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EVOLUTIONARY HOMOLOGS

Table of contents

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, 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).

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).

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 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).

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