Alcohol metabolism contributes to brain histone acetylation.

Emerging evidence suggests that epigenetic regulation is dependent on metabolic state, and implicates specific metabolic factors in neural functions that drive behaviour1. In neurons, acetylation of histones relies on the metabolite acetyl-CoA, which is produced from acetate by chromatin-bound acetyl-CoA synthetase 2 (ACSS2)2. Notably, the breakdown of alcohol in the liver leads to a rapid increase in levels of blood acetate3, and alcohol is therefore a major source of acetate in the body. Histone acetylation in neurons may thus be under the influence of acetate that is derived from alcohol4, with potential effects on alcohol-induced gene expression in the brain, and on behaviour5. Here, using in vivo stable-isotope labelling in mice, we show that the metabolism of alcohol contributes to rapid acetylation of histones in the brain, and that this occurs in part through the direct deposition of acetyl groups that are derived from alcohol onto histones in an ACSS2-dependent manner. A similar direct deposition was observed when mice were injected with heavy-labelled acetate in vivo. In a pregnant mouse, exposure to labelled alcohol resulted in the incorporation of labelled acetyl groups into gestating fetal brains. In isolated primary hippocampal neurons ex vivo, extracellular acetate induced transcriptional programs related to learning and memory, which were sensitive to ACSS2 inhibition. We show that alcohol-related associative learning requires ACSS2 in vivo. These findings suggest that there is a direct link between alcohol metabolism and gene regulation, through the ACSS2-dependent acetylation of histones in the brain.

Gene activation by histone and factor acetyltransferases.

Persuasive evidence has emerged that acetyltransferases appear to truly function to acetylate both histones and transcription factors in vivo to effect gene activation. In the cell, acetyltransferases have been identified as components of large, multifunctional and evolutionarily conserved macromolecular assemblies, whose components and structures suggest complex functions. In addition, the first atomic resolution structures of HATs have revealed conserved mechanisms of acetyl-CoA interaction among the superfamily of GNATs (Gcn5-related N-acetyltransferases). Finally, enzymatic acetyltransferase activities are themselves regulated by phosphorylation and interaction with other proteins.

The SAGA unfolds: convergence of transcription regulators in chromatin-modifying complexes.

Several previously characterized transcriptional adaptors and coactivators are now known to be histone acetyltransferases (HATs). Recent studies in Saccharomyces cerevisiae indicate that the Gcn5p HAT exists in large complexes containing several phenotypic classes of transcription factors. Genetic and biochemical studies of these transcription factors and their functions within HAT complexes suggest that acetylation of histones is one function of an integrated system of modular activities. These activities include interaction with activators, histone acetylation and interaction with basal factors. Coordination of these functions may well be an important component of gene activation in vivo.

Snf1--a histone kinase that works in concert with the histone acetyltransferase Gcn5 to regulate transcription.

Modification of histones is an important element in the regulation of gene expression. Previous work suggested a link between acetylation and phosphorylation, but questioned its mechanistic basis. We have purified a histone H3 serine-10 kinase complex from Saccharomyces cerevisiae and have identified its catalytic subunit as Snf1. The Snf1/AMPK family of kinases function in conserved signal transduction pathways. Our results show that Snf1 and the acetyltransferase Gcn5 function in an obligate sequence to enhance INO1 transcription by modifying histone H3 serine-10 and lysine-14. Thus, phosphorylation and acetylation are targeted to the same histone by promoter-specific regulation by a kinase/acetyltransferase pair, supporting models of gene regulation wherein transcription is controlled by coordinated patterns of histone modification.

Acetylation of histones and transcription-related factors.

The state of chromatin (the packaging of DNA in eukaryotes) has long been recognized to have major effects on levels of gene expression, and numerous chromatin-altering strategies-including ATP-dependent remodeling and histone modification-are employed in the cell to bring about transcriptional regulation. Of these, histone acetylation is one of the best characterized, as recent years have seen the identification and further study of many histone acetyltransferase (HAT) proteins and their associated complexes. Interestingly, most of these proteins were previously shown to have coactivator or other transcription-related functions. Confirmed and putative HAT proteins have been identified from various organisms from yeast to humans, and they include Gcn5-related N-acetyltransferase (GNAT) superfamily members Gcn5, PCAF, Elp3, Hpa2, and Hat1: MYST proteins Sas2, Sas3, Esa1, MOF, Tip60, MOZ, MORF, and HBO1; global coactivators p300 and CREB-binding protein; nuclear receptor coactivators SRC-1, ACTR, and TIF2; TATA-binding protein-associated factor TAF(II)250 and its homologs; and subunits of RNA polymerase III general factor TFIIIC. The acetylation and transcriptional functions of these HATs and the native complexes containing them (such as yeast SAGA, NuA4, and possibly analogous human complexes) are discussed. In addition, some of these HATs are also known to modify certain nonhistone transcription-related proteins, including high-mobility-group chromatin proteins, activators such as p53, coactivators, and general factors. Thus, we also detail these known factor acetyltransferase (FAT) substrates and the demonstrated or potential roles of their acetylation in transcriptional processes.

Nucleotides in the polyomavirus enhancer that control viral transcription and DNA replication.

The polyomavirus enhancer is required in cis for high-level expression of the viral early region and for replication of the viral genome. We introduced multiple mutations in the enhancer which reduced transcription and DNA replication. Polyomaviruses with these mutant enhancers formed very small plaques in whole mouse embryo cells. Revertants of the viral mutants were isolated and characterized. Reversion occurred by any of the following events: restoration of guanosines at nucleotide (nt) 5134 and nt 5140 within the adenovirus 5 E1A enhancer core AGGAAGTGACT; acquisition of an A----G mutation at nt 5258, which is the same mutation that enables polyomavirus to grow in embryonal carcinoma F9 cells; duplication of mutated sequences between nt 5146 and 5292 (including sequences homologous with immunoglobulin G, simian virus 40, and bovine papillomavirus enhancer elements). Reversion restored both the replicative and transcriptional functions of the viruses. Revertants that acquired the F9 mutation at nt 5258 grew at least 20-fold better than the original mutant in whole mouse embryo cells, but replicated only marginally better than the original mutant in 3T6 cells. Viruses with a reversion of the mutation at nt 5140 replicated equally well in both types of cells. Since individual nucleotides in the polyomavirus enhancer simultaneously altered DNA replication and transcription in specific cell types, it is likely that these processes rely upon a common element, such as an enhancer-binding protein.

Do products of the myc proto-oncogene play a role in transcriptional regulation of the prothymosin alpha gene?

The Myc protein has been reported to activate transcription of the rat prothymosin alpha gene by binding to an enhancer element or E box (CACGTG) located in the first intron (S. Gaubatz et al., Mol. Cell. Biol. 14:3853-3862, 1994). The human prothymosin alpha gene contains two such motifs: in the promoter region at kb -1.2 and in intron 1, approximately 2 kb downstream of the transcriptional start site in a region which otherwise bears little homology to the rat gene. Using chloramphenicol acetyltransferase (CAT) reporter constructs driven either by the 5-kb human prothymosin alpha promoter or by a series of truncated promoters, we showed that removal of the E-box sequence had no effect on transient expression of CAT activity in mouse L cells. When intron 1 of the prothymosin alpha gene was inserted into the most extensive promoter construct downstream of the CAT coding region, a diminution in transcription, which remained virtually unchanged upon disruption of the E boxes, was observed. CAT constructs driven by the native prothymosin alpha promoter or the native promoter and intron were indifferent to Myc; equivalent CAT activity was observed in the presence of ectopic normal or mutant Myc genes. Similarly, expression of a transiently transfected wild-type prothymosin alpha gene as the reporter was not affected by a repertoire of myc-derived genes, including myc itself and dominant or recessive negative myc mutants. In COS-1 cells, equivalent amounts of the protein were produced from transfected prothymosin alpha genes regardless of whether genomic E boxes were disrupted, intron 1 was removed, or a repertoire of myc-derived genes was included in the transfection cocktail. More importantly, cotransfection of a dominant negative Max gene failed to reduce transcription of the endogenous prothymosin alpha gene in COS cells or the wild-type transfected gene in COS or L cells. Taken together, the data do not support the idea that Myc activates transcription of the intact human prothymosin alpha gene or reporter constructs that mimic its structure. Rather, they suggest that the human prothymosin alpha promoter and downstream elements are buffered so as to respond poorly, if at all, to transient fluctuations in transcription factors which regulate other genes.

Identification of human proteins functionally conserved with the yeast putative adaptors ADA2 and GCN5.

Transcriptional adaptor proteins are required for full function of higher eukaryotic acidic activators in the yeast Saccharomyces cerevisiae, suggesting that this pathway of activation is evolutionarily conserved. Consistent with this view, we have identified possible human homologs of yeast ADA2 (yADA2) and yeast GCN5 (yGCN5), components of a putative adaptor complex. While there is overall sequence similarity between the yeast and human proteins, perhaps more significant is conservation of key sequence features with other known adaptors. We show several functional similarities between the human and yeast adaptors. First, as shown for yADA2 and yGCN5, human ADA2 (hADA2) and human GCN5 (hGCN5) interacted in vivo in a yeast two-hybrid assay. Moreover, hGCN5 interacted with yADA2 in this assay, suggesting that the human proteins form similar complexes. Second, both yADA2 and hADA2 contain cryptic activation domains. Third, hGCN5 and yGCN5 had similar stabilizing effects on yADA2 in vivo. Furthermore, the region of yADA2 that interacted with yGCN5 mapped to the amino terminus of yADA2, which is highly conserved in hADA2. Most striking, is the behavior of the human proteins in human cells. First, GAL4-hADA2 activated transcription in HeLa cells, and second, either hADA2 or hGCN5 augmented GAL4-VP16 activation. These data indicated that the human proteins correspond to functional homologs of the yeast adaptors, suggesting that these cofactors play a key role in transcriptional activation.

Histone acetyltransferase activity is conserved between yeast and human GCN5 and is required for complementation of growth and transcriptional activation.

Yeast and human ADA2 and GCN5 (y- and hADA2 and y- and hGCN5, respectively) have been shown to potentiate transcription in vivo and may function as adaptors to bridge physical interactions between DNA-bound activators and the basal transcriptional machinery. Recently it was shown that yGCN5 is a histone acetyltransferase (HAT), suggesting a link between enzymatic modification of nucleosomes and transcriptional activation. In this report, we demonstrate that hGCN5 is also an HAT and has the same substrate specificity as yGCN5. Since hGCN5 does not complement functional defects caused by deletion of yGCN5, we constructed a series of hGCN5-yGCN5 chimeras to identify human regions capable of activity in yeast. Interestingly, only the putative HAT domain of hGCN5, when fused to the remainder of yGCN5, complemented gcn5- cells for growth and transcriptional activation. Moreover, an amino acid substitution mutation within the HAT domain reduced both HAT activity in vitro and transcription in vivo. These findings directly link enzymatic histone acetylation and transcriptional activation and show evolutionary conservation of this potentially crucial pathway in gene regulation.

Inhibition of TATA-binding protein function by SAGA subunits Spt3 and Spt8 at Gcn4-activated promoters.

SAGA is a 1.8-MDa yeast protein complex that is composed of several distinct classes of transcription-related factors, including the adaptor/acetyltransferase Gcn5, Spt proteins, and a subset of TBP-associated factors. Our results indicate that mutations that completely disrupt SAGA (deletions of SPT7 or SPT20) strongly reduce transcriptional activation at the HIS3 and TRP3 genes and that Gcn5 is required for normal HIS3 transcriptional start site selection. Surprisingly, mutations in Spt proteins involved in the SAGA-TBP interaction (Spt3 and Spt8) cause derepression of HIS3 and TRP3 transcription in the uninduced state. Consistent with this finding, wild-type SAGA inhibits TBP binding to the HIS3 promoter in vitro, while SAGA lacking Spt3 or Spt8 is not inhibitory. We detected two distinct forms of SAGA in cell extracts and, strikingly, one lacks Spt8. Conditions that induce HIS3 and TRP3 transcription result in an altered balance between these complexes strongly in favor of the form without Spt8. These results suggest that the composition of SAGA may be dynamic in vivo and may be regulated through dissociable inhibitory subunits.

Repression of GCN5 histone acetyltransferase activity via bromodomain-mediated binding and phosphorylation by the Ku-DNA-dependent protein kinase complex.

GCN5, a putative transcriptional adapter in humans and yeast, possesses histone acetyltransferase (HAT) activity which has been linked to GCN5's role in transcriptional activation in yeast. In this report, we demonstrate a functional interaction between human GCN5 (hGCN5) and the DNA-dependent protein kinase (DNA-PK) holoenzyme. Yeast two-hybrid screening detected an interaction between the bromodomain of hGCN5 and the p70 subunit of the human Ku heterodimer (p70-p80), which is the DNA-binding component of DNA-PK. Interaction between intact hGCN5 and Ku70 was shown biochemically using recombinant proteins and by coimmunoprecipitation of endogenous proteins following chromatography of HeLa nuclear extracts. We demonstrate that the catalytic subunit of DNA-PK phosphorylates hGCN5 both in vivo and in vitro and, moreover, that the phosphorylation inhibits the HAT activity of hGCN5. These findings suggest a possible regulatory mechanism of HAT activity.

The ADA complex is a distinct histone acetyltransferase complex in Saccharomyces cerevisiae.

We have identified two Gcn5-dependent histone acetyltransferase (HAT) complexes from Saccharomyces cerevisiae, the 0.8-MDa ADA complex and the 1.8-MDa SAGA complex. The SAGA (Spt-Ada-Gcn5-acetyltransferase) complex contains several subunits which also function as part of other protein complexes, including a subset of TATA box binding protein-associated factors (TAFIIs) and Tra1. These observations raise the question of whether the 0.8-MDa ADA complex is a subcomplex of SAGA or whether it is a distinct HAT complex that also shares subunits with SAGA. To address this issue, we sought to determine if the ADA complex contained subunits that are not present in the SAGA complex. In this study, we report the purification of the ADA complex over 10 chromatographic steps. By a combination of mass spectrometry analysis and immunoblotting, we demonstrate that the adapter proteins Ada2, Ada3, and Gcn5 are indeed integral components of ADA. Furthermore, we identify the product of the S. cerevisiae gene YOR023C as a novel subunit of the ADA complex and name it Ahc1 for ADA HAT complex component 1. Biochemical functions of YOR023C have not been reported. However, AHC1 in high copy numbers suppresses the cold sensitivity caused by particular mutations in HTA1 (I. Pinto and F. Winston, personal communication), which encodes histone H2A (J. N. Hirschhorn et al., Mol. Cell. Biol. 15:1999-2009, 1995). Deletion of AHC1 disrupted the integrity of the ADA complex but did not affect SAGA or give rise to classic Ada(-) phenotypes. These results indicate that Gcn5, Ada2, and Ada3 function as part of a unique HAT complex (ADA) and represent shared subunits between this complex and SAGA.

Activation of chromosomal DNA replication in Saccharomyces cerevisiae by acidic transcriptional activation domains.

A large body of evidence from viral systems has established that transcription factors play an important and direct role in activating viral DNA replication. Among the transcriptional activation domains that can stimulate viral DNA replication are acidic domains such as those derived from herpes simplex virus VP16 and the tumor suppressor p53. Here we show that acidic activation domains can also activate a cellular origin of replication in a chromosomal context. When tethered to the yeast ARS1 (autonomously replicating sequence 1) origin of replication, both VP16 and p53 activation domains can enhance origin function. In addition, the C-terminal acidic region of the yeast transcription factor ABF1, which normally activates the ARS1 origin, is sufficient for activating ARS1 function when tethered to the origin. Mutations at residues Trp-53 and Phe-54 of a 20-residue (41 to 60) activation region of p53 abolish the activation of both replication and transcription, suggesting that the same structural determinants may be employed to activate both processes in yeast. Furthermore, using a two-dimensional gel electrophoresis method, we demonstrate that the GAL4-p53 chimeric activator can activate initiation of chromosomal replication from an origin inserted at the native ARS1 locus. These findings strongly suggest functional conservation of the mechanisms used by the acidic activation domains to activate viral DNA replication in mammalian cells and chromosomal replication in yeast.

Functional organization of the yeast SAGA complex: distinct components involved in structural integrity, nucleosome acetylation, and TATA-binding protein interaction.

SAGA, a recently described protein complex in Saccharomyces cerevisiae, is important for transcription in vivo and possesses histone acetylation function. Here we report both biochemical and genetic analyses of members of three classes of transcription regulatory factors contained within the SAGA complex. We demonstrate a correlation 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. In addition, we have investigated biochemical functions suggested by the moderate phenotypic classes and find that first, 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. Second, SAGA interacts with TBP, the TATA-binding protein, and this interaction requires Spt8 in vitro. Overall, our 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.

p53 sites acetylated in vitro by PCAF and p300 are acetylated in vivo in response to DNA damage.

The p53 tumor suppressor protein is a sequence-specific transcription factor that modulates the response of cells to DNA damage. Recent studies suggest that full transcriptional activity of p53 requires the coactivators CREB binding protein (CBP)/p300 and PCAF. These coactivators interact with each other, and both possess intrinsic histone acetyltransferase activity. Furthermore, p300 acetylates p53 to activate its sequence-specific DNA binding activity in vitro. In this study, we demonstrate that PCAF also acetylates p53 in vitro at a lysine residue distinct from that acetylated by p300 and thereby increases p53's ability to bind to its cognate DNA site. We have generated antibodies to acetylated p53 peptides at either of the two lysine residues that are targeted by PCAF or p300 and have demonstrated that these antibodies are highly specific for both acetylation and the particular site. Using these antibodies, we detect acetylation of these sites in vivo, and interestingly, acetylation at both sites increases in response to DNA-damaging agents. These data indicate that site-specific acetylation of p53 increases under physiological conditions that activate p53 and identify CBP/p300 and PCAF as the probable enzymes that modify p53 in vivo.

Histone post-translational modifications regulate transcription and silent chromatin in Saccharomyces cerevisiae.

Regulation of chromatin structure is important for the control of DNA-templated processes such as gene expression and silencing, and its dysregulation is implicated in diverse developmental and cell proliferative defects such as tumorigenesis. Covalent post-translational modifications of histones are one of the prominent means to regulate the chromatin structure. Here, we summarize findings from our lab and others regarding the interactions between different covalent modifications of histones in the budding yeast Saccharomyces cerevisiae. First, we describe the effect of histone H3 phosphorylation at residue serine 10 in transcriptional gene activation, and its histone H3 acetylation dependent and independent modes of action and downstream effects on TATA-binding protein (TBP) recruitment. Further, we review how ubiquitylation of histone H2B and its deubiquitylation by ubiquitin proteases Ubp8 and Ubp10 regulate histone H3 methylations, and consequently affect co-activator-dependent gene transcription and silent chromatin, respectively.

CREB-binding protein and p300/CBP-associated factor are transcriptional coactivators of the p53 tumor suppressor protein.

The structurally related transcriptional coactivators p300 and CBP possess histone acetyltransferase activity and associate with P/CAF, which is also a histone acetyltransferase. CBP and p300 have properties of tumor suppressor proteins; their interaction with P/CAF is disrupted by the adenoviral E1A oncoprotein, and the genes encoding CBP and p300 are mutated in human cancer. We observed a physical interaction between the transactivation domain of the p53 tumor suppressor protein and CBP. Furthermore, CBP and P/CAF enhanced the ability of p53 to activate expression of the endogenous p21(cip1/waf1) gene, whereas E1A and dominant negative CBP mutants suppressed p53-dependent p21(cip1/waf1) expression. These studies link two tumor suppressor families and provide a framework for understanding the molecular mechanism by which p53 activates transcription.

Exploring the Dynamic Relationship Between Cellular Metabolism and Chromatin Structure Using SILAC-Mass Spec and ChIP-Sequencing.

Metabolic state and chromatin structure are tightly linked, enabling adaptation of gene expression to changing environment and metabolism. The bioenergetic pathways and enzymes that provide metabolic cofactors for histone modification have recently emerged as central regulators of chromatin. Current research therefore focuses on the dynamic interface of cellular metabolism and chromatin structure. Here, we provide an adaptable approach to examine broadly in changing physiological states, how chromatin structure is dynamically modulated by metabolic activity. We employ two complementary methods: high-throughput sequencing to establish the location of epigenetic changes, and stable isotope tracing using mass spectrometry to evaluate chromatin modification dynamics. Our two-pronged approach is of particular advantage when interrogating how metabolic and oncogenic mutations influence the dynamic relationship between metabolism, nutritional environment, and chromatin regulation.

Two tandem and independent sub-activation domains in the amino terminus of p53 require the adaptor complex for activity.

The ability of p53 to function as a tumor suppressor is linked to its function as a transcriptional activator, since p53 mutants that do not transactivate are unable to suppress tumor cell growth. Previous studies identified an activation domain in the amino terminal 40 residues of the protein, a region that binds to several general transcription factors and to some oncogene products. For example, mdm-2, a cellular oncoprotein, binds to this region and represses p53 transactivation. Here we describe a new activation domain within the amino terminus of p53 that maps between amino acids 40-83, and whose residues trp-53 and phe-54 are critical for function both in yeast and in mammalian cells. In vivo studies in yeast show that the new activation subdomain, unlike the previously described, is mdm-2 independent. Both p53 activation subdomains (1-40 and 40-83) require the yeast adaptor complex ADA2/ADA3/GCN5 for transcriptional activation. Moreover, since activation by p53 requires GCN5's enzymatic histone acetyltransferase domain, p53 may regulate gene expression by influencing chromatin modification.

BAP1: a novel ubiquitin hydrolase which binds to the BRCA1 RING finger and enhances BRCA1-mediated cell growth suppression.

We have identified a novel protein, BAP1, which binds to the RING finger domain of the Breast/Ovarian Cancer Susceptibility Gene product, BRCA1. BAP1 is a nuclear-localized, ubiquitin carboxy-terminal hydrolase, suggesting that deubiquitinating enzymes may play a role in BRCA1 function. BAP1 binds to the wild-type BRCA1-RING finger, but not to germline mutants of the BRCA1-RING finger found in breast cancer kindreds. BAP1 and BRCA1 are temporally and spatially co-expressed during murine breast development and remodeling, and show overlapping patterns of subnuclear distribution. BAP1 resides on human chromosome 3p21.3; intragenic homozygous rearrangements and deletions of BAP1 have been found in lung carcinoma cell lines. BAP1 enhances BRCA1-mediated inhibition of breast cancer cell growth and is the first nuclear-localized ubiquitin carboxy-terminal hydrolase to be identified. BAP1 may be a new tumor suppressor gene which functions in the BRCA1 growth control pathway.