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1.
Genes Dev ; 35(23-24): 1678-1692, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34819351

RESUMO

How transcription programs rapidly adjust to changing metabolic and cellular cues remains poorly defined. Here, we reveal a function for the Yaf9 component of the SWR1-C and NuA4 chromatin regulatory complexes in maintaining timely transcription of metabolic genes across the yeast metabolic cycle (YMC). By reading histone acetylation during the oxidative and respiratory phase of the YMC, Yaf9 recruits SWR1-C and NuA4 complexes to deposit H2A.Z and acetylate H4, respectively. Increased H2A.Z and H4 acetylation during the oxidative phase promotes transcriptional initiation and chromatin machinery occupancy and is associated with reduced RNA polymerase II levels at genes-a pattern reversed during transition from oxidative to reductive metabolism. Prevention of Yaf9-H3 acetyl reading disrupted this pattern of transcriptional and chromatin regulator recruitment and impaired the timely transcription of metabolic genes. Together, these findings reveal that Yaf9 contributes to a dynamic chromatin and transcription initiation factor signature that is necessary for the proper regulation of metabolic gene transcription during the YMC. They also suggest that unique regulatory mechanisms of transcription exist at distinct metabolic states.

2.
J Biol Chem ; 297(4): 101145, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34473995

RESUMO

Bromodomains (BD) are conserved reader modules that bind acetylated lysine residues on histones. Although much has been learned regarding the in vitro properties of these domains, less is known about their function within chromatin complexes. SWI/SNF chromatin-remodeling complexes modulate transcription and contribute to DNA damage repair. Mutations in SWI/SNF subunits have been implicated in many cancers. Here we demonstrate that the BD of Caenorhabditis elegans SMARCA4/BRG1, a core SWI/SNF subunit, recognizes acetylated lysine 14 of histone H3 (H3K14ac), similar to its Homo sapiens ortholog. We identify the interactions of SMARCA4 with the acetylated histone peptide from a 1.29 Å-resolution crystal structure of the CeSMARCA4 BD-H3K14ac complex. Significantly, most of the SMARCA4 BD residues in contact with the histone peptide are conserved with other proteins containing family VIII bromodomains. Based on the premise that binding specificity is conserved among bromodomain orthologs, we propose that loop residues outside of the binding pocket position contact residues to recognize the H3K14ac sequence. CRISPR-Cas9-mediated mutations in the SMARCA4 BD that abolish H3K14ac binding in vitro had little or no effect on C. elegans viability or physiological function in vivo. However, combining SMARCA4 BD mutations with knockdown of the SWI/SNF accessory subunit PBRM-1 resulted in severe developmental defects in animals. In conclusion, we demonstrated an essential function for the SWI/SNF bromodomain in vivo and detected potential redundancy in epigenetic readers in regulating chromatin remodeling. These findings have implications for the development of small-molecule BD inhibitors to treat cancers and other diseases.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Histonas/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Histonas/genética , Humanos , Ligação Proteica , Fatores de Transcrição/genética
3.
Elife ; 102021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34581668

RESUMO

Measuring protein-protein interaction (PPI) affinities is fundamental to biochemistry. Yet, conventional methods rely upon the law of mass action and cannot measure many PPIs due to a scarcity of reagents and limitations in the measurable affinity ranges. Here, we present a novel technique that leverages the fundamental concept of friction to produce a mechanical signal that correlates to binding potential. The mechanically transduced immunosorbent (METRIS) assay utilizes rolling magnetic probes to measure PPI interaction affinities. METRIS measures the translational displacement of protein-coated particles on a protein-functionalized substrate. The translational displacement scales with the effective friction induced by a PPI, thus producing a mechanical signal when a binding event occurs. The METRIS assay uses as little as 20 pmols of reagents to measure a wide range of affinities while exhibiting a high resolution and sensitivity. We use METRIS to measure several PPIs that were previously inaccessible using traditional methods, providing new insights into epigenetic recognition.


Assuntos
Bioensaio/métodos , Imunoadsorventes/química , Mapeamento de Interação de Proteínas , Proteínas/metabolismo , Fenômenos Biofísicos , Magnetismo , Ligação Proteica , Proteômica
4.
Nat Commun ; 12(1): 2490, 2021 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-33941775

RESUMO

DNA methylation and trimethylated histone H4 Lysine 20 (H4K20me3) constitute two important heterochromatin-enriched marks that frequently cooperate in silencing repetitive elements of the mammalian genome. However, it remains elusive how these two chromatin modifications crosstalk. Here, we report that DNA methyltransferase 1 (DNMT1) specifically 'recognizes' H4K20me3 via its first bromo-adjacent-homology domain (DNMT1BAH1). Engagement of DNMT1BAH1-H4K20me3 ensures heterochromatin targeting of DNMT1 and DNA methylation at LINE-1 retrotransposons, and cooperates with the previously reported readout of histone H3 tail modifications (i.e., H3K9me3 and H3 ubiquitylation) by the RFTS domain to allosterically regulate DNMT1's activity. Interplay between RFTS and BAH1 domains of DNMT1 profoundly impacts DNA methylation at both global and focal levels and genomic resistance to radiation-induced damage. Together, our study establishes a direct link between H4K20me3 and DNA methylation, providing a mechanism in which multivalent recognition of repressive histone modifications by DNMT1 ensures appropriate DNA methylation patterning and genomic stability.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA/genética , Heterocromatina/metabolismo , Histonas/metabolismo , Elementos Nucleotídeos Longos e Dispersos/genética , Animais , Linhagem Celular , Cristalografia por Raios X , Genoma/genética , Instabilidade Genômica/genética , Heterocromatina/genética , Camundongos
5.
J Biol Chem ; 296: 100721, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33933452

RESUMO

DNA double-strand breaks (DSBs) are among the deleterious lesions that are both endogenous and exogenous in origin and are repaired by nonhomologous end joining or homologous recombination. However, the molecular mechanisms responsible for maintaining genome stability remain incompletely understood. Here, we investigate the role of two E3 ligases, Dma1 and Dma2 (homologs of human RNF8), in the maintenance of genome stability in budding yeast. Using yeast spotting assays, chromatin immunoprecipitation and plasmid and chromosomal repair assays, we establish that Dma1 and Dma2 act in a redundant and a catalysis-dependent manner in the maintenance of genome stability, as well as localize to transcribed regions of the genome and increase in abundance upon phleomycin treatment. In addition, Dma1 and Dma2 are required for the normal kinetics of histone H4 acetylation under DNA damage conditions, genetically interact with RAD9 and SAE2, and are in a complex with Rad53 and histones. Taken together, our results demonstrate the requirement of Dma1 and Dma2 in regulating DNA repair pathway choice, preferentially affecting homologous recombination over nonhomologous end joining, and open up the possibility of using these candidates in manipulating the repair pathways toward precision genome editing.


Assuntos
Biocatálise , Proteínas de Ciclo Celular/metabolismo , Instabilidade Genômica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Reparo do DNA
7.
Cell Rep ; 34(3): 108638, 2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33472068

RESUMO

Histone acetylation levels are regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs) that antagonistically control the overall balance of this post-translational modification. HDAC inhibitors (HDACi) are potent agents that disrupt this balance and are used clinically to treat diseases including cancer. Despite their use, little is known about their effects on chromatin regulators, particularly those that signal through lysine acetylation. We apply quantitative genomic and proteomic approaches to demonstrate that HDACi robustly increases a low-abundance histone 4 polyacetylation state, which serves as a preferred binding substrate for several bromodomain-containing proteins, including BRD4. Increased H4 polyacetylation occurs in transcribed genes and correlates with the targeting of BRD4. Collectively, these results suggest that HDAC inhibition functions, at least in part, through expansion of a rare histone acetylation state, which then retargets lysine-acetyl readers associated with changes in gene expression, partially mimicking the effect of bromodomain inhibition.

8.
Biochim Biophys Acta Gene Regul Mech ; 1864(2): 194600, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32645359

RESUMO

Fueled by key technological innovations during the last several decades, chromatin-based research has greatly advanced our mechanistic understanding of how genes are regulated by epigenetic factors and their associated histone-modifying activities. Most notably, the landmark finding that linked histone acetylation by Gcn5 of the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex to gene activation ushered in a new area of chromatin research and a realization that histone-modifying activities have integral genome functions. This review will discuss past and recent studies that have shaped our understanding of how the histone-modifying activities of SAGA are regulated by, and modulate the outcomes of, other histone modifications during gene transcription. Because much of our understanding of SAGA was established with budding yeast, we will focus on yeast as a model. We discuss the actions of cis- and trans-histone crosstalk pathways that involve the histone acetyltransferase, deubiquitylase, and reader domains of SAGA. We conclude by considering unanswered questions about SAGA and related complexes.


Assuntos
Epigênese Genética , Regulação Fúngica da Expressão Gênica , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transativadores/metabolismo , Transcrição Genética , Acetilação , Cromatina/metabolismo , Enzimas Desubiquitinantes/metabolismo , Histona Acetiltransferases/metabolismo , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Ubiquitinação
9.
Science ; 370(6522)2020 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-33303586

RESUMO

Determining structures of protein complexes is crucial for understanding cellular functions. Here, we describe an integrative structure determination approach that relies on in vivo measurements of genetic interactions. We construct phenotypic profiles for point mutations crossed against gene deletions or exposed to environmental perturbations, followed by converting similarities between two profiles into an upper bound on the distance between the mutated residues. We determine the structure of the yeast histone H3-H4 complex based on ~500,000 genetic interactions of 350 mutants. We then apply the method to subunits Rpb1-Rpb2 of yeast RNA polymerase II and subunits RpoB-RpoC of bacterial RNA polymerase. The accuracy is comparable to that based on chemical cross-links; using restraints from both genetic interactions and cross-links further improves model accuracy and precision. The approach provides an efficient means to augment integrative structure determination with in vivo observations.


Assuntos
Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Mapas de Interação de Proteínas/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Histonas/química , Histonas/genética , Mutação , Conformação Proteica , Mapeamento de Interação de Proteínas , Saccharomyces cerevisiae/genética
10.
Nat Genet ; 52(12): 1384-1396, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33139953

RESUMO

Trimethylated histone H3 lysine 27 (H3K27me3) regulates gene repression, cell-fate determination and differentiation. We report that a conserved bromo-adjacent homology (BAH) module of BAHCC1 (BAHCC1BAH) 'recognizes' H3K27me3 specifically and enforces silencing of H3K27me3-demarcated genes in mammalian cells. Biochemical, structural and integrated chromatin immunoprecipitation-sequencing-based analyses demonstrate that direct readout of H3K27me3 by BAHCC1 is achieved through a hydrophobic trimethyl-L-lysine-binding 'cage' formed by BAHCC1BAH, mediating colocalization of BAHCC1 and H3K27me3-marked genes. BAHCC1 is highly expressed in human acute leukemia and interacts with transcriptional corepressors. In leukemia, depletion of BAHCC1, or disruption of the BAHCC1BAH-H3K27me3 interaction, causes derepression of H3K27me3-targeted genes that are involved in tumor suppression and cell differentiation, leading to suppression of oncogenesis. In mice, introduction of a germline mutation at Bahcc1 to disrupt its H3K27me3 engagement causes partial postnatal lethality, supporting a role in development. This study identifies an H3K27me3-directed transduction pathway in mammals that relies on a conserved BAH 'reader'.


Assuntos
Carcinogênese/genética , Código das Histonas/genética , Histonas/metabolismo , Leucemia/genética , Proteínas/genética , Proteínas/metabolismo , Animais , Diferenciação Celular/genética , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Regulação da Expressão Gênica/genética , Inativação Gênica/fisiologia , Células HEK293 , Células HeLa , Humanos , Células Jurkat , Leucemia/patologia , Metilação , Camundongos , Camundongos Transgênicos , Transplante de Neoplasias , Processamento de Proteína Pós-Traducional/genética , Transplante Heterólogo
11.
Epigenetics Chromatin ; 13(1): 44, 2020 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-33097091

RESUMO

The chromatin-binding E3 ubiquitin ligase ubiquitin-like with PHD and RING finger domains 1 (UHRF1) contributes to the maintenance of aberrant DNA methylation patterning in cancer cells through multivalent histone and DNA recognition. The tandem Tudor domain (TTD) of UHRF1 is well-characterized as a reader of lysine 9 di- and tri-methylation on histone H3 (H3K9me2/me3) and, more recently, lysine 126 di- and tri-methylation on DNA ligase 1 (LIG1K126me2/me3). However, the functional significance and selectivity of these interactions remain unclear. In this study, we used protein domain microarrays to search for additional readers of LIG1K126me2, the preferred methyl state bound by the UHRF1 TTD. We show that the UHRF1 TTD binds LIG1K126me2 with high affinity and selectivity compared to other known methyllysine readers. Notably, and unlike H3K9me2/me3, the UHRF1 plant homeodomain (PHD) and its N-terminal linker (L2) do not contribute to multivalent LIG1K126me2 recognition along with the TTD. To test the functional significance of this interaction, we designed a LIG1K126me2 cell-penetrating peptide (CPP). Consistent with LIG1 knockdown, uptake of the CPP had no significant effect on the propagation of DNA methylation patterning across the genomes of bulk populations from high-resolution analysis of several cancer cell lines. Further, we did not detect significant changes in DNA methylation patterning from bulk cell populations after chemical or genetic disruption of lysine methyltransferase activity associated with LIG1K126me2 and H3K9me2. Collectively, these studies identify UHRF1 as a selective reader of LIG1K126me2 in vitro and further implicate the histone and non-histone methyllysine reader activity of the UHRF1 TTD as a dispensable domain function for cancer cell DNA methylation maintenance.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Código das Histonas , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/química , Epigênese Genética , Células HCT116 , Células HeLa , Histonas/química , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilação , Processamento de Proteína Pós-Traducional , Domínio Tudor , Ubiquitina-Proteína Ligases/química
12.
Genome Res ; 30(11): 1605-1617, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33020206

RESUMO

Histone H3 lysine 36 methylation (H3K36me) is a conserved histone modification associated with transcription and DNA repair. Although the effects of H3K36 methylation have been studied, the genome-wide dynamics of H3K36me deposition and removal are not known. We established rapid and reversible optogenetic control for Set2, the sole H3K36 methyltransferase in yeast, by fusing the enzyme with the light-activated nuclear shuttle (LANS) domain. Light activation resulted in efficient Set2-LANS nuclear localization followed by H3K36me3 deposition in vivo, with total H3K36me3 levels correlating with RNA abundance. Although genes showed disparate levels of H3K36 methylation, relative rates of H3K36me3 accumulation were largely linear and consistent across genes, suggesting that H3K36me3 deposition occurs in a directed fashion on all transcribed genes regardless of their overall transcription frequency. Removal of H3K36me3 was highly dependent on the demethylase Rph1. However, the per-gene rate of H3K36me3 loss weakly correlated with RNA abundance and followed exponential decay, suggesting H3K36 demethylases act in a global, stochastic manner. Altogether, these data provide a detailed temporal view of H3K36 methylation and demethylation that suggests transcription-dependent and -independent mechanisms for H3K36me deposition and removal, respectively.


Assuntos
Histonas/metabolismo , Metiltransferases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcrição Genética , Genoma Fúngico , Código das Histonas , Histona Desmetilases/metabolismo , Histonas/química , Lisina/metabolismo , Metilação , Modelos Estatísticos , Optogenética , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
13.
Nat Commun ; 11(1): 5466, 2020 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-33122719

RESUMO

Human Microrchidia 4 (MORC4) is associated with acute and chronic pancreatitis, inflammatory disorders and cancer but it remains largely uncharacterized. Here, we describe the structure-function relationship of MORC4 and define the molecular mechanism for MORC4 activation. Enzymatic and binding assays reveal that MORC4 has ATPase activity, which is dependent on DNA-binding functions of both the ATPase domain and CW domain of MORC4. The crystal structure of the ATPaseCW cassette of MORC4 and mutagenesis studies show that the DNA-binding site and the histone/ATPase binding site of CW are located on the opposite sides of the domain. The ATPase and CW domains cooperate in binding of MORC4 to the nucleosome core particle (NCP), enhancing the DNA wrapping around the histone core and impeding binding of DNA-associated proteins, such as transcription factors, to the NCP. In cells, MORC4 mediates formation of nuclear bodies in the nucleus and has a role in the progression of S-phase of the cell cycle, and both these functions require CW and catalytic activity of MORC4. Our findings highlight the mechanism for MORC4 activation, which is distinctly different from the mechanisms of action observed in other MORC family members.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas Nucleares , Sítios de Ligação , Ciclo Celular , Cristalografia por Raios X , DNA/metabolismo , Células HEK293 , Histonas/metabolismo , Humanos , Corpos de Inclusão Intranuclear/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Ligação Proteica , Domínios Proteicos/fisiologia , Pontos de Checagem da Fase S do Ciclo Celular , Espectrometria de Fluorescência , Fatores de Transcrição/metabolismo , Nucleases de Dedos de Zinco/química , Nucleases de Dedos de Zinco/metabolismo
14.
Proc Natl Acad Sci U S A ; 117(31): 18439-18447, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32675241

RESUMO

In mammals, repressive histone modifications such as trimethylation of histone H3 Lys9 (H3K9me3), frequently coexist with DNA methylation, producing a more stable and silenced chromatin state. However, it remains elusive how these epigenetic modifications crosstalk. Here, through structural and biochemical characterizations, we identified the replication foci targeting sequence (RFTS) domain of maintenance DNA methyltransferase DNMT1, a module known to bind the ubiquitylated H3 (H3Ub), as a specific reader for H3K9me3/H3Ub, with the recognition mode distinct from the typical trimethyl-lysine reader. Disruption of the interaction between RFTS and the H3K9me3Ub affects the localization of DNMT1 in stem cells and profoundly impairs the global DNA methylation and genomic stability. Together, this study reveals a previously unappreciated pathway through which H3K9me3 directly reinforces DNMT1-mediated maintenance DNA methylation.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA , Heterocromatina/metabolismo , Histonas/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , Heterocromatina/genética , Histonas/química , Histonas/genética , Humanos , Lisina/genética , Lisina/metabolismo , Metilação , Processamento de Proteína Pós-Traducional
15.
Cell Rep ; 31(10): 107751, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32521276

RESUMO

Set2 co-transcriptionally methylates lysine 36 of histone H3 (H3K36), producing mono-, di-, and trimethylation (H3K36me1/2/3). These modifications recruit or repel chromatin effector proteins important for transcriptional fidelity, mRNA splicing, and DNA repair. However, it was not known whether the different methylation states of H3K36 have distinct biological functions. Here, we use engineered forms of Set2 that produce different lysine methylation states to identify unique and shared functions for H3K36 modifications. Although H3K36me1/2 and H3K36me3 are functionally redundant in many SET2 deletion phenotypes, we found that H3K36me3 has a unique function related to Bur1 kinase activity and FACT (facilitates chromatin transcription) complex function. Further, during nutrient stress, either H3K36me1/2 or H3K36me3 represses high levels of histone acetylation and cryptic transcription that arises from within genes. Our findings uncover the potential for the regulation of diverse chromatin functions by different H3K36 methylation states.


Assuntos
Histonas/metabolismo , Processamento de Proteína Pós-Traducional/genética , Transcrição Genética/genética , Humanos , Metilação
16.
J Biol Chem ; 295(19): 6561-6569, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32245891

RESUMO

Histone H2B monoubiquitylation (H2Bub1) has central functions in multiple DNA-templated processes, including gene transcription, DNA repair, and replication. H2Bub1 also is required for the trans-histone regulation of H3K4 and H3K79 methylation. Although previous studies have elucidated the basic mechanisms that establish and remove H2Bub1, we have only an incomplete understanding of how H2Bub1 is regulated. We report here that the histone H4 basic patch regulates H2Bub1. Yeast cells with arginine-to-alanine mutations in the H4 basic patch (H42RA) exhibited a significant loss of global H2Bub1. H42RA mutant yeast strains also displayed chemotoxin sensitivities similar to, but less severe than, strains containing a complete loss of H2Bub1. We found that the H4 basic patch regulates H2Bub1 levels independently of interactions with chromatin remodelers and separately from its regulation of H3K79 methylation. To measure H2B ubiquitylation and deubiquitination kinetics in vivo, we used a rapid and reversible optogenetic tool, the light-inducible nuclear exporter, to control the subcellular location of the H2Bub1 E3 ligase, Bre1. The ability of Bre1 to ubiquitylate H2B was unaffected in the H42RA mutant. In contrast, H2Bub1 deubiquitination by SAGA-associated Ubp8, but not by Ubp10, increased in the H42RA mutant. Consistent with a function for the H4 basic patch in regulating SAGA deubiquitinase activity, we also detected increased SAGA-mediated histone acetylation in H4 basic patch mutants. Our findings uncover that the H4 basic patch has a regulatory function in SAGA-mediated histone modifications.


Assuntos
Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transativadores/metabolismo , Ubiquitinação , Acetilação , Cromatina/genética , Cromatina/metabolismo , Endopeptidases/genética , Endopeptidases/metabolismo , Histonas/genética , Mutação , Optogenética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transativadores/genética
17.
ACS Infect Dis ; 6(7): 1719-1733, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32347704

RESUMO

A hallmark of human immunodeficiency type-1 (HIV) infection is the integration of the viral genome into host chromatin, resulting in a latent reservoir that persists despite antiviral therapy or immune response. Thus, key priorities toward eradication of HIV infection are to understand the mechanisms that allow HIV latency and to develop latency reversal agents (LRAs) that can facilitate the clearance of latently infected cells. The repressive H3K27me3 histone mark, catalyzed by the PRC2 complex, plays a pivotal role in transcriptional repression at the viral promoter in both cell line and primary CD4+ T cell models of latency. EZH2 inhibitors which block H3K27 methylation have been shown to act as LRAs, suggesting other PRC2 components could also be potential targets for latency reversal. EED, a core component of PRC2, ensures the propagation of H3K27me3 by allosterically activating EZH2 methyltransferase activity. Therefore, we sought to investigate if inhibition of EED would also reverse latency. Inhibitors of EED, EED226 and A-395, demonstrated latency reversal activity as single agents, and this activity was further enhanced when used in combination with other known LRAs. Loss of H3K27me3 following EED inhibition significantly increased the levels of H3K27 acetylation globally and at the HIV LTR. These results further confirm that PRC2 mediated repression plays a significant role in the maintenance of HIV latency and suggest that EED may serve as a promising new target for LRA development.


Assuntos
Infecções por HIV , Complexo Repressor Polycomb 2 , Infecções por HIV/tratamento farmacológico , Histonas/metabolismo , Humanos , Metilação , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Processamento de Proteína Pós-Traducional
18.
Epigenetics Chromatin ; 13(1): 3, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980037

RESUMO

BACKGROUND: Plant homeodomain (PHD) fingers are central "readers" of histone post-translational modifications (PTMs) with > 100 PHD finger-containing proteins encoded by the human genome. Many of the PHDs studied to date bind to unmodified or methylated states of histone H3 lysine 4 (H3K4). Additionally, many of these domains, and the proteins they are contained in, have crucial roles in the regulation of gene expression and cancer development. Despite this, the majority of PHD fingers have gone uncharacterized; thus, our understanding of how these domains contribute to chromatin biology remains incomplete. RESULTS: We expressed and screened 123 of the annotated human PHD fingers for their histone binding preferences using reader domain microarrays. A subset (31) of these domains showed strong preference for the H3 N-terminal tail either unmodified or methylated at H3K4. These H3 readers were further characterized by histone peptide microarrays and/or AlphaScreen to comprehensively define their H3 preferences and PTM cross-talk. CONCLUSIONS: The high-throughput approaches utilized in this study establish a compendium of binding information for the PHD reader family with regard to how they engage histone PTMs and uncover several novel reader domain-histone PTM interactions (i.e., PHRF1 and TRIM66). This study highlights the usefulness of high-throughput analyses of histone reader proteins as a means of understanding how chromatin engagement occurs biochemically.


Assuntos
Histonas/metabolismo , Proteínas de Homeodomínio/metabolismo , Sítios de Ligação , Histonas/química , Proteínas de Homeodomínio/química , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Metilação , Ligação Proteica , Processamento de Proteína Pós-Traducional
19.
ACS Chem Biol ; 15(1): 103-111, 2020 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-31634430

RESUMO

Histone post-translational modifications (PTMs) are crucial for many cellular processes including mitosis, transcription, and DNA repair. The cellular readout of histone PTMs is dependent on both the chemical modification and histone site, and the array of histone PTMs on chromatin is dynamic throughout the eukaryotic life cycle. Accordingly, methods that report on the presence of PTMs are essential tools for resolving open questions about epigenetic processes and for developing therapeutic diagnostics. Reader domains that recognize histone PTMs have shown potential as advantageous substitutes for anti-PTM antibodies, and engineering efforts aimed at enhancing reader domain affinities would advance their efficacy as antibody alternatives. Here we describe engineered chromodomains from Drosophila melanogaster and humans that bind more tightly to H3K9 methylation (H3K9me) marks and result in the tightest reported reader-H3K9me interaction to date. Point mutations near the binding interface of the HP1 chromodomain were screened in a combinatorial fashion, and a triple mutant was found that binds 20-fold tighter than the native scaffold without any loss in PTM-site selectivity. The beneficial mutations were then translated to a human homologue, CBX1, resulting in an even tighter interaction with H3K9me3. Furthermore, we show that these engineered readers (eReaders) increase detection of H3K9me marks in several biochemical assays and outperform a commercial anti-H3K9me antibody in detecting H3K9me-containing nucleosomes in vitro, demonstrating the utility of eReaders to complement antibodies in epigenetics research.


Assuntos
Cromatina/química , Histonas/química , Lisina/química , Processamento de Proteína Pós-Traducional/genética , Proteínas Recombinantes/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Clonagem Molecular , Drosophila melanogaster/química , Drosophila melanogaster/genética , Epigênese Genética/genética , Regulação da Expressão Gênica , Humanos , Metilação , Modelos Moleculares , Mutação , Nucleossomos/metabolismo , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/genética , Especificidade por Substrato
20.
Mol Cell ; 76(6): 909-921.e3, 2019 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-31676231

RESUMO

Metabolic signaling to chromatin often underlies how adaptive transcriptional responses are controlled. While intermediary metabolites serve as co-factors for histone-modifying enzymes during metabolic flux, how these modifications contribute to transcriptional responses is poorly understood. Here, we utilize the highly synchronized yeast metabolic cycle (YMC) and find that fatty acid ß-oxidation genes are periodically expressed coincident with the ß-oxidation byproduct histone crotonylation. Specifically, we found that H3K9 crotonylation peaks when H3K9 acetylation declines and energy resources become limited. During this metabolic state, pro-growth gene expression is dampened; however, mutation of the Taf14 YEATS domain, a H3K9 crotonylation reader, results in de-repression of these genes. Conversely, exogenous addition of crotonic acid results in increased histone crotonylation, constitutive repression of pro-growth genes, and disrupted YMC oscillations. Together, our findings expose an unexpected link between metabolic flux and transcription and demonstrate that histone crotonylation and Taf14 participate in the repression of energy-demanding gene expression.


Assuntos
Acil Coenzima A/metabolismo , Metabolismo Energético , Regulação Fúngica da Expressão Gênica , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fator de Transcrição TFIID/metabolismo , Metabolismo Energético/genética , Ácidos Graxos/metabolismo , Histonas/genética , Homeostase , Lisina , Oxirredução , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais , Fator de Transcrição TFIID/genética , Transcrição Genética
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