the acetyltransferase activities of PCAF, human GCN5 and an enzymatically active PCAF deletion mutant containing the C-terminal half of the protein. The stabilization requires the continuous presence of coenzyme, suggesting that the acetyltransferase-coenzyme complexes are stable, while the isolated apoenzymes are not. Human GCN5 and the N-terminal deletion mutant of PCAF are stabilized equally well by preincubation with either CoA or acetyl-CoA, while intact PCAF is better stabilized by acetyl-CoA than by CoA. Intact PCAF, but not the N-terminal truncation mutant or human GCN5, is autoacetylated. These findings raise the possibility that the intracellular concentrations of the coenzymes affect the stability and therefore the nuclear activity of these acetyltransferases (Herrera, 1997).

Steroid receptors and coactivator proteins are thought to stimulate gene expression by facilitating the assembly of basal transcription factors into a stable preinitiation complex. What is not clear, however, is how these transcription factors gain access to transcriptionally repressed chromatin to modulate the transactivation of specific gene networks in vivo. The available evidence indicates that acetylation of chromatin in vivo is coupled to transcription and that specific histone acetyltransferases (HATs) target histones bound to DNA and overcome the inhibitory effect of chromatin on gene expression. SRC-1 possesses intrinsic histone acetyltransferase activity; it also interacts with another HAT, p300/CBP-associated factor (PCAF). The HAT activity of SRC-1 maps to its carboxy-terminal region and is primarily specific for histones H3 and H4. Acetylation by SRC-1 and PCAF of histones bound at specific promoters may result from ligand binding to steroid receptors and could be a mechanism by which the activation functions of steroid receptors and associated coactivators enhance formation of a stable preinitiation complex, thereby increasing transcription of specific genes from transcriptionally repressed chromatin templates (Spencer, 1997).

CREB binding protein (CBP) acts as a transcriptional adaptor for many different transcription factors by directly contacting DNA-bound activators. Known also as p300 protein, it was identified intitially as a cellular target of adenoviral E1A oncoprotein. CBP forms a complex with TBP in vivo and it acts as a coactivator for the transcription factor MyoD (Drosophila homolog: Nautilus), as well as CREB, c-Jun and cFOS. In the case of c-Fos, CBP- induced stimulation of c-Fos activity is abrogated by adenovirus E1A protein, which has the capacity to modulate AP1-site-containing promoters. One mechanism by which CBP is thought to stimulate transcriptions is the recruitment of the histone acetyltransferase (HAT) P/CAF to the promoter. It is now clear that CBP possesses intrinsic HAT activity. The HAT domain of CBP is adjacent to the binding site for the transcriptional activator E1A. All four nucleosomal proteins are substrates for CBP HAT activity. Although E1A displaces P/CAF from CBP, it does not disrupt the CBP-associated HAT activity. Thus E1A carries HAT activity when complexed with CBP. Targeting CBP-associated HAT activity to specific promoters may therefore be a mechanism by which E1A acts as a transcriptional activator. The HAT domain of CBP does not show any overt sequence similarity to the HAT domain of the GCN5 family of proteins to which P/CAF belong. This raises the possibility that CBP can accomplish distinct functions by taking advantage of two different HAT activities: one intrinsic to CBP and one associated with the P/CAF protein (Bannister, 1996 and references).

The yeast transcriptional adapter Gcn5p serves as a histone acetyltransferase, directly linking chromatin modification to transcriptional regulation. Two human homologs of Gcn5p have been reported previously, hsGCN5 and hsP/CAF (p300/CREB binding protein [CBP]-associated factor). While hsGCN5 is predicted to be close to the size of the yeast acetyltransferase, hsP/CAF contains an additional 356 amino-terminal residues of unknown function. Surprisingly, it was found that in mouse, both the GCN5 and the P/CAF genes encode proteins containing this extended amino-terminal domain. Moreover, while a shorter version of GCN5 might be generated upon alternative or incomplete splicing of a longer transcript, mRNAs encoding the longer protein are much more prevalent in both mouse and human cells, and larger proteins are detected by GCN5-specific antisera in both mouse and human cell extracts. Mouse GCN5 (mmGCN5) and mmP/CAF genes are ubiquitously expressed, but maximum expression levels are found in different, complementary sets of tissues. Both mmP/CAF and mmGCN5 interact with CBP/p300. Interestingly, mmGCN5 maps to chromosome 11 and cosegregates with BRCA1, and mmP/CAF maps to a central region of chromosome 17. As expected, recombinant mmGCN5 and mmP/CAF both exhibit histone acetyltransferase activity in vitro with similar substrate specificities. However, in contrast to yeast Gcn5p and the previously reported shorter form of hsGCN5, mmGCN5 readily acetylates nucleosomal substrates as well as free core histones. Thus, the unique amino-terminal domains of mammalian P/CAF and GCN5 may provide additional functions important to recognition of chromatin substrates and the regulation of gene expression (W. Xu, 1998).

A purified recombinant chromatin assembly system, including ACF (Acf-1 + ISWI) and NAP-1, has been used to examine the role of histone acetylation in ATP-dependent chromatin remodeling. The binding of a transcriptional activator (Gal4-VP16) to chromatin assembled using this recombinant assembly system dramatically enhances the acetylation of nucleosomal core histones by the histone acetyltransferase p300. This effect requires both the presence of Gal4-binding sites in the template and the VP16-activation domain. Order-of-addition experiments indicate that prior activator-meditated, ATP-dependent chromatin remodeling by ACF is required for the acetylation of nucleosomal histones by p300. Thus, chromatin remodeling, which requires a transcriptional activator, ACF and ATP, is an early step in the transcriptional process that regulates subsequent core histone acetylation. Glycerol gradient sedimentation and immunoprecipitation assays demonstrate that the acetylation of histones by p300 facilitates the transfer of H2A-H2B from nucleosomes to NAP-1. The results from these biochemical experiments suggest that (1) transcriptional activators (e.g., Gal4-VP16) and chromatin remodeling complexes (e.g., ACF) induce chromatin remodeling in the absence of histone acetylation; (2) transcriptional activators recruit histone acetyltransferases (e.g., p300) to promoters after chromatin remodeling has occurred; and (3) histone acetylation is important for a step subsequent to chromatin remodeling and results in the transfer of histone H2A-H2B dimers from nucleosomes to a histone chaperone such as NAP-1. These results indicate a precise role for histone acetylation, namely to alter the structure of nucleosomes (e.g., facilitate the loss of H2A-H2B dimers) that have been remodeled previously by the action of ATP-dependent chromatin remodeling complexes. Thus, transcription from chromatin templates is ordered and sequential, with precise timing and roles for ATP-dependent chromatin remodeling, subsequent histone acetylation, and alterations in nucleosome structure. The presence of altered (i.e., H2A-H2B-depleted) nucleosomes at a transcriptionally active, chromatin-remodeled promoter may help to maintain an open chromatin structure conducive to multiple rounds of activated transcription (Ito, 2000).

CREB-binding protein (CBP) and CBP-associated factor (P/CAF) are coactivators possessing an intrinsic histone acetyltransferase (HAT) activity. They are positioned at promoter regions via association with sequence-specific DNA-binding factors and stimulate transcription in a gene-specific manner. The current view suggests that coactivator function depends mainly on the strength and specificity of transcription factor-coactivator interactions. Two dominant-negative mutants of hepatocyte nuclear factor-1alpha (HNF-1alpha), P447L and P519L, occurring in maturity onset diabetes of the young (MODY3) patients, exhibit paradoxically stronger interactions than the wild-type protein with either CBP or P/CAF. However, CBP and P/CAF recruited by these mutants lack HAT activity. In contrast, wild-type HNF-1alpha and other transcription factors, such as Sp1 or HNF-4, stimulate the HAT activity of CBP. The results suggest a more dynamic role for DNA-binding proteins in the transcription process than was considered previously. They are not only required for the recruitment of coactivators to the promoter but they may also modulate their enzymatic activity (Soutoglou, 2001).

The tumor suppressor protein, p53, plays a critical role in mediating cellular response to stress signals by regulating genes involved in cell cycle arrest and apoptosis. p53 is believed to be inactive for DNA binding unless its C terminus is modified or structurally altered. Unmodified p53 actively binds to two sites at -1.4 and -2.3 kb within the chromatin-assembled p21 promoter and requires the C terminus and the histone acetyltransferase, p300, for transcription. Acetylation of the C terminus by p300 is not necessary for binding or promoter activation. Instead, p300 acetylates p53-bound nucleosomes in the p21 promoter with spreading to the TATA box. Thus, p53 is an active DNA and chromatin binding protein that may selectively regulate its target genes by recruitment of specific cofactors to structurally distinct binding sites (Espinosa, 2001).

Surprisingly, p300 does not function by facilitating p53 binding to its DNA recognition sites within chromatin. Instead, p300 acts at a later step in the transcription process by acetylating nucleosomes within the proximal and distal p21 promoter when targeted by bound p53. This presumably renders the nucleosomes sufficiently fluid to allow interaction with other components of the transcription machinery. p300-mediated transcriptional activation has been described for other chromatin-assembled genes. These experiments demonstrate that a mechanism by which p300 can regulate the activity of natural promoters operates by acetylating chromatin over a long-range when recruited by a distal transcription factor. In the absence of p53, p300 cannot acetylate nucleosomes due to lack of template targeting, and the p21 promoter remains inactive. p53 proteins containing mutations in lysine residues acetylated by p300 are as active as wild-type p53 in regulating p21 transcription in vitro. This indicates that acetylation of p53 does not contribute to its transactivation potential, and that p300 does not mediate transcription by this mechanism in biochemical assays. This conclusion is in agreement with previous in vivo analyses in which p53 mutants lacking these lysine residues does not show a significant decrease in transcriptional activity. However, p53 acetylation may play a role in protein stabilization or subnuclear localization (Espinosa, 2001).

The human ISWI-containing factor RSF (remodeling and spacing factor) mediates nucleosome deposition and, in the presence of ATP, generates regularly spaced nucleosome arrays. Using this system, recombinant chromatin was reconstituted with bacterially produced histones. Acetylation of the histone tails was found to play an important role in establishing regularly spaced nucleosome arrays. Recombinant chromatin lacking histone acetylation is impaired in directing transcription. Histone-tail modifications regulate transcription from the recombinant chromatin. Acetylation of the histone tails by p300 increases transcription. Methylation of the histone H3 tail by Suv39H1 represses transcription in an HP1-dependent manner. The effects of histone-tail modifications were observed in nuclear extracts. A highly reconstituted RNA polymerase II transcription system is refractory to the effect imposed by acetylation and methylation (Loyola, 2001).

The establishment of conditions that permit the reconstitution of recombinant chromatin allows for the analysis of the effect of the different histone tail modifications in transcription. Toward this goal, the ability of the recombinant chromatin to be used as template for transcription was analyzed and the effect of two histone-tail modifications was specifically analyzed: p300-mediated acetylation and Suv39H1-mediated methylation (Loyola, 2001).

Although these two modifications can have opposite effects on transcription, these modifications were not recognized in a highly reconstituted transcription system; their effect was observed only in crude extracts. There are different explanations for findings. The most logical explanation is that acetylation and/or methylation per se does not affect template utilization but affects the ability of the chromatin templates to be recognized by the transcription machinery. It is likely that these modifications provide marks on the histone tails that are recognized by factors present in extracts but missing in the reconstituted system that affects transcription. This hypothesis is supported by the findings with methylation and transcription. It was found that HP1-mediated repression of transcription requires Suv39H1-mediated methylation of histone H3. This finding is in perfect agreement with results obtained in vivo showing that the binding of HP1 to chromatin requires methylation of histone H3-Lys 9. Surprisingly, however, chromatin, H3-Lys 9 methylation, and HP1 are not sufficient to establish repression, since this could not be reproduced in a reconstituted transcription system. It is likely that other factors are required to establish repression. Studies in yeast have shown that histone deacetylation is required to establish the appropriate substrate for methylation by Suv39H1. Although the use of chromatin without pre-existing modification bypasses the requirement for the histone deacetylase enzymatic activity, it is possible that the histone deacetylases that target histone H3-lysines 9 and 14 not only function to generate the appropriate substrate but also might be active components of the Suv39H1-repressive complex (Loyola, 2001).

With regards to acetylation, it was observed that chromatin reconstituted with hypoacetylated human histone polypeptides is not optimal for transcription in crude extracts; however, the reconstituted system is indifferent to acetylation of the histone polypeptides. This finding is in agreement with the histone-code hypothesis and strongly suggests that factors in the extract, but lacking in the reconstituted system, might recognize the acetylated mark(s) to stimulate transcription. Using recombinant chromatin, it was observed that acetylation of histone tails, specifically by p300, stimulates transcription in extracts. In agreement with the results obtained using chromatin reconstituted with hypo/hyperacetylated human histones, no effect was observed in a reconstituted transcription system. Although a possible explanation to this observation is the absence of a factor in the reconstituted system, the inability of the reconstituted transcription system to respond to acetylation of the recombinant chromatin might also be the result of the inability of p300 to acetylate specific residues on the histone tails. The recombinant chromatin is devoid of histone-tail modifications, and it is likely that p300-mediated acetylation of a specific residue might require other histone modifications. This possibility is supported by studies showing that phosphorylation of histone H3-Ser 10 modulates acetylation of histone H3-Lys 14. The presence of a specific kinase in the extract might phosphorylate histone H3-Ser 10, resulting in efficient acetylation. Elucidation of the factors necessary for p300-mediated acetylation to result in optimal transcription and of the factor(s) required for Suv39-H1-mediated methylation to result in repression of transcription, and their exact mechanism of action, require further studies. The development of the system described in the present study, capable of generating recombinant chromatin will permit the setting of biochemical complementation assays to isolate the different factors involved in these processes as well as the elucidation of their mechanism of action (Loyola, 2001).

The N-terminal tails of the core histones play important roles in transcriptional regulation, but their mechanism(s) of action are poorly understood. Pure chromatin templates assembled with varied combinations of recombinant wild-type and mutant core histones have been employed to ascertain the role of individual histone tails, both in overall acetylation patterns and in transcription. In vitro assays show an indispensable role for H3 and H4 tails, especially major lysine substrates, in p300-dependent transcriptional activation, as well as activator-targeted acetylation of promoter-proximal histone tails by p300. These results indicate, first, that constraints to transcription are imposed by nucleosomal histone components other than histone N-terminal tails and, second, that the histone N-terminal tails have selective roles, which can be modulated by targeted acetylation, in transcriptional activation by p300 (An, 2002).

The first significant conclusion from these results is that the tails do not simply and uniquely impose constraints to the binding and function of either gene-specific transcriptional activators or components of the general transcriptional machinery. Instead, it seems clear that the globular domains themselves maintain a repressed state and that specific N-terminal tails and corresponding natural acetylatable lysine residues are actively required for the reversal of these effects. Another significant conclusion from the present study is that the H3 and H4 tails are selectively required for the observed derepression and net activation by Gal4-VP16 and p300 and, that these tails are not redundant for transcription. These results are consistent with differential effects of H3 versus H4 tail mutations on the transcriptional regulation of specific genes and differential functions for H3 and H4 tails versus H2A and H2B tails both in transcription and in higher-order chromatin structure (An, 2002 and references therein).

These results also establish a direct link between activator-dependent acetylation of histones by p300 and activator-dependent transcription. Beyond the fact that activator-dependent transcription requires activator- and p300-dependent histone tail acetylation, the selective requirement for H3 and H4 tails and corresponding acetylation sites for transcription correlates with the observations (1) that H3 is the preferred p300 substrate in chromatin, (2) that optimal H3 and H4 acetylation occurs independently of H2A and H2B tails, whereas maximal H2A and H2B acetylation is dependent upon H3 and H4 tails, and (3) that there is a strong activator-mediated targeting of acetylation to promoter-proximal H3 and H4 (An, 2002).

Dynamic changes in the modification pattern of histones, such as acetylation, phosphorylation, methylation, and ubiquitination, are thought to provide a code for the correct regulation of gene expression mostly by affecting chromatin structure and interactions of non-histone regulatory factors with chromatin. Recent studies have suggested the existence of an interplay between histone modifications during transcription. The CBP/p300 acetylase and cofactor-associated arginine [R] methyltransferase 1 (CARM1) can positively regulate the expression of estrogen-responsive genes, but the existence of a crosstalk between lysine acetylation and arginine methylation on chromatin has not yet been established in vivo. By following the in vivo pattern of modifications on histone H3, following estrogen stimulation of the pS2 promoter, it has been shown that arginine methylation follows prior acetylation of H3. Within 15 min after estrogen stimulation, CBP is bound to chromatin, and acetylation of K18 takes place. Following these events, K23 is acetylated, CARM1 associates with chromatin, and methylation at R17 takes place. Exogenous expression of CBP is sufficient to drive the association of CARM1 with chromatin and methylation of R17 in vivo, whereas an acetylase-deficient CBP mutant is unable to induce these events. A mechanism for the observed cooperation between acetylation and arginine methylation comes from the finding that acetylation at K18 and K23, but not K14, tethers recombinant CARM1 to the H3 tail and allows it to act as a more efficient arginine methyltransferase. These results reveal an ordered and interdependent deposition of acetylation and arginine methylation during estrogen-regulated transcription and provides support for a combinatorial role of histone modifications in gene expression (Daujat, 2002).

c-Myb plays important roles in cell survival and differentiation in immature hematopoietic cells. c-Myb is acetylated at the carboxyl-terminal conserved domain by histone acetyltransferase p300 both in vitro and in vivo. The acetylation sites in vivo have been located at the lysine residues of the conserved domain (K471, K480, K485) by the use of the mutant Myb (Myb-KAmut), in which all three lysine residues are substituted into alanine. Electrophoretic mobility shift assay reveals that Myb-KAmut shows higher DNA binding activity than wild type c-Myb and that acetylation of c-Myb in vitro by p300 causes dramatic increase in DNA binding activity. Accordingly, transactivation activity of both mim-1 and CD34 promoters by Myb-KAmut is higher than that driven by wild type c-Myb. Furthermore, the bromodomain of p300, in addition to the histone acetyltransferase (HAT) domain, is required for effective acetylation of c-Myb; hGCN5 is revealed to be an acetyl-transferase for c-Myb in vitro (Tomita, 2000).

The multifunctional Creb-binding protein (CBP) protein plays a pivotal role in many critical cellular processes. This study demonstrate that the bromodomain of CBP binds to histone H3 acetylated on lysine 56 (K56Ac) with higher affinity than to its other monoacetylated binding partners. Autoacetylation of CBP is critical for the bromodomain-H3 K56Ac interaction, and it is proposed that this interaction occurs via autoacetylation-induced conformation changes in CBP. Unexpectedly, the bromodomain promotes acetylation of H3 K56 on free histones. The CBP bromodomain also interacts with the histone chaperone anti-silencing function 1 (ASF1) via a nearby but distinct interface. This interaction is necessary for ASF1 to promote acetylation of H3 K56 by CBP, indicating that the ASF1-bromodomain interaction physically delivers the histones to the histone acetyl transferase domain of CBP. A CBP bromodomain mutation manifested in Rubinstein-Taybi syndrome has compromised binding to both H3 K56Ac and ASF1, suggesting that these interactions are important for the normal function of CBP (Das, 2014).

Interaction of CBP with the basal transcriptional apparatus

How does the CREB stimulate target gene expression following CREB's phosphorylation at Ser-133. Two signals are required for target gene activation: a phospho(Ser-133)-dependent interaction of CREB with RNA polymerase II via the coactivator CBP and a glutamine-rich domain interaction with TFIID via hTAFII130. The adenovirus E1A oncoprotein is found to inhibit phospho(Ser-133) CREB activity by binding to CBP, specifically blocking recruitment of RNA Pol II to the promoter. These results suggest that the recruitment of CBP-RNA Pol II complexes per se is not sufficient for transcriptional activation and that activator-mediated recruitment of TFIID is additionally required for induction of signal-dependent genes (Nakajima, 1997a).

The coactivator CBP has been proposed to stimulate the expression of certain signal-dependent genes via its association with RNA polymerase II complexes. Complex formation between CBP and RNA polymerase II requires RNA helicase A (RHA), a nuclear DNA/RNA helicase that is related to the Drosophila male dosage compensation factor Mle. In transient transfection assays, RHA is found to cooperate with CBP in mediating target gene activation via the CAMP responsive factor CREB. Since a mutation in RHA that compromises its helicase activity correspondingly reduces CREB-dependent transcription, it is proposed that RHA may induce local changes in chromatin structure that promote engagement of the transcriptional apparatus on signal responsive promoters. The involvement of a DNA helicase such as RHA in signal-dependent transcription is intriguiing because it suggests that recruitment of CBP complexes may promote local unwinding of promoter DNA via RHA and thereby permit engagement of the transcriptional apparatus (Nakajima, 1997b).

CREB-mediated constitutive transcription requires only CREB-binding sites and a minimal promoter region (containing the TATA through start sequences), indicating that CREB interacts directly with components of the general transcription machinery. Human CREB specifically binds to TFIIB. CREB binds TFIID complexes containing TBP, but does not directly interact with TBP. TFIIB interacts with TBP but CREB does not form a stable ternary complex with TFIIB and TBP. CREB interacts independently with TFIIB and TFIID, but not directly with TBP. Both wild-type CREB and a protein kinase A phosphorylation site mutant of CREB exhibit equivalent interactions with TFIIB, indicating that this phosphorylation is not required for interaction. Consistent with the role of CREB in promoting constitutive or basal transcription, the constitutive activation domain of CREB is sufficient for interaction with both TFIIB and TFIID (Xing, 1995).

A specific mutation in TAFII250 (see Drosophila TBP-associated factor 250kD), the largest subunit of the transcription factor TFIID, disrupts cell growth control in the temperature-sensitive mutant hamster cell line ts13. Transcription from the cyclin A and D1(but not the c-fos and myc promoters) is also dramatically reduced in ts13 cells at the nonpermissive temperature. These findings provide an intriguing link between TAF-mediated transcriptional regulation and cell cycle progression. An enhancer element in the cyclin A (see Drosophila Cyclin A) promoter (TSRE) has been mapped that responds to mutations in TAFII250. An analysis of chimeric promoter constructs reveals that the cyclin A TSRE can confer TAFII250 dependence to the core promoter of c-fos. Reciprocal hybrid promoter constructs suggest that TAFII250 also contributes to the transcriptional properties of the cyclin A core promoter. The cellular activators that specifically bind to the TSRE and mediate transcription in a TAFII250-dependent manner have been purified and identified. TSRE-binding proteins include members of the activating transcription factor (ATF) family. These results suggest that the ts13 mutation of TAFII250 has compromised the ability of TFIID to mediate activation of transcription by specific enhancer factors such as ATF, as well as its ability to perform certain core promoter functions. These defects in TAFII250 apparently result in the down-regulation of key molecules, such as cyclin A, which may be responsible for the ts13 cell cycle arrest phenotype (Wang, 1997).

An human RNA polymerase II complex has been isolated that contains chromatin structure remodeling activity and histone acetyltransferase activity. This complex contains the Srb proteins, the Swi-Snf complex, and the histone acetyltransferases CBP and PCAF in addition to RNA polymerase II. Notably, the general transcription factors are absent from this complex. The complex was purified by two different methods: conventional chromatography and affinity chromatography using antibodies directed against CDK8 (see Drosophila Cdk8), the human homolog of the yeast Srb10 protein. Protein interaction studies demonstrate a direct interaction between RNA polymerase II and the histone acetyltransferases p300 and PCAF. Importantly, p300 interacts specifically with the nonphosphorylated, initiation-competent form of RNA polymerase II. In contrast, PCAF interacts with the elongation-competent, phosphorylated form of RNA polymerase II (Cho, 1998).

Enhancers provide critical information directing cell-type-specific transcriptional programs, regulated by binding of signal-dependent transcription factors and their associated cofactors. This study reports that the most strongly activated estrogen (E2)-responsive enhancers are characterized by trans-recruitment and in situ assembly of a large 1-2 MDa complex of diverse DNA-binding transcription factors by ERalpha at ERE-containing enhancers. Enhancers recruiting these factors are referred to as mega transcription factor-bound in trans (MegaTrans) enhancers. The MegaTrans complex is a signature of the most potent functional enhancers and is required for activation of enhancer RNA transcription and recruitment of coactivators, including p300 (see Drosophila CBP) and Med1. The MegaTrans complex functions, in part, by recruiting specific enzymatic machinery, exemplified by DNA-dependent protein kinase. Thus, MegaTrans-containing enhancers represent a cohort of functional enhancers that mediate a broad and important transcriptional program and provide a molecular explanation for transcription factor clustering and hotspots noted in the genome (Liu, 2014).

CBP as a coactivator of nuclear receptors

The CREB-binding protein (CBP) and its homolog P300 act as cofactors mediating nuclear-receptor-activated gene transcription (See Drosophila Ecdysone receptor). The role of CBP/P300 in the transcriptional response to cyclic AMP, phorbol esters, serum, the lipophilic hormones and as the target of the E1A oncoprotein suggests they may serve as integrators of extracellular and intracellular signaling pathways leading to gene activation. Since CBP is known to be a histone acetyltransferase gene activation carried out by nuclear receptors is likely to involve chromatin modification (Chakravarti, 1996).

The binding of lipophilic hormones, retinoids and vitamins to members of the nuclear-receptor superfamily modifies the DNA-binding and transcriptional properties of these receptors, resulting in the activation or repression of target genes. Ligand binding induces conformational changes in nuclear receptors and promotes their association with a diverse group of nuclear proteins, including SRC-1/p160, TIF-2/GRIP-1 and CBP/p300, which function as co-activators of transcription, and RIP-140, TIF-1 and TRIP-1/SUG-1 whose functions are unclear. A short sequence motif LXXLL (where L is leucine and X is any amino acid) present in RIP-140, SRC-1 and CBP is necessary and sufficient to mediate the binding of these proteins to liganded nuclear receptors. The ability of SRC-1 to bind the estrogen receptor and enhance its transcriptional activity is dependent upon the integrity of the LXXLL motifs and on key hydrophobic residues in a conserved helix (helix 12) of the estrogen receptor that are required for its ligand-induced activation function. It is proposed that the LXXLL motif is a signature sequence that facilitates the interaction of different proteins with nuclear receptors, and is thus a defining feature of a new family of nuclear proteins (Heery, 1997).

The functionally conserved proteins CBP and p300 act in conjunction with other factors to activate transcription of DNA. A new factor, p/CIP, has been discovered that is present in the cell as a complex with CBP and is required for transcriptional activity of nuclear receptors and other CBP/p300-dependent transcription factors. The highly related nuclear-receptor co-activator protein NCoA-1 is also specifically required for ligand-dependent activation of genes by nuclear receptors. p/CIP, NCoA-1 and CBP all contain related leucine-rich charged helical interaction motifs that are required for receptor-specific mechanisms of gene activation, and that allow the selective inhibition of distinct signal-transduction pathways (Torchia, 1997).

Estrogen- and antiestrogen-regulated, AF-2-dependent transcriptional activation by purified full-length human estrogen receptor (ER) was carried out with chromatin templates in vitro. With this system, the ability of purified human p300 to function as a transcriptional coactivator was examined. In the absence of ligand-activated ER, p300 is found to have little effect (less than twofold increase) on transcription, whereas, in contrast, p300 is observed to act synergistically with ligand-activated ER to enhance transcription. When transcription is limited to a single round, p300 and ER are found to enhance the efficiency of transcription initiation in a cooperative manner. When transcription reinitiation is allowed to occur, ER, but not p300, is able to increase the number of rounds of transcription. These results suggest a two-stroke mechanism for transcriptional activation by ligand-activated ER and p300. In the first stroke, ER and p300 function cooperatively to increase the efficiency of productive transcription initiation. In the second stroke, ER promotes the reassembly of the transcription preinitiation complex. Therefore, ER exhibits distinct, dual functions in transcription initiation and reinitiation (Kraus, 1998).

The nuclear receptor (NR) coactivator TIF2 possesses a single NR interaction domain (NID) and two autonomous activation domains, AD1 and AD2. The TIF2 NID is composed of three NR-interacting modules each containing the NR box motif LxxLL. Mutation of boxes I, II and III abrogates TIF2-NR interaction and stimulation, in transfected cells, of the ligand-induced activation function-2 (AF-2) present in the ligand-binding domains (LBDs) of several NRs. The presence of an intact NR interaction module II in the NID is sufficient for both efficient interaction with NR holo-LBDs and stimulation of AF-2 activity. Modules I and III are poorly efficient on their own, but synergistically can promote interaction with NR holo-LBDs and AF-2 stimulation. TIF2 AD1 activity appears to be mediated through CBP, since AD1 could not be separated mutationally from the CBP interaction domain. In contrast, TIF2 AD2 activity apparently does not involve interaction with CBP. TIF2 exhibits the characteristics expected for a bona fide NR coactivator, in both mammalian and yeast cells. Moreover, in mammalian cells, a peptide encompassing the TIF2 NID inhibits the ligand-induced AF-2 activity of several NRs, indicating that NR AF-2 activity is either mediated by endogenous TIF2 or by coactivators recognizing a similar surface on NR holo-LBDs (Voegel, 1998).

In CV-1 cells CREB-binding protein (CBP) enhances the androgen receptor (AR)-dependent transcription under transient transfection conditions. The ligand binding domain (LBD) and residues 38-296 of the N-terminal region of AR are not required because the activity of a receptor mutant devoid of these domains is augmented by coexpressed CBP. There is physical interaction between AR and CBP in vivo, as judged by coimmunoprecipitation experiments from cell extracts. Consistent with the role of CBP as a coactivator for AR, the 12S E1A adenoviral protein that inactivates CBP function strongly inhibits AR-dependent transactivation. Exogenous CBP is also capable of overcoming the inhibitory effect of AR on AP-1 activity and diminishes the mutual transcriptional repression between AR and NF-kappaB (RelA). Collectively, these data imply that transcriptional interference between AR and AP-1 or NF-kappaB is mediated, at least in part, through competition for intracellular CBP; this coactivator serves as an integrator between androgen-mediated and other signaling pathways (Aarnisalo,1998).

The role of the transcriptional coactivator p300 in gene activation by thyroid hormone receptor (TR) upon the addition of ligand has been investigated. The ligand-bound TR targets chromatin disruption, independent of gene activation. Exogenous p300 facilitates transcription from a disrupted chromatin template, but does not itself disrupt chromatin in the presence or absence of ligand-bound receptor. Nevertheless, the acetyltransferase activity of p300 is required to facilitate transcription from a disrupted chromatin template. Expression of E1A prevents aspects of chromatin remodeling and transcriptional activation dependent on TR and p300. E1A selectively inhibits the acetylation of non-histone substrates. E1A does not prevent the assembly of a DNase I-hypersensitive site induced by TR, but does inhibit topological alterations and the loss of canonical nucleosome arrays dependent on the addition of ligand. Mutants of E1A incompetent for interaction with p300 partially inhibit chromatin disruption but still allow nuclear receptors to activate transcription. It is concluded that p300 has no essential role in chromatin disruption, but makes use of acetyltransferase activity to stimulate transcription at a subsequent step (Li, 1999).

Nuclear hormone receptors are ligand-activated transcription factors that regulate the expression of genes that are essential for development, reproduction and homeostasis. The hormone response is mediated through recruitment of p160 receptor coactivators and the general transcriptional coactivator CBP/p300, which function synergistically to activate transcription. These coactivators exhibit intrinsic histone acetyltransferase activity, function in the remodelling of chromatin, and facilitate the recruitment of RNA polymerase II and the basal transcription machinery. The activities of the p160 coactivators are dependent on CBP. Both coactivators are essential for proper cell-cycle control, differentiation and apoptosis, and are implicated in cancer and other diseases. To elucidate the molecular basis of assembling the multiprotein activation complex, a structural and thermodynamic analysis was undertaken of the interaction domains of CBP and the activator for thyroid hormone and retinoid receptors (ACTR). Although the isolated domains are intrinsically disordered, they combine with high affinity to form a cooperatively folded helical heterodimer. This study uncovers a unique mechanism, called 'synergistic folding', through which p160 coactivators recruit CBP/p300 to allow transmission of the hormonal signal to the transcriptional machinery (Demarest, 2002).

Extracellular signals and cell-intrinsic transcription factors cooperatively instruct generation of diverse neurons. However, little is known about how neural progenitors integrate both cues and orchestrate chromatin changes for neuronal specification. This paper reports that extrinsic signal retinoic acid (RA) and intrinsic transcription factor Neurogenin2 (Ngn2) collaboratively trigger transcriptionally active chromatin in spinal motor neuron genes during development. Retinoic acid receptor (RAR) binds Ngn2 and is thereby recruited to motor neuron genes targeted by Ngn2. RA then facilitates the recruitment of a histone acetyltransferase CBP to the Ngn2/RAR-complex, markedly inducing histone H3/H4-acetylation. Correspondingly, timely inactivation of CBP and its paralog p300 results in profound defects in motor neuron specification and motor axonal projection, accompanied by significantly reduced histone H3-acetylation of the motor neuron enhancer. This study uncovers the mechanism by which extrinsic RA-signal and intrinsic transcription factor Ngn2 cooperate for cell fate specification through their synergistic activity to trigger transcriptionally active chromatin (Lee, 2009).

Multiple signaling pathways ultimately modulate the epigenetic information embedded in the chromatin of gene promoters by recruiting epigenetic enzymes. In estrogen-regulated gene programming the acetyltransferase CREB-binding protein (CBP) is specifically and exclusively methylated by the coactivator-associated arginine methyltransferase (CARM1) in vivo. CARM1-dependent CBP methylation and p160 coactivators are required for estrogen-induced recruitment to chromatin targets. Notably, methylation increases the histone acetyltransferase (HAT) activity of CBP and stimulates its autoacetylation. Comparative genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) studies revealed a variety of patterns by which p160, CBP, and methyl-CBP (meCBP) are recruited (or not) by estrogen to chromatin targets. Moreover, significant target gene-specific variation in the recruitment of (1) the p160 RAC3 protein, (2) the fraction of a given meCBP species within the total CBP, and (3) the relative recruitment of different meCBP species suggests the existence of a target gene-specific 'fingerprint' for coregulator recruitment. Crossing ChIP-seq and transcriptomics profiles revealed the existence of meCBP 'hubs' within the network of estrogen-regulated genes. Together, these data provide evidence for an unprecedented mechanism by which CARM1-dependent CBP methylation results in gene-selective association of estrogen-recruited meCBP species with different HAT activities and specifies distinct target gene hubs, thus diversifying estrogen receptor programming (Ceschin, 2011).

CBP as a coactivator of miscellaneous zinc finger transcription factors

Modification of histones (DNA-binding proteins found in chromatin) by addition of acetyl groups occurs to a greater degree when the histones are associated with transcriptionally active DNA. A breakthrough in understanding how this acetylation is mediated was the discovery that various transcriptional co-activator proteins have intrinsic histone acetyltransferase activity (for example, Gcn5p, PCAF, TAF(II)250 and p300/CBP. These acetyltransferases also modify certain transcription factors (TFIIEbeta, TFIIF, EKLF and p53). GATA-1 is an important transcription factor in the hematopoietic lineage and is essential for terminal differentiation of erythrocytes and megakaryocytes. It is associated in vivo with the acetyltransferase p300/CBP. GATA-1 is acetylated in vitro by p300. This significantly increases the amount of GATA-1 bound to DNA and alters the mobility of GATA-1-DNA complexes. This is suggestive of a conformational change in GATA-1. GATA-1 is also acetylated in vivo and acetylation directly stimulates GATA-1-dependent transcription. Mutagenesis of important acetylated residues shows that there is a relationship between the acetylation and in vivo function of GATA-1. It is proposed that acetylation of transcription factors can alter interactions between these factors and DNA and among different transcription factors, and is an integral part of the transcription and differentiation processes (Boyes, 1997).

In nonhematopoietic cells, CREB-binding protein (CBP) markedly stimulates GATA-1's transcriptional activity in transient transfection experiments. GATA-1 and CBP also coimmunoprecipitate from nuclear extracts of erythroid cells. Interaction mapping pinpoints contact sites to the zinc finger region of GATA-1 and to the E1A-binding region of CBP. Expression of a conditional form of adenovirus E1A in murine erythroleukemia cells blocks differentiation and expression of endogenous GATA-1 target genes, whereas mutant forms of E1A, unable to bind CBP/p300, have no effect. These findings add GATA-1, and very likely other members of the GATA family, to the growing list of molecules implicated in the complex regulatory network surrounding CBP/p300 (Blobel, 1998).

The transcription factor GATA-1 is a key regulator of erythroid-cell differentiation and survival. The transcriptional cofactor CREB-binding protein (CBP) binds to the zinc finger domain of GATA-1, markedly stimulates the transcriptional activity of GATA-1, and is required for erythroid differentiation. CBP, but not p/CAF, acetylates GATA-1 at two highly conserved lysine-rich motifs present at the C-terminal tails of both zinc fingers. GATA-1 is acetylated in vivo at the same sites acetylated by CBP in vitro. In addition, CBP stimulates GATA-1 acetylation in vivo in an E1A-sensitive manner, thus establishing a correlation between acetylation and transcriptional activity of GATA-1. Acetylation in vitro does not alter the ability of GATA-1 to bind DNA, and mutations in either motif do not affect DNA binding of GATA-1 expressed in mammalian cells. Since certain functions of GATA-1 are revealed only in an erythroid environment, GATA-1 constructs were examined for their ability to trigger terminal differentiation when introduced into a GATA-1-deficient erythroid cell line. Mutations in either acetylation motif partially impairs the ability of GATA-1 to induce differentiation while mutations in both motifs abrogate it completely. Taken together, these data indicate that CBP is an important cofactor for GATA-1 and suggest a novel mechanism in which acetylation by CBP regulates GATA-1 activity in erythroid cells (Hung, 1999).

Erythroid Kruppel-like factor (EKLF) is a red cell-specific transcriptional activator that is crucial for consolidating the switch to high levels of adult beta-globin expression during erythroid ontogeny. EKLF is required for integrity of the chromatin structure at the beta-like globin locus, and it interacts with a positive-acting factor in vivo. EKLF is an acetylated transcription factor, and it interacts in vivo with CBP, p300, and P/CAF. However, its interactions with these histone acetyltransferases are not equivalent, since CBP and p300, but not P/CAF, utilize EKLF as a substrate for in vitro acetylation within EKLF's trans-activation regions. The functional effects of these interactions are that CBP and p300, but not P/CAF, enhance EKLF's transcriptional activation of the beta-globin promoter in erythroid cells. These results establish EKLF as a tissue-specific transcription factor that undergoes post-translational acetylation and suggest a mechanism by which EKLF is able to alter chromatin structure and induce beta-globin expression within the beta-like globin cluster (Zhang, 1998).

The erythroid cell-specific transcription factor erythroid Krüppel-like factor (EKLF) is an important activator of ß-globin gene expression. EKLF achieves this by binding to the CACCC element at the ß-globin promoter via its zinc finger domain. The coactivators CBP and P300 interact with acetylate, and enhance its activity, helping to explain ELKF's role as a transcription activator. EKLF can also interact with the corepressors mSin3A (see Drosophila Sin3A) and HDAC1 (histone deacetylase 1) through its zinc finger domain. When linked to a GAL4 DNA binding domain, full-length EKLF or its zinc finger domain alone can repress transcription in vivo. This repressive activity can be relieved by the HDAC inhibitor trichostatin A. Although recruitment of EKLF to a promoter is required to show repression, its zinc finger domain cannot bind directly to DNA and repress transcription simultaneously. In addition, the target promoter configuration is important for enabling EKLF to exhibit any repressive activity. These results suggest that EKLF may function in vivo as a transcription repressor and play a previously unsuspected additional role in regulating erythroid gene expression and differentiation (Chen, 2001).

CBP interactions with E1A

CREB binding protein (CBP) functions as an essential coactivator of transcription factors that are inhibited by the adenovirus early gene product E1A. Transcriptional activation by the signal transducer and activator of transcription-1 (STAT1) protein requires the C/H3 domain in CBP, which is the primary target of E1A inhibition. The C/H3 domain is not required for retinoic acid receptor (RAR) function, nor is it involved in E1A inhibition. Instead, E1A inhibits RAR function by preventing the assembly of CBP-nuclear receptor coactivator complexes, revealing differences in required CBP domains for transcriptional activation by RAR and STAT1 (Kurokawa, 1998).

Transforming viral proteins such as E1A force cells through the restriction point of the cell cycle into S phase by forming complexes with two cellular proteins: the retinoblastoma protein (Rb), a transcriptional co-repressor, and CBP/p300, a transcriptional co-activator. These two proteins locally influence chromatin structure: Rb recruits a histone deacetylase, whereas CBP is a histone acetyltransferase. Progression through the restriction point is triggered by phosphorylation of Rb, leading to disruption of Rb-associated repressive complexes and allowing the activation of S-phase genes. CBP, like Rb, is controlled by phosphorylation at the G1/S boundary, increasing its histone acetyltransferase activity. This enzymatic activation is mimicked by E1A (Ait-Si-Ali, 1998)

CBP, P/CAF (p300/CBP associated factor) and p/CIP (p300/CBP interacting protein)

The role of the nuclear protein P/CAF in regulating the transcription of the gene for human heavy (H) ferritin in given cell types was analyzed. P/CAF is a histone acetylase, recruited to specific promoters via interaction with the co-activator molecule p300/CREB-binding protein (CBP). Histone acetylation promoted by P/CAF destabilizes the nucleosome structure, thus contributing to activation of transcription. The transcription of the H ferritin gene is regulated by the transcription factor B-box-binding factor (Bbf), which bridges RNA polymerase II via p300/CBP. H ferritin gene is expressed at high levels in cells containing high levels of the P/CAF transcript. Transient overexpression of P/CAF in cells constitutively expressing low levels of this protein activates transcription driven by the region of the H promoter interacting with Bbf. The involvement of p300/CBP in the possible P/CAF-mediated regulation of H promoter was also explored by evaluating the phenomenon in the presence of the oncoprotein E1A. The results of these experiments demonstrate that P/CAF activates the H promoter also in the presence of limited amounts of p300/CBP. It is argued that P/CAF is a component of the basal transcription apparatus of the H ferritin gene and that the relative amounts of the P/CAF protein in different cell types could account for the cell-specific control of the H ferritin gene transcription (Bevilacqua, 1998).

Cyclin D1 is overexpressed in a significant percentage of human breast cancers, particularly in those that also express the estrogen receptor (ER). Experimentally overexpressed cyclin D1 can associate with the ER and stimulate its transcriptional functions in the absence of estrogen. This effect is separable from the established function of cyclin D1 as a regulator of cyclin-dependent kinases. Cyclin D1 can also interact with the histone acetyltransferase, p300/CREB-binding protein-associated protein (P/CAF), thereby facilitating an association between P/CAF and the ER. Ectopic expression of P/CAF potentiates cyclin D1-stimulated ER activity in a dose-dependent manner. This effect is largely dependent on the acetyltransferase activity of P/CAF. These results suggest that cyclin D1 may trigger the activation of the ER through the recruitment of P/CAF, by providing histone acetyltransferase activity and, potentially, links to additional P/CAF-associated transcriptional coactivators (McMahon, 1999).

CREB-binding proteins (CBP) and p300 are essential transcriptional coactivators for a large number of regulated DNA-binding transcription factors, including CREB, nuclear receptors, and STATs. CBP and p300 function in part by mediating the assembly of multiprotein complexes that contain additional cofactors such as p300/CBP interacting protein (p/CIP), a member of the p160/SRC family of coactivators, and the p300/CBP associated factor p/CAF. In addition to serving as molecular scaffolds, CBP and p300 each possess intrinsic acetyltransferase activities that are required for their function as coactivators. The adenovirus E1A protein inhibits the acetyltransferase activity of CBP on binding to the C/H3 domain, whereas binding of CREB, or a CREB/E1A fusion protein to the KIX domain, fails to inhibit CBP acetyltransferase activity. Surprisingly, p/CIP can either inhibit or stimulate CBP acetyltransferase activity depending on the specific substrate evaluated and the functional domains present in the p/CIP protein. While the CBP interaction domain of p/CIP inhibits acetylation of histones H3, H4, or high mobility group by CBP, it enhances acetylation of other substrates, such as Pit-1. These observations suggest that the acetyltransferase activities of CBP/p300 and p/CAF can be differentially modulated by factors binding to distinct regions of CBP/p300. Because these interactions are likely to result in differential effects on the coactivator functions of CBP/p300 for different classes of transcription factors, regulation of CBP/p300 acetyltransferase activity may represent a mechanism for integration of diverse signaling pathways (Perissi, 1999).

The basic helix-loop-helix transcription factor TAL1 (or SCL) is a critical regulator of hematopoietic and vascular development and is misexpressed in the majority of patients with T-cell acute lymphoblastic leukemia. TAL1 (Potential Drosophila homolog: Helix loop helix protein 3B) can interact with transcriptional co-activator and co-repressor complexes possessing histone acetyltransferase and deacetylase activities, respectively. TAL1 is subject to acetylation in vivo and can be acetylated by p300 and the p300/CBP-associated factor P/CAF in vitro. P/CAF-mediated acetylation, which maps to a lysine-rich motif in the loop region, increases TAL1 binding to DNA while selectively inhibiting its interaction with the transcriptional co-repressor mSin3A. Furthermore, P/CAF protein, TAL1-P/CAF interaction and TAL1 acetylation increase significantly in murine erythroleukemia cells induced to differentiate in culture, while enforced expression of an acetylation-defective P/CAF mutant inhibits endogenous TAL1 acetylation, TAL1 DNA-binding activity, TAL1-directed transcription and terminal differentiation of these cells. These results reveal a novel mechanism by which TAL1 activity is regulated and implicate acetylation of this transcription factor in promotion of erythroid differentiation (Huang, 2000).

An important finding of this work is that TAL1's interaction with the co-repressor mSin3A is significantly, and selectively, destabilized by P/CAF-mediated acetylation. Both co-activators and co-repressors have been shown to interact physically and functionally with TAL1. In fact, TAL1 interacts reciprocally with mSin3A and with the co-activators p300 and P/CAF in the same cells, according to their stage of differentiation. While a decline in mSin3A concentration is likely to be responsible for this co-regulator switch in MEL cells, it was not evident why TAL1 and mSin3A fail to interact in differentiating primary erythroid cells that continue to express both proteins. These studies provide a potential explanation and define a novel mechanism for regulating transcription factor activity, involving acetylation-induced destabilization of transcription factor-co-repressor interaction. This contrasts with the reduced affinity of the transcriptional co-activator ACTR for the estrogen receptor as a result of its hormone-induced acetylation, and the enhanced affinity of HNF-4 for CBP as the result of its acetylation by CBP. That TSA treatment alone destabilizes the TAL1-mSin3A interaction, without the requirement for P/CAF overexpression, suggests that, as for histones, a dynamic equilibrium exists in cells between acetylation and deacetylation of non-histone substrates (Huang, 2000).

CBP interaction with bHLH transcription factors

NeuroD1/BETA2 is a key regulator of pancreatic islet morphogenesis and insulin hormone gene transcription in islet beta cells. This factor also appears to be involved in neurogenic differentiation, because NeuroD1/BETA2 is able to induce premature differentiation of neuronal precursors and convert ectoderm into fully differentiated neurons upon ectopic expression in Xenopus embryos. Amino acid sequences in mammalian and Xenopus NeuroD1/BETA2 have been identified that are necessary for insulin gene expression and ectopic neurogenesis. Evolutionarily conserved sequences spanning the basic helix-loop-helix (amino acids [aa] 100 to 155) and C-terminal (aa 156 to 355) regions are important for both of these processes. The transactivation domains (AD1, aa 189 to 299; AD2, aa 300 to 355) are within the carboxy-terminal region, as analyzed by using GAL4:NeuroD1/BETA2 chimeras. Selective activation of mammalian insulin gene enhancer-driven expression and ectopic neurogenesis in Xenopus embryos is regulated by two independent and separable domains of NeuroD1/BETA2, located between aa 156 to 251 and aa 252 to 355. GAL4:NeuroD1/BETA2 constructs spanning these sequences demonstrate that only aa 252 to 355 contain activation domain function, although both aa 156 to 251 and 300 to 355 are found to interact with the p300/CREB binding protein (CBP) coactivator. These results implicate p300/CBP in NeuroD1/BETA2 function and further suggest that comparable mechanisms are utilized to direct target gene transcription during differentiation and in adult islet beta cells (Sharma, 1999).

PCAF is a histone acetyltransferase that associates with p300/CBP and competes with E1A for access to them. While exogenous expression of PCAF potentiates both MyoD-directed transcription and myogenic differentiation, PCAF inactivation by anti-PCAF antibody microinjection prevents differentiation. MyoD interacts directly with both p300/CBP and PCAF, forming a multimeric protein complex on the promoter elements. Viral transforming factors that interfere with muscle differentiation disrupt this complex without affecting the MyoD-DNA interaction, indicating functional significance of the complex formation. Exogenous expression of PCAF or p300 promotes p21 expression and terminal cell-cycle arrest. Both of these activities are dependent on the histone acetyltransferase activity of PCAF, but not on that of p300. These results indicate that recruitment of histone acetyltransferase activity of PCAF by MyoD, through p300/CBP, is crucial for activation of the myogenic program (Puri, 1997).

In response to decreased cellular oxygen concentrations, the basic helix-loop-helix (bHLH)/PAS (Per, Arnt, Sim) hypoxia-inducible transcription factor, HIF-1alpha, mediates the activation of networks of target genes involved in angiogenesis, erythropoiesis and glycolysis. The mechanism for activation of HIF-1alpha has been demonstrated to be a multi-step process that includes hypoxia-dependent nuclear import and activation (derepression) of the transactivation domain, resulting in recruitment of the CREB-binding protein (CBP)/p300 coactivator. Inducible nuclear accumulation is dependent on a nuclear localization signal (NLS) within the C-terminal end of HIF-1alpha which also harbors the hypoxia-inducible transactivation domain. Nuclear import of HIF-1alpha is inhibited by either deletion or a single amino acid substitution within the NLS sequence motif and, within the context of the full-length protein, these mutations also resulted in inhibition of the transactivation activity of HIF-1alpha and recruitment of CBP. However, nuclear localization per se is not sufficient for transcriptional activation, since fusion of HIF-1alpha to the heterologous GAL4 DNA-binding domain generates a protein that shows constitutive nuclear localization but requires hypoxic stimuli to function as a CBP-dependent transcription factor. Thus, hypoxia-inducible nuclear import and transactivation by recruitment of CBP can be functionally separated from one another and both can play critical roles in signal transduction by HIF-1alpha (Kallio, 1998).

Recruitment of p300/CBP by the hypoxia-inducible factor, HIF-1, is essential for the transcriptional response to hypoxia and requires an interaction between the p300/CBP CH1 region and HIF-1alpha. A new p300-CH1 interacting protein, p35srj, has been identified and cloned. p35srj is an alternatively spliced isoform of MRG1, a human protein of unknown function. Virtually all endogenous p35srj is bound to p300/CBP in vivo, and it inhibits HIF-1 transactivation by blocking the HIF-1alpha/p300 CH1 interaction. p35srj did not affect transactivation by transcription factors that bind p300/CBP outside the CH1 region. Endogenous p35srj is up-regulated markedly by the HIF-1 activators hypoxia or deferoxamine, suggesting that it could operate in a negative-feedback loop. In keeping with this notion, a p300 CH1 mutant domain, defective in HIF-1 but not p35srj binding, enhances endogenous HIF-1 function. In hypoxic cells, p35srj may regulate HIF-1 transactivation by controlling access of HIF-1alpha to p300/CBP, and may keep a significant portion of p300/CBP available for interaction with other transcription factors by partially sequestering and functionally compartmentalizing cellular p300/CBP (Bhattacharya, 1999).

Histone acetyltransferases (HATs) play a critical role in transcriptional control by relieving the repressive effects of chromatin, and yet how HATs themselves are regulated remains largely unknown. Here, it is shown that Twist directly binds two independent HAT domains of acetyltransferases, p300 and p300/CBP-associated factor (PCAF), and directly regulates their HAT activities. Twist strongly binds the C-terminal fragment (amino acids 1257-2414) of p300 spanning the HAT domain as well as the CH3 domain. Further deletion reveals that this interaction requires the CH3 domain (compare 1572-2414 and 1869-2414), which is known to interact with other proteins. Of particular interest, Twist retains an interaction with a HAT domain even in the absence of the CH3 domain (1257-1572). Twist also binds the N terminus of p300 (1-566 and 1-744), although these interactions are 5- to 10-fold weaker than those with the CH3 and HAT domains. Twist shows strong binding to PCAF. Intriguingly, experiments using a series of PCAF internal deletion mutants reveal that this interaction required the presence of the intact HAT domain and bromodomain. Thus, Twist interacts independently with the HAT domains of two different proteins, p300 and PCAF, suggesting that Twist may recognize common motifs present in these HAT domains. The N terminus of Twist is a primary domain interacting with both acetyltransferases, and the same domain is required for the inhibition of p300-dependent transcription by Twist. Taken together, these findings support the view that Twist suppresses the coactivator functions of p300 and PCAF through physical interactions mediated by the N terminus of Twist. Adenovirus E1A protein mimics the effects of Twist by inhibiting the HAT activities of p300 and PCAF. These findings establish a cogent argument for considering the HAT domains as a direct target for acetyltransferase regulation by both a cellular transcription factor and a viral oncoprotein (Hamamori, 1999).

E1A has been shown to bind the CH3 domain of p300/CBP and to displace PCAF from this domain. The effect of E1A has been interpreted as a simple competition between E1A and PCAF for the CH3 domain. The present study adds a further level of complexity by demonstrating that E1A and Twist may exert their inhibition not only by physically disrupting the p300-PCAF complex formation but also through suppression of their enzymatic activities. The interaction of Twist at the CH3 domain raises the intriguing possibility that Twist might also prevent PCAF association with p300/CBP by competing with PCAF for the common CH3 domain. These two mechanisms may not necessarily work simultaneously, and cells would have exquisite control mechanisms that determine how these two mechanisms of p300 and PCAF regulation may be differentially utilized in a given situation. Individual histone acetyltransferases have distinct roles. For instance, myogenic transcription and differentiation are dependent on the HAT activity of PCAF but not on that of p300/CBP. Similar observations are made in other systems, indicating that the transcriptional activities of the HAT domains of p300 and PCAF are highly promoter dependent. The dual inhibitory mechanisms involving the HAT inhibition as well as the competitive displacement of cofactors would allow E1A and possibly Twist to regulate a broad range of transcriptional activators that are differentially dependent on p300 and PCAF and their HAT activities (Hamamori, 1999 and references).

The mechanisms by which neural stem cells give rise to neurons, astrocytes, or oligodendrocytes are beginning to be elucidated. However, it is not known how the specification of one cell lineage results in the suppression of alternative fates. In addition to inducing neurogenesis, the bHLH transcription factor neurogenin (Ngn1) inhibits the differentiation of neural stem cells into astrocytes. While Ngn1 promotes neurogenesis by functioning as a transcriptional activator, Ngn1 inhibits astrocyte differentiation by sequestering the CBP-Smad1 transcription complex away from astrocyte differentiation genes, and by inhibiting the activation of STAT transcription factors that are necessary for gliogenesis. Thus, two distinct mechanisms are involved in the activation and suppression of gene expression during cell-fate specification by neurogenin (Sun, 2001).

Neuronal differentiation is promoted by both platelet-derived growth factor (PDGF) and by neurotrophin-3 (NT3). The cytokines leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) are potent inducers of astrocyte production, and thyroid hormone induces oligodendrocyte differentiation. LIF and CNTF exert their effects primarily via the JaK/STAT signaling pathway. LIF and CNTF bind to related receptors, which activate a receptor-associated tyrosine kinase, the Janus kinase (JaK1). Activated JaK1 phosphorylates two cytoplasmic proteins, the signal transducers and activators of transcription 1 and 3 (STAT1 and STAT3). This leads to STAT dimerization and translocation to the nucleus where the STATs activate cell type and stimulus-specific programs of gene expression (Sun, 2001 and references therein).

Other factors, such as bone morphogenetic protein (BMP), can enhance both neuronal and astrocyte differentiation, depending on the age of the stimulated cortical progenitors. BMP-induced astrocyte differentiation appears to be mediated by the downstream Smad signaling proteins. BMPs bind a multimeric receptor, which in turn results in the direct phosphorylation of Smad1. This permits Smad1 to dimerize with Smad4 and to translocate to the nucleus, where these factors cooperate with STATs to activate glial-specific programs of gene expression (Sun, 2001 and references therein).

The cooperation between Smads and STATs on glial promoters such as the glial fibrillary acidic protein (GFAP) promoter appears to be facilitated by a family of coactivator proteins termed p300/CBP. CBP (CREB binding protein) and p300 are ubiquitously expressed and are involved in the transcriptional coactivation of many different transcription factors. STATs and Smads bind to different domains of CBP/p300, and the STAT/p300/Smad complex, acting at the STAT binding element in the astrocyte-specific GFAP promoter, is particularly effective at inducing astrocyte differentiation in neural stem cells (Sun, 2001 and references therein).

Many transcription factors require CBP/p300 in order to activate transcription, and there is evidence that the levels of CBP/p300 are limiting, i.e., that there is competition among the various families of transcription factors for CBP/p300 binding. For example, nuclear steroid receptors indirectly inhibit AP-1-dependent transcription by sequestering CBP/p300 away from AP-1 and onto sites where the nuclear receptors are bound. Similarly, the anti-adenoviral actions of interferon are attributed to interferon's ability to activate STATs, which then sequester CBP/p300 away from the adenoviral transcription factor E1A. During early cortical development, endogenous Ngn1 associates with both CBP and Smad1, and the presence of neurogenin blocks STAT binding to CBP. Xenopus neurogenin has been shown to recruits CBP/p300 to the NeuroD promoter to activate transcription and induce neurogenesis. The characterization of the domains of CBP that interact with neurogenin reveal that both an N- and a C-terminal domain are involved. Interestingly, the neurogenin binding domains of CBP overlap with the STAT binding sites on CBP (but not with the Smad binding sites). This is consistent with the finding that neurogenin competes with STAT proteins for binding to CBP. By sequestering CBP, neurogenin may not only inhibit STAT-mediated transcription, but may also inhibit the function of other CBP-dependent transcription factors. Ngn1 also inhibits AP-1-dependent transcription. This may be relevant to Ngn's ability to inhibit astrocyte differentiation since the analysis of the GFAP promoter identifies multiple sites, including an AP-1 site, that contribute to neurogenin's inhibition of the GFAP promoter. Taken together, these findings suggest that CBP/p300 may orchestrate broad programs of gene expression that are relevant to cell fate determination. The effect of CBP/p300 on cell fate may then be determined by the relative binding affinity and abundance of different transcription factors that either compete or cooperate with one another for binding to CBP/p300 (Sun, 2001 and references therein).

In addition to sequestering the CBP-Smad1 complex, neurogenin also inhibits the activation of astrocyte-specific genes by blocking STAT activation. The mechanism by which Ngn1 reduces the level of phospho-STAT1 and -STAT3 is unknown. The Ngn1 deficient in binding DNS can also inhibit STAT phosphorylation, though not to the extent seen with wild-type Ngn1. This suggests that Ngn1 inhibits STAT phosphorylation only in part by a mechanism that is independent of Ngn1 binding to DNA (Sun, 2001).

An intricate array of heterogeneous transcription factors participate in programming tissue-specific gene expression through combinatorial interactions that are unique to a given cell-type. The zinc finger-containing transcription factor GATA4, which is widely expressed in mesodermal and endodermal derived tissues, is thought to regulate cardiac myocyte-specific gene expression through combinatorial interactions with other semi-restricted transcription factors such as myocyte enhancer factor 2, nuclear factor of activated T-cells, serum response factor, and Nkx2.5. GATA4 also interacts with the cardiac-expressed basic helix-loop-helix transcription factor dHAND (also known as HAND2). GATA4 and dHAND synergistically activate expression of cardiac-specific promoters from the atrial natriuretic factor gene, the b-type natriuretic peptide gene, and the alpha-myosin heavy chain gene. Using artificial reporter constructs this functional synergy was shown to be GATA site-dependent, but E-box site-independent. A mechanism for the transcriptional synergy is suggested by the observation that the bHLH domain of dHAND physically interacted with the C-terminal zinc finger domain of GATA4 forming a higher order complex. This transcriptional synergy observed between GATA4 and dHAND is associated with p300 recruitment, but not with alterations in DNA binding activity of either factor. Moreover, the bHLH domain of dHAND directly interacts with the CH3 domain of p300 suggesting the existence of a higher order complex between GATA4, dHAND, and p300. These results suggest the existence of an enhanceosome complex comprised of p300 and multiple semi-restricted transcription factors that together specify tissue-specific gene expression in the heart (Dai, 2002).

CBP interaction with NFkappaB

The nuclear factor kappaB (NF-kappaB) transcription factor is responsive to specific cytokines and stress and is often activated in association with cell damage and growth arrest in eukaryotes. NF-kappaB is a heterodimeric protein, typically composed of 50- and 65-kilodalton subunits of the Rel family, of which RelA(p65) stimulates transcription of diverse genes. Specific cyclin-dependent kinases (CDKs) were found to regulate transcriptional activation by NF-kappaB through interactions with the coactivator p300. The transcriptional activation domain of RelA(p65) interacts with an amino-terminal region of p300 distinct from a carboxyl-terminal region of p300 required for binding to the cyclin E-Cdk2 complex. The CDK inhibitor p21 or a dominant negative Cdk2, which inhibits p300-associated cyclin E-Cdk2 activity, stimulates kappaB-dependent gene expression, which is also enhanced by expression of p300 in the presence of p21. The interaction of NF-kappaB and CDKs through the p300 and CBP coactivators provides a mechanism for the coordination of transcriptional activation with cell cycle progression (Perkins, 1996).

The transcriptional activity of NF-kappa B is stimulated upon phosphorylation of its p65 subunit on serine 276 by protein kinase A (PKA). The transcriptional coactivator CPB/p300 associates with NF-kappa B p65 through two sites, an N-terminal domain that interacts with the C-terminal region of unphosphorylated p65, and a second domain that only interacts with p65 phosphorylated on serine 276. Accessibility to both sites is blocked in unphosphorylated p65 through an intramolecular masking of the N terminus by the C-terminal region of p65. Phosphorylation by PKA both weakens the interaction between the N- and C-terminal regions of p65 and creates an additional site for interaction with CBP/p300. Therefore, PKA regulates the transcriptional activity of NF-kappa B by modulating its interaction with CBP/p300 (Zhong, 1998).

Homodimers of the NF-kappaB p50 subunit are transcriptionally repressive in cells, whereas they can promote transcription in vitro, suggesting that their endogenous effects are mediated by association with other factors. Transcriptionally inactive nuclear NF-kappaB in resting cells consists of homodimers of either p65 or p50 complexed with the histone deacetylase HDAC-1. Only the p50-HDAC-1 complexes bind to DNA and suppress NF-kappaB-dependent gene expression in unstimulated cells. Appropriate stimulation causes nuclear localization of NF-kappaB complexes containing phosphorylated p65 that associates with CBP and displaces the p50-HDAC-1 complexes. Phosphorylation of p65 determines whether it associates with either CBP or HDAC-1, ensuring that only p65 entering the nucleus from cytoplasmic NF-kappaB:IkappaB complexes can activate transcription (Zhong, 2002).

The effects of HDAC-1 and CBP/p300 underscore the importance of acetylation in regulating NF-kappaB activity, although the identity of CBP/p300 targets remains to be fully determined. CBP/p300 can acetylate the four core histones, loosening chromatin and facilitating transcription. Histones associated with NF-kappaB-dependent genes are acetylated following stimulation. Alternative targets include p53, where acetylation is important for CBP-mediated p53-dependent transcription, although unlike p53 and despite many attempts, p65 acetylation by CBP/p300 has not been detected. Thus, it appears that the major effect of CBP/p300 on NF-kappaB-dependent transcription is via acetylation of histones or other proteins in the chromatin remodeling and transcriptional apparatus (Zhong, 2002).

In summary, it has been shown that p65 phosphorylation determines whether nuclear NF-kappaB associates with HDAC-1 (inactive) or CBP/p300 (active) and that p50-HDAC-1 represses NF-kappaB-dependent gene expression in resting cells. Such a regulatory mechanism ensures that only stimulus-induced NF-kappaB activates transcription, and NF-kappaB in the nucleus for any other reason is transcriptionally silent. This mechanism is unique among the inducible transcription factors, since it imposes an additional layer of control on NF-kappaB that most likely reflects the necessity of maintaining it as a true inducible transcription factor (Zhong, 2002).

The nuclear function of the heterodimeric NF-kappaB transcription factor is regulated in part through reversible acetylation of its RelA subunit. The p300 and CBP acetyltransferases play a major role in the in vivo acetylation of RelA, principally targeting lysines 218, 221 and 310 for modification. Analysis of the functional properties of hypoacetylated RelA mutants containing lysine-to-arginine substitutions at these sites and of wild-type RelA co-expressed in the presence of a dominantly interfering mutant of p300 reveals that acetylation at lysine 221 in RelA enhances DNA binding and impairs assembly with IkappaBa. Conversely, acetylation of lysine 310 is required for full transcriptional activity of RelA in the absence of effects on DNA binding and IkappaBa assembly. Together, these findings highlight how site-specific acetylation of RelA differentially regulates distinct biological activities of the NF-kappaB transcription factor complex (Chen, 2002).

In summary, these studies demonstrate that acetylation of RelA at distinct sites differentially regulates various biological functions of NF-kappaB. Acetylation of lysine 310 of RelA is required for full transactivation by the NF-kappaB complex, most likely by recruiting an unidentified cofactor. Acetylation of lysine 221 enhances RelA binding to the kappaB enhancer,while acetylation of lysine 221 alone or in combination with lysine 218 impairs the assembly of RelA with IkappaBa. Lysines 218 and 221 are highly conserved within all Rel family members, including Dorsal from Drosophila. The possibility that these evolutionarily conserved lysine residues are targets for reversible acetylation and contribute to the regulation of the biological functions of other Rel factors remains an intriguing possibility (Chen, 2002).

CBP and the Notch pathway

Signaling through the Notch pathway activates the proteolytic release of the Notch intracellular domain (ICD), a dedicated transcriptional coactivator of CSL enhancer-binding proteins. Chromatin-dependent transactivation by the recombinant Notch ICD-CBF1 enhancer complex in vitro requires an additional coactivator, Mastermind (MAM). MAM provides two activation domains necessary for Notch signaling in mammalian cells and in Xenopus embryos. The central MAM activation domain (TAD1) recruits CBP/p300 to promote nucleosome acetylation at Notch enhancers and activate transcription in vitro. MAM expression induces phosphorylation and relocalization of endogenous CBP/p300 proteins to nuclear foci in vivo. Moreover, coexpression with MAM and CBF1 strongly enhances phosphorylation and proteolytic turnover of the Notch ICD in vivo. Enhanced phosphorylation of the ICD and p300 requires a glutamine-rich region of MAM (TAD2) that is essential for Notch transcription in vivo. Thus MAM may function as a timer to couple transcription activation with disassembly of the Notch enhancer complex on chromatin (Fryer, 2002).

Unexpectedly, expression of MAM induces endogenous CBP/p300 proteins to accumulate in multiple nuclear foci in vivo. These structures do not form upon expression of a mutant MAM protein lacking the C-terminal TAD2 region (1-301MM). Thus, binding of MAM to CBP/p300, which is mediated through TAD1, is not sufficient to cause CBP/p300 to accumulate in these structures. Expression of other Notch components (ICD, CBF1) did not affect the subnuclear localization of CBP/p300, indicating that these foci are not a consequence of high levels of Notch signaling in the nucleus. One possibility is that MAM may regulate the expression or modification of CBP/p300 independently of Notch signaling. Indeed, the MAM-induced foci are accompanied by increased phosphorylation of CBP, and this phosphorylation requires the C-terminal TAD2 domain of MAM. Consequently, overexpression of MAM in the nucleus may promote widespread phosphorylation of CBP, which may cause the CBP/p300 proteins to concentrate in these structures. Changes in CBP/p300 phosphorylation have been shown to alter its activity and differentially affect its interactions with other transcription factors. It will therefore be important to assess whether MAM promotes CBP/p300 phosphorylation within the Notch enhancer complex, and whether phosphorylation of CBP/p300 is important for transcriptional activation by Notch (Fryer, 2002).

The timing of Notch signaling is tightly controlled in developmental processes such as somite formation, during which Notch target genes such as cHairy1 and mHES1 undergo periodic cycles of expression at the direction of a molecular oscillator, or vertebrate segmentation clock. This clock may be established through the intrinsic timing of Notch signaling as well as the half-life of Notch-induced transcriptional repressors. The Notch ICD is subject to proteolytic degradation in the nucleus through the action of the ubiquitin ligases such as Sel-10. Rapid turnover of the ICD may be required to allow genes to respond rapidly to subsequent cycles of Notch signaling. Coexpression with MAM and CBF1 promotes the phosphorylation and proteolytic turnover of the ICD in vivo, indicating that MAM couples transcription activation with degradation of the ICD. In this respect, MAM may act as a timer to control the length of time that the Notch complex remains associated with the enhancer. By extension, MAM might contribute to the periodic expression of Notch target genes during somitogenesis through its potential effects on the disassembly of the Notch enhancer complex (Fryer, 2002).

The data indicate that CBF1 acts in concert with MAM to control the proteolytic turnover of the ICD in vivo. Importantly, both MAM and CBF1 appear to be stable upon coexpression with the ICD, and thus it appears that the ICD can be destabilized independently of its interacting partners. The requirement for CBF1 may reflect its ability to enhance binding of MAM to the ICD, or alternatively CBF1 might be needed to target the Notch enhancer complex to DNA. Stability of a mutant ICD protein lacking the PEST domain is unaffected by coexpression with MAM and CBF1, and turnover is accompanied by increased phosphorylation of the ICD. Importantly, the MAM TAD2 domain is necessary for both enhanced phosphorylation and turnover of the ICD. Because p300 has been shown to be critical for the regulated turnover of the p53 transactivator by MDM2, it will be important to assess whether recruitment of p300 by MAM may similarly be required for proteolytic degradation of the ICD. Nevertheless, it is clear that recruitment of CBP/p300 through the MAM TAD1 region is not sufficient to couple activation with turnover of the Notch ICD under the conditions examined in this study (Fryer, 2002).

Thus the TAD2 region is required for MAM to promote the phosphorylation of its two associated factors, CBP/p300 and the Notch ICD. Because MAM does not possess intrinsic ICD protein kinase activity, it is attractive to consider that the Notch ICD and CBP/p300 may instead be targeted for phosphorylation by cyclin-dependent kinases that associate with the transcription complex and are recruited to the promoter by MAM. Phosphorylation events mediated by CDK7 and Srb10 (the CDK8 homolog in yeast) have been implicated in the proteolytic destruction of other enhancer factors. The CDK9 subunit of the positive transcription elongation factor, P-TEFb, also associates with RNAPII, whereas CDK8 interacts with RNAPII as a component of human and yeast mediator complexes that have been variously implicated in activation and repression of transcription. Another possibility is that the ICD is phosphorylated by a protein kinase that associates with MAM directly. It remains to be determined whether the MAM-induced phosphorylation is accompanied by increased ubiquitination of the ICD, and whether the degradation of the ICD observed is caused by ubiquitin-dependent proteolysis such as that described for the nuclear Sel-10 ubiquitin ligase. It will also be important to learn whether modification of the ICD regulates its transcriptional activity, as has been observed for other transcription factors, and whether these steps may ultimately be coupled to disassembly of the Notch enhancer complex and turnover of the Notch ICD (Fryer, 2002).

In summary, MAM is an essential component of the Notch enhancer complex in vitro as well as in vivo. The human MAM protein recruits p300/CBP to the Notch enhancer complex and controls the stability of the Notch ICD through the action of its unique C-terminal activation domain. Further studies will be needed to evaluate whether these properties are shared among the various MAM proteins in different species, and to learn how MAM-induced phosphorylation of the ICD and CBP/p300 proteins is coordinated with the regulation of Notch transcription (Fryer, 2002).

Cyclin D1 belongs to the core cell cycle machinery, and it is frequently overexpressed in human cancers. The full repertoire of cyclin D1 functions in normal development and oncogenesis is unclear at present. This study developed Flag- and haemagglutinin-tagged cyclin D1 knock-in mouse strains that allowed a high-throughput mass spectrometry approach to search for cyclin D1-binding proteins in different mouse organs. In addition to cell cycle partners, several proteins involved in transcription were uncovered. Genome-wide location analyses (chromatin immunoprecipitation coupled to DNA microarray; ChIP-chip) showed that during mouse development cyclin D1 occupies promoters of abundantly expressed genes. In particular, it was found that in developing mouse retinas - an organ that critically requires cyclin D1 function - cyclin D1 binds the upstream regulatory region of the Notch1 gene, where it serves to recruit CREB binding protein (CBP) histone acetyltransferase. Genetic ablation of cyclin D1 resulted in decreased CBP recruitment, decreased histone acetylation of the Notch1 promoter region, and led to decreased levels of the Notch1 transcript and protein in cyclin D1-null (Ccnd1-/-) retinas. Transduction of an activated allele of Notch1 into Ccnd1-/- retinas increased proliferation of retinal progenitor cells, indicating that upregulation of Notch1 signalling alleviates the phenotype of cyclin D1-deficiency. These studies show that in addition to its well-established cell cycle roles, cyclin D1 has an in vivo transcriptional function in mouse development. This approach, which termed 'genetic-proteomic', can be used to study the in vivo function of essentially any protein (Bienvenu, 2010).

c-Myb, through interaction with p300, controls the proliferation and differentiation of hematopoietic stem and progenitor cells

Precise control of hematopoietic stem cell (HSC) proliferation and differentiation is needed to maintain a lifetime supply of blood cells. Using genome-wide ENU mutagenesis and phenotypic screening, a mouse line has been identified that harbors a point mutation in the transactivation (TA) domain of the transcription factor c-Myb (M303V) that reduces c-Myb-dependent TA by disrupting its interaction with the transcriptional coactivator p300. The biological consequences of the c-MybM303V/M303V mutation include thrombocytosis, megakaryocytosis, anemia, lymphopenia, and the absence of eosinophils. Detailed analysis of hematopoiesis in c-MybM303V/M303V mice reveals distinct blocks in T cell, B cell, and red blood cell development, as well as a remarkable 10-fold increase in the number of HSCs. Cell cycle analyses show that twice as many HSCs from c-MybM303V/M303V animals are actively cycling. Thus c-Myb, through interaction with p300, controls the proliferation and differentiation of hematopoietic stem and progenitor cells (Sandberg, 2005).

The present study supports a critical role for c-Myb-p300 interaction throughout the hematopoietic hierarchy. Importantly, c-Myb is not required at every step and for every lineage; rather, there are distinct steps and lineages that are critically dependent on c-Myb function. These include negative regulation of HSC proliferation, megakaryocyte numbers, and essential roles in erythropoiesis, eosinophil, T, and B cell development. Although previous studies have provided clues that c-Myb may play a role in some of these processes, the severe phenotype of c-Myb-/- animals has precluded a detailed examination of c-Myb function in adult animals (Sandberg, 2004).

Several lines of evidence support a critical role for c-Myb in directing megakaryocyte and erythrocyte development. (1) c-Myb-/- mice die of anemia in utero at day 15 due to a failure to initiate fetal liver hematopoiesis. megakaryocyte/erythrocyte progenitors (MEPs) are likely formed in c-Myb-/- mice, since megakaryocytes are present in the fetal liver of c-Myb-/- animals. c-Myb-/- animals contain low numbers of definitive stem/progenitor cells that can give rise to early thymic precursors and initiate myeloid cell development. Thus, the failure to initiate fetal erythropoiesis is likely to result from an inability of later progenitor cells to differentiate into RBCs. (2) Neonatal mice containing ~20% the normal amount of c-Myb also have anemia and elevated numbers of megakaryocytes and platelets. (3) The c-MybM303V/M303V mice and other ENU-induced alleles of c-Myb contain elevated numbers of platelets and CD41+ megakaryocytes and reduced numbers of RBCs. Thus, multiple independent alleles of c-Myb give an identical phenotype: reduced numbers of RBCs and elevated numbers of megakaryocytes. Since these two cell types arise from a common precursor, the simplest explanation is that c-Myb is required for the MEP to initiate the RBC program and also participates in turning off the megakaryocyte program. In support of this view, only modest reductions in the number of MEPs are found in c-MybM303V/M303V animals compared to normal littermates; however, these cells are unable to make RBCs following culture. Thus, these bipotential cells are present, but their differentiation potential is limited. Collectively, these data suggest that c-Myb normally limits the ability of the HSC to differentiate toward the megakaryocyte lineage while promoting the RBC program (Sandberg, 2004).

The low numbers of B and T cells in the blood of c-Myb-/- mice suggest that c-Myb may be regulating both B and T cell development. c-MybM303V/M303V mice contained normal numbers of phenotypic Lin-Kit+Sca1+IL7R+ common lymphoid progenitors (CLPs), suggesting that these early precursor cells are present in normal numbers. Consistent with this observation, normal numbers of the earliest pre-pro B cells were found. However, the numbers of pro-B cells were reduced 3-fold, and the numbers of pre-B cells were down more than 10-fold in c-MybM303V/M303V mice. Importantly, the percentage of B cells at subsequent stages of development was not different, although the absolute numbers were reduced in c-MybM303V/M303V mice, and their functional capacity remains to be tested. Reduced numbers of pro-B and pre-B cells in c-MybM303V/M303V mice suggest that c-Myb may play a role in directing IgH chain rearrangement or in the expansion of those cells that have successfully rearranged IgH (Sandberg, 2004).

The ability of c-Myb to regulate T cell development is well established; however, identification of the precise points that c-Myb regulates has been controversial. Early studies demonstrated a requirement for c-Myb at the pre-DN1 stage of T cell development, while later studies with a dominant-negative form of c-Myb showed a role for c-Myb in the regulation of DN3 and DN4 cell expansion post-TCR β chain selection. A recent study with neonatal mice with partial reductions in c-Myb levels showed that in fetal thymus development c-Myb is required for DN1 to DN2 maturation and the transition from DN2 to DN3, with the former step requiring higher threshold levels of c-Myb. Very recent data generated with conditional null alleles in which Myb is deleted early or late in T cell development in the thymus support a roll for c-Myb in directing the DN3-to-DN4 transition, survival of preselection double-positive cells, and differentiation of CD4 SP cells. In agreement with these data, the c-MybM303V/M303V mice described in this study show 2- to 10-fold reductions in thymocyte number with 3-fold reductions in the number of both DN1 and DN4 cells in mature animals. Interestingly, competitive bone marrow reconstitution experiments show that cells harboring the c-MybM303V/M303V allele contain normal numbers of DN1 cells and more than 4-fold reductions in DN4 cells. Based on the current work and previous studies, it seems that c-Myb can regulate T cell development at many points; however, the mechanistic details remain an area of active research (Sandberg, 2004).

Reconstitution studies with mixed bone marrow chimeras show a 10- to 20-fold increase in the number of functional HSCs in bone marrow from c-MybM303V/M303V mice, and 5-fold elevations in the absolute number of HSCs in primary recipients; thus, these phenotypic HSCs are functional. Secondary and tertiary transplant experiments of these bone marrow chimeras show that these HSCs are capable of self-renewal and long-term multilineage reconstitution, thus confirming that these represent bona fide long term HSCs. These experiments also reveal that the defects in hematopoiesis in c-MybM303V/M303V mice are cell intrinsic; c-MybM303V/M303V bone marrow gives rise to normal numbers of myeloid and granulocytes, while the numbers of T and B cells are decreased. Importantly, WT cells cotransplanted into these same animals differentiate normally, showing that the defects in hematopoiesis result from direct effects of c-MybM303V/M303V on the development of HSC and progenitors and not from secondary effects of the lymphopenia, anemia, or megakaryocytosis. HSCs from c-MybM303V/M303V mice contain an increase in the number of actively cycling cells, providing a mechanistic framework for the elevated numbers of HSCs. Thus, c-Myb-p300 normally acts to repress the proliferation of HSCs or links proliferation with subsequent differentiation (Sandberg, 2004).

Previous data have shown that c-Myb is required to generate definitive HSCs, that c-Myb-/- animals die at E14.5 as a result of a lack of definitive erythropoiesis, and that few HSCs are found in early embryos. In contrast, partial loss of c-Myb results in increased numbers of fetal liver progenitor cells expressing CD34+ or Sca-1+, supporting a negative role for c-Myb in the control of early progenitors. Interestingly, many of the phenotypes described in the c-MybM303V/M303V mice are also found in mice homozygous for a triple point mutation in the KIX domain of the transcriptional coactivator p300 (p300KIX/KIX) that disrupts the association of p300 with c-Myb. It will interesting to determine if the p300KIX/KIX and other c-Myb mutants have similar alterations in the number of HSCs and progenitor cells (Sandberg, 2004).

The current study shows that the c-Myb-p300 interaction controls hematopoiesis at many distinct points, both promoting and repressing proliferation and differentiation and thus highlighting c-Myb as a key regulator of hematopoiesis. Determining how a single transcription factor is able to control the diverse processes of selfrenewal, proliferation, and differentiation at distinct points in hematopoiesis and defining the molecular interactions that control c-Myb activity remain important areas of future research (Sandberg, 2004).

Myc regulation by p300

The c-Myc oncoprotein (Myc) controls cell fate by regulating gene transcription in association with a DNA-binding partner, Max. While Max lacks a transcription regulatory domain, the N terminus of Myc contains a transcription activation domain (TAD) that recruits cofactor complexes containing the histone acetyltransferases (HATs) GCN5 (see Drosophila Pcaf) and Tip60. This study reports a novel functional interaction between Myc TAD and the p300 coactivator-acetyltransferase. p300 associates with Myc in mammalian cells and in vitro through direct interactions with Myc TAD residues 1 to 110 and acetylates Myc in a TAD-dependent manner in vivo at several lysine residues located between the TAD and DNA-binding domain. Moreover, the Myc:Max complex is differentially acetylated by p300 and GCN5 and is not acetylated by Tip60 in vitro, suggesting distinct functions for these acetyltransferases. Whereas p300 and CBP can stabilize Myc independent of acetylation, p300-mediated acetylation results in increased Myc turnover. In addition, p300 functions as a coactivator that is recruited by Myc to the promoter of the human telomerase reverse transcriptase gene; also, p300/CBP stimulates Myc TAD-dependent transcription in a HAT domain-dependent manner. These results suggest dual roles for p300/CBP in Myc regulation: as a Myc coactivator that stabilizes Myc and as an inducer of Myc instability via direct Myc acetylation (Faiola, 2005).

Other CBP interactions

Growth factors such as epidermal growth factor (EGF) and insulin regulate development and metabolism via genes containing both POU homeodomain (Pit-1) and phorbol ester (AP-1) response elements. Although CREB binding protein (CBP) functions as a coactivator on these elements, the mechanism of transactivation has been unclear. CBP is recruited to these elements only after it is phosphorylated at serine 436 by growth factor-dependent signaling pathways. In contrast, p300, a protein closely related to CBP that lacks this phosphorylation site, binds only weakly to the transcription complex and in a growth factor-independent manner. A small region of CBP (amino acids 312-440), which has been termed the GF box, contains a potent transactivation domain and mediates this effect. Direct phosphorylation represents a novel mechanism controlling coactivator recruitment to the transcription complex (Zanger, 2001).

Previous in vitro studies have suggested that CBP and p300 interact constitutively with both c-Fos and c-Jun and mediate activation on an AP-1 element. These constitutive interactions are confirmed in this study, where both CBP and p300 activate the AP-1 element 2- to 3-fold in the absence of growth factor stimulation. CBP is found to bind to c-Jun through its CREB binding domain. Although the constitutive interactions between p300/CBP and c-Jun may be functionally significant on the AP-1 element, the growth factor-dependent recruitment of CBP to the AP-1 complex may prove to be quantitatively more important (Zanger, 2001).

The molecular mechanisms of transcriptional activation by beta-catenin are only poorly understood. The closely related acetyltransferases p300 and CBP potentiate beta-catenin-mediated activation of the siamois promoter, a known Wnt target. beta-catenin and p300 also synergize to stimulate a synthetic reporter gene construct, whereas activation of the cyclin D1 promoter by beta-catenin is refractory to p300 stimulation. Axis formation and activation of the beta-catenin target genes siamois and Xnr-3 in Xenopus embryos are sensitive to the E1A oncoprotein, a known inhibitor of p300/CBP. The C-terminus of beta-catenin interacts directly with a region overlapping the CH-3 domain of p300. p300 could participate in alleviating promoter repression imposed by chromatin structure and in recruiting the basal transcription machinery to promoters of particular Wnt target genes (Hecht, 2000).

The finding that p300/CBP serves as a coactivator for beta-catenin in vertebrates is unexpected given that in Drosophila dCBP has been shown to negatively regulate Wingless-signaling. The apparent discrepancy between the function of vertebrate p300/CBP and dCBP could be explained most easily if vertebrate CBP and p300, or dCBP and its mammalian orthologs, were functionally different from one another. Species-specific differences are known for certain aspects of Wnt signaling. Also, CBP and p300 are differentially engaged in retinoic acid and cAMP responses in mammalian cells. However, the results presented here show that CBP and p300 can both serve as cofactors for ß-catenin. An interaction between LEF-1 and the CH-3 domain of p300, which corresponds to the dCBP-2 region used in the Drosophila studies, could not be detected. Rather, ß-catenin can interact with p300 in the absence of TCFs (Drosophila homolog, Pangolin). These findings indicate that dCBP differs from p300 and CBP and that in vertebrates p300 or CBP enter the beta-catenin-TCF complex through an interaction with beta-catenin, whereas dCBP may be brought to a promoter by its interaction with dTCF (Hecht, 2000).

Aside from the difference in the TCF interaction, the genetic studies in Drosophila and the experiments in vertebrates may also reveal a more complicated involvement of p300/CBP in Wnt signaling. For example, upon binding to dTCF, Armadillo may form a ternary complex and reprogram the activity of dCBP that is already present. Alternatively, p300 or CBP may function as coactivators of beta-catenin only initially. Over time their activity could change and lead to the downregulation of target genes as reported for the interferon-beta enhanceosome, which eventually is destabilized and disassembled by CBP through acetylation of an architectural component, the HMG I(Y) protein. Similarly, coactivator complexes associated with the promoter-bound estrogen receptor are dissociated after acetylation by p300/CBP, which leads to the attenuation of the hormone response. Thus, one could hypothesize that p300 performs a stimulatory function and also provides a shut-off mechanism in Wnt signaling (Hecht, 2000).

ß-catenin plays a pivotal role in the transcriptional activation of Wnt-responsive genes by binding to TCF/LEF transcription factors. Although it has been suggested that the COOH-terminal region of ß-catenin functions as an activation domain, the mechanisms of activation remain unclear. To screen for potential transcriptional coactivators that bind to the COOH-terminal region of ß-catenin, a novel yeast two-hybrid system, the Ras recruitment system (RRS) that detects protein-protein interactions at the inner surface of the plasma membrane, was used. RRS is based on the ability of mammalian Ras to rescue the growth defect of the yeast temperature-sensitive cdc25-2 strain, in which the endogenous Ras is inactive at the nonpermissive temperature (37°C) due to the lack of a functional Cdc25 guanyl nucleotide exchange factor. For RRS screening, a bait protein of interest is fused at the COOH terminus of mammalian activated Ras. This activated Ras lacks the membrane localization signal [Ras(61)deltaF], whereas library cDNAs are fused to the v-Src myristoylation sequence targeted to the plasma membrane. A protein-protein interaction between the bait and library protein results in the recruitment of Ras to the membrane and complementation of the cdc25-2 mutation. Using this system, the CREB-binding protein (CBP) was isolated. From armadillo (arm) repeat 10 to the COOH terminus of ß-catenin is involved in binding to CBP, whereas ß-catenin interacts directly with the CREB-binding domain of CBP. ß-Catenin synergizes with CBP to stimulate the activity of a synthetic reporter in vivo. Conversely, ß-catenin-dependent transcriptional activation is repressed by E1A, an antagonist of CBP function, but not by an E1A mutant that does not bind to CBP. The activation of Wnt target genes such as siamois and Xnr3 in Xenopus embryos is also sensitive to E1A. These findings suggest that CBP provides a link between ß-catenin and the transcriptional machinery, and possibly mediates the oncogenic function of ß-catenin (Takemaru, 2000).

ß-catenin plays a pivotal role in the transcriptional activation of Wnt-responsive genes by binding to TCF/LEF transcription factors. Although it has been suggested that the COOH-terminal region of ß-catenin functions as an activation domain, the mechanisms of activation remain unclear. To screen for potential transcriptional coactivators that bind to the COOH-terminal region of ß-catenin, a novel yeast two-hybrid system, the Ras recruitment system (RRS) that detects protein-protein interactions at the inner surface of the plasma membrane, was used. RRS is based on the ability of mammalian Ras to rescue the growth defect of the yeast temperature-sensitive cdc25-2 strain, in which the endogenous Ras is inactive at the nonpermissive temperature (37°C) due to the lack of a functional Cdc25 guanyl nucleotide exchange factor. For RRS screening, a bait protein of interest is fused at the COOH terminus of mammalian activated Ras. This activated Ras lacks the membrane localization signal [Ras(61)deltaF], whereas library cDNAs are fused to the v-Src myristoylation sequence targeted to the plasma membrane. A protein-protein interaction between the bait and library protein results in the recruitment of Ras to the membrane and complementation of the cdc25-2 mutation. Using this system, the CREB-binding protein (CBP) was isolated. From armadillo (arm) repeat 10 to the COOH terminus of ß-catenin is involved in binding to CBP, whereas ß-catenin interacts directly with the CREB-binding domain of CBP. ß-Catenin synergizes with CBP to stimulate the activity of a synthetic reporter in vivo. Conversely, ß-catenin-dependent transcriptional activation is repressed by E1A, an antagonist of CBP function, but not by an E1A mutant that does not bind to CBP. The activation of Wnt target genes such as siamois and Xnr3 in Xenopus embryos is also sensitive to E1A. These findings suggest that CBP provides a link between ß-catenin and the transcriptional machinery, and possibly mediates the oncogenic function of ß-catenin (Takemaru, 2000).

The human T-cell leukemia virus type 1 (HTLV-1)-encoded Tax protein activates viral transcription through interaction with the cellular transcription factor CREB (cyclic AMP response element [CRE] binding protein). Although Tax stabilizes the binding of CREB to the Tax-responsive viral CREs in the HTLV-1 promoter, the precise molecular mechanism by which Tax mediates strong transcriptional activation through CREB remains unclear. In this report, it is shown that Tax promotes high-affinity binding of the KIX domain of CREB binding protein (CBP) to CREB-viral CRE complexes, increasing the stability of KIX in these nucleoprotein complexes by up to 4.4 kcal/mol. Comparable KIX binding affinities were measured for both phosphorylated and unphosphorylated forms of CREB; in all cases high-affinity binding is dependent upon both Tax and the viral CRE. Tax also promoted association of KIX to a truncated form of CREB containing only the 73-amino-acid basic leucine zipper (bZIP) domain, indicating that the entire amino-terminal CBP-interacting domain of CREB is nonessential in the presence of Tax. Functional studies upholds the binding studies, as expression of the bZIP domain of CREB is sufficient to support Tax transactivation of HTLV-1 transcription in vivo. Transfection of a KIX expression plasmid, which lacks activation properties, inhibits Tax transactivation in vivo. This suggests that KIX occupies the CBP binding site on Tax, and therefore CBP is likely to be a cofactor in mediating Tax stimulation of HTLV-1 transcription. Together, these data support a model in which Tax anchors CBP to the HTLV-1 promoter, with strong transcriptional activation resulting from the CBP-associated activities of nucleosome remodeling and recruitment of the general transcription machinery (Giebler, 1997).

A unique aspect of the retrovirus life cycle is the obligatory integration of the provirus into host cell chromosomes. Unlike viruses that do not integrate, retroviruses must conserve an ability to activate transcription from a chromatin context. Human immunodeficiency virus (HIV)-1 encodes an unusual and an unusually potent transcriptional transactivator, Tat, which binds to a nascent viral leader RNA, TAR. The action of Tat has been well studied in various reductive model systems; however, the physiological mechanism through which Tat gains access to chromatin-associated proviral long terminal repeats (LTRs) is not understood. A nuclear histone acetyltransferase activity is shown to associate with Tat. Intracellularly, Tat forms a ternary complex with p300 and P/CAF, two histone acetyltransferases (HATs). A murine cell defect in Tat transactivation of the HIV-1 LTR is linked to the reduced abundance of p300 and P/CAF. Thus, overexpression of p300 and P/CAF reconstituted Tat transactivation of the HIV-1 LTR in NIH3T3 cells to a level similar to that observed for human cells. By using transdominant p300 or P/CAF mutants that lack enzymatic activity, a requirement for the HAT component was delineated from the latter but not the former in Tat function. Tat-associated HAT is preferentially important for transactivation of integrated, but not unintegrated, HIV-1 LTR (Benkirane, 1998).

Nucleosomal histone modification is believed to be a critical step in the activation of RNA polymerase II-dependent transcription. p300/CBP and PCAF (see Drosophila Pcaf) histone acetyltransferases (HATs) are coactivators for several transcription factors, including nuclear hormone receptors, p53, and Stat1alpha, and participate in transcription by forming an activation complex and by promoting histone acetylation. The adenoviral E1A oncoprotein represses transcriptional signaling by binding to p300/CBP and displacing PCAF and p/CIP proteins from the complex. E1A directly represses the HAT activity of both p300/CBP and PCAF in vitro and p300-dependent transcription in vivo. Additionally, E1A inhibits nucleosomal histone modifications by the PCAF complex and blocks p53 acetylation. These results demonstrate the modulation of HAT activity as a novel mechanism of transcriptional regulation (Chakravarti, 1999).

The implications of these studies are several. (1) These results provide a novel mechanism of E1A action and delineate a novel transcriptional repressor function of the carboxyl half of the protein. (2) Perhaps more importantly, the mechanism of action of repression is novel and establishes a block of the HAT activity as an additional mode of RNA polymerase II control. (3) This study provides evidence to explain the inhibitory activity of E1A for transcription factors that do not depend on both p300/CBP and PCAF-HAT activity. These results help explain why E1A blocks Myo D function during muscle differentiation, a process that does not require the p300/CBP-HAT activity. (4) The ability of E1A to disrupt activation complexes and to inhibit HAT activity are the properties that map to separable domains. Thus, E1A mutants can be used as valuable probes to study both HAT activity-dependent and -independent transcriptional processes. At this stage the mechanism of inhibition of the HAT activity remains obscure. It is possible that the regulation of acetylases would be analogous to that of kinases by cellular kinase inhibitors. However, since there are no obvious structural similarities among the tails of histones, p53, and E1A, this possibility remains purely speculative. In the future, it will be important to determine whether other coactivation/HAT complexes, including the SAGA, p300, and TFIID complexes as well as other nonhistone substrates, are also sensitive to E1A inhibition. Additionally, it is suggested that other viral proteins (e.g., SV40 large T antigen) could have similar activity, which leads to the intriguing speculation that cellular factors with corresponding activities may exist. In conclusion, this work demonstrates that the acetyltransferase activity of coactivators can be modulated and thus reveals a novel pathway for controlling hormonal signaling by modulating RNA polymerase II transcription (Chakravarti, 1999).

A novel cofactor, ACTR, directly binds nuclear receptors and stimulates their transcriptional activities in a hormone-dependent fashion. ACTR has sequence homology to SRC-1 and TIF2, factors that associate with members of the nuclear receptor family and augment the transcription activity of steroid receptors such as glucocorticoid receptor and estrogen receptor. ACTR possesses an N-terminal PAS/bHLH domain, a central receptor-interaction domain able to interact with RAR, RXR (Drosophila homolog: Ultraspiracle) and TR (See Ecdysone Receptor for more information) in a hormone dependent fashion, and a C-terminal histone acetyl transferase domain. ACTR also recruits two other nuclear factors, CBP and P/CAF, and thus plays a central role in creating a multisubunit coactivator complex. In addition, and unexpectedly, purified ACTR is a potent histone acetyltransferase and appears to define a distinct evolutionary branch to this recently described family. ACTR is able to recruit P/CAF. Thus, hormonal activation by nuclear receptors involves the mutual recruitment of at least three classes of histone acetyltransferases that may act cooperatively as an enzymatic unit to reverse the effects of histone deacetylase shown to be part of the nuclear receptor corepressor complex. Interestingly, while all currently identified histone acetyltransferases (including ACTR) are capable of acetylating core histones H3 and H4, ACTR displays an unusual additional activity as an effective acetylator of mononucleosomes, similar only to that observed in CBP/p300 (Chen, 1997).

The mechanism has been examined by which growth factor-mediated induction of the Ras pathway interferes with signaling via the second messenger cAMP. Activation of cellular Ras with insulin or NGF stimulates recruitment of the S6 kinase pp90RSK to the signal-dependent coactivator CBP. Formation of the pp90RSK-CBP complex occurs with high stoichiometry and persists for 6-8 hr following growth factor addition. pp90RSK specifically recognizes the E1A-binding domain of the coactivator CBP. In addition, like E1A, binding of pp90RSK to CBP is sufficient to repress transcription of cAMP-responsive genes via the cAMP-inducible factor CREB. By contrast with its effects on the cAMP pathway, formation of the pp90RSK-CBP complex is required for induction of Ras-responsive genes. These results provide a demonstration of cross-coupling between two signaling pathways that occurs at the level of a signal-dependent coactivator (Nakajima, 1996).

Cells respond to either viral infection or exposure to double-stranded RNA by carrying out the transcriptional induction of a subset of alpha/beta interferon-stimulated genes, employing a pathway distinct from the interferon signal pathway. The transcriptional induction is mediated through a DNA sequence containing the alpha/beta interferon-stimulated response element (ISRE). A novel transcription factor recognizes this response element and has been designated double-stranded RNA-activated factor 1 (DRAF1). The DNA-binding specificity of DRAF1 correlates with transcriptional induction, thereby distinguishing it as a positive regulator of alpha/beta interferon-stimulated genes. Two of the components of DRAF1 have now been identified as interferon regulatory factor 3 (IRF-3) and the transcriptional coactivator CREB-binding protein (CBP)/p300. IRF-3 preexists in the cytoplasm of uninfected cells and translocates to the nucleus following viral infection. Translocation of IRF-3 is accompanied by an increase in serine and threonine phosphorylation. Coimmunoprecipitation analyses of endogenous proteins demonstrate an association of IRF-3 with the transcriptional coactivators CBP and p300 only subsequent to infection. Antibodies to the IRF-3, CBP, and p300 molecules react with DRAF1 bound to the ISRE target site of induced genes. The cellular response that leads to DRAF1 activation and specific gene expression may serve to increase host survival during viral infection (Weaver, 1998).

The AML1 transcription factor and the transcriptional coactivators p300 and CBP are the targets of chromosome translocations associated with acute myeloid leukemia and myelodysplastic syndrome. In the t(8;21) translocation, the AML1 (CBFA2/PEBP2alphaB) gene becomes fused to the MTG8 (ETO) gene. The terminal differentiation step leading to mature neutrophils in response to granulocyte colony-stimulating factor (G-CSF) is inhibited by the ectopic expression of the AML1-MTG8 fusion protein in L-G murine myeloid progenitor cells. Overexpression of normal AML1 proteins reverses this inhibition and restores the competence to differentiate. Immunoprecipitation analysis shows that p300 and CREB-binding protein (CBP) interact with AML1. The C-terminal region of AML1 is responsible for the induction of cell differentiation and for the interaction with p300. Overexpression of p300 stimulates AML1-dependent transcription and the induction of cell differentiation. These results suggest that p300 plays critical roles in AML1-dependent transcription during the differentiation of myeloid cells. Thus, AML1 and its associated factors p300 and CBFbeta, all of which are targets of chromosomal rearrangements in human leukemia, function cooperatively in the differentiation of myeloid cells (Kitabayashi, 1998).

The complex between AML1 (Runx1) and CBFß is the most frequent target of specific chromosome translocations in human leukemia. The MOZ gene, which encodes a histone acetyltransferase (HAT), is also involved in some leukemia-associated translocations. MOZ is part of the AML1 complex and strongly stimulates AML1-mediated transcription. The stimulation of AML1-mediated transcription is independent of the inherent HAT activity of MOZ. Rather, a potent transactivation domain within MOZ appears to be essential for stimulation of AML1-mediated transcription. MOZ, as well as CBP and MOZ-CBP, can acetylate AML1 in vitro. The amount of AML1-MOZ complex increases during the differentiation of M1 myeloid cells into monocytes/macrophages, suggesting that the AML1-MOZ complex might play a role in cell differentiation. However, the MOZ-CBP fusion protein, which is created by the t(8;16) translocation associated with acute monocytic leukemia, inhibits AML1-mediated transcription and differentiation of M1 cells. These results suggest that MOZ-CBP might induce leukemia by antagonizing the function of the AML1 complex (Kitabayashi, 2001).

p300 is a multifunctional transcriptional coactivator that serves as an adapter for several transcription factors including nuclear steroid hormone receptors. p300 possesses an intrinsic histone acetyltransferase (HAT) activity that may be critical for promoting steroid-dependent transcriptional activation. The vitamin D receptor (VDR) is a member of the steroid and nuclear hormone receptor superfamily of eukaryotic transcription factors and binds target DNA, or response elements, as a homodimer or heterodimer with the 9-cis retinoid X receptor (RXR). In osteoblastic cells, transcription of the bone-specific osteocalcin (OC) gene is principally regulated by the Runx2/Cbfa1 transcription factor and is stimulated in response to vitamin D3 via the vitamin D3 receptor complex. Therefore, p300 control of basal and vitamin D3-enhanced activity of the OC promoter was addressed. Transient overexpression of p300 was found to result in a significant dose-dependent increase of both basal and vitamin D3-stimulated OC gene activity. This stimulatory effect requires intact Runx2/Cbfa1 binding sites and the vitamin D-responsive element. In addition, by coimmunoprecipitation, it has been shown that the endogenous Runx2/Cbfa1 and p300 proteins are components of the same complexes within osteoblastic cells under physiological concentrations. It has also been demonstrated, by chromatin immunoprecipitation assays, that p300, Runx2/Cbfa1, and 1alpha,25-dihydroxyvitamin D3 receptor interact with the OC promoter in intact osteoblastic cells expressing this gene. The effect of p300 on the OC promoter is independent of its intrinsic HAT activity, since a HAT-deficient p300 mutant protein up-regulates expression and cooperates with P/CAF to the same extent as the wild-type p300. On the basis of these results, it is proposed that p300 interacts with key transcriptional regulators of the OC gene and bridges distal and proximal OC promoter sequences to facilitate responsiveness to vitamin D3 (Sierra, 2003).

The Ets-1 transcription factor plays a critical role in cell growth and development, but the means by which it activates transcription remain unclear. It has been shown that Ets-1 binds the transcriptional coactivators CREB binding protein (CBP) and the related p300 protein (together referred to as CBP/p300) and that this interaction is required for specific Ets-1 transactivation functions. The Ets-1- and c-Myb-dependent aminopeptidase N (CD13/APN) promoter and an Ets-1-dependent artificial promoter are repressed by adenovirus E1A, a CBP/p300-specific inhibitor. Ets-1 activity is potentiated by CBP and p300 overexpression. The transactivation function of Ets-1 correlates with its ability to bind an N-terminal cysteine- and histidine-rich region spanning CBP residues 313 to 452. Ets-1 also binds a second cysteine- and histidine-rich region of CBP, between residues 1449 and 1892. Both Ets-1 and CBP/p300 form a stable immunoprecipitable nuclear complex, independent of DNA binding. This Ets-1-CBP/p300 immunocomplex possesses histone acetyltransferase activity, consistent with previous findings that CBP/p300 is associated with such enzyme activity. These results indicate that CBP/p300 may mediate antagonistic and synergistic interactions between Ets-1 and other transcription factors that use CBP/p300 as a coactivator, including c-Myb and AP-1 (Yang, 1998).

POU-domain proteins, such as the pituitary-specific factor Pit-1, are members of the homeodomain family of proteins which are important in development and homeostasis, acting constitutively or in response to signal-transduction pathways to either repress or activate the expression of specific genes. Whereas homeodomain-containing repressors such as Rpx2 seem to recruit only a co-repressor complex, the activity of Pit-1 is determined by a regulated balance between a co-repressor complex that contains N-CoR/SMRT, mSin3A/B and histone deacetylases, and a co-activator complex that includes the CREB-binding protein (CBP) and p/CAF. Activation of Pit-1 by cyclic AMP or growth factors depends on distinct amino- and carboxy-terminal domains of CBP, respectively. Furthermore, the histone acetyltransferase functions of CBP or p/CAF are required for Pit-1 function that is stimulated by cyclic AMP or growth factors, respectively. These data show that there is a switch in specific requirements for histone acetyltransferases and CBP domains in mediating the effects of different signal-transduction pathways on specific DNA-bound transcription factors (L. Xu, 1998).

The transcriptional coactivators CBP and P/CAF are required for activation of transcription from the IFN beta enhanceosome. CBP and P/CAF acetylate HMG I(Y), the essential architectural component required for enhanceosome assembly. Acetylation takes place at distinct lysine residues, causing distinct effects on transcription. Thus, in the context of the enhanceosome, acetylation of HMG I by CBP, but not by P/CAF, leads to enhanceosome destabilization and disassembly. Acetylation of HMG I(Y) by CBP is essential for turning off IFN beta gene expression. The acetyltransferase activities of CBP and P/CAF modulate both the strength of the transcriptional response and the kinetics of virus-dependent activation of the IFN beta gene (Munshi, 1998).

Xenopus NF-Y is identified as a key regulator of acetylation responsiveness for the Xenopus hsp70 promoter within chromatin assembled in Xenopus oocyte nuclei. Y-box sequences are required for the assembly of DNase I-hypersensitive sites in the hsp70 promoter, and for transcriptional activation both by inhibitors of histone deacetylase and by the p300 acetyltransferase. The viral oncoprotein E1A interferes with both of these activation steps. Xenopus NF-YA, NF-YB and NF-YC have been cloned and NF-Y has been established as the predominant Y-box-binding protein in Xenopus oocyte nuclei. NF-Y interacts with p300 in vivo and is itself a target for acetylation by p300. Transcription from the hsp70 promoter in chromatin can be enhanced further by heat shock factor. Two steps in chromatin modification at the Xenopus hsp70 promoter are suggested: first the binding of NF-Y to the Y-boxes to pre-set chromatin and second the recruitment of p300 to modulate transcriptional activity (Li, 1998).

The activity of c-Jun, the major component of the transcription factor AP-1, is potentiated by amino-terminal phosphorylation on serines 63 and 73 (Ser-63/73). This phosphorylation is mediated by the Jun amino-terminal kinase (JNK) and required to recruit the transcriptional coactivator CREB-binding protein (CBP). AP-1 function is antagonized by activated members of the steroid/thyroid hormone receptor superfamily. Recently, a competition for CBP has been proposed as a mechanism for this antagonism. Hormone-activated nuclear receptors prevent c-Jun phosphorylation on Ser-63/73, and consequently, AP-1 activation as well, by blocking the induction of the JNK signaling cascade. Consistently, nuclear receptors also antagonize other JNK-activated transcription factors such as Elk-1 and ATF-2. It is shown here that dexamethasone, a glucocorticoid receptor agonist, and two other nuclear hormone receptors, the retinoic acid receptor and the thyroid hormone receptor, also block c-Jun activation by a mechanism that is (1) independent of the c-Jun DNA binding domain and is one which (2) relies specifically on the c-Jun amino-terminal phosphorylation step. Interference with the JNK signaling pathway represents a novel mechanism by which nuclear hormone receptors antagonize AP-1. This mechanism is based on the blockade of the AP-1 activation step, which is a requisite for interaction with CBP. In addition to acting directly on gene transcription, regulation of the JNK cascade activity constitutes an alternative mode whereby steroids and retinoids may control cell fate and conduct their pharmacological actions as immunosupressive, anti-inflammatory, and antineoplastic agents. Nuclear receptor interference would rely on the inhibition of MEKK activity or a downstream step in the pathway. Dexamethasone can also inhibit a constitutively active MAPK pathway (Caelles, 1997).

Calcium is the principal second messenger in the control of gene expression by electrical activity in neurons. Recruitment of the coactivator CREB-binding protein, CBP, by the prototypical calcium-responsive transcription factor, CREB and stimulation of CBP activity by nuclear calcium signals is one mechanism through which calcium influx into excitable cells activates gene expression. Another CBP-interacting transcription factor, c-Jun, can mediate transcriptional activation upon activation of L-type voltage-gated calcium channels. Calcium-activated transcription mediated by c-Jun functions in the absence of stimulation of the c-Jun N-terminal protein kinase (JNK/SAPK1) signaling pathway and does not require c-Jun amino acid residues Ser63 and Ser73, the two major phosphorylation sites that regulate c-Jun activity in response to stress signals. Similar to CREB-mediated transcription, activation of c-Jun-mediated transcription by calcium signals requires calcium/calmodulin-dependent protein kinases and is dependent on CBP function. These results identify c-Jun as a calcium-regulated transcriptional activator and suggest that control of coactivator function (i.e. recruitment of CBP and stimulation of CBP activity) is a general mechanism for gene regulation by calcium signals (Cruzalegui, 1999).

p300 and the closely related CREB binding protein (CBP) are transcriptional adaptors that are present in intracellular complexes with TATA binding protein (TBP) and bind to upstream activators including p53 and nuclear hormone receptors. They have intrinsic and associated histone acetyltransferase activity, suggesting that chromatin modification is an essential part of their role in regulating transcription. Detailed characterization of a panel of antibodies raised against p300/CBP has revealed the existence of a 270-kDa cellular protein, p270, distinct from p300 and CBP but sharing at least two independent epitopes with p300. The subset of p300/CBP-derived antibodies that cross-reacts with p270 consistently coprecipitates a series of cellular proteins with relative molecular masses ranging from 44 to 190 kDa. Purification and analysis of various proteins in this group reveals that they are components of the human SWI/SNF complex and that p270 is an integral member of this complex (Dallas, 1998).

Using the coiled-coil region of Stat5b as the bait in a yeast two-hybrid screen, the association of Nmi, a protein of unknown function previously reported as an N-Myc interactor, was identifed. Nmi interacts with all STATs except Stat2. Two cytokine systems, IL-2 and IFNgamma, were evaluated and Nmi was demonstrated to augment STAT-mediated transcription in response to these cytokines. Interestingly, Nmi lacks an intrinsic transcriptional activation domain; instead, Nmi enhances the association of CBP/p300 coactivator proteins with Stat1 and Stat5, and together with CBP/p300 augments IL-2- and IFNgamma-dependent transcription. Therefore, these data not only reveal that Nmi can potentiate STAT-dependent transcription, but also suggest that it can augment coactivator protein recruitment to at least some members of a group of sequence-specific transcription factors (Zhu, 1999).

Disaggregation of the spherical nuclear bodies termed promyelocytic (PML) oncogenic domains (PODs) is a characteristic of acute promyelocytic leukemia. The cAMP enhancer binding protein (CREB)-binding protein (CBP) associates with PML in vitro and is recruited to the PODs in vivo. Through its association with CBP, wild-type PML dramatically stimulates nuclear receptor transcriptional activity. These results demonstrate that a fraction of CBP is compartmentalized to the POD through its association with PML and thus suggest that PML and other POD-associated proteins may play an unexpectedly broad role in aspects of transcriptional regulation and human disease (Doucas, 1999).

The orphan nuclear receptor, steroidogenic factor-1 (SF-1), plays an important role in the development of the adrenal gland and in sexual differentiation. SF-1 regulates the transcription of variety of genes, including several steroidogenic enzymes, Mullerian inhibiting substance, and gonadotropin genes. Attempts have been made to identify domains in SF-1 that are required for transactivation and to determine whether SF-1 interacts with a subset of known coactivators. Natural variants of the FTZ-F1 locus include embryonal long terminal repeat-binding protein (ELP)-1, ELP-2, and SF-1, all of which share the DNA-binding domain. Analyses of the transcriptional activity of these variants reveal that the activity of ELP-2 and SF-1 is much greater than ELP-1, which contains a distinct carboxy terminus. Further studies were performed using GAL4-SF-1 fusion proteins that were constructed by replacement of the zinc finger region and FTZ-F1 box of SF-1 with the DNA-binding domain of GAL4. Elimination of the putative AF-2 domain at the carboxy terminus of GAL4-SF-1 proteins results in a complete loss of transactivation. Several lines of evidence demonstrate that SF-1 interacts with steroid receptor coactivator-1 (SRC-1). Full-length SRC-1 enhances GAL4-SF-1-mediated transactivation, whereas a dominant negative form of SRC-1, consisting of its interaction domain alone, inhibits the activity of GAL4-SF-1. In mammalian two-hybrid assays, fusion of the VP16 activation domain to the interaction domain of SRC-1 confirms the interaction between SRC-1 and GAL4-SF-1 and demonstrates that the AF-2 domain is required for interaction with SRC-1. Furthermore, SRC-1, together with the cAMP responsive element binding protein (CBP) or a closely related factor, p300, synergistically enhance transcriptional activity of GAL4-SF-1. It is concluded that the carboxy-terminal AF-2 region of SF-1 functions as an activation domain and that SRC-1 and CBP/p300 are components of the coactivator complex with SF-1 (Ito, 1998)

The co-activators CBP and p300 are important for normal cell differentiation and cell cycle progression and are the targets for viral proteins that dysregulate these cellular processes. The E6 protein from the oncogenic human papillomavirus type 16 (HPV-16) binds to three regions (C/H1, C/H3 and the C-terminus) of both CBP and p300. The interaction of E6 with CBP/p300 is direct and independent of proteins known to bind the co-activators, such as p53. The E6 protein from low-risk HPV type 6 does not interact with C/H3 or the C-terminus but associates with the C/H1 domain at 50% of the level of HPV-16. HPV-16 E6 inhibits the intrinsic transcriptional activity of CBP/p300 and decreases the ability of p300 to activate p53- and NFkappa-B-responsive promoter elements. Interestingly, some mutations in HPV-16 E6 abrogate C/H3-E6 interactions, but do not alter the ability of E6 to associate with the C/H1 domain, suggesting that these modified proteins could be used to delineate the functional significance of the C/H1 and C/H3 domains of CBP/p300 (Patel, 1999).

What are the advantages for the virus to target CBP/p300? The human papillomaviruses infect basal epithelial cells, which are destined to terminally differentiate. Papillomaviruses have a small coding capacity and do not code for any of the replicative machinery; differentiating cells contain very little or none of the cell's replicative components. Therefore the virus needs to stimulate cells into S-phase to have on hand a supply of replicative enzymes. CBP/p300 is known to be important for cell differentiation, and transcription factors such as members of the AP-1 family, which are thought to be important for keratinocyte differentiation, bind to the regions bound by E6. Therefore disruption of AP-1 transactivation may have an effect on keratinocyte differentiation. In the natural infection HPV-16 causes disruption of differentiation and hyperproliferation of the stratum spinosum and granulosum. In addition, HPV-16 E6 and E7 proteins can independently modulate the ability of primary human keratinocytes to differentiate. In contrast, HPV-6 does not disrupt differentiation, but does cause a hyperproliferation of the epithelial cells. The HPV-16 E6 protein may therefore have a role in inhibiting differentiation, resulting in hyperproliferation. The papillomavirus group cause persistent infections with lesions remaining for months, even years. It has been suggested that the viruses can down-regulate the local immune response to allow persistence of the infection. In fact studies have shown a reduced class I MHC expression and reduced numbers of Langerhans and T cells in the immediate vicinity of the lesion. The fact that HPV-16 E6 can inhibit the activation of NF-kappaB by CBP/p300 may help to explain these findings, since this transcription factor is activated by a number of stimuli, including viral infection. In addition, binding sites for NF-kappaB are found in a number of promoters including those for class I MHC, cytokine modulators of the immune response including IL-2, IL-6, IL-8 and GM-CSF. Keratinocytes synthesize a variety of these chemotactic factors, including IL-6, IL-8 and GM-CSF, when stimulated by viral infection. Therefore inhibition of the co-activation of NF-kappaB may help the virus escape immune recognition in the epithelium. In summary, E6 of HPV-16 binds to three regions of CBP/p300, resulting in the abrogation of the co-activation functions. These co-activators are important for cell differentiation and cell cycle control and are central to the activation of a number of genes that modulate immune responses. Therefore the outcome of the interaction between E6 and CBP/p300 may involve the inhibition of differentiation of the epithelial cells harboring the virus and the down-regulation of the immune recognition machinery to permit the persistence of the viral infection (Patel, 1999).

It was asked whether the acetylase CREB-binding protein (CBP) can acetylate proteins not directly involved in transcription. A large panel of proteins, involved in a variety of cellular processes, were tested as substrates for recombinant CBP. This screen identified two proteins involved in nuclear import, Rch1 (human importin-alpha) and importin-alpha7, as targets for CBP. The acetylation site within Rch1 was mapped to a single residue, Lys22. By comparing the context of Lys22 with the sequences of other known substrates of CBP and the closely related acetylase p300, G/SK (in the single-letter amino acid code) was identified as a consensus acetylation motif. Mutagenesis of the glycine, as well as the lysine, severely impairs Rch1 acetylation, supporting the view that GK is part of a recognition motif for acetylation by CBP/p300. Using an antibody raised against an acetylated Rch1 peptide, it was show that Rch1 is acetylated at Lys22 in vivo and that CBP or p300 can mediate this reaction. Lys22 lies within the binding site for a second nuclear import factor, importin-beta. Acetylation of Lys22 promotes interaction with importin-beta in vitro. Collectively, these results demonstrate that acetylation is not unique to proteins involved in transcription. Acetylation may regulate a variety of biological processes, including nuclear import (Bannister, 2000).

The CCAAT displacement protein/cut homolog (CDP/cut) is a divergent homeodomain protein that is highly conserved through evolution and has properties of a potent transcriptional repressor. CDP/cut contains three conserved cut-repeat domains and a conserved homeobox, each involved in directing binding specificity to unique nucleotide sequence elements. Furthermore, CDP/cut may play a role as a structural component of chromatin through its direct interaction with nucleosomal DNA and association with nuclear matrix attachment regions. CDP/cut is cell-cycle regulated through interactions with Rb, p107, specific kinases and phosphatases directing the transcriptional activity of CDP/cut on such genes encoding p21WAF1,CIP1, c-myc, thymidine kinase, and histones. CDP/cut is associated with histone deacetylase activity and is associated with a corepressor complex through interactions with histone deacetylases. The interaction of CDP/cut with CBP and p300/CREB-binding protein-associated factor (PCAF) is reported along with the modification of CDP/cut by the histone acetyltransferase PCAF. Acetylation of CDP/cut by PCAF is directed at conserved lysine residues near the homeodomain region and regulates CDP/cut function. These observations are consistent with the ability of CDP/cut to regulate genes as a transcriptional repressor, suggesting acetylation as a mechanism that regulates CDP/cut function (Li, 2000).

Complexes containing p300, but not CBP, and the nuclear proto-oncoprotein SYT were detected in confluent cultures of G1-arrested cells but not in sparse cells or during S or G2. SYT sequences constitute the N-terminal segment of a fusion oncogene product, SYT-SSX, routinely detected in synovial sarcoma, an aggressive human tumor. SYT/p300 complex formation promotes cell adhesion to a fibronectin matrix, as reflected by compromise of this process in cells expressing SYT deletion mutants that retain p300 binding activity and in the primary fibroblasts of p300 but not CBP heterozygous null mice. The mechanism linking the action of SYT/p300 complexes to adhesion function is, at least in part, transcription activation-independent and results in proper activation of beta1 integrin, a major adhesion receptor (Eid, 2000).

Indirect evidence, based on studies of E1A and SV40 T Ag function, implies that p300/CBP actively suppresses the emergence in cultured cells of multiple neoplastic characteristics, one of which is loss of contact inhibition of cell growth. Results presented here reveal an unexpected function of p300. Both p300 and a known human proto-oncoprotein, SYT, interact in the nucleus. Formation of SYT/p300 complexes is confined to G1-arrested, confluent cells, is absent in sparse G1 cells, and appears to translate into proper cell adhesion to a fibronectin matrix. What seems likely to be a failure to form a sufficient quantity of these nuclear complexes, as in p300+/- embryonic cells and in cells synthesizing certain dominant negative SYT mutants, leads to a major defect in cell adhesion/spreading. Taken together, these results are consistent with the hypothesis that SYT/p300 complex formation participates in the control of contact inhibition of cell growth, a G1-specific phenomenon, and an adhesion-dependent process (Eid, 2000).

The level of ribosomal gene transcription has been shown to be finely regulated in response to changes in cell growth rate and the state of differentiation. This regulation is believed, at least in part, to be due to a change in the number of actively transcribed genes. Transcription of the ribosomal genes by RNA polymerase I (PolI) is activated both in vitro and in vivo by UBF. The recruitment of this protein to the PolI promoter is in fact the first step in ribosomal gene activation, permitting the subsequent association of the TATA-binding protein (TBP)-containing complex SL-1, and hence of the polymerase. UBF contains multiple tandem homologies to the DNA binding domain of high mobility group 1 (HMG-1), the HMG box, and loops approximately 140 bp of ribosomal DNA into a single turn, a structure that has been called the ribosomal enhancesome. Data on the promotion of PolI transcription in vertebrates are compatible with the formation of two precisely juxtaposed enhancesomes on the PolI promoter as a prerequisite to promoter recognition by SL-1. Mammalian and Xenopus UBFs are functionally interchangeable for this task in vivo. However, enhancesome formation is clearly incompatible with the nucleosomal chromatin structure of the inactive genes. Therefore, the transition from the inactive to active ribosomal gene state requires the replacement of one or more nucleosomes with enhancesomes. Chromatin remodeling has been shown to be facilitated by the recruitment of co-activators with acetyltransferase activity. Further, the HMG box of Drosophila TCF/LEF functionally recruits the histone acetyltransferase (HAT) CREB-binding protein (CBP). The potential of CBP to activate ribosomal transcription by PolI has been investigated (Pelletier, 2000 and references therein).

In the canonical Wnt signaling pathway, ß-catenin activates target genes through its interactions with Tcf/Lef-family transcription factors and additional transcriptional coactivators. The crystal structure of ICAT, an inhibitor of ß-catenin-mediated transcription, bound to the armadillo repeat domain of ß-catenin, has been determined. ICAT contains an N-terminal helilical domain that binds to repeats 11 and 12 of ß-catenin, and an extended C-terminal region that binds to repeats 5-10 in a manner similar to that of Tcfs and other ß-catenin ligands. Full-length ICAT dissociates complexes of ß-catenin, Lef-1, and the transcriptional coactivator p300, whereas the helical domain alone selectively blocks binding to p300. The C-terminal armadillo repeats of ß-catenin may be an attractive target for compounds designed to disrupt aberrant ß-catenin-mediated transcription associated with various cancers (Daniels, 2002).

RNA polymerase I (PolI) transcription is activated by the HMG box architectural upstream binding factor (UBF), which loops approximately 140 bp of DNA into the enhancesome, necessitating major chromatin remodeling. The acetyltransferase CBP is recruited to and acetylates UBF both in vitro and in vivo. CBP activates PolI transcription in vivo through its acetyltransferase domain and acetylation of UBF facilitates transcription derepression and activation in vitro. CBP activation and Rb suppression of ribosomal transcription by recruitment to UBF are mutually exclusive, regulating in vivo PolI transcription through an acetylation-deacetylation 'flip-flop.' Thus, PolI transcription is regulated by protein acetylation, and the competitive recruitment of CBP and Rb (Pelletier, 2000).

Rb suppresses ribosomal transcription in vitro via a direct interaction with UBF. An interaction has been identified in vitro between Rb aa 379-928 and the HMG boxes 1 and/or 2 of mUBF. Other data further suggest that the Rb 'pocket' (aa 379-792) is sufficient for transcriptional suppression in vitro and for the Rb-UBF interaction in vivo. Since HMG boxes 1 and 2 also bind CBP, it is possible that Rb and CBP binding to UBF are exclusive events. If this is the case, suppression by Rb and activation by CBP would also be exclusive (Pelletier, 2000).

Rb suppresses ribosomal transcription in vivo. Consistent with a role for the Rb pocket in this suppression, it has also been found that the Rb-related pocket protein p107 is equally effective in this suppression. It was next determined if the pocket domain of Rb (aa 379-792) could bind HMG boxes 1 and/or 2 of xUBF. Rb(379-792) binds a polypeptide containing HMG boxes 1 and 2, but does not bind to sequences C-terminal of HMG box 2. Thus the pocket region of Rb is sufficient to bind xUBF. It was also found that the individual HMG boxes 1 and 2 bind the Rb pocket region less efficiently than does the box12 combination. Rb and CBP were then placed in competition for xUBF. Preincubation of full-length xUBF with the interaction domain CBP2 is sufficient to inhibit subsequent binding to Rb(379-792). Conversely, pre-incubation of xUBF with Rb(379-792) inhibits its subsequent binding to CBP2. These data strongly suggest that suppression of PolI transcription by Rb results, at least in part, from its capacity to interfere with the recruitment of CBP to UBF. However, in other systems, Rb has also been shown to suppress transcription by the recruitment of histone deacetylase 1 (HDAC1). Hence, Rb could also potentially reverse the catalytic effects of recruiting CBP to UBF. It was therefore asked if HDAC1 deacetylates xUBF acetylated with the CBP-HAT domain and whether the presence of Rb enhances this deacetylation. The rate of deacetylation of xUBF by HDAC1 in the presence of Rb(379-792) is nearly two times more rapid than in its absence. Suppression of PolI transcription in vivo by Rb (and p107) is at least partly relieved by inhibiting deacetylation with TSA. The fact that Rb suppression can not be reversed with TSA alone is consistent with Rb also preventing CBP recruitment to UBF. In fact, the Rb-induced suppression of PolI transcription can be completely relieved, and indeed reversed, by the coexpression of CBP in combination with TSA treatment (Pelletier, 2000).

The recruitment of CBP to UBF could activate transcription (1) by the acetylation of UBF, (2) by the acetylation of local chromatin, (3) by displacing Rb, or (4) by a combination of these effects. The fact that Rb can cooperate in the deacetylation of UBF suggests that acetylated UBF can effectively bind Rb and this was confirmed in pulldown experiments. The role of CBP, Rb, and acetylation in DNA binding by UBF was also investigated. Neither an excess of CBP2 nor saturation acetylation of UBF with the HAT domain of CBP has any detectable effect on UBF's capacity to bind the ribosomal promoter DNA. Rb(379-928), [or the pocket domain Rb(379-792) or GST-Rb(379-928)], has no effect on DNA binding by UBF (Pelletier, 2000).

Since the DNA binding of UBF is unaffected by CBP or Rb binding or by acetylation, it was asked if acetylated UBF is necessary for transcription activation in vitro. Bacterially produced UBF, which is necessarily unacetylated, has been found in many laboratories to be refractory for in vitro transcription. However, UBF produced in mammalian and insect cells or by in vitro translation has been found to be functional. This suggests that post-transcriptional modification of UBF may be important, and indeed this has been shown to be the case for UBF phosphorylation. Rat and mouse nuclear extracts were therefore depleted of endogenous UBF and used to study the capacity of bacterially expressed (i.e., unacetylated) UBF to activate transcription from the rat or mouse PolI promoters. Bacterially produced rUBF and xUBF were either acetylated with matrix-immobilized active CBP HAT domain or mock acetylated (unacetylated) with the immobilized inactive HAT domain and then the HAT protein was removed by centrifugation. UBF has been shown both to derepress PolI transcription in vitro as well as to activate it. The derepression properties of rUBF in the rat extract was investigated in competition with added histone H1. As expected, addition of H1 to the rat extract represses transcription of the rat promoter, and this repression is even more pronounced after UBF depletion. Addition of unacetylated rUBF does not relieve H1 repression, and even increases it somewhat. However, the acetylated rUBF relieves H1 repression and gives about a 2-fold increase in transcription (or more than 4-fold the level observed in the presence of the same amount of unacetylated UBF). The capacity of UBF to activate transcription from the mouse promoter was also tested in a UBF-depleted mouse nuclear extract. Here the unacetylated rUBF gives a small degree of transcription activation (1.7 times), but the acetylated rUBF activates much more effectively (3.5 times). Xenopus UBF has been shown to activate the mouse promoter in vivo, although it has also been shown to be ineffective in activating the rat or human promoters in vitro. Bacterial unacetylated xUBF has a clear repressive (0.5 times) effect on the mouse promoter in vitro, but after acetylation, this repression is completely relieved and transcription is somewhat activated (Pelletier, 2000).

These data strongly support a 'flip-flop' model for the regulation of ribosomal transcription by CBP and Rb-HDAC1. (The term 'flip-flop' is used to describe a system with two alternative semistable states, here CBP-bound or Rb-bound UBF.) The formation of a UBF-CBP complex activates transcription by acetylation of UBF itself, and perhaps also by opening up the adjacent ribosomal chromatin, allowing further UBF ingression and gene activation. Excess Rb prevents formation of a UBF-CBP complex and, by recruiting HDAC1, catalyses UBF deacetylation and hence its inactivation. Acetylation of UBF significantly enhances its ability to activate PolI transcription in vitro. Although changes in DNA binding and the ability of UBF to bind Rb have been excluded, the mechanism by which UBF acetylation functions remains unknown. One possible explanation being actively pursued is that acetylation induces a structural change in UBF. Quite possibly Rb recruitment to UBF has roles other than just to promote UBF deacetylation. Rb can inhibit SL-1 recruitment to UBF. Rb may also cooperate in deacetylation of adjacent histones. Enhancesome structure, with its single 140 bp loop of DNA, could accommodate the core histones in a weak association with the DNA. Yet, xUBF can also associate stably with nucleosomes. Thus, the CBP/Rb flip-flop could catalyze the transition between a predominantly nucleosomal and a predominantly enhancesomal gene state, the transition not necessarily requiring complete displacement of either core histones or UBF. It has in fact been observed that the core histones remain associated with the active ribosomal genes, but only via their N-terminal domains. Whether a nucleosome-enhancesome transition is facilitated by UBF acetylation, histone acetylation, or a combination of the two must now be determined (Pelletier, 2000).

Recent data suggest that both acetylation and phosphorylation can cooperate to activate transcription in vivo. UBF is known to be multiply phosphorylated, mainly within the C-terminal acidic domain but also in HMG box 5. Each of these modifications has been shown to activate transcription in vitro and, in the case of the acidic domain, phosphorylation has been shown to enhance recruitment of SL-1. Here acetylation is also important for UBF function. In future work attempts will be made to test whether a functional link exists between the phosphorylation and the acetylation of UBF (Pelletier, 2000).

Spinal and bulbar muscular atrophy (SBMA) is one of eight inherited neurodegenerative diseases known to be caused by CAG repeat expansion. The expansion results in an expanded polyglutamine tract, which likely confers a novel, toxic function to the affected protein. Cell culture and transgenic mouse studies have implicated the nucleus as a site for pathogenesis, suggesting that a critical nuclear factor or process is disrupted by the polyglutamine expansion. In this report evidence is presented that CREB-binding protein (CBP), a transcriptional co-activator that orchestrates nuclear response to a variety of cell signaling cascades, is incorporated into nuclear inclusions formed by polyglutamine-containing proteins in cultured cells, transgenic mice and tissue from patients with SBMA. CBP incorporation into nuclear inclusions form in a cell culture model of another polyglutamine disease, spinocerebellar ataxia type 3. Evidence is presented that soluble levels of CBP are reduced in cells expressing expanded polyglutamine despite increased levels of CBP mRNA. Finally, it is demonstrated that over-expression of CBP rescues cells from polyglutamine-mediated toxicity in neuronal cell culture. These data support a CBP-sequestration model of polyglutamine expansion disease (McCampbell, 2000).

Huntington's Disease (HD) is caused by an expansion of a polyglutamine tract within the huntingtin (htt) protein. Pathogenesis in HD appears to include the cytoplasmic cleavage of htt and release of an amino-terminal fragment capable of nuclear localization. Potential consequences to nuclear function of a pathogenic amino-terminal region of htt (httex1p) have been investigated including aggregation, protein-protein interactions, and transcription. httex1p coaggregates with p53 in inclusions generated in cell culture and interacts with p53 in vitro and in cell culture. Expanded httex1p represses transcription of the p53-regulated promoters, p21(WAF1/CIP1) and MDR-1. httex1p also interacts in vitro with CREB-binding protein (CBP) and mSin3a, and CBP to localize to neuronal intranuclear inclusions in a transgenic mouse model of HD. These results raise the possibility that expanded repeat htt causes aberrant transcriptional regulation through its interaction with cellular transcription factors that may result in neuronal dysfunction and cell death in HD (Steffan, 2000).

Expanded polyglutamine repeats have been proposed to cause neuronal degeneration in Huntington's disease (HD) and related disorders, through abnormal interactions with other proteins containing short polyglutamine tracts such as the transcriptional coactivator CREB binding protein, CBP. CBP is depleted from its normal nuclear location and os present in polyglutamine aggregates in HD cell culture models, HD transgenic mice, and human HD postmortem brain. Expanded polyglutamine repeats specifically interfere with CBP-activated gene transcription, and overexpression of CBP rescues polyglutamine-induced neuronal toxicity. Thus, polyglutamine-mediated interference with CBP-regulated gene transcription may constitute a genetic gain of function, underlying the pathogenesis of polyglutamine disorders (Nicifora, 2001).

Insulin negatively regulates expression of the insulin-like growth factor binding protein 1 (IGFBP-1) gene by means of an insulin-responsive element (IRE) that also contributes to glucocorticoid stimulation of this gene. The Caenorhabditis elegans protein DAF-16 binds the IGFBP-1-IRE with specificity similar to that of the forkhead (FKH) factor(s) that act both to enhance glucocorticoid responsiveness and to mediate the negative effect of insulin at this site. In HepG2 cells, DAF-16 and its mammalian homologs, FKHR, FKHRL1, and AFX (Drosophila homolog: Foxo), activate transcription through the IGFBP-1.IRE; this effect is inhibited by the viral oncoprotein E1A, but not by mutants of E1A that fail to interact with the coactivator p300/CREB-binding protein (CBP). DAF-16 and FKHR can interact with both the KIX and E1A/SRC interaction domains of p300/CBP, as well as the steroid receptor coactivator (SRC). A C-terminal deletion mutant of DAF-16 that is nonfunctional in C. elegans fails to bind the KIX domain of CBP, fails to activate transcription through the IGFBP-1.IRE, and inhibits activation of the IGFBP-1 promoter by glucocorticoids. Thus, the interaction of DAF-16 homologs with the KIX domain of CBP is essential to basal and glucocorticoid-stimulated transactivation. Although AFX interacts with the KIX domain of CBP, it does not interact with SRC and does not respond to glucocorticoids or insulin. Thus, it is concluded that DAF-16 and FKHR act as accessory factors to the glucocorticoid response, by recruiting the p300/CBP/SRC coactivator complex to an FKH factor site in the IGFBP-1 promoter, which allows the cell to integrate the effects of glucocorticoids and insulin on genes that carry this site (Nasrin, 2000).

The IFN-beta enhanceosome activates transcription by directing the ordered recruitment of chromatin modifying and general transcription factors to the IFN-beta promoter. The enhanceosome is assembled in the nucleosome-free enhancer region of the IFN-beta gene, leading to the modification and remodeling of a strategically positioned nucleosome that masks the TATA box and the start site of transcription. Initially, the GCN5 complex is recruited, which acetylates the nucleosome, and this is followed by recruitment of the CBP-PolII holoenzyme complex. Nucleosome acetylation in turn facilitates SWI/SNF recruitment by CBP, resulting in chromatin remodeling. This program of recruitment culminates in the binding of TFIID to the promoter and the activation of transcription (Agalioti, 2002)

Hypoxia-inducible factor 1alpha (HIF1alpha) plays a pivotal role in embryogenesis, angiogenesis, and tumorigenesis. HIF1alpha-mediated transcription requires the coactivator p300, at least in part, through interaction with the cysteine- and histidine-rich 1 domain of p300. To understand the molecular basis of this interaction, a random mutagenesis screen in yeast has been employed for efficient identification of residues that are functionally critical for protein interactions. As a result, four residues (Leu-795, Cys-800, Leu-818, and Leu-822) in the C-terminal activation domain of HIF1alpha have been identified as crucial for HIF1 transactivation in mammalian systems. Moreover, data from residue substitution experiments indicate the stringent necessity of leucine and hydrophobic cysteine for C-terminal activation domain function. Likewise, Leu-344, Leu-345, Cys-388, and Cys-393 in the cysteine- and histidine-rich 1 domain of p300 have also been shown to be essential for the functional interaction. It is proposed that hypoxia-induced HIF1alpha-p300 interaction relies upon a leucine-rich hydrophobic interface that is regulated by the hydrophilic and hydrophobic sulfhydryls of HIF1alpha Cys-800 (Gu, 2001).

DNA repair in chromatin is subject to topological constraints, suggesting a requirement for chromatin modification and remodeling activities. Thymine DNA glycosylase (TDG) initiates repair of G/T and G/U mismatches, commonly associated with CpG islands, by removing thymine and uracil moieties. TDG associates with transcriptional coactivators CBP and p300 and the resulting complexes are competent for both the excision step of repair and histone acetylation. Furthermore, TDG stimulates CBP transcriptional activity in transfected cells and reciprocally serves as a substrate for CBP/p300 acetylation. Remarkably, this acetylation triggers release of CBP from DNA ternary complexes and also regulates recruitment of repair endonuclease APE. These observations reveal a potential regulatory role for protein acetylation in base mismatch repair and a role for CBP/p300 in maintaining genomic stability (Tini, 2002).

The histone acetyltransferases CREB binding protein (CBP) and the related p300 protein function as key transcriptional co-activators in multiple pathways. In the case of transcriptional activation by nuclear receptors, ligand promotes the recruitment of co-activators of the p160 family, such as GRIP-1. Subsequently, the p160 co-activators recruit other co-activators via two activation domains, AD1 and AD2. AD1 binds CBP or p300, whereas AD2 has been shown to activate transcription through the recruitment of the arginine methyltransferase CARM1. The KIX domain of CBP has been shown to be methylated by CARM1 in vitro. Another domain of CBP is specifically methylated by CARM1 on conserved arginine residues both in vitro and in vivo. Functional evidence is provided that arginine residues methylated by CARM1 play a critical role in GRIP-1-dependent transcriptional activation and in hormone-induced gene activation. Altogether, these data provide strong evidence that arginine methylation represents an important mechanism for modulating co-activator transcriptional activity (Chevillard-Briet, 2002).

What then could be the biochemical consequence of CBP methylation? One obvious possibility is that CBP methylation regulates its HAT activity. However, significant change was found in CBP HAT activity following CBP methylation by CARM1. Another interesting possibility is that methylation could change CBP conformation and/or regulate some protein-protein interactions responsible for CBP-mediated transcriptional activation on steroid hormone receptors. Interestingly, methylation of two proteins has been shown to link arginine methylation to the regulation of protein-protein interaction. To date, no protein has been shown to bind directly to the domain of CBP that is methylated by CARM1 (Chevillard-Briet, 2002).

p300 and CREB binding protein can both activate and repress transcription. The CRD1 transcriptional repression domain has been localized between residues 1017 and 1029 of p300. This region contains two copies of the sequence ψKxE that are modified by the ubiquitin-like protein SUMO-1 (see Drosophila SUMO). Mutations that reduce SUMO modification increase p300-mediated transcriptional activity and expression of a SUMO-specific protease or catalytically inactive Ubc9 relieves repression, demonstrating that p300 repression is mediated by SUMO conjugation. SUMO-modified CRD1 domain binds HDAC6 in vitro, and p300 repression is relieved by histone deacetylase inhibition and siRNA-mediated ablation of HDAC6 expression. These results reveal a mechanism controlling p300 function and suggest that SUMO-dependent repression is mediated by recruitment of HDAC6 (Girdwood, 2003).

Transcriptional coactivators showing physical and functional interactions with PPARgamma include the protein acetyl transferase p300, the TRAP/Mediator complex that interacts with the general transcription machinery, and the highly regulated PGC-1alpha. PGC-1alpha directly interacts with TRAP/Mediator, through the PPARgamma-interacting subunit TRAP220, and stimulates TRAP/Mediator-dependent function on DNA templates. Further, while ineffective by itself, PGC-1alpha stimulates p300-dependent histone acetylation and transcription on chromatin templates in response to PPARgamma. These functions are mediated by largely independent PPARgamma, p300, and TRAP220 interaction domains in PGC-1alpha, whereas p300 and TRAP220 show ligand-dependent interactions with a common region of PPARgamma. Apart from showing PGC-1gamma functions both in chromatin remodeling and in preinitiation complex formation or function (transcription), these results suggest a key role for PGC-1gamma, through concerted but dynamic interactions, in coordinating these steps (Wallberg, 2003).

Nuclear receptors are ligand-inducible transcription factors that specifically regulate the expression of target genes involved in metabolism, development, and reproduction. After binding to their cognate DNA response elements, ligand-bound nuclear receptors activate target gene transcription through interactions with various coactivators and/or components of the basal transcription machinery. Coactivators are thought either to effect chromatin remodeling or to act as a bridge between gene-specific activators and the general transcription machinery (RNA polymerase II and cognate initiation or elongation factors) (Wallberg, 2003 and references therein).

Cofactors that modify chromatin structure fall mainly into two broad classes: those that regulate accessibility of nucleosomal DNA in an ATP-dependent manner and those that alter nucleosome/chromatin structure through covalent modifications (including acetylation, methylation, and phosphorylation) of the N-terminal tails of histones. Of relevance here, the latter category includes the well-characterized histone acetyltransferases (HATs) CBP/p300, PCAF/GCN5, and members of the p160/SRC family. These proteins have been shown to mediate transcriptional activity of many nuclear hormone receptors, and were originally isolated as targets of the ligand binding domains of nuclear receptors (Wallberg, 2003 and references therein).

Coactivators that act more directly on the general transcription machinery are evidenced by their ability to stimulate transcription from naked DNA templates. Among such coactivators, the phylogenetically conserved Mediator complex appears to provide the main conduit for communication between DNA-bound activators and the general transcriptional machinery. Several of the subunits in the Mediator complex have been shown to be direct targets for different activators. The human TRAP/Mediator complex (essentially equivalent to later-described complexes such as SMCC, DRIP, ARC, and NAT) was first identified through ligand-dependent interactions with thyroid hormone receptor, and subsequently shown to mediate the function of a number of nuclear receptors and other activators. Relevant to the present study, the TRAP220 subunit was shown to interact, through two NR boxes (containing LXXLL motifs), with a number of receptors that include TR and PPARγ. The involvement of TRAP220 (via the TRAP/Mediator) in TR and PPARγ function was shown most convincingly in cell-free systems reconstituted with purified factors and by assays in TRAP220 null fibroblasts (Wallberg, 2003 and references therein).

PGC-1α, now documented as a coactivator for many nuclear hormone receptors, was originally identified as a PPARγ-interacting coactivator in brown adipose tissue and has been implicated in diverse physiological processes related, in large part, to cellular metabolism. Moreover, it is highly regulated, being induced by cold in brown fat and by fasting or diabetes in the liver. PGC-1α in turn induces tissue-specific programs of metabolic control, including thermogenesis in brown fat, hepatic gluconeogenesis, and fiber-type switching in skeletal muscle. The PGC-1α interaction with PPARγ is ligand independent and mediated through an N-terminal domain (residues 200-400) and an LXXLL motif, whereas its interactions with other nuclear receptors are ligand dependent and mediated through the LXXLL motif (Wallberg, 2003 and references therein).

Although the precise mechanisms involved in its transcriptional coactivator function are not clear, PGC-1α binds, through its N-terminal 200 amino acids, to both p300/CBP and SRC-1. The PGC-1α C-terminal domain has been implicated in coupling of transcription and RNA processing on PGC-1α target genes, through interactions with splicing factors and an elongating form of RNA polymerase II, suggesting that PGC-1α might reside in both transcription preinitiation and transcription elongation complexes. However, it has not been clear whether the TRAP/Mediator complex, which also interacts with RNA polymerase II, might also play some role in facilitating PGC-1α function and, related, whether PGC-1α, like TRAP/Mediator, might act more directly to stimulate transcription by RNA polymerase II (Wallberg, 2003 and references therein).

The present study of coactivator functions in PPARγ-dependent transcription demonstrates direct interactions of PGC-1α with TRAP/Mediator via the TRAP220 subunit, TRAP/Mediator-dependent effects of PGC-1α on transcription from DNA templates, and p300-dependent effects of PGC-1α on histone acetylation and transcription from chromatin templates. These results document functions for PGC-1α, in both chromatin modification and preinitiation complex assembly/function steps, and further suggest that PGC-1α plays a key role in coordinating these events (Wallberg, 2003).

Drosophila sine oculis, eyes absent, and dachshund are essential for compound eye formation and form a gene network with direct protein interaction and genetic regulation. The vertebrate homologues of these genes, Six, Eya, and Dach, also form a similar genetic network during muscle formation. To elucidate the molecular mechanism underlying the network among Six, Eya, and Dach, the molecular interactions among the encoded proteins was examined. Eya interacts directly with Six but never with Dach. Dach transactivates a multimerized GAL4 reporter gene by coproduction of GAL4-Eya fusion proteins. Transactivation by Eya and Dach is repressed by overexpression of VP16 or E1A but not by E1A mutation, which is defective for CREB binding protein (CBP) binding. Recruitment of CBP to the immobilized chromatin DNA template is dependent on FLAG-Dach and GAL4-Eya3. These results indicate that CBP is a mediator of the interaction between Eya and Dach. Contrary to expectations, Dach binds to chromatin DNA by itself, not being tethered by GAL4-Eya3. Dach also binds to naked DNA with lower affinity. The conserved DD1 domain is responsible for binding to DNA. Transactivation was also observed by coproduction of GAL4-Six, Eya, and Dach, indicating that Eya and Dach synergy is relevant when Eya is tethered to DNA through Six protein. These results demonstrate that synergy is mediated through direct interaction of Six-Eya and through the interaction of Eya-Dach with CBP and explain the molecular basis for the genetic interactions among Six, Eya, and Dach. This work provides fundamental information on the role and the mechanism of action of this gene cassette in tissue differentiation and organogenesis (Ikeda, 2002).

Cell signaling affects gene expression by regulating the activity of transcription factors. Mitogen-activated protein kinase (MAPK) phosphorylation of Ets-1 and Ets-2 occurs at a conserved site N terminal to their Pointed (PNT) domains. This results in enhanced transactivation by preferential recruitment of the coactivators CREB binding protein (CBP) and p300. This phosphorylation-augmented interaction was discovered in an unbiased affinity chromatography screen of HeLa nuclear extracts by using either mock-treated or ERK2-phosphorylated ETS proteins as ligands. Binding between purified proteins has demonstrated a direct interaction. Both the phosphoacceptor site, which lies in an unstructured region, and the PNT domain are required for the interaction. Minimal regions that are competent for induced CBP/p300 binding in vitro also support MAPK-enhanced transcription in vivo. CBP coexpression potentiates MEK1-stimulated Ets-2 transactivation of promoters with Ras-responsive elements. Furthermore, CBP and Ets-2 interact in a phosphorylation-enhanced manner in vivo. This study describes a distinctive interface for a transcription factor-coactivator complex and demonstrates a functional role for inducible CBP/p300 binding. In addition, these findings decipher the mechanistic link between Ras/MAPK signaling and two specific transcription factors that are relevant to both normal development and tumorigenesis (Foulds, 2004).

The STAT3 NH2-terminal domain stabilizes enhanceosome assembly by interacting with the p300 bromodomain

Signal transducer and activator of transcription 3 (STAT3) is a latent transcription factor mainly activated by the interleukin-6 cytokine family. Previous studies have shown that activated STAT3 recruits p300, a coactivator whose intrinsic histone acetyltransferase activity is essential for transcription. This study investigated the function of the STAT3 NH(2)-terminal domain and how its interaction with p300 regulates STAT3 signal transduction. In STAT3(-/-) mouse embryonic fibroblasts, a stably expressed NH(2) terminus-deficient STAT3 mutant (STAT3-DeltaN) was unable to efficiently induce either STAT3-mediated reporter activity or endogenous mRNA expression. Chromatin immunoprecipitation assays were performed to determine whether the NH(2)-terminal domain regulates p300 recruitment or stabilizes enhanceosome assembly. Despite equivalent levels of STAT3 binding, cells expressing the STAT3-DeltaN mutant were unable to recruit p300 and RNA polymerase II to the native socs3 promoter as efficiently as those expressing STAT3-full length. It has been previously reported that the STAT3 NH(2)-terminal domain is acetylated by p300 at Lys-49 and Lys-87. By introducing K49R/K87R mutations, this study found that the acetylation status of the STAT3 NH(2)-terminal domain regulates its interaction with p300. In addition, the STAT3 NH(2)-terminal binding site maps to the p300 bromodomain, a region spanning from amino acids 995 to 1255. Finally a p300 mutant lacking the bromodomain (p300-DeltaB) exhibited a weaker binding to STAT3, and the enhanceosome formation on the socs3 promoter was inhibited when p300-DeltaB was overexpressed. Taken together, these data suggest that the STAT3 NH(2)-terminal domain plays an important role in the interleukin-6 signaling pathway by interacting with the p300 bromodomain, thereby stabilizing enhanceosome assembly (Hou, 2008).

STATs are cytoplasmic transcription factors that can be activated by the IL-6 cytokine family, a group of homologous peptides that include IL-6, oncostatin M (OSM), IL-11, and leukemia-inhibitory factor. The classic signaling pathway initiated by the IL-6 cytokine family is via ligand binding to cognate high affinity α chain receptors, e.g. IL-6Rα or OSMRα, that then complex with the ubiquitously expressed transmembrane protein gp130 β-subunit. The ligand-Rα-gp130 complex activates the cytoplasmic Janus and Tyk tyrosine kinases that phosphorylate the cytoplasmic domain of gp130, thereby providing docking sites for STAT1 and STAT3 isoforms. The recruited STATs are then phosphorylated by the same Janus/Tyk kinases at a specific tyrosine on the COOH-terminal transactivation domain (TAD), inducing their subsequent dimerization, nuclear translocation, and specific binding to IL-6 response elements. After the nuclear translocation of hetero- or homodimeric STATs, the magnitude of IL-6 signaling is further regulated by the recruitment of coactivators with histone acetyltransferase activity (Hou, 2008).

The crucial role of histone acetyltransferases in inducing chromatin remodeling and transcription activation has long been recognized. Several proteins with intrinsic histone acetyltransferase activity have been identified, including GCN5, p300/CREB-binding protein (CBP) homologs, p300/CBP-associated factor, and TAFII250. Histone acetyltransferases activate transcription by one or more of the following ways. (1) They are able to relax core nucleosome structure by acetylating the NH2-terminal histone tails. (2) They can directly acetylate transcription factors and alter their transcription activities (3) They function as scaffold proteins to recruit other coactivators to the transcriptional apparatus. (4) They serve as bridging factors to physically connect sequence-specific transcription factors with multiple components in the basal transcription machinery. p300 and its homolog CBP are potent transcriptional coactivators that are actively involved in all of the four processes mentioned above. They have been shown to interact with several transcription factors, such as MyoD, p53, and E2F1, and regulate their activity by reversible acetylation (Hou, 2008).

There is strong evidence demonstrating that the histone acetyltransferase activity of p300 is required for STAT3 target gene activation. Previous studies have shown that overexpression of the p300 inhibitor adenovirus 12S E1A significantly inhibits the IL-6-induced activation of human angiotensinogen (hAGT), a vasoactive peptide and acute phase protein controlled by STAT3. The ectopic expression of p300 enhances the induction of hAGT reporter gene stimulated by IL-6. Conversely expression of a p300 mutant deficient in histone acetyltransferase activity functions as a dominant-negative inhibitor and strongly inhibits STAT3-dependent transcription. Moreover IL-6-inducible p300-STAT3 association causes an increase in histone H4 acetylation on the hAGT promoter, indicating that p300 recruitment augments STAT3 transactivation by acetylating histone tails, thereby relaxing chromatin structure (Hou, 2008).

p300 interacts with STAT3 within both its COOH-terminal TAD and NH2-terminal domain, and this phenomenon has also been confirmed for STAT1 and STAT2. The STAT family shares a highly conserved modular structure that includes an NH2-terminal domain, a coiled coil domain, a DNA-binding domain, a linker domain, an SH2 domain, and a COOH-terminal TAD. The coiled coil domain is actively involved in protein-protein interaction, and the SH2 domain mediates the STAT3 dimerization via intermolecular Tyr(P)-SH2 interactions. The COOH-terminal TAD contains a conserved single tyrosine residue that is phosphorylated in STAT activation and facilitates transcriptional activation. The function of the NH2-terminal domain in STAT3, however, is poorly understood. In this study the STAT3 NH2-terminal function was investigated by stably expressing an NH2 terminus-deleted mutant (STAT3-ΔN) in STAT3-/- MEFs. Both OSM-inducible γ-FBG reporter gene and endogenous mRNA expression including socs3, c-fos, and p21 were significantly reduced in response to STAT3-ΔN expression. Because the STAT3 NH2-terminal domain is involved in p300 binding, the defective activity observed in STAT3-ΔN is probably caused by the reduced cooperation between STAT3 and p300. This hypothesis was then tested in native chromatin by chromatin immunoprecipitation (ChIP) assays that revealed a reduction in OSM-inducible p300 recruitment to the socs3 promoter in MEFs stably expressing STAT3-ΔN. At the same time, there was a decrease in RNA pol II binding to the socs3 promoter, indicating the STAT3 NH2-terminal domain not only stabilizes coactivator association but also facilitates the assembly of transcription preinitiation complex (Hou, 2008).

Recent studies identified STAT3 not only as a binding partner of p300 but also as a substrate for its acetylation where p300 modifies STAT3 at multiple sites. A single acetylation on Lys residue Lys-685 localized in the COOH-terminal TAD is required for STAT3 dimerization and the subsequent DNA binding activity. This laboratory independently identified two other Lys residues, Lys-49 and Lys-87, in the STAT3 NH2-terminal domain that are also inducibly acetylated by p300 in response to IL-6 and OSM. Although these NH2-terminal acetylations have no effect on STAT3 DNA binding activity, they are essential for STAT3-dependent transcription because K49R/K87R substitutions significantly inhibit STAT3 target gene expression. It was also noticed that the K49R/K87R mutations decrease the association between p300 and STAT3, indicating that the inducible NH2-terminal acetylations may augment STAT3-p300 interaction. This study further investigated the interaction between the STAT3 NH2-terminal domain and p300 and found that the acetyl-Lys mimic substitutions (K49Q/K87Q) increase the STAT3 NH2-terminal binding to p300, confirming the hypothesis that the NH2-terminal acetylations stabilize the STAT3-p300 interaction. It was also discovered that the STAT3 NH2-terminal binding site maps to the p300 bromodomain. The deletion of the bromodomain in p300 molecule decreased its ability to cooperate with STAT3. In addition, the bromodomain-deficient p300 mutant (p300-ΔB) exhibited weaker chromatin binding. Taken together, a model is proposed in which the IL-6- or OSM-inducible acetylations of STAT3 on Lys residues 49 and 87 trigger the recognition of the NH2-terminal domain by the p300 bromodomain, resulting in a strengthened recruitment of p300 to the promoter of the STAT3 target gene, thereby facilitating subsequent enhanceosome assembly (Hou, 2008).

Structure of p300 bound to MEF2 on DNA reveals a mechanism of enhanceosome assembly

Transcription co-activators CBP and p300 are recruited by sequence-specific transcription factors to specific genomic loci to control gene expression. A highly conserved domain in CBP/p300, the TAZ2 domain, mediates direct interaction with a variety of transcription factors including the myocyte enhancer factor 2 (MEF2). This study reports the crystal structure of a ternary complex of the p300 TAZ2 domain bound to MEF2 on DNA at 2.2A resolution. The structure reveals three MEF2:DNA complexes binding to different sites of the TAZ2 domain. Using structure-guided mutations and a mammalian two-hybrid assay, this study shows that all three interfaces contribute to the binding of MEF2 to p300, suggesting that p300 may use one of the three interfaces to interact with MEF2 in different cellular contexts and that one p300 can bind three MEF2:DNA complexes simultaneously. These studies, together with previously characterized TAZ2 complexes bound to different transcription factors, demonstrate the potency and versatility of TAZ2 in protein-protein interactions. These results also support a model wherein p300 promotes the assembly of a higher-order enhanceosome by simultaneous interactions with multiple DNA-bound transcription factors (He, 2011).

CBP and development

A Caenorhabditis elegans gene closely related to CBP/p300, referred to as cbp-1, is required during early embryogenesis to specify several major differentiation pathways. There is extensive sequence conservation throughout the whole protein between CBP/p300 and CBP-1. The largest block of a highly conserved region between human CBP/p300 and CBP-1 spans amino acids 861-1757 of CBP-1. This region contains several important functional domains defined in CBP/p300 including the histone acetyltransferase (HAT) domain, the region that is critical for binding to E1A and the HAT, P/CAF, as well as the bromodomain and the C/H 2 domain that mediate p300 interaction with ATF-2. Between CBP and CBP-1, sequence comparison shows a 51% amino acid identity in the HAT domain and 70% in the region important for E1A and P/CAF interactions. The bromodomain and the C/H2 domain show 61% and 50% amino acid identity, respectively. Another functional domain that is highly conserved is the KIX domain of CBP, which binds transcription factor CREB; it shares 70% amino acid identity with CBP-1 (Shi, 1998).

CBP-1 protein is first detectable in nuclei beginning at the two-cell stage; this ubiquitous nuclear staining persists at least through the 100-cell stage of embryogenesis. The fact that CBP-1 protein is present at the two-cell stage, before the earliest detected zygotic transcription, suggests that CBP-1 is likely to be a maternally expressed protein in C. elegans (Shi, 1998).

A reverse genetic assay, termed RNAi, was used to determine the phenotypic consequences of blocking CBP-1 expression. Several recent studies have shown that an RNA injection procedure induces phenotypes that appear identical to those that are known to result from strong or null genetic mutations in the genes tested. These RNA-induced phenotypes have been correlated with a lack of protein expression from the targeted genes. For the RNAi assay, in vitro transcribed RNA from a cbp-1 cDNA clone was injected into wild-type adult hermaphrodites. Beginning a few hours after injection, wild-type hermaphrodites that receive cbp-1 RNA produce exclusively inviable embryos. Strikingly, these terminal embryos arrest development with nearly twice the normal complement of cells, and most embryos completely lack evidence of muscle, intestinal, and hypodermal differentiation. In contrast, the germ cells seem unaffected and appear morphologically normal, showing proper localizations for the germ-line-specific P-granules. The numerous small cells produced in these embryos most closely resemble neurons, which led to the hypothesis that many of the cells that would have differentiated into other tissues in normal embryos have instead adopted neuronal fates. Analysis of this phenotype suggests a critical role for CBP-1 in promoting all non-neuronal pathways of somatic differentiation in the C. elegans embryo (Shi, 1998).

Interfering with expression of HDAC1and RbAp48-related genes can partially suppress the differentiation defects of worms subject to the RNAi treatment. C. elegans hda-1 is related to the mammalian HDAC1 component of histone deacetylase. Two open reading frames were identified that share extensive sequence homology with RbAp48, RbAp46, an RbAp48-related protein in mammalian cells, and with p55, the RbAp48 homolog in Drosophila (known as Chromatin assembly factor 1 subunit). These two C. elegans genes are referred to as rba-1 and rba-2, for RbAp48-related genes. RBA-1 shares 53% amino acid identity and 63% amino acid similarity with RbAp48, 52% and 63% with RbAp46, and 53% and 62% with Drosophila Chromatin assembly factor 1 subunit. RBA-2 shares 72% amino acid identity and 79% amino acid similarity with RbAp48, 71% and 80% with RbAp46, and 72% and 81% with Drosophila Chromatin assembly factor 1 subunit (Shi, 1998).

The endoderm differentiation defects observed in embryos subject to cbp-1 RNAi treatment are similar to those caused by mutations in the transcription factor SKN-1, a maternally expressed gene required to specify the fate of ventral blastomeres in C. elegans embryos. This suggests that CBP-1 may function with SKN-1 or downstream transcription factors to specify endoderm differentiation. Such a hypothesis would predict that inhibiting HDA-1 expression in the skn-1 mutant background might suppress the skn-1 mutant phenotype. Endoderm differentiation is restored completely by inhibition of hda-1 expression in skn-1 mutant embryos. Similarly, inhibiting the expression of either rba-1 or rba-2 also completely restored endoderm differentiation in the skn-1 mutant. These findings suggest a model in which CBP-1 may activate transcription and differentiation in C. elegans by directly or indirectly antagonizing a repressive effect for histone deacetylase (Shi, 1998).

Terminal differentiation of muscle cells follows a precisely orchestrated program of transcriptional regulatory events at the promoters of both muscle-specific and ubiquitous genes. Two distinct families of transcriptional co-activators, GCN5/PCAF and CREB-binding protein (CBP)/p300, are crucial to this process. While both possess histone acetyl-transferase (HAT) activity, previous studies have failed to identify a requirement for CBP/p300 HAT function in myogenic differentiation. This issue has been addressed directly using a chemical inhibitor of CBP/p300 in addition to a negative transdominant mutant. CBP/p300 HAT activity is critical for myogenic terminal differentiation. Furthermore, this requirement is restricted to a subset of events in the differentiation program: cell fusion and specific gene expression. These data help to define the requirements for enzymatic function of distinct coactivators at different stages of the muscle cell differentiation program (Polesskaya, 2001).

The coactivators CBP and its paralog p300 are thought to supply adaptor molecule and protein acetyltransferase functions to many transcription factors that regulate gene expression. Normal development requires CBP and p300, and mutations in these genes are found in hematopoietic and epithelial tumors. It is unclear, however, which functions of CBP and p300 are essential in vivo. The protein-binding KIX domains of CBP and p300 have nonredundant functions in mice. In mice homozygous for point mutations in the KIX domain of p300 designed to disrupt the binding surface for the transcription factors c-Myb and CREB, multilineage defects occur in hematopoiesis, including anaemia, B-cell deficiency, thymic hypoplasia, megakaryocytosis and thrombocytosis. By contrast, age-matched mice homozygous for identical mutations in the KIX domain of CBP are essentially normal. There is a synergistic genetic interaction between mutations in c-Myb and mutations in the KIX domain of p300, which suggests that the binding of c-Myb to this domain of p300 is crucial for the development and function of megakaryocytes. Thus, conserved domains in two highly related coactivators have contrasting roles in hematopoiesis (Kasper, 2002).

Impaired terminal differentiation is found in interfollicular keratinocytes of p107/p130-double-null mice epidermis. A decreased number of hair follicles and a clear developmental delay is seen in hair, whiskers and tooth germs. Skin grafts of p107/p130-deficient epidermis onto NOD/scid mice show altered differentiation and hyperproliferation of the interfollicular keratinocytes, thus demonstrating that the absence of p107 and p130 results in the deficient control of differentiation in keratinocytes in a cell-autonomous manner. Follicular cysts, misoriented and dysplastic follicles, together with aberrant hair cycling, are also observed in the p107/p130 skin transplants, in addition to normal hair formation. Finally, the hair abnormalities in p107/p130-null skin are associated with altered Bmp4-dependent signaling, including decreased DeltaNp63 expression, an ectodermal specific Bmp4 target. These results indicate an essential role for p107 and p130 in the epithelial-mesenchimal interactions (Ruiz, 2003).

CBP regulates the differentiation of interneurons from ventral forebrain neural precursors during murine development

The mechanisms that regulate appropriate genesis and differentiation of interneurons in the developing mammalian brain are of significant interest not only because interneurons play key roles in the establishment of neural circuitry, but also because when they are deficient, this can cause epilepsy. In this regard, one genetic syndrome that is associated with deficits in neural development and epilepsy is Rubinstein-Taybi Syndrome (RTS), where the transcriptional activator and histone acetyltransferase CBP is mutated and haploinsufficient. This study has asked whether CBP is necessary for the appropriate genesis and differentiation of interneurons in the murine forebrain, since this could provide an explanation for the epilepsy that is associated with RTS. CBP is expressed in neural precursors within the embryonic medial ganglionic eminence (MGE), an area that generates the vast majority of interneurons for the cortex. Using primary cultures of MGE precursors, this study shows that knockdown of CBP causes deficits in differentiation of these precursors into interneurons and oligodendrocytes, and that overexpression of CBP is by itself sufficient to enhance interneuron genesis. Moreover, it was shown that levels of the neurotransmitter synthesis enzyme GAD67, which is expressed in inhibitory interneurons, are decreased in the dorsal and ventral forebrain of neonatal CBP+/- mice, indicating that CBP plays a role in regulating interneuron development in vivo. Thus, CBP normally acts to ensure the differentiation of appropriate numbers of forebrain interneurons, and when its levels are decreased, this causes deficits in interneuron development, providing a potential explanation for the epilepsy seen in individuals with RTS (Tsui, 2104).

Transcriptional integration of Wnt and Nodal pathways in establishment of the Spemann organizer: the histone acetyltransferase p300 is recruited to organizer promoters in a Wnt and Nodal effector-dependent manner

Signaling inputs from multiple pathways are essential for the establishment of distinct cell and tissue types in the embryo. Therefore, multiple signals must be integrated to activate gene expression and confer cell fate, but little is known about how this occurs at the level of target gene promoters. During early embryogenesis, Wnt and Nodal signals are required for formation of the Spemann organizer, which is essential for germ layer patterning and axis formation. Signaling by both Wnt and Nodal pathways is required for the expression of multiple organizer genes, suggesting that integration of these signals is required for organizer formation. This study demonstrates transcriptional cooperation between the Wnt and Nodal pathways in the activation of the organizer genes Goosecoid (Gsc), Cerberus (Cer), and Chordin (Chd). Combined Wnt and Nodal signaling synergistically activates transcription of these organizer genes. Effectors of both pathways occupy the Gsc, Cer and Chd promoters and effector occupancy is enhanced with active Wnt and Nodal signaling. This suggests that, at organizer gene promoters, a stable transcriptional complex containing effectors of both pathways forms in response to combined Wnt and Nodal signaling. Consistent with this idea, the histone acetyltransferase p300 is recruited to organizer promoters in a Wnt and Nodal effector-dependent manner. Taken together, these results offer a mechanism for spatial and temporal restriction of organizer gene transcription by the integration of two major signaling pathways, thus establishing the Spemann organizer domain (Reid, 2012).

The Wnt and Nodal pathways cooperate to activate transcription of the organizer genes Gsc, Cer, and Chd utilizing adjacent Wnt and Nodal responsive cis-regulatory elements present in the proximal promoters close to the start site of transcription. Functional conservation of these promoters is apparent in the sequence of the response elements, the proximity of the two elements, and their distance from the start site of transcription. The Sia/Twn response is mediated by defined P3 elements present in each of the promoters. Elements mediating the FoxH1-dependent response to Nodal signals have been identified in close proximity to the Sia/Twn elements of each promoter, but are less conserved in sequence. For Gsc, Cer and Chd, the two response elements are in close proximity and are separated by no more than 43 bp. And in each case, the pair of response elements has a strikingly similar location within 250 bp of the start site of transcription. These similar features of three organizer gene promoters argue for functional conservation in mediating the transcriptional response to Wnt and Nodal signaling inputs (Reid, 2012).

At enhancer regions, multiple bound transcription factors may interact to synergistically activate a strong transcriptional output. A number of mechanisms may account for synergy, including cooperative binding to regulatory elements, cooperative recruitment of coactivators, as well as alterations in DNA conformation or nucleosome deposition. The synergy in activation of Gsc, Cer, and Chd may reflect one or several of these mechanisms. While it remains unclear whether cooperative binding is occurring among the Wnt and Nodal effectors, the data clearly demonstrate that the steady state binding of transcriptional effectors is increased when both Wnt and Nodal pathway effectors occupy these promoters . This suggests that the presence of Sia/Twn with FoxH1 and Smad2/3 at organizer gene promoters facilitates enhanced occupancy, which is suggestive of cooperative binding (Reid, 2012).

The common coactivator and lysine acetyltransferase, p300, is recruited to organizer gene promoters in response to both the Wnt and Nodal pathways. The role that p300 plays in the synergistic transcription of organizer genes in response to Wnt and Nodal is not yet understood. Overexpression of p300 alone has no apparent phenotype, suggesting that increasing p300 levels does not alter expression of target genes. The results demonstrate a requirement for p300 activity in the expression of a Gsc reporter, as well as increased occupancy of p300 at organizer promoters in the presence of Sia/Twn or Nodal signals. However, no further enhancement of p300 occupancy is observed in response to the combination of Wnt and Nodal. Perhaps p300 provides a permissive function for transcription, while other recruited coactivators provide an activating function. Similarly, p300 could be acting as a scaffolding protein, either stabilizing a transcriptional complex of both Wnt and Nodal effectors, or allowing effectors to interact with other coactivators and/or the basal transcriptional machinery. The combined effects of Wnt and Nodal inputs could enhance p300 enzymatic activity, resulting in more extensive modification of local histones or transcription factors and increased transcription. In the context of organizer gene expression, changes in histone H3K9/14 or H4K5/8/12/16 acetylation have not been observed in response to Wnt or Nodal signals. However, p300 is also known to modify other lysine residues in histone tails, such as H3K18/27, as well as transcription factors. Activated Smad2/3 is acetylated by p300, which increases transcriptional activity. Preliminary results indicate that Sia is acetylated, however, it is unclear what role acetylation might play in Sia-dependent transcription, or whether other Nodal or Wnt effectors might be acetylated in a signal-dependent manner (Reid, 2012).

It is difficult to relate the experimental induction of organizer gene expression with combinations of Sia/Twn and Nodal to the natural activation of these genes in the intact embryo. It is hypothesized that the temporal and spatial restriction of organizer gene expression is due, at least in part, to the presence of Sia, Twn and Nodal effectors in the cells of the organizer. However, the increase in organizer gene expression observed in response to Sia+Xnr1 or Twn+Xnr1 is much greater than the endogenous expression levels of Gsc, Chd or Cer in the whole embryo. Similarly, expression of the Gsc-luciferase reporter in dorsal blastomeres results in an approximately 10-fold increase in luciferase activity, which is much lower than the nearly 36 to 48-fold induction observed in response to Sia+Xnr1 or Twn+Xnr1. It is hypothesized that an increase in ectopic axis formation would be observed in response to low doses of Sia+Xnr1 or Twn+Xnr1, but consistent results were not obtained. This issue might be more clearly addressed by timed loss of function experiments to specifically inhibit Sia/Twn or Nodal activity during organizer formation. It also seems likely that a number of other transcription factors, such as specific repressors of organizer gene expression may be involved in the formation of the organizer domain (Reid, 2012).

This work has defined a molecular mechanism for the transcriptional integration of Wnt and Nodal signals at organizer gene promoters in the Xenopus gastrula. It is further proposed that this mechanism is likely utilized in multiple vertebrate species to establish the organizer transcriptional domain. Support for the conservation of this mechanism across vertebrates comes from regulatory similarities in organizer formation, organizer gene expression and organizer gene promoter structure. Wnt and Nodal signals are essential for organizer gene expression and organizer formation in Xenopus, zebrafish, chick and mouse. The functional organization of organizer gene promoters is also conserved to an extent. Most strikingly in the case of Gsc, highly conserved DE and PE elements are present in the Xenopus, zebrafish, chick, mouse, and human Gsc genes. For Cer, conserved response elements are present in Xenopus, zebrafish and mouse, but their organization differs among species. For Chd, the available genomic information is insufficient for a conclusive comparison. The effectors of Nodal signaling, FoxH1 and Smad2/3, are also utilized in the control of organizer gene transcription in these vertebrate systems (Reid, 2012).

In contrast to these many conserved features of organizer gene regulation, Sia and Twn are only found in amphibian species, and not in other vertebrates. Given that Wnt inputs and the PE element are conserved across species, it is likely that functional homologs of Sia/Twn, mediating the Wnt-dependent transcriptional activation via the PE, exist in other vertebrate species. Alternatively, Sia/Twn may serve a regulatory function that is unique to organizer gene regulation in Xenopus; if this is the case, conservation of the PE may reflect distinct regulatory requirements among species. It should be noted that Sia/Twn are not the only species-specific regulators of organizer formation. In zebrafish, the transcriptional repressor bozozok is a direct target of the Wnt pathway, is expressed early in organizer formation, and is essential for organizer gene expression and organizer formation. However, as is the case for Sia/Twn, no vertebrate orthologs of bozozok have been identified. Whether functional homologs of Sia/Twn and bozozok exist in other species or whether these factors carry out species-specific regulatory functions remains to be seen. Given the dramatically different sizes and developmental rates for vertebrate embryos, and the non-autonomous function of the organizer, temporal and spatial constraints for organizer formation may differ among species. The non-conserved regulatory components found in Xenopus and zebrafish may be necessary for the unique regulatory demands of organizer formation in distinct species (Reid, 2012).

A number of important aspects of organizer gene regulation remain undefined. The full composition and structure of the activating protein complex, which forms at organizer gene promoters, is yet to be defined. How the Wnt and Nodal pathway effectors interact physically, what modifications occur in response to cofactor recruitment, and how together these result in enhanced, yet spatially restricted transcriptional output, are important mechanistic questions to pursue. The results offer a molecular mechanism for the initiation of organizer gene expression in a spatially and temporally precise manner. However, organizer gene expression is a dynamic process with changing regulatory inputs as development proceeds. Within 60 min of the initiation of organizer gene expression it is likely that promoter occupancy and regulatory complex composition changes dramatically as the initiation phase gives way to the maintenance phase or cell lineage specification. Whether the mechanism that are proposed in this paper for the initiation of organizer gene expression is broadly applicable to the many known organizer genes, and across species as well, will require genome wide analyses of effector occupancy, coregulator recruitment, and chromatin modification in several vertebrate species. Ongoing studies such as these will provide profound mechanistic insight at the interface of transcriptional control and embryonic pattern formation (Reid, 2012).

Cells within the organizer domain receive Wnt and Nodal signals and integrate these signals to generate temporally and spatially specific transcriptional responses. Wnt and Nodal inputs are directly received at multiple organizer gene promoters, and functional interactions among pathway effectors result in strong transcriptional activation of organizer genes. Integration of these signals is accomplished by assembly of an activating complex, consisting of Sia, Twn, FoxH1, Smad2/3, and p300 at the Gsc, Cer, and Chd promoters. In the late blastula, cells receiving both Wnt and Nodal inputs integrate these signals at the level of organizer gene promoters, thus establishing a temporally and spatially distinct transcription domain, resulting in formation of the Spemann organizer (Reid, 2012).

CBP and memory

The stabilization of learned information into long-term memories requires new gene expression. CREB binding protein (CBP) is a coactivator of transcription that can be independently regulated in neurons. CBP functions both as a platform for recruiting other required components of the transcriptional machinery and as a histone acetyltransferase (HAT) that alters chromatin structure. To dissect the chromatin remodeling versus platform function of CBP or the developmental versus adult role of this gene, transgenic mice were generated that express CBP in which HAT activity is eliminated. Acquisition of new information and short-term memory still occurs in these mice, while the stabilization of short-term memory into long-term memory is impaired. The behavioral phenotype is due to an acute requirement for CBP HAT activity in the adult; it is rescued by both suppression of transgene expression or by administration of the histone deacetylase inhibitor Trichostatin A (TSA) in adult animals (Korzus, 2004).

It is suggested that CBP acetyltransferase activity is critical for activation of genes controlling memory consolidation. It is well known that the transcription factor CREB is required for long-term memory consolidation and that CREB phosphorylation at S133 is necessary to recruit CBP and for subsequent transcriptional activation. However, behaviorally induced CREB phosphorylation is transient and does not correlate with peak c-fos induction. Moreover, S133 phosphorylation of CREB alone is not sufficient to induce transcription. NMDA-dependent phosphorylation of S301 on CBP has been also shown to be required for CBP-dependent gene expression. This suggests that a separate signaling pathway directly to CBP must be activated in neurons to allow normal CREB-mediated gene activation, as well as other non-CREB-dependent genes that use CBP as a coactivator. The current results suggest that this CBP pathway is also critical for memory consolidation by recruitment of functional CBP histone acetyltransferase activity and possible alteration of local chromatin structure. In addition to its chromatin remodeling function, it is possible that nuclear nonhistone substrates for CBP acetyltransferase activity might be critical during memory consolidation. This activation of CBP-mediated acetylation at specific gene targets could serve to alter the subsequent requirements for transcriptional activation of those genes in response to future cellular signals. This sort of the acetylation-mediated covalent modification would open up a temporal window in which cellular signals, which did not recruit an active acetyltransferase to the promoter, would nevertheless stimulate transcription as shown in Figure 6C. It is possible that after initial CBP-mediated integration of short-lasting neuronal signals leading to acetylation-dependent alteration of local chromatin structure, CBP HAT or even CBP platform function may become dispensable for subsequent steps. In fact, CBP-independent activation of CREB (or CREM, a CREB family member), which is mediated by other coactivators and bypasses the classical requirements for phosphorylation of CREB/CREM and interaction with CBP, has been described. This could allow for prolonged elevation of transcription in response to an initial learning event by maintaining transcription even after signals to CREB and CBP were no longer present (Korzus, 2004).


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date revised: 10 March 2018

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