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
|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
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.
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).
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).
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).
The deduced amino-acid sequence of Drosophila CBP predicts a protein of 332K relative molecular mass, which includes all the motifs found in the mammalian CBP/p300 homologs (Akimaru, 1997a).
date revised: 20 January 99
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