Crystal structure and mechanism of histone acetylation of the yeast GCN5 transcriptional coactivator.
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ABSTRACT:
SUBMITTER: Trievel RC
PROVIDER: S-EPMC17710 | biostudies-literature | 1999 Aug
REPOSITORIES: biostudies-literature
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Project description:The histone acetylation of post-translational modification can be highly dynamic and play a crucial role in regulating cellular proliferation, survival, differentiation and motility. Of the enzymes that mediate post-translation modifications, the GCN5 of the histone acetyltransferase (HAT) proteins family that add acetyl groups to target lysine residues within histones, has been most extensively studied. According to the mechanism studies of GCN5 related proteins, two key processes, deprotonation and acetylation, must be involved. However, as a fundamental issue, the structure of hGCN5/AcCoA/pH3 remains elusive. Although biological experiments have proved that GCN5 mediates the acetylation process through the sequential mechanism pathway, a dynamic view of the catalytic process and the molecular basis for hGCN5/AcCoA/pH3 are still not available and none of theoretical studies has been reported to other related enzymes in HAT family. To explore the molecular basis for the catalytic mechanism, computational approaches including molecular modeling, molecular dynamic (MD) simulation and quantum mechanics/molecular mechanics (QM/MM) simulation were carried out. The initial hGCN5/AcCoA/pH3 complex structure was modeled and a reasonable snapshot was extracted from the trajectory of a 20 ns MD simulation, with considering post-MD analysis and reported experimental results. Those residues playing crucial roles in binding affinity and acetylation reaction were comprehensively investigated. It demonstrated Glu80 acted as the general base for deprotonation of Lys171 from H3. Furthermore, the two-dimensional QM/MM potential energy surface was employed to study the sequential pathway acetylation mechanism. Energy barriers of addition-elimination reaction in acetylation obtained from QM/MM calculation indicated the point of the intermediate ternary complex. Our study may provide insights into the detailed mechanism for acetylation reaction of GCN5, and has important implications for the discovery of regulators against GCN5 enzymes and related HAT family enzymes.
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Project description:In the last several years, a number of studies have shown that spliceosome assembly and splicing catalysis can occur co-transcriptionally. However, it has been unclear which specific transcription factors play key roles in coupling splicing to transcription and the mechanisms through which they act. Here we report the discovery that Gcn5, which encodes the histone acetyltransferase (HAT) activity of the SAGA complex, has genetic interactions with the genes encoding the heterodimeric U2 snRNP proteins Msl1 and Lea1. These interactions are dependent upon the HAT activity of Gcn5, suggesting a functional relationship between Gcn5 HAT activity and Msl1/Lea1 function. To understand the relationship between Gcn5 and Msl1/Lea1, we carried out an analysis of Gcn5's role in co-transcriptional recruitment of Msl1 and Lea1 to pre-mRNA and found that Gcn5 HAT activity is required for co-transcriptional recruitment of the U2 snRNP (and subsequent snRNP) components to the branchpoint, while it is not required for U1 recruitment. Although previous studies suggest that transcription elongation can alter co-transcriptional pre-mRNA splicing, we do not observe evidence of defective transcription elongation for these genes in the absence of Gcn5, while Gcn5-dependent histone acetylation is enriched in the promoter regions. Unexpectedly, we also observe Msl1 enrichment in the promoter region for wild-type cells and cells lacking Gcn5, indicating that Msl1 recruitment during active transcription can occur independently of its association at the branchpoint region. These results demonstrate a novel role for acetylation by SAGA in co-transcriptional recruitment of the U2 snRNP and recognition of the intron branchpoint.
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Project description:Changes in expression levels of genes encoding for proteins that control metabolic pathways are essential to maintain nutrient and energy homeostasis in individual cells as well as in organisms. An important regulated step in this process is accomplished through covalent chemical modifications of proteins that form complexes with the chromatin of gene promoters. The peroxisome proliferators gamma co-activator 1 (PGC-1) family of transcriptional co-activators comprises important components of a number of these complexes and participates in a large array of glucose and lipid metabolic adaptations. Here, we show that PGC-1beta is acetylated on at least 10 lysine residues distributed along the length of the protein by the acetyl transferase general control of amino-acid synthesis (GCN5) and that this acetylation reaction is reversed by the deacetylase sirtuin 1 (SIRT1). GCN5 strongly interacts with PGC-1beta and represses its transcriptional activity associated with transcription factors such as ERRalpha, NRF-1, and HNF4alpha, however acetylation and transcriptional repression do not occur when a catalytically inactive GCN5 is co-expressed. Transcriptional repression coincides with PGC-1beta redistribution to nuclear foci where it co-localizes with GCN5. Furthermore, knockdown of GCN5 ablates PGC-1beta acetylation and increases transcriptional activity. In primary skeletal muscle cells, PGC-1beta induction of endogenous target genes, including MCAD and GLUT4, is largely repressed by GCN5. Functionally, this translates to a blunted response to PGC-1beta-induced insulin-mediated glucose transport. These results suggest that PGC-1beta acetylation by GCN5 might be an important step in the control of glucose and lipid pathways and its dysregulation could contribute to metabolic diseases.
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Project description:Epigenetic mechanisms play diverse roles in the regulation of genome stability in eukaryotes. In Arabidopsis thaliana, genome stability is maintained during DNA replication by the H3.1K27 methyltransferases ARABIDOPSIS TRITHORAX-RELATED PROTEIN 5 (ATXR5) and ATXR6, which catalyze the deposition of K27me1 on replication-dependent H3.1 variants. The loss of H3.1K27me1 in atxr5 atxr6 double mutants leads to heterochromatin defects, including transcriptional de-repression and genomic instability, but the molecular mechanisms involved remain largely unknown. In this study, we identified the transcriptional co-activator and conserved histone acetyltransferase GCN5 as a mediator of transcriptional de-repression and genomic instability in the absence of H3.1K27me1. GCN5 is part of a SAGA-like complex in plants that requires the GCN5-interacting protein ADA2b and the chromatin remodeler CHR6 to mediate the heterochromatic defects in atxr5 atxr6 mutants. Our results also indicate that Arabidopsis GCN5 acetylates multiple lysine residues on H3.1 variants, but H3.1K27 and H3.1K36 play essential functions in inducing genomic instability in the absence of H3.1K27me1. Finally, we show that H3.1K36 acetylation by GCN5 is negatively regulated by H3.1K27me1 in vitro. Overall, this work reveals a key molecular role for H3.1K27me1 in maintaining transcriptional silencing and genome stability in heterochromatin by restricting GCN5-mediated histone acetylation in plants.
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Project description:Molecular regulation of stem cell differentiation is exerted through both genetic and epigenetic determinants over distal regulatory or enhancer regions. Understanding the mechanistic action of active or poised enhancers is therefore imperative for control of stem cell differentiation. Based on the genome-wide co-occurrence of different epigenetic marks in committed proliferating myoblasts, we have previously generated a 14-state chromatin state model to profile rexinoid-responsive histone acetylation in early myoblast differentiation. Here, we delineate the functional mode of transcription regulators during early myogenic differentiation using genome-wide chromatin state association. We define a role of transcriptional coactivator p300, when recruited by muscle master regulator MyoD, in the establishment and regulation of myogenic loci at the onset of myoblast differentiation. In addition, we reveal an enrichment of loci-specific histone acetylation at p300 associated active or poised enhancers, particularly when enlisted by MyoD. We provide novel molecular insights into the regulation of myogenic enhancers by p300 in concert with MyoD. Our studies present a valuable aptitude for driving condition-specific chromatin state or enhancers pharmacologically to treat muscle-related diseases and for the identification of additional myogenic targets and molecular interactions for therapeutic development. ABBREVIATIONS:MRF: Muscle regulatory factor; HAT: Histone acetyltransferase; CBP: CREB-binding protein; ES: Embryonic stem; ATCC: American type culture collection; DM: Differentiation medium; DMEM: Dulbecco's Modified Eagle Medium; GM: Growth medium; GO: Gene ontology; GREAT: Genomic regions enrichment of annotations tool; FPKM: Fragments per kilobase of transcript per million; GEO: Gene expression omnibus; MACS: Model-based analysis for ChIP-seq.
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Project description:During mammalian spermatogenesis, germ cell chromatin undergoes dramatic histone acetylation-mediated reorganization, whereby 90%-99% of histones are evicted. Given the potential role of retained histones in fertility and embryonic development, the genomic location of retained nucleosomes is of great interest. However, the ultimate position and mechanisms underlying nucleosome eviction or retention are poorly understood, including several studies utilizing micrococcal-nuclease sequencing (MNase-seq) methodologies reporting remarkably dissimilar locations. We utilized assay for transposase accessible chromatin sequencing (ATAC-seq) in mouse sperm and found nucleosome enrichment at promoters but also retention at inter- and intragenic regions and repetitive elements. We further generated germ-cell-specific, conditional knockout mice for the key histone acetyltransferase Gcn5, which resulted in abnormal chromatin dynamics leading to increased sperm histone retention and severe reproductive phenotypes. Our findings demonstrate that Gcn5-mediated histone acetylation promotes chromatin accessibility and nucleosome eviction in spermiogenesis and that loss of histone acetylation leads to defects that disrupt male fertility and potentially early embryogenesis.
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Project description:Histone acetylation marks are written by histone acetyltransferases (HATs) and read by bromodomains (BrDs), and less commonly by other protein modules. These proteins regulate many transcription-mediated biological processes, and their aberrant activities are correlated with several human diseases. Consequently, small molecule HAT and BrD inhibitors with therapeutic potential have been developed. Structural and biochemical studies of HATs and BrDs have revealed that HATs fall into distinct subfamilies containing a structurally related core for cofactor binding, but divergent flanking regions for substrate-specific binding, catalysis, and autoregulation. BrDs adopt a conserved left-handed four-helix bundle to recognize acetyllysine; divergent loop residues contribute to substrate-specific acetyllysine recognition.
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Project description:Candida glabrata is poised to adapt to drug pressure rapidly and acquire antifungal resistance leading to therapeutic failure. Given the limited antifungal armamentarium, there is an unmet need to explore new targets or therapeutic strategies for antifungal treatment. The lysine acetyltransferase Gcn5 has been implicated in the pathogenesis of C. albicans. Yet how Gcn5 functions and impacts antifungal resistance in C. glabrata is unknown. Disrupting GCN5 rendered C. glabrata cells more sensitive to various stressors, partially reverted resistance in drug-resistant mutants, and attenuated the emergence of resistance compared to wild-type cells. RNA sequencing (RNA-seq) analysis revealed transcriptomic changes involving multiple biological processes and different transcriptional responses to antifungal drugs in gcn5Δ cells compared to wild-type cells. GCN5 deletion also resulted in reduced intracellular survival within THP-1 macrophages. In summary, Gcn5 plays a critical role in modulating the virulence of C. glabrata and regulating its response to antifungal pressure and host defense. IMPORTANCE As an important and successful human pathogen, Candida glabrata is known for its swift adaptation and rapid acquisition of resistance to the most commonly used antifungal agents, resulting in therapeutic failure in clinical settings. Here, we describe that the histone acetyltransferase Gcn5 is a key factor in adapting to antifungal pressure and developing resistance in C. glabrata. The results provide new insights into epigenetic control over the drug response in C. glabrata and may be useful for drug target discovery and the development of new therapeutic strategies to combat fungal infections.
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Project description:Dynamic disruption and reassembly of promoter-proximal nucleosomes is a conserved hallmark of transcriptionally active chromatin. Histone H3-K56 acetylation (H3K56Ac) enhances these turnover events and promotes nucleosome assembly during S phase. Here we sequence nascent transcripts to investigate the impact of H3K56Ac on transcription throughout the yeast cell cycle. We find that H3K56Ac is a genome-wide activator of transcription. While H3K56Ac has a major impact on transcription initiation, it also appears to promote elongation and/or termination. In contrast, H3K56Ac represses promiscuous transcription that occurs immediately following replication fork passage, in this case by promoting efficient nucleosome assembly. We also detect a stepwise increase in transcription as cells transit S phase and enter G2, but this response to increased gene dosage does not require H3K56Ac. Thus, a single histone mark can exert both positive and negative impacts on transcription that are coupled to different cell cycle events.
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