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EVOLUTIONARY HOMOLOGS

Table of contents

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.

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