Histone acetyltransferases, which are able to acetylate histone and non-histone proteins, play important roles in gene regulation. Many histone acetyltransferases are related to yeast Gcn5, a component of two transcription regulatory complexes SAGA (Spt-Ada-Gcn5-Acetyltransferase) and ADA. In this work, by characterizing a mutation in the Arabidopsis GCN5 gene (AtGCN5) the regulatory function of this gene was studied in controlling floral meristem activity. In addition to pleiotropic effects on plant development, this mutation also leads to the production of terminal flowers. The flowers show homeotic transformations of petals into stamens and sepals into filamentous structures and produce ectopic carpels. The phenotypes correlate to an expansion of the expression domains within floral meristems of the key regulatory genes WUSCHEL (WUS) and AGAMOUS (AG). These results suggest that AtGCN5 is required to regulate the floral meristem activity through the WUS/AG pathway. This study brings new elements on the elucidation of specific developmental pathways regulated by AtGCN5 and on the control mechanism of meristem regulatory gene expression (Bertrand, 2003).
Arabidopsis genes homologous with the yeast ADA2 and GCN5 genes have been identified that encode components of the ADA and SAGA histone acetyltransferase complexes. The biological roles of the Arabidopsis ADA2b and GCN5 genes have been explored. T-DNA insertion mutations in ADA2b and GCN5 have pleiotropic effects on plant growth and development, including dwarf size, aberrant root development, and short petals and stamens in flowers. Approximately 5% of the 8200 genes assayed by DNA microarray analysis showed changes of expression in the mutants, three-fourths of which were upregulated and only half of which were altered similarly in the two mutant strains. In cold acclimation experiments, C-repeat binding factors (CBFs) were induced in the mutants as in wild-type plants, but subsequent transcription of cold-regulated (COR) genes was reduced in both mutants. Remarkably, nonacclimated ada2b-1 (but not gcn5-1) mutant plants were more freezing tolerant than nonacclimated wild-type plants, suggesting that ADA2b may directly or indirectly repress a freezing tolerance mechanism that does not require the expression of CBF or COR genes. It is concluded that the Arabidopsis ADA2b and GCN5 proteins have both similar and distinct functions in plant growth, development, and gene expression and may be components of both a common coactivator complex and separate complexes with distinct biological activities (Vlachonasios, 2003).
The cloning of a transcription-associated histone acetyltransferase type A(HAT A) is reported. This Tetrahymena enzyme is strikingly homologous to the yeast protein Gcn5, a putative transcriptional adaptor, and recombinant Gcn5p is demonstrated to possess HAT activity. Both the ciliate enzyme and Gcn5p contain potential active site residues found in other acetyltransferases and a highly conserved bromodomain. The presence of this domain in nuclear A-type HATs, but not in cytoplasmic B-type HATs, suggests a mechanism whereby HAT A is directed to chromatin to facilitate transcriptional activation. These findings shed light on the biochemical function of the evolutionarily conserved Gcn5p-Ada complex, directly linking histone acetylation to gene activation, and indicate that histone acetylation is a targeted phenomenon (Brownell, 1996).
A selection for yeast mutants resistant to GAL4-VP16-induced toxicity previously identified two genes, ADA2 and ADA3, which may function as adaptors for some transcriptional activation domains and thereby facilitate activation. Two new genes has been identified by the same selection, one of which is identical to GCN5. gcn5 mutants share properties with ada mutants, including slow growth, temperature sensitivity and reduced activation by the VP16 and GCN4 activation domains. Double mutant studies suggest that ADA2 and GCN5 function together in a complex or pathway. Moreover, GCN5 binds to ADA2 both by the two-hybrid assay in vivo and by co-immunoprecipitation in vitro. This suggests that ADA2 and GCN5 are part of a heteromeric complex that mediates transcriptional activation. Finally, this study demonstrates the functional importance of the bromodomain of GCN5, a sequence found in other global transcription factors such as the SWI/SNF complex and the TATA binding protein-associated factors. This domain is not required for the interaction between GCN5 and ADA2 and thus may mediate a more general activity of transcription factors (Marcus, 1994).
Putative transcriptional adaptor proteins are found in eukaryotes from yeast to humans and are required for full function of many eukaryotic acidic activators. To study their functional interactions, deletion mutations in the yeast adaptors ADA2, GCN5, and ADA3 were created. A region has been defined within the middle of GCN5 required for interaction with ADA2 in vitro. Regions of ADA2 required for function in vivo have been defined and whether these same regions are involved in physical interaction of ADA2 with GCN5 or ADA3 has been determined in vitro. Two regions are crucial for ADA2 function in vivo, the amino terminus and a middle region. Immunoprecipitation analysis shows that the amino terminus of ADA2 is required for interaction with GCN5, while a region in the middle of ADA2 is necessary for interaction with ADA3. Deletions of the region that is required for interaction with ADA3 abolishes dependence of lexA-ADA2 transcriptional activity on ADA3. Moreover, using coimmunoprecipitation analysis, physical interaction between ADA2, ADA3, and GCN5 has been demonstrated in yeast extracts. Taken together, the physical interaction in vivo, along with the correlation observed between regions of ADA2 required for in vitro interaction with GCN5 and ADA3, and regions required for function in vivo, argue for the existence of a physiologically relevant adaptor complex (Candau, 1996).
Yeast GCN5 is one component of a putative adaptor complex that includes ADA2 and ADA3 and functionally connects DNA-bound transcriptional activators with general transcription factors. GCN5 possesses histone acetyltransferase (HAT) activity, conceptually linking transcriptional activation with enzymatic modification at chromatin. The minimal catalytic domain within GCN5 necessary to confer HAT activity has been identified; in vivo activity of GCN5 is shown to require this domain. However, complementation of growth and transcriptional activation in gcn5- cells requires not only the HAT domain of GCN5, but also interaction with ADA2. The bromodomain in GCN5 is dispensable for HAT activity and for transcriptional activation by strong activators; however, it was required for full complementation in other assays. Fusion of GCN5 to the bacterial lexA DNA binding domain activates transcription in vivo, and requires both the HAT domain and the ADA2 interaction domain. These results suggest that both functions of GCN5, HAT activity and interaction with ADA2, are necessary for targeting and acetylation of nucleosomal histones (Candau, 1997).
The transcriptional adaptor protein Gcn5 has been identified as a nuclear histone acetyltransferase (HAT). Although recombinant yeast Gcn5 efficiently acetylates free histones, it fails to acetylate histones contained in nucleosomes, indicating that additional components are required for acetylation of chromosomal histones. Gcn5 functions as a catalytic subunit in two high-molecular-mass native HAT complexes, with apparent molecular masses of 0.8 and 1.8 megadalton (MD), respectively, which acetylate nucleosomal histones. Both the 0.8- and 1.8-MD Gcn5-containing complexes cofractionate with Ada2 and are lost in gcn5delta, ada2delta, or ada3delta yeast strains, illustrating that these HAT complexes are bona fide native Ada-transcriptional adaptor complexes. Importantly, the 1.8-MD adaptor/HAT complex also contains Spt gene products that are linked to TATA-binding protein (TBP) function. This complex is lost in spt20/ada5delta and spt7delta strains and Spt3, Spt7, Spt20/Ada5, Ada2, and Gcn5 all copurify with this nucleosomal HAT complex. Therefore, the 1.8-MD adaptor/HAT complex illustrates an interaction between Ada and Spt gene products and confirms the existence of a complex containing the TBP group of Spt proteins as demonstrated by genetic and biochemical studies. This novel transcription regulatory complex has been named SAGA (Spt-Ada-Gcn5-Acetyltransferase). The function of Gcn5 as a histone acetyltransferase within the Ada and SAGA adaptor complexes indicates the importance of histone acetylation during steps in transcription activation mediated by interactions with transcription activators and general transcription factors (i.e., TBP) (Grant, 1997).
The yeast transcriptional adaptor, Gcn5p, is a catalytic subunit of a nuclear (type A) histone acetyltransferase linking histone acetylation to gene activation. Gcn5p acetylates histones H3 and H4 non-randomly at specific lysines in the amino-terminal domains. Lysine 14 of H3 and lysines 8 and 16 of H4 are highly preferred acetylation sites for Gcn5p. Lysine 9 is the preferred position of acetylation in newly synthesized yeast H3 in vivo. This finding, along with the fact that lysines 5 and 12 in H4 are predominant acetylation sites during chromatin assembly of many organisms, indicates that Gcn5p acetylates a distinct set of lysines that do not overlap with those sites characteristically used by type B histone acetyltransferases for histone deposition and chromatin assembly (Kuo, 1996).
The coactivator/adaptor protein Gcn5 is a conserved histone acetyltransferase, which functions as the catalytic subunit in multiple yeast transcriptional regulatory complexes. The ability of Gcn5 to acetylate nucleosomal histones is significantly reduced relative to its activity on free histones, where it predominantly modifies histone H3 at lysine 14. However, the association of Gcn5 in multisubunit complexes potentiates its nucleosomal histone acetyltransferase activity. The association of Gcn5 with other proteins in two native yeast complexes, Ada and SAGA (Spt-Ada-Gcn5-acetyltransferase), directly confers upon Gcn5 the ability to acetylate an expanded set of lysines on H3. Furthermore Ada and SAGA have overlapping, yet distinct, patterns of acetylation, suggesting that the association of specific subunits determines site specificity (Grant, 1999).
Whereas the histone acetyltransferase activity of yeast Gcn5p has been widely studied, its structural interactions with the histones and the role of the carboxy-terminal bromodomain are still unclear. Using a glutathione S-transferase pull down assay this study shows that Gcn5p binds the amino-terminal tails of histones H3 and H4, but not H2A and H2B. The deletion of bromodomain abolishes this interaction and bromodomain alone is able to interact with the H3 and H4 N termini. The amino acid residues of the H4 N terminus involved in the binding with Gcn5p have been studied by site-directed mutagenesis. The substitution of amino acid residues R19 or R23 of the H4 N terminus with a glutamine (Q) abolishes the interaction with Gcn5p and the bromodomain. These residues differ from those known to be acetylated or to be involved in binding the SIR proteins. This evidence and the known dispensability of the bromodomain for Gcn5p acetyltransferase activity suggest a new structural role for the highly evolutionary conserved bromodomain (Ornaghi, 1999).
SAGA is important for transcription in vivo and possesses histone acetylation function. This study reports both biochemical and genetic analyses of members of three classes of transcription regulatory factors contained within the SAGA complex. A correlation is demonstrated between the phenotypic severity of SAGA mutants and SAGA structural integrity. Specifically, null mutations in the Gcn5/Ada2/Ada3 or Spt3/Spt8 classes cause moderate phenotypes and subtle structural alterations, while mutations in a third subgroup, Spt7/Spt20, as well as Ada1, disrupt the complex and cause severe phenotypes. Interestingly, double mutants (gcn5Delta spt3Delta and gcn5Delta spt8Delta) causing loss of a member of each of the moderate classes have severe phenotypes, similar to spt7Delta, spt20Delta, or ada1Delta mutants. Two biochemical functions suggested by the moderate phenotypic classes were investigated. (1) Normal nucleosomal acetylation by SAGA requires a specific domain of Gcn5, termed the bromodomain. Deletion of this domain also causes specific transcriptional defects at the HIS3 promoter in vivo. (2) SAGA interacts with TBP, the TATA-binding protein, and this interaction requires Spt8 in vitro. Overall, these data demonstrate that SAGA harbors multiple, distinct transcription-related functions, including direct TBP interaction and nucleosomal histone acetylation. Loss of either of these causes slight impairment in vivo, but loss of both is highly detrimental to growth and transcription (Sterner, 1999).
Regulation of eukaryotic gene expression requires ATP-dependent chromatin remodeling enzymes, such as SWI/SNF, and histone acetyltransferases, such as Gcn5p. SWI/SNF remodeling controls recruitment of Gcn5p HAT activity to many genes in late mitosis and these chromatin remodeling enzymes play a role in regulating mitotic exit. In contrast, interphase expression of GAL1, HIS3, PHO5, and PHO8 is accompanied by SWI/SNF-independent recruitment of Gcn5p HAT activity. Surprisingly, prearresting cells in late mitosis imposes a requirement for SWI/SNF in recruiting Gcn5p HAT activity to the GAL1 promoter, and GAL1 expression also becomes dependent on both chromatin remodeling enzymes. It is proposed that SWI/SNF and Gcn5p are globally required for mitotic gene expression due to the condensed state of mitotic chromatin (Krebs, 2000).
Elp3 and Gcn5 are histone acetyltransferases (HATs) that function in transcription as subunits of Elongator and SAGA/ADA, respectively. Nutations that impair the in vitro HAT activity of Elp3 confer typical elp phenotypes such as temperature sensitivity. Combining an elp3Delta mutation with histone H3 or H4 tail mutations confers lethality or sickness, supporting a role for Elongator in chromatin remodelling in vivo. gcn5Deltaelp3Delta double mutants display a number of severe phenotypes, and similar phenotypes result from combining the elp mutation with mutation in a gene encoding a SAGA-specific, but not an ADA-specific subunit, indicating that Elongator functionally overlaps with SAGA. Because concomitant active site alterations in Elp3 and Gcn5 are sufficient to confer severe phenotypes, the redundancy must be specifically related to the HAT activity of these complexes. In support of this conclusion, gcn5Deltaelp3Delta phenotypes are suppressed by concomitant mutation of the HDA1 and HOS2 histone deacetylases. These results demonstrate functional redundancy among transcription-associated HAT and deacetylase activities, and indicate the importance of a fine-tuned acetylation-deacetylation balance during transcription in vivo (Wittschieben, 2000).
Promoter-specific recruitment of histone acetyltransferase activity is often critical for transcriptional activation. A detailed study of the interaction between the histone acetyltransferase complexes SAGA and NuA4, and transcription activators is presented. It is demonstrated by affinity chromatography and photo-cross-linking label transfer that acidic activators directly interact with Tra1p, a shared subunit of SAGA and NuA4. Mutations within the COOH-terminus of Tra1p disrupted its interaction with activators and resulted in gene-specific transcriptional defects that correlated with lowered promoter-specific histone acetylation. These data demonstrate that the essential Tra1 protein serves as a common target for activators in both SAGA and NuA4 acetyltransferases (Brown, 2001).
Transcription is regulated through chromatin remodeling and histone modification, mediated by large protein complexes. Histone and nucleosome interaction has been shown to be mediated by specific chromatin domains called bromodomains and chromodomains. Evidence for a similar function of two additional domains within the yeast SAGA complex, containing the histone acetyltransferase Gcn5. Deletion and substitution mutations within Gcn5 and Ada2, an interacting protein within SAGA, have been studied, substrate recognition functions have been identified within the SANT domain of Ada2 and regions of the histone acetyltransferase domain of Gcn5 that are distinct from catalytic function itself. These results suggest that histone and nucleosomal substrate recognition by SAGA involves multiple conserved domains and proteins, beyond those that have been previously identified (Sterner, 2002).
The SANT domain is a novel motif found in a number of eukaryotic transcriptional regulatory proteins that was identified based on its homology to the DNA binding domain of c-myb. The SANT domain is essential for the in vivo functions of yeast Swi3p, Ada2p, and Rsc8p, subunits of three distinct chromatin remodeling complexes. The Ada2p SANT domain is essential for histone acetyltransferase activity of native, Gcn5p-containing HAT complexes. Furthermore, kinetic analyses indicate that an intact SANT domain is required for an Ada2p-dependent enhancement of histone tail binding and enzymatic catalysis by Gcn5p. These results are consistent with a general role for SANT domains in functional interactions with histone N-terminal tails (Boyer, 2002).
Histone phosphorylation influences transcription, chromosome condensation, DNA repair and apoptosis. Histone H3 Ser10 phosphorylation (pSer10) by the yeast Snf1 kinase regulates INO1 gene activation in part via Gcn5/SAGA complex-mediated Lys14 acetylation (acLys14). How such chromatin modification patterns develop is largely unexplored. This study examines the mechanisms surrounding pSer10 at INO1, and at GAL1, which herein is identified as a new regulatory target of Snf1/pSer10. Snf1 behaves as a classic coactivator in its recruitment by DNA-bound activators, and in its role in modifying histones and recruiting TATA-binding protein (TBP). However, one important difference in Snf1 function in vivo at these promoters is that SAGA recruitment at INO1 requires histone phosphorylation via Snf1, whereas at GAL1, SAGA recruitment is independent of histone phosphorylation. In addition, the GAL1 activator physically interacts with both Snf1 and SAGA, whereas the INO1 activator interacts only with Snf1. Thus, at INO1, pSer10's role in recruiting SAGA may substitute for recruitment by DNA-bound activator. These results emphasize that histone modifications share general functions between promoters, but also acquire distinct roles tailored for promoter-specific requirements (Lo, 2005).
Chromatin creates transcriptional barriers that are overcome by coactivator activities such as histone acetylation by Gcn5 and ATP-dependent chromatin remodeling by SWI/SNF. Factors defining the differential coactivator requirements in the transactivation of various promoters remain elusive. Induction of the Saccharomyces cerevisiae PHO5 promoter does not require Gcn5 or SWI/SNF under fully inducing conditions of no phosphate. PHO5 activation is highly dependent on both coactivators at intermediate phosphate concentrations, conditions that reduce the nuclear concentration of the Pho4 transactivator and severely diminish its association with PHO5 in the absence of Gcn5 or SWI/SNF. Conversely, physiological increases in Pho4 nuclear concentration and binding at PHO5 suppress the need for both Gcn5 and SWI/SNF, suggesting that coactivator redundancy is established at high Pho4 binding site occupancy. Consistent with this, it is demonstrated, using chromatin immunoprecipitation, that Gcn5 and SWI/SNF are directly recruited to PHO5, as well as other strongly transcribed promoters, including GAL1-10, RPL19B, RPS22B, PYK1, and EFT2, none of which do not require either coactivator for expression. These results show that activator concentration and binding site occupancy play crucial roles in defining the extent to which transcription requires individual chromatin remodeling enzymes. In addition, Gcn5 and SWI/SNF associate with many more genomic targets than previously appreciated (Dhasarathy, 2005).
Previous work suggests that the Nhp6 HMGB protein stimulates RNA polymerase II transcription via the TATA-binding protein TBP, and Nhp6 functions in the same functional pathway as the Gcn5 histone acetyltransferase. This report examines the genetic relationship between Nhp6 and Gcn5 with the Mot1 and Ccr4-Not complexes, which have both been implicated in regulating DNA-binding by TBP. Combining either a nhp6ab or a gcn5 mutation with mot1, ccr4, not4 or not5 mutations results in lethality. Combining TBP point mutations with either mot1 or ccr4 also results in either a growth defect or lethality. Several of these synthetic lethalities can be suppressed by overexpression of TFIIA, TBP, or Nhp6, suggesting these genes facilitate formation of the TBP-TFIIA-DNA complex. The growth defect of a not5 mutant can be suppressed by a mot1 mutant. HO gene expression is reduced by nhp6ab, gcn5, or mot1 mutations, and the additive decreases in HO mRNA levels in nhp6ab mot1 and gcn5 mot1 strains suggest different modes of action. Chromatin immunoprecipitation experiments show decreased binding of TBP to promoters in mot1 mutants, and a further decrease when combined with either nhp6ab or gcn5 mutations (Biswas, 2005).
Gcn5 protein is a prototypical histone acetyltransferase that controls transcription of multiple yeast genes. To identify molecular functions that act downstream of or in parallel with Gcn5 protein, a screen was performed for suppressors that rescue the transcriptional defects of HIS3 caused by a catalytically inactive mutant Gcn5, the E173H mutant. One bypass of Gcn5 requirement gene (BGR) suppressor was mapped to the REG1 locus that encodes a semidominant mutant truncated after amino acid 740. Reg1(1-740) protein does not rescue the complete knockout of GCN5, nor does it suppress other gcn5- defects, including the inability to utilize nonglucose carbon sources. Reg1(1-740) enhances HIS3 transcription while HIS3 promoter remains hypoacetylated, indicating that a noncatalytic function of Gcn5 is targeted by this suppressor protein. Reg1 protein is a major regulator of Snf1 kinase that phosphorylates Ser10 of histone H3. However, whereas Snf1 protein is important for HIS3 expression, replacing Ser10 of H3 with alanine or glutamate neither attenuates nor augments the BGR phenotypes. Overproduction of Snf1 protein also preferentially rescues the E173H allele. Biochemically, both Snf1 and Reg1(1-740) proteins copurify with Gcn5 protein. Snf1 can phosphorylate recombinant Gcn5 in vitro. Together, these data suggest that Reg1 and Snf1 proteins function in an H3 phosphorylation-independent pathway that also involves a noncatalytic role played by Gcn5 protein (Liu, 2005).
The bromodomain is an approximately 110 amino acid module found in histone acetyltransferases and the ATPase component of certain nucleosome remodelling complexes. This study reports the crystal structure at 1.9 Å resolution of the Saccharomyces cerevisiae Gcn5p bromodomain complexed with a peptide corresponding to residues 15-29 of histone H4 acetylated at the zeta-N of lysine 16. This bromodomain preferentially binds to peptides containing an N-acetyl lysine residue. Only residues 16-19 of the acetylated peptide interact with the bromodomain. The primary interaction is the N-acetyl lysine binding in a cleft with the specificity provided by the interaction of the amide nitrogen of a conserved asparagine with the oxygen of the acetyl carbonyl group. A network of water-mediated H-bonds with protein main chain carbonyl groups at the base of the cleft contributes to the binding. Additional side chain binding occurs on a shallow depression that is hydrophobic at one end and can accommodate charge interactions at the other. These findings suggest that the Gcn5p bromodomain may discriminate between different acetylated lysine residues depending on the context in which they are displayed (Owen, 2000).
The solution structure of the bromodomain from the human transcriptional coactivator GCN5 has been determined using NMR methods. The structure has a left-handed four-helix bundle topology, with two short additional helices in a long connecting loop. A hydrophobic groove and deep hydrophobic cavity are formed by loops at one end of the molecule. NMR binding experiments show that the cavity forms a specific binding pocket for the acetamide moiety. Peptides containing an N(epsilon)-acetylated lysine residue bind in this pocket with modest affinity (K(D) approximately 0.9 mM); no comparable binding occurs with unacetylated peptides. The GCN5 bromodomain binds the small ligands N(omega)-acetylhistamine and N-methylacetamide, confirming specificity for the alkyl acetamide moiety and showing that the primary element of recognition is simply the sterically unhindered terminal acetamide moiety of an acetylated lysine residue. Additional experiments show that binding is enhanced if the acetyl-lysine residue occurs within the context of a basic peptide and is inhibited by the presence of nearby acidic residues and by the carboxyl group of the free acetyl-lysine amino acid. The binding of the GCN5 bromodomain to acetylated peptides appears to have little additional sequence dependence, although weak interactions with other regions of the peptide are implicated by the binding data. Discrimination between ligands of positive and negative charge is attributed to the presence of several acidic residues located on the loops that form the sides of the binding pocket. Unlike the residues forming the acetamide binding cavity, these acidic side-chains are not conserved in other bromodomain sequences, suggesting that bromodomains might display differences in substrate selectivity and specificity as well as differences in function in vivo (Hudson, 2000).
The Saccharomyces cerevisiae SAGA complex is a multifunctional coactivator that regulates transcription by RNA polymerase II. The 3D structure of SAGA, revealed by electron microscopy, is formed by five modular domains and shows a high degree of structural conservation to human TFTC, reflecting their related subunit composition. The positions of several SAGA subunits were mapped by immunolabeling and by analysis of mutant complexes. The Taf (TBP-associated factor) subunits, shared with TFIID, occupy a central region in SAGA and form a similar structure in both complexes. The locations of two histone fold-containing core subunits, Spt7 and Ada1, are consistent with their role in providing a SAGA-specific interface with the Tafs. Three components that perform distinct regulatory functions, Spt3, Gcn5, and Tra1, are spatially separated, underscoring the modular nature of the complex. These data provide insights into the molecular architecture of SAGA and imply a functional organization to the complex (Wu, 2004).
PCAF and GCN5 are histone acetyltransferase (HAT) paralogs that play roles in the remodeling of chromatin in health and disease. A conformationally flexible loop in the catalytic domain had been observed in the X-ray structures of GCN5 in different liganded states. Conformation and dynamics of this PCAF/GCN5 alpha5-beta6 loop were investigated in solution using tryptophan fluorescence. A mutant human PCAF HAT domain [PCAF(Wloop)] was created in which the natural tryptophan (Trp-514) remote from the alpha5-beta6 loop was replaced with tyrosine, and a glutamate within the loop (Glu-641) was substituted with tryptophan. This PCAF(Wloop) protein exhibits catalytic parameters within 3-fold of those of the wild-type PCAF catalytic domain, suggesting that the loop mutation is not deleterious for HAT activity. While saturating CoASH induces a 30% quenching of Trp fluorescence in PCAF(Wloop), binding of the high-affinity bisubstrate analogue H3-CoA-20 leads to a 2-fold fluorescence increase. These different effects correlate with the different alpha5-beta6 loop conformations seen previously in X-ray structures. On the basis of stopped-flow fluorescence studies, binding of H3-CoA-20 to PCAF(Wloop) proceeds via a rapid association step followed by a slower conformational change involving loop movement. Time-resolved fluorescence measurements support a model in which the alpha5-beta6 loop in the H3-CoA-20-PCAF(Wloop) complex exists in a narrower ensemble of conformations compared to free PCAF(Wloop). The relevance of loop dynamics to PCAF/GCN5 catalysis and substrate specificity are discussed (Zheng, 2005).
The access of transcription factors to eukaryotic promoters often requires modification of their chromatin structure, which is accomplished by the action of two general classes of multiprotein complexes. One class contains histone acetyltransferases (HATs), such as Gcn5 in the SAGA complex, that acetylates nucleosomal histones. The second class contains ATPases, such as Swi2 in the Swi/Snf complex, that provide the energy for nucleosome remodelling. In several promoters these two complexes cooperate but their functional linkage is unknown. A protein module that is present in all nuclear HATs, the bromodomain, could provide such a link. The recently reported in vitro binding of a HAT bromodomain with acetylated lysines within H3 and H4 amino-terminal peptides indicates that this interaction may constitute a targeting step for events that follow histone acetylation. A suitable promoter has been used to show that bromodomain residues essential for acetyl-lysine binding are not required in vivo for Gcn5-mediated histone acetylation but are fundamental for the subsequent Swi2-dependent nucleosome remodelling and consequent transcriptional activation. The Gcn5 bromodomain stabilizes the Swi/Snf complex on this promoter (Syntichaki, 2000).
The functions of the SAGA and SWI/SNF complexes are interrelated and can form stable 'epigenetic marks' on promoters in vivo. Stable promoter occupancy by SWI/SNF and SAGA in the absence of transcription activators requires the bromodomains of the Swi2/Snf2 and Gcn5 subunits, respectively, and nucleosome acetylation. This acetylation can be brought about by either the SAGA or NuA4 HAT complexes. The bromodomain in the Spt7 subunit of SAGA is dispensable for this activity but will anchor SAGA if it is swapped into Gcn5, indicating that specificity of bromodomain function is determined in part by the subunit it occupies. Thus, bromodomains within the catalytic subunits of SAGA and SWI/SNF anchor these complexes to acetylated promoter nucleosomes (Hassan, 2002).
Acetylation and other modifications on histones comprise histone codes that govern transcriptional regulatory processes in chromatin. Yet little is known of how different histone codes are translated and put into action. Using fluorescence resonance energy transfer, it has been shown that bromodomain-containing proteins recognize different patterns of acetylated histones in intact nuclei of living cells. The bromodomain protein Brd2 selectively interacts with acetylated lysine 12 on histone H4, whereas TAF(II)250 and PCAF recognized H3 and other acetylated histones, indicating fine specificity of histone recognition by different bromodomains. This hierarchy of interactions was also seen in direct peptide binding assays. Interaction with acetylated histone was essential for Brd2 to amplify transcription. Moreover association of Brd2, but not other bromodomain proteins, with acetylated chromatin persisted on chromosomes during mitosis. Thus the recognition of histone acetylation code by bromodomains is selective, is involved in transcription, and potentially conveys transcriptional memory across cell divisions (Kanno, 2004).
GCN5 is a histone acetyltransferase (HAT) originally identified in Saccharomyces cerevisiae and required for transcription of specific genes within chromatin as part of the SAGA (SPT-ADA-GCN5 acetylase) coactivator complex. Mammalian cells have two distinct GCN5 homologs (PCAF and GCN5L) that have been found in three different SAGA-like complexes (PCAF complex, TFTC [TATA-binding-protein-free TAF(II)-containing complex], and STAGA [SPT3-TAF(II)31-GCN5L acetylase]). The composition and roles of these mammalian HAT complexes are still poorly characterized. This study describes the purification and characterization of the human STAGA complex. STAGA contains homologs of most yeast SAGA components, including two novel human proteins with histone-like folds and sequence relationships to yeast SPT7 and ADA1. Furthermore, it is demonstrated that STAGA has acetyl coenzyme A-dependent transcriptional coactivator functions from a chromatin-assembled template in vitro and associates in HeLa cells with spliceosome-associated protein 130 (SAP130) and DDB1, two structurally related proteins. SAP130 is a component of the splicing factor SF3b that associates with U2 snRNP and is recruited to prespliceosomal complexes. DDB1 (p127) is a UV-damaged-DNA-binding protein that is involved, as part of a complex with DDB2 (p48), in nucleotide excision repair and the hereditary disease xeroderma pigmentosum. These results thus suggest cellular roles of STAGA in chromatin modification, transcription, and transcription-coupled processes through direct physical interactions with sequence-specific transcription activators and with components of the splicing and DNA repair machineries (Martinez, 2001).
P/CAF is shown to be able to regulate transcription: this function is independent of its binding to CBP. The HAT domain of P/CAF has transcriptional activation potential in yeast. In mammalian cells P/CAF can stimulate transcription of the RSV promoter, using the activity of its HAT domain. The adenovirus protein E1A is shown to target P/CAF and sequester its transcriptional activity. Binding of E1A to P/CAF is direct, independent of CBP and requires residues within E1A conserved region 1. The P/CAF binding residues in E1A are within a motif shown to be essential for efficient disruption of myogenesis by E1A. The fact that E1A can directly bind and regulate the activity of P/CAF, independently of its regulation of CBP, highlights an important role for P/CAF in the process of cell differentiation (Reid, 1998).
A novel human (h) multiprotein complex, called TATA-binding protein (TBP)-free TAFII-containing complex (TFTC), has been identified that is able to nucleate RNA polymerase II transcription and can mediate transcriptional activation. TFTC, similar to other TBP-free TAFII complexes (yeast SAGA, hSTAGA, and hPCAF) contains the acetyltransferase hGCN5 and is able to acetylate histones in both a free and a nucleosomal context. The recently described TRRAP cofactor for oncogenic transcription factor pathways was also characterized as a TFTC subunit. Furthermore, four other previously uncharacterized subunits of TFTC have been identified: hADA3, hTAFII150, hSPT3, and hPAF65beta. Thus, the polypeptide composition of TFTC suggests that TFTC is recruited to chromatin templates by activators to acetylate histones and thus may potentiate initiation and activation of transcription (Brand, 1999).
The specific post-translational modifications to histones influence many nuclear processes including gene regulation, DNA repair and replication. Recent studies have identified effector proteins that recognize patterns of histone modification and transduce their function in downstream processes. For example, histone acetyltransferases (HATs) have been shown to participate in many essential cellular processes, particularly those associated with activation of transcription. Yeast SAGA (Spt-Ada-Gcn5 acetyltransferase) and SLIK (SAGA-like) are two highly homologous and conserved multi-subunit HAT complexes, which preferentially acetylate histones H3 and H2B and deubiquitinate histone H2B. This study identifies the chromatin remodelling protein Chd1 (chromo-ATPase/helicase-DNA binding domain 1; see Drosophila Chd1) as a component of SAGA and SLIK. These findings indicate that one of the two chromodomains of Chd1 specifically interacts with the methylated lysine 4 mark on histone H3 that is associated with transcriptional activity. Furthermore, the SLIK complex shows enhanced acetylation of a methylated substrate and this activity is dependent upon a functional methyl-binding chromodomain, both in vitro and in vivo. This study identifies the first chromodomain that recognizes methylated histone H3 (Lys 4) and possibly identifies a larger subfamily of chromodomain proteins with similar recognition properties (Pray-Grant, 2005).
PCAF 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).
Whereas the histone acetylase PCAF has been suggested to be part of a coactivator complex mediating transcriptional activation by the nuclear hormone receptors, the physical and functional interactions between nuclear receptors and PCAF have remained unclear. Efforts to clarify these relationships have revealed two novel properties of nuclear receptors (Blanco, 1998).
(1) First, the RXR/RAR heterodimer directly recruits PCAF from mammalian cell extracts in a ligand-dependent manner and increased expression of PCAF leads to enhanced retinoid-responsive transcription. Of the two domains present in PCAF, the carboxy-terminal domain (from amino acid position 352 to 832) represents the region homologous to the yeast GCN5 and contains histone acetylase activity. The amino-terminal region (composed of amino acids 1-351) shares little homology with known genes, and its function has not been fully elucidated. To determine a region of PCAF involved in binding to the heterodimer, truncated recombinant PCAFs lacking either the amino-terminal or carboxy-terminal domain were examined. Recombinant deltaN1 or deltaN2, lacking either the amino-terminal domain alone or the amino-terminal region plus the additional 113 amino acids of the carboxy-terminal region, binds to the heterodimer-RARE complex, although the binding of deltaN2 is slightly weaker than that of deltaN1 and full-length PCAF. In contrast to this, deltaC shows little binding to the complex. Ligand has no effect on the binding activity of the truncated PCAF. These results indicate that the conserved carboxy-terminal domain is required for binding to the heterodimer-RARE complex (Blanco, 1998).
(2) With respect to the second novel property of nuclear receptors, PCAF directly associates with the DNA-binding domain of nuclear receptors, independently of p300/CBP binding, and therefore defines a novel cofactor interaction surface. These results show that dissociation of corepressors enables ligand-dependent PCAF binding to the receptors. This observation illuminates how a ligand-dependent receptor function can be propagated to regions outside the ligand-binding domain itself. To evaluate whether PCAF enhances ligand-dependent promoter activity through its histone acetylase activity, two additional deletion constructs were examined in which the recently identified catalytic domain of PCAF was deleted (deltaHAT1 and deltaHAT2). Histone acetylase activity is found to be completely abrogated in these deletion constructs in vitro. deltaHAT1 and deltaHAT2, similar to deltaC and deltaN2, fails to give full enhancement in promoter activity attained by the intact PCAF. These results support the idea that PCAF potentiates retinoid-dependent transcription at least partly through its histone acetylase activity. Immunoblot analysis performed with transfected cells shows that exogenous PCAF is expressed in a dose-dependent manner, while the expression of the endogenous p300 remains unchanged. On the basis of these observations, it is suggested that PCAF may play a more central role in nuclear receptor function than previously anticipated (Blanco, 1998).
It was found that corepressors from the N-CoR-SMRT family inhibit binding of recombinant PCAF to the nuclear receptor heterodimer in the absence of ligand, but that this binding is restored upon addition of ligand, concomitant with repressor release. These results suggest that corepressors, by virtue of their dissociation from the receptor, confer ligand dependence on PCAF binding. It has been shown that N-CoR and SMRT bind to the hinge region of receptors. Because the hinge region present in the ligand-binding domain is only ~30 amino acids away from the DNA-binding domain, corepressors could cause either a steric block of PCAF binding or induce a local conformational change that precludes PCAF binding. Adding to this passive regulation of activity exclusion, corepressors have recently been shown to be associated with the histone deacetylase HDAC-1 and mSin3, which are thought to establish transcriptional repression via modification of chromatin. A model depicting how ligand reverses this process in two steps is presented: first the ligand promotes the dissociation of the repressor complex, which in turn enables the second step of PCAF recruitment. Like the repressors, PCAF itself also functions in at least two ways: (1) as a histone acetylase it has the direct capacity to modify chromatin to reverse repression, and (2) via its p300/CBP- and SRC-interaction domains, it serves to recruit additional activators (Blanco, 1998).
Histone acetyltransferases regulate transcription, but little is known about the role of these enzymes in developmental processes. Gcn5 (encoded by Gcn5l2) and Pcaf, mouse histone acetyltransferases, share similar sequences and enzymatic activities. Both interact with p300 and CBP (encoded by Ep300 and Crebbp, respectively), two other histone acetyltransferases that integrate multiple signalling pathways. Pcaf is thought to participate in many of the cellular processes regulated by p300/CBP, but the functions of Gcn5 are unknown in mammalian cells. The gene Pcaf is dispensable in mice. In contrast, Gcn5l2-null embryos die during embryogenesis. These embryos develop normally to 7.5 days post coitum (d.p.c.), but their growth is severely retarded by 8.5 d.p.c. and they fail to form dorsal mesoderm lineages, including chordamesoderm and paraxial mesoderm. Differentiation of extra-embryonic and cardiac mesoderm seems to be unaffected. Loss of the dorsal mesoderm lineages is due to a high incidence of apoptosis in the Gcn5l2 mutants that begins before the onset of morphological abnormality. Embryos null for both Gcn5l2 and Pcaf show even more severe defects, indicating that these histone acetyltransferases have overlapping functions during embryogenesis. These studies are the first to demonstrate that specific acetyltransferases are required for cell survival and mesoderm formation during mammalian development (Xu, 2000).
Histone acetyltransferases (HATs) are involved in the acetylation of core histones, which is an important event for transcription regulation through alterations in the chromatin structure in eukaryotes. To clarify participatory in vivo roles for two such enzymes known as GCN5 and PCAF, homozygous cell culture mutants, DeltaGCN5 and DeltaPCAF, were generated devoid of two alleles of each of the GCN5 and PCAF genes, respectively, with the help of gene targeting technique. While the PCAF-deficiency exhibits no effect on growth rate, the GCN5-deficiency caused delayed growth rate of cultured DT40 cells. FACS analyses revealed not only that the number of cells in S phase decreases, but also that the cell cycle progression is suppressed at G1/S phase transition for DeltaGCN5. RT-PCR analyses revealed that the GCN5-deficiency exhibits opposite influences on transcriptions of G1/S phase transition-related genes, i.e., repressions for E2F-1, E2F-3, E2F-4, E2F-6, DP-2, cyclin A, cyclin D3, PCNA, cdc25B and p107, and, activations for p27, c-myc, cyclin D2 and cyclin G1. Similarly, the deficiency influences oppositely transcription of apoptosis-related genes, i.e., decreased expression of bcl-xL and increased expression of bcl-2. Immunoblotting analyses using a number of anti-acetylated histone antisera revealed that the GCN5-deficiency leads to decreased acetylation levels of K16/H2B and K9/H3, and increases the acetylation levels of K7/H2A, K18/H3, K23/H3, K27/H3, K8/H4 and K12/H4. These results indicate that GCN5 preferentially acts as a supervisor in the normal cell cycle progression having comprehensive control over expressions of these cell cycle-related genes, as well as apoptosis-related genes, probably via alterations in the chromatin structure, mimicked by changing acetylation status of core histones, surrounding these widely distributed genes (Kikuchi, 2005).
Deletion of genes encoding the histone acetyltransferases GCN5, p300, or CBP results in embryonic lethality in mice. PCAF and GCN5 physically interact with p300 and CBP in vitro. To determine whether these two groups of histone acetyltransferases interact functionally in vivo, mice lacking one or more alleles of p300, GCN5, or PCAF were generated. As expected, it was found that mice heterozygous for any single null allele are viable. The majority of GCN5(+/-)p300(+/-) mice also survive to adulthood with no apparent abnormalities. However, approximately 25% of these mice die before birth. These embryos are developmentally stunted and exhibit increased apoptosis compared with wild-type or single GCN5(+/-) or p300(+/-) littermates at embryonic day 8.5. In contrast, no abnormalities were observed in PCAF(-/-) p300(+/-) mice. Of interest, it was found that p300 protein levels vary in different mouse genetic backgrounds, which likely contributes to the incomplete penetrance of the abnormal phenotype of GCN5(+/-) p300(+/-) mice. These data indicate that p300 cooperates specifically with GCN5 to provide essential functions during early embryogenesis (Phan, 2005).
The c-Myc oncoprotein (Myc) controls cell fate by regulating gene transcription in association with a DNA-binding partner, Max. While Max lacks a transcription regulatory domain, the N terminus of Myc contains a transcription activation domain (TAD) that recruits cofactor complexes containing the histone acetyltransferases (HATs) GCN5 and Tip60. This study reports a novel functional interaction between Myc TAD and the p300 coactivator-acetyltransferase. p300 associates with Myc in mammalian cells and in vitro through direct interactions with Myc TAD residues 1 to 110 and acetylates Myc in a TAD-dependent manner in vivo at several lysine residues located between the TAD and DNA-binding domain. Moreover, the Myc:Max complex is differentially acetylated by p300 and GCN5 and is not acetylated by Tip60 in vitro, suggesting distinct functions for these acetyltransferases. Whereas p300 and CBP can stabilize Myc independent of acetylation, p300-mediated acetylation results in increased Myc turnover. In addition, p300 functions as a coactivator that is recruited by Myc to the promoter of the human telomerase reverse transcriptase gene; also, p300/CBP stimulates Myc TAD-dependent transcription in a HAT domain-dependent manner. These results suggest dual roles for p300/CBP in Myc regulation: as a Myc coactivator that stabilizes Myc and as an inducer of Myc instability via direct Myc acetylation (Faiola, 2005).
The c-MYC oncoprotein functions as a sequence-specific transcription factor. The ability of c-MYC to activate transcription relies in part on the recruitment of cofactor complexes containing the histone acetyltransferases mammalian GCN5 (mGCN5)/PCAF and TIP60. In addition to acetylating histones, these enzymes have been shown to acetylate other proteins involved in transcription, including sequence-specific transcription factors. This study was initiated in order to determine whether c-MYC is a direct substrate of mGCN5 and TIP60. mGCN5/PCAF and TIP60 are shown to acetylate c-MYC in vivo. By using nanoelectrospray tandem mass spectrometry to examine c-MYC purified from human cells, the major mGCN5-induced acetylation sites have been mapped. Acetylation of c-MYC by either mGCN5/PCAF or TIP60 results in a dramatic increase in protein stability. The data reported here suggest a conserved mechanism by which acetyltransferases regulate c-MYC function by altering its rate of degradation (Patel, 2005).
Myc oncoproteins promote cell cycle progression in part through the transcriptional up-regulation of the cyclin D2 gene. Myc is bound to the cyclin D2 promoter in vivo. Binding of Myc induces cyclin D2 expression and histone acetylation at a single nucleosome in a MycBoxII/TRRAP-dependent manner. TRRAP is a component of TIP60 and PCAF/GCN5 histone acetyl transferase (HAT) complexes. Down-regulation of cyclin D2 mRNA expression in differentiating HL60 cells is preceded by a switch of promoter occupancy from Myc/Max to Mad/Max complexes, loss of TRRAP binding, increased HDAC1 binding, and histone deacetylation. Thus, recruitment of TRRAP and regulation of histone acetylation are critical for transcriptional activation by Myc (Bouchard, 2001).
The aim of this study was to resolve the role of MBII (an effector domain of Myc that binds TRRAP) and TRRAP in gene activation by Myc, using an endogenous target gene of Myc, cyclin D2, as model. Upon binding to the cyclin D2 promoter, Myc recruits TRRAP and induces the preferential acetylation of histone H4 at a single nucleosome. Conversely, loss of endogenous Myc binding correlates with histone deacetylation and loss of TRRAP binding during the TPA-induced differentiation of a human promyelocytic cell line, HL60. The integrity of MBII is required for TRRAP recruitment, histone acetylation, and transcriptional activation at the cyclin D2 locus. Therefore, previous suggestions that MBII has no role in transcriptional activation based on transient reporter assays need to be reevaluated. Deletion of the entire N terminus of Myc up to MBII (s-Myc) renders Myc unable to induce cell cycle progression and expression of either cyclin A or cyclin D2 in 3T3 fibroblasts, consistent with recent results that the N terminus of Myc is required for regulation of proliferation and induction of gene expression in a cell-type-dependent manner. Most likely, this is because stable association with TRRAP requires sequences in the N terminus of Myc in addition to MBII (Bouchard, 2001 and references therein).
Mad proteins are thought to antagonize the function of Myc by recruiting a repressor complex that contains histone deacetylase activity. Observations suggest that this model applies to the cyclin D2 promoter: (1) repression of the cyclin D2 promoter by Mad1 requires the integrity of an N-terminal domain, which mediates recruitment of histone deacetylase complexes through interaction with Sin3A; (2) during HL60 differentiation, Mad1 and HDAC1 are corecruited to the cyclin D2 promoter, correlating with histone deacetylation of both histones H3 and H4 at the cyclin D2 promoter. Taken together, these data strongly support a model in which endogenous Myc/Max and Mad/Max complexes contribute to the regulation of transcription of the cyclin D2 gene through their antagonistic effects on histone acetylation. In addition, these findings show the functional relevance of the switch between Myc/Max and Mad/Max complexes during differentiation of hematopoietic cells. Recent work on the gene encoding the catalytic subunit of telomerase, htert, suggests that this model also may apply to this promoter (Bouchard, 2001 and references therein).
Up-regulation of the CAD (carbamoyl phosphate synthase, aspartate transcarbamylase, dihydroorotase) gene by Myc does not involve changes in histone acetylation. Instead, high levels of histone acetylation at the promoter were found in both quiescent and proliferating cells, showing that Myc can control at least one step in addition to histone acetylation to promote active transcription. Additional proteins have been identified that bind to different domains of Myc and that are candidates for such an activity: for example, the C terminus of Myc binds to Ini1, a component of the Swi/Snf family of chromatin-remodeling complexes. Clearly, a detailed analysis of the role of Myc in activation of individual promoters will be required before the role of each interaction in Myc biology can be resolved fully (Bouchard, 2001 and references therein).
During the G(1) phase of the cell cycle, an E2F-RB complex represses transcription, via the recruitment of histone deacetylase activity. Phosphorylation of RB at the G(1)/S boundary generates a pool of 'free' E2F, which then stimulates transcription of S-phase genes. Given that E2F1 activity is stimulated by p300/CBP acetylase and repressed by an RB-associated deacetylase, it was asked if E2F1 is subject to modification by acetylation. The p300/CBP-associated factor P/CAF, and to a lesser extent p300/CBP itself, can acetylate E2F1 in vitro, and intracellular E2F1 is acetylated. The acetylation sites lie adjacent to the E2F1 DNA-binding domain and involve lysine residues highly conserved in E2F1, 2 and 3. Acetylation by P/CAF has three functional consequences on E2F1 activity: increased DNA-binding ability, activation potential and protein half-life. These results suggest that acetylation stimulates the functions of the non-RB bound 'free' form of E2F1. Consistent with this, it is found that the RB-associated histone deacetylase can deacetylate E2F1. These results identify acetylation as a novel regulatory modification that stimulates E2F1's activation functions (Martinez-Balbas, 2000).
One of the predictions of the histone code hypothesis is the existence of functional interactions between chromatin remodeling complexes, such as SWI/SNF, and histone acetylase complexes, such as GCN5. Recent studies have elucidated the temporal sequence in which these coactivators of transcription are recruited to promoters in vivo and how their enzymatic properties contribute to gene activation. The best-characterized example in mammals is provided by the human IFN-ß gene. The gene is switched on by three transcription factors (NF-kappaB, IRFs, and ATF-2/c-Jun), and an architectural protein [HMG I(Y)], all of which bind cooperatively to the nucleosome-free enhancer DNA to form an enhanceosome. The enhanceosome targets the modification and repositioning of a nucleosome that blocks the formation of a transcriptional preinitiation complex on the IFN-ß promoter. This is accomplished by the ordered recruitment of HATs, SWI/SNF, and basal transcription factors. Initially, the GCN5 HAT-containing complex is recruited, and it acetylates the nucleosome. This is followed by the recruitment of the CBP-PolII holoenzyme complex. Next, the SWI/SNF remodeling machine arrives at the promoter via bivalent interactions with CBP and the acetylated histone N tails. SWI/SNF alters the structure of the nucleosome via an unknown mechanism, thus allowing recruitment and DNA binding of TFIID to the TATA box. The DNA bending induced upon TFIID binding to the promoter causes sliding of the SWI/SNF-modified nucleosome to a new position 36 bp downstream, thus allowing the initiation of transcription. This ordered recruitment and nucleosome sliding is consistent with the view that histone acetylation sets the stage for ATP-dependent remodeling by establishing a recognition surface for the bromodomains present in SWI/SNF-like remodeling machines. Furthermore, since histone acetylation precedes the recruitment of additional complexes bearing bromodomains, such as TFIID, it is possible that this modification also regulates recruitment of TFIID to promoters (Agalioti, 2002).
Experiments were carried out to test the histone code hypothesis. Only a small subset of lysines in histones H4 and H3 are acetylated in vivo during viral infection, and this modification is carried out by the GCN5 transcriptional coactivator complex. Reconstitution of recombinant nucleosomes bearing mutations in these lysine residues reveals a biochemical cascade for gene activation via a point-by-point interpretation of the histone code through the ordered recruitment of bromodomain transcription complexes. More specifically, acetylation of H4 lysine 8 is required for recruitment of the SWI/SNF complex, whereas acetylation of lysines 9 and 14 in histone H3 is critical for the recruitment of the general transcription factor TFIID. Thus, the information contained in the DNA address of the enhancer is extended (transferred) to the histone N termini by generating novel adhesive surfaces that participate in the recruitment of transcription complexes (Agalioti, 2002).
The precision by which the histone code is decoded is remarkable. Most likely, the code is translated via specific interactions of bromodomains with the acetylated histone N termini. The bromodomain in BRG1 associates with the H4 tail acetylated at K8, whereas the double bromodomain in TAFII250 binds to the doubly acetylated (at K9 and K14) H3 tail. The competition assays using either acetylated histone N termini peptides or bromodomain polypeptides as competitors revealed an unprecedented level of specificity for the interactions between bromodomains and acetyl-lysine histone N termini. Again, this remarkable degree of specificity may be dictated by the conformational changes forced upon these complexes by their interactions with other proteins in the complex. Thus, the point-by-point interpretation of the histone acetylation code may rely on the precise allosteric changes induced in many proteins upon their association with transcription factor complexes. For example, the initial recruitment of SWI/SNF via its association with CBP is stabilized on the promoter through the association of the BRG1 bromodomain with the H4 K8 acetylated tail. Although, BRG1's bromodomain could interact at least in principle with other acetylated lysine residues on H3, these interactions may not be of sufficient strength to ensure stable binding of the SWI/SNF complex to the promoter. Similarly, recruitment of TFIID to the SWI/SNF-modified promoter is stabilized via two types of interactions. The first with various enhanceosome components and the second with the interaction between the two bromodomains of TAFII250 and the two acetyl groups on residues K9 and K14 of histone H3. Several observations support this notion: (1) both TBP and TAFII250 are simultaneously recruited to the promoter with almost identical kinetics in vivo; (2) recruitment of TFIID in vivo occurs only when both H3 K9 and K14 are acetylated; (3) mutations in either K9 or K14 abrogate TFIID recruitment. The data show that the TAFII250 double bromodomain, when recruited to the natural IFN-ß promoter, interacts specifically with the H3 K9 and K14 acetylated residues and not with the acetylated H4 tails. However, when tested in isolation and out of the promoter/chromatin natural context, the double TAFII250 bromodomain interacts with similar affinities to both H4 and H3 acetylated tails, a result consistent with in vitro observations. It is proposed that the inordinate set of interactions occurring with purified bromodomains and acetylated histone tails is 'fixed' when present in natural promoter/chromatin contexts. In the latter case, it is possible that the competing interactions cannot take place either because the alternative target is occupied (e.g., the H4 tail is already bound by SWI/SNF) or the specific three-dimensional conformation of the transcription complex does not permit these interactions to occur (Agalioti, 2002).
A model is presented that depicts the ordered biochemical cascade decoding the DNA and histone acetylation code during activation of human IFN-ß gene transcription following Sendai virus infection. It is thought that the promoter DNA code contains all the information for the assembly of the enhanceosome in response to virus infection. The enhanceosome that assembles at the promoter element recruits the GCN5 histone acetyltransferase. Subsequently GCN5 acetylates initially H4K8 and H3K9. An unknown kinase recruited by the enhanceosome phosphorylates H3 Ser 10, a prerequisite for H3K14 acetylation by GCN5. The histone code is subsequently translated by recruiting of additional components required for transcription. The bromodomain containing transcription complexes SWI/SNF and TFIID are recruited to the promoter via bivalent interactions between the enhanceosome and specifically acetylated histone N termini, and this subsequently stimulates transcription of the IFN-ß gene (Agalioti, 2002).
Changes in histone acetylation at promoters correlate with transcriptional activation and repression, but whether acetylation of histones in the coding region of genes is important for transcription is less clear. Cells lacking the histone acetyltransferases Gcn5 and Elp3 have widespread and severe histone H3 hypoacetylation in chromatin. Surprisingly, severe hypoacetylation in the promoter does not invariably affect the ability of TBP to bind the TATA element, or transcription of the gene. By contrast, similar hypoacetylation of the coding region correlates with inhibition of transcription, and inhibition correlates better with the overall charge of the histone H3 tail than with hypoacetylation of specific lysine residues. These data provide insights into the effects of histone H3 hypoacetylation in vivo and underscore the importance of the overall charge of the histone tail for transcription (Kristjuhan, 2002).
These data suggest that only very severe H3 hypoacetylation in the coding region of an active gene correlates with effects on transcription. For example, reducing the average overall acetylation level in the coding region to about half of that of wild-type did not show a relationship with reduced transcription, while the 4- to 5-fold reduction introduced at some genes invariably correlated with a dramatic transcription effect. Interestingly, similarly severe reductions in histone H3 promoter acetylation did not always correlate with reduced transcription, and the levels of promoter acetylation and coding region acetylation did not change together. For example, transcription at genes such as FAB1 and TRA1 was not significantly affected, yet their average promoter acetylation was reduced 5-fold. In contrast to the promoter, acetylation in the coding region of these genes was only slightly reduced. This suggests that histone H3 acetylation in the promoter and coding region can be deposited independently and that it can differentially affect the initial association of TBP with promoters and 'postrecruitment events' such as, for example, the movement of RNAPII through DNA (Kristjuhan, 2002).
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