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1.
Cell ; 187(4): 861-881.e32, 2024 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-38301646

RESUMEN

Genomic instability can trigger cancer-intrinsic innate immune responses that promote tumor rejection. However, cancer cells often evade these responses by overexpressing immune checkpoint regulators, such as PD-L1. Here, we identify the SNF2-family DNA translocase SMARCAL1 as a factor that favors tumor immune evasion by a dual mechanism involving both the suppression of innate immune signaling and the induction of PD-L1-mediated immune checkpoint responses. Mechanistically, SMARCAL1 limits endogenous DNA damage, thereby suppressing cGAS-STING-dependent signaling during cancer cell growth. Simultaneously, it cooperates with the AP-1 family member JUN to maintain chromatin accessibility at a PD-L1 transcriptional regulatory element, thereby promoting PD-L1 expression in cancer cells. SMARCAL1 loss hinders the ability of tumor cells to induce PD-L1 in response to genomic instability, enhances anti-tumor immune responses and sensitizes tumors to immune checkpoint blockade in a mouse melanoma model. Collectively, these studies uncover SMARCAL1 as a promising target for cancer immunotherapy.


Asunto(s)
Antígeno B7-H1 , ADN Helicasas , Inmunidad Innata , Melanoma , Escape del Tumor , Animales , Ratones , Antígeno B7-H1/metabolismo , Inestabilidad Genómica , Melanoma/inmunología , Melanoma/metabolismo , ADN Helicasas/metabolismo
2.
Nature ; 589(7841): 299-305, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33299181

RESUMEN

Linker histone H1 proteins bind to nucleosomes and facilitate chromatin compaction1, although their biological functions are poorly understood. Mutations in the genes that encode H1 isoforms B-E (H1B, H1C, H1D and H1E; also known as H1-5, H1-2, H1-3 and H1-4, respectively) are highly recurrent in B cell lymphomas, but the pathogenic relevance of these mutations to cancer and the mechanisms that are involved are unknown. Here we show that lymphoma-associated H1 alleles are genetic driver mutations in lymphomas. Disruption of H1 function results in a profound architectural remodelling of the genome, which is characterized by large-scale yet focal shifts of chromatin from a compacted to a relaxed state. This decompaction drives distinct changes in epigenetic states, primarily owing to a gain of histone H3 dimethylation at lysine 36 (H3K36me2) and/or loss of repressive H3 trimethylation at lysine 27 (H3K27me3). These changes unlock the expression of stem cell genes that are normally silenced during early development. In mice, loss of H1c and H1e (also known as H1f2 and H1f4, respectively) conferred germinal centre B cells with enhanced fitness and self-renewal properties, ultimately leading to aggressive lymphomas with an increased repopulating potential. Collectively, our data indicate that H1 proteins are normally required to sequester early developmental genes into architecturally inaccessible genomic compartments. We also establish H1 as a bona fide tumour suppressor and show that mutations in H1 drive malignant transformation primarily through three-dimensional genome reorganization, which leads to epigenetic reprogramming and derepression of developmentally silenced genes.


Asunto(s)
Transformación Celular Neoplásica/genética , Cromatina/química , Cromatina/genética , Histonas/deficiencia , Histonas/genética , Linfoma/genética , Linfoma/patología , Alelos , Animales , Linfocitos B/metabolismo , Linfocitos B/patología , Autorrenovación de las Células , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina/genética , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Genes Supresores de Tumor , Centro Germinal/patología , Histonas/metabolismo , Humanos , Linfoma/metabolismo , Ratones , Mutación , Células Madre/metabolismo , Células Madre/patología
3.
Mol Cell ; 72(1): 162-177.e7, 2018 10 04.
Artículo en Inglés | MEDLINE | ID: mdl-30244833

RESUMEN

Histone post-translational modifications (PTMs) are important genomic regulators often studied by chromatin immunoprecipitation (ChIP), whereby their locations and relative abundance are inferred by antibody capture of nucleosomes and associated DNA. However, the specificity of antibodies within these experiments has not been systematically studied. Here, we use histone peptide arrays and internally calibrated ChIP (ICeChIP) to characterize 52 commercial antibodies purported to distinguish the H3K4 methylforms (me1, me2, and me3, with each ascribed distinct biological functions). We find that many widely used antibodies poorly distinguish the methylforms and that high- and low-specificity reagents can yield dramatically different biological interpretations, resulting in substantial divergence from the literature for numerous H3K4 methylform paradigms. Using ICeChIP, we also discern quantitative relationships between enhancer H3K4 methylation and promoter transcriptional output and can measure global PTM abundance changes. Our results illustrate how poor antibody specificity contributes to the "reproducibility crisis," demonstrating the need for rigorous, platform-appropriate validation.


Asunto(s)
Anticuerpos/genética , Inmunoprecipitación de Cromatina/métodos , Heterocromatina/genética , Histonas/genética , Anticuerpos/química , Anticuerpos/inmunología , Especificidad de Anticuerpos , Heterocromatina/química , Heterocromatina/inmunología , Código de Histonas/genética , Histonas/química , Histonas/inmunología , Humanos , Metilación , Nucleosomas/genética , Regiones Promotoras Genéticas/genética , Procesamiento Proteico-Postraduccional/genética
4.
Nature ; 573(7773): 281-286, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-31485078

RESUMEN

Enzymes that catalyse CpG methylation in DNA, including the DNA methyltransferases 1 (DNMT1), 3A (DNMT3A) and 3B (DNMT3B), are indispensable for mammalian tissue development and homeostasis1-4. They are also implicated in human developmental disorders and cancers5-8, supporting the critical role of DNA methylation in the specification and maintenance of cell fate. Previous studies have suggested that post-translational modifications of histones are involved in specifying patterns of DNA methyltransferase localization and DNA methylation at promoters and actively transcribed gene bodies9-11. However, the mechanisms that control the establishment and maintenance of intergenic DNA methylation remain poorly understood. Tatton-Brown-Rahman syndrome (TBRS) is a childhood overgrowth disorder that is defined by germline mutations in DNMT3A. TBRS shares clinical features with Sotos syndrome (which is caused by haploinsufficiency of NSD1, a histone methyltransferase that catalyses the dimethylation of histone H3 at K36 (H3K36me2)8,12,13), which suggests that there is a mechanistic link between these two diseases. Here we report that NSD1-mediated H3K36me2 is required for the recruitment of DNMT3A and maintenance of DNA methylation at intergenic regions. Genome-wide analysis shows that the binding and activity of DNMT3A colocalize with H3K36me2 at non-coding regions of euchromatin. Genetic ablation of Nsd1 and its paralogue Nsd2 in mouse cells results in a redistribution of DNMT3A to H3K36me3-modified gene bodies and a reduction in the methylation of intergenic DNA. Blood samples from patients with Sotos syndrome and NSD1-mutant tumours also exhibit hypomethylation of intergenic DNA. The PWWP domain of DNMT3A shows dual recognition of H3K36me2 and H3K36me3 in vitro, with a higher binding affinity towards H3K36me2 that is abrogated by TBRS-derived missense mutations. Together, our study reveals a trans-chromatin regulatory pathway that connects aberrant intergenic CpG methylation to human neoplastic and developmental overgrowth.


Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , ADN Intergénico/metabolismo , Histonas/metabolismo , Animales , Línea Celular , ADN Metiltransferasa 3A , Estudio de Asociación del Genoma Completo , Trastornos del Crecimiento/genética , Trastornos del Crecimiento/fisiopatología , Humanos , Ratones , Unión Proteica , Dominios Proteicos , Transporte de Proteínas , Síndrome de Sotos/genética , Síndrome de Sotos/fisiopatología
5.
Biochem J ; 481(4): 219-244, 2024 Feb 21.
Artículo en Inglés | MEDLINE | ID: mdl-38353483

RESUMEN

The role of histone post-translational modifications (PTMs) in chromatin structure and genome function has been the subject of intense debate for more than 60 years. Though complex, the discourse can be summarized in two distinct - and deceptively simple - questions: What is the function of histone PTMs? And how should they be studied? Decades of research show these queries are intricately linked and far from straightforward. Here we provide a historical perspective, highlighting how the arrival of new technologies shaped discovery and insight. Despite their limitations, the tools available at each period had a profound impact on chromatin research, and provided essential clues that advanced our understanding of histone PTM function. Finally, we discuss recent advances in the application of defined nucleosome substrates, the study of multivalent chromatin interactions, and new technologies driving the next era of histone PTM research.


Asunto(s)
Cromatina , Histonas , Cromatina/genética , Histonas/metabolismo , Nucleosomas/genética , Procesamiento Proteico-Postraduccional
6.
Nat Methods ; 18(3): 303-308, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33589837

RESUMEN

Current proteomic approaches disassemble and digest nucleosome particles, blurring readouts of the 'histone code'. To preserve nucleosome-level information, we developed Nuc-MS, which displays the landscape of histone variants and their post-translational modifications (PTMs) in a single mass spectrum. Combined with immunoprecipitation, Nuc-MS quantified nucleosome co-occupancy of histone H3.3 with variant H2A.Z (sixfold over bulk) and the co-occurrence of oncogenic H3.3K27M with euchromatic marks (for example, a >15-fold enrichment of dimethylated H3K79me2). Nuc-MS is highly concordant with chromatin immunoprecipitation-sequencing (ChIP-seq) and offers a new readout of nucleosome-level biology.


Asunto(s)
Histonas/metabolismo , Nucleosomas/metabolismo , Proteómica/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Línea Celular , Inmunoprecipitación de Cromatina/métodos , Células HEK293 , Código de Histonas , Humanos , Metilación
7.
Cell ; 136(5): 952-63, 2009 Mar 06.
Artículo en Inglés | MEDLINE | ID: mdl-19269370

RESUMEN

Reversible protein phosphorylation is a signaling mechanism involved in all cellular processes. To create a systems view of the signaling apparatus in budding yeast, we generated an epistatic miniarray profile (E-MAP) comprised of 100,000 pairwise, quantitative genetic interactions, including virtually all protein and small-molecule kinases and phosphatases as well as key cellular regulators. Quantitative genetic interaction mapping reveals factors working in compensatory pathways (negative genetic interactions) or those operating in linear pathways (positive genetic interactions). We found an enrichment of positive genetic interactions between kinases, phosphatases, and their substrates. In addition, we assembled a higher-order map from sets of three genes that display strong interactions with one another: triplets enriched for functional connectivity. The resulting network view provides insights into signaling pathway regulation and reveals a link between the cell-cycle kinase, Cak1, the Fus3 MAP kinase, and a pathway that regulates chromatin integrity during transcription by RNA polymerase II.


Asunto(s)
Fosforilación , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Acetilación , Histonas/metabolismo , Proteínas Quinasas/metabolismo
8.
Anal Chem ; 93(12): 5151-5160, 2021 03 30.
Artículo en Inglés | MEDLINE | ID: mdl-33749242

RESUMEN

We report a novel platform [native capillary zone electrophoresis-top-down mass spectrometry (nCZE-TDMS)] for the separation and characterization of whole nucleosomes, their histone subunits, and post-translational modifications (PTMs). As the repeating unit of chromatin, mononucleosomes (Nucs) are an ∼200 kDa complex of DNA and histone proteins involved in the regulation of key cellular processes central to human health and disease. Unraveling the covalent modification landscape of histones and their defined stoichiometries within Nucs helps to explain epigenetic regulatory mechanisms. In nCZE-TDMS, online Nuc separation is followed by a three-tier tandem MS approach that measures the intact mass of Nucs, ejects and detects the constituent histones, and fragments to sequence the histone. The new platform was optimized with synthetic Nucs to significantly reduce both sample requirements and cost compared to direct infusion. Limits of detection were in the low-attomole range, with linearity of over ∼3 orders of magnitude. The nCZE-TDMS platform was applied to endogenous Nucs from two cell lines distinguished by overexpression or knockout of histone methyltransferase NSD2/MMSET, where analysis of constituent histones revealed changes in histone abundances over the course of the CZE separation. We are confident the nCZE-TDMS platform will help advance nucleosome-level research in the fields of chromatin and epigenetics.


Asunto(s)
Electroforesis Capilar , Nucleosomas , Histonas/metabolismo , Humanos , Espectrometría de Masas , Procesamiento Proteico-Postraduccional
9.
Addict Biol ; 26(1): e12816, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-31373129

RESUMEN

Epigenetic enzymes oversee long-term changes in gene expression by integrating genetic and environmental cues. While there are hundreds of enzymes that control histone and DNA modifications, their potential roles in substance abuse and alcohol dependence remain underexplored. A few recent studies have suggested that epigenetic processes could underlie transcriptomic and behavioral hallmarks of alcohol addiction. In the present study, we sought to identify epigenetic enzymes in the brain that are dysregulated during protracted abstinence as a consequence of chronic and intermittent alcohol exposure. Through quantitative mRNA expression analysis of over 100 epigenetic enzymes, we identified 11 that are significantly altered in alcohol-dependent rats compared with controls. Follow-up studies of one of these enzymes, the histone demethylase KDM6B, showed that this enzyme exhibits region-specific dysregulation in the prefrontal cortex and nucleus accumbens of alcohol-dependent rats. KDM6B was also upregulated in the human alcoholic brain. Upregulation of KDM6B protein in alcohol-dependent rats was accompanied by a decrease of trimethylation levels at histone H3, lysine 27 (H3K27me3), consistent with the known demethylase specificity of KDM6B. Subsequent epigenetic (chromatin immunoprecipitation [ChIP]-sequencing) analysis showed that alcohol-induced changes in H3K27me3 were significantly enriched at genes in the IL-6 signaling pathway, consistent with the well-characterized role of KDM6B in modulation of inflammatory responses. Knockdown of KDM6B in cultured microglial cells diminished IL-6 induction in response to an inflammatory stimulus. Our findings implicate a novel KDM6B-mediated epigenetic signaling pathway integrated with inflammatory signaling pathways that are known to underlie the development of alcohol addiction.


Asunto(s)
Alcoholismo/genética , Histona Demetilasas con Dominio de Jumonji/genética , Animales , Células Cultivadas , Epigénesis Genética , Etanol/metabolismo , Histona Demetilasas/genética , Histonas/metabolismo , Humanos , Corteza Prefrontal/metabolismo , Ratas , Transducción de Señal , Regulación hacia Arriba
10.
Mol Cell ; 46(5): 691-704, 2012 Jun 08.
Artículo en Inglés | MEDLINE | ID: mdl-22681890

RESUMEN

To date, cross-species comparisons of genetic interactomes have been restricted to small or functionally related gene sets, limiting our ability to infer evolutionary trends. To facilitate a more comprehensive analysis, we constructed a genome-scale epistasis map (E-MAP) for the fission yeast Schizosaccharomyces pombe, providing phenotypic signatures for ~60% of the nonessential genome. Using these signatures, we generated a catalog of 297 functional modules, and we assigned function to 144 previously uncharacterized genes, including mRNA splicing and DNA damage checkpoint factors. Comparison with an integrated genetic interactome from the budding yeast Saccharomyces cerevisiae revealed a hierarchical model for the evolution of genetic interactions, with conservation highest within protein complexes, lower within biological processes, and lowest between distinct biological processes. Despite the large evolutionary distance and extensive rewiring of individual interactions, both networks retain conserved features and display similar levels of functional crosstalk between biological processes, suggesting general design principles of genetic interactomes.


Asunto(s)
Epistasis Genética , Evolución Molecular , Genes Fúngicos , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Regulación Fúngica de la Expresión Génica , Redes Reguladoras de Genes , Genoma Fúngico , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Especificidad de la Especie
11.
Eukaryot Cell ; 12(5): 654-64, 2013 May.
Artículo en Inglés | MEDLINE | ID: mdl-23457193

RESUMEN

Rtt109 is a fungal histone acetyltransferase (HAT) that catalyzes histone H3 acetylation functionally associated with chromatin assembly. Rtt109-mediated H3 acetylation involves two histone chaperones, Asf1 and Vps75. In vivo, Rtt109 requires both chaperones for histone H3 lysine 9 acetylation (H3K9ac) but only Asf1 for full H3K56ac. In vitro, Rtt109-Vps75 catalyzes both H3K9ac and H3K56ac, whereas Rtt109-Asf1 catalyzes only H3K56ac. In this study, we extend the in vitro chaperone-associated substrate specificity of Rtt109 by showing that it acetylates vertebrate linker histone in the presence of Vps75 but not Asf1. In addition, we demonstrate that in Saccharomyces cerevisiae a short basic sequence at the carboxyl terminus of Rtt109 (Rtt109C) is required for H3K9ac in vivo. Furthermore, through in vitro and in vivo studies, we demonstrate that Rtt109C is required for optimal H3K56ac by the HAT in the presence of full-length Asf1. When Rtt109C is absent, Vps75 becomes important for H3K56ac by Rtt109 in vivo. In addition, we show that lysine 290 (K290) in Rtt109 is required in vivo for Vps75 to enhance the activity of the HAT. This is the first in vivo evidence for a role for Vps75 in H3K56ac. Taken together, our results contribute to a better understanding of chaperone control of Rtt109-mediated H3 acetylation.


Asunto(s)
Histona Acetiltransferasas/fisiología , Histonas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/enzimología , Acetilación , Secuencia de Aminoácidos , Animales , Proteínas Aviares/química , Dominio Catalítico , Proteínas de Ciclo Celular/química , Pollos , Técnicas de Inactivación de Genes , Histona Acetiltransferasas/química , Histonas/química , Lisina/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo
12.
Epigenomes ; 8(2)2024 May 12.
Artículo en Inglés | MEDLINE | ID: mdl-38804369

RESUMEN

Nucleosomes are non-uniformly distributed across eukaryotic genomes, with stretches of 'open' chromatin strongly associated with transcriptionally active promoters and enhancers. Understanding chromatin accessibility patterns in normal tissue and how they are altered in pathologies can provide critical insights to development and disease. With the advent of high-throughput sequencing, a variety of strategies have been devised to identify open regions across the genome, including DNase-seq, MNase-seq, FAIRE-seq, ATAC-seq, and NicE-seq. However, the broad application of such methods to FFPE (formalin-fixed paraffin-embedded) tissues has been curtailed by the major technical challenges imposed by highly fixed and often damaged genomic material. Here, we review the most common approaches for mapping open chromatin regions, recent optimizations to overcome the challenges of working with FFPE tissue, and a brief overview of a typical data pipeline with analysis considerations.

13.
bioRxiv ; 2024 Mar 20.
Artículo en Inglés | MEDLINE | ID: mdl-38562823

RESUMEN

During tumor development, promoter CpG islands (CGIs) that are normally silenced by Polycomb repressive complexes (PRCs) become DNA hypermethylated. The molecular mechanism by which de novo DNA methyltransferase(s) catalyze CpG methylation at PRC-regulated regions remains unclear. Here we report a cryo-EM structure of the DNMT3A long isoform (DNMT3A1) N-terminal region in complex with a nucleosome carrying PRC1-mediated histone H2A lysine 119 monoubiquitination (H2AK119Ub). We identify regions within the DNMT3A1 N-terminus that bind H2AK119Ub and the nucleosome acidic patch. This bidentate interaction is required for effective DNMT3A1 engagement with H2AK119Ub-modified chromatin in cells. Furthermore, aberrant redistribution of DNMT3A1 to Polycomb target genes inhibits their transcriptional activation during cell differentiation and recapitulates the cancer-associated DNA hypermethylation signature. This effect is rescued by disruption of the DNMT3A1-acidic patch interaction. Together, our analyses reveal a binding interface critical for countering promoter CGI DNA hypermethylation, a major molecular hallmark of cancer.

14.
J Biol Chem ; 287(3): 1709-18, 2012 Jan 13.
Artículo en Inglés | MEDLINE | ID: mdl-22128187

RESUMEN

RNA polymerase II initiates from low complexity sequences so cells must reliably distinguish "real" from "cryptic" promoters and maintain fidelity to the former. Further, this must be performed under a range of conditions, including those found within inactive and highly transcribed regions. Here, we used genome-scale screening to identify those factors that regulate the use of a specific cryptic promoter and how this is influenced by the degree of transcription over the element. We show that promoter fidelity is most reliant on histone gene transactivators (Spt10, Spt21) and H3-H4 chaperones (Asf1, HIR complex) from the replication-independent deposition pathway. Mutations of Rtt106 that abrogate its interactions with H3-H4 or dsDNA permit extensive cryptic transcription comparable with replication-independent deposition factor deletions. We propose that nucleosome shielding is the primary means to maintain promoter fidelity, and histone replacement is most efficiently mediated in yeast cells by a HIR/Asf1/H3-H4/Rtt106 pathway.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Histonas/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas/fisiología , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transcripción Genética/fisiología , Proteínas de Ciclo Celular/genética , Histonas/genética , Chaperonas Moleculares/genética , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/genética , Nucleosomas/genética , Nucleosomas/metabolismo , Proteínas Represoras/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
15.
EMBO J ; 28(3): 205-12, 2009 Feb 04.
Artículo en Inglés | MEDLINE | ID: mdl-19131970

RESUMEN

As RNA polymerase II (RNApII) transitions from initiation to elongation, Mediator and the basal transcription factors TFIID, TFIIA, TFIIH, and TFIIE remain at the promoter as part of a scaffold complex, whereas TFIIB and TFIIF dissociate. The yeast Ctk1 kinase associates with elongation complexes and phosphorylates serine 2 in the YSPTSPS repeats of the Rpb1 C-terminal domain, a modification that couples transcription to mRNA 3'-end processing. The higher eukaryotic kinase Cdk9 not only performs a similar function, but also functions at the 5'-end of genes in the transition from initiation to elongation. In strains lacking Ctk1, many basal transcription factors cross-link throughout transcribed regions, apparently remaining associated with RNApII until it terminates. Consistent with this observation, preinitiation complexes formed on immobilized templates with transcription extracts lacking Ctk1 leave lower levels of the scaffold complex behind after escape. Taken together, these results suggest that Ctk1 is necessary for the release of RNApII from basal transcription factors. Interestingly, this function of Ctk1 is independent of its kinase activity, suggesting a structural function of the protein.


Asunto(s)
Proteínas Quinasas/metabolismo , ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Factores de Transcripción/metabolismo , Transcripción Genética , Modelos Genéticos , Mutación/genética , Estabilidad Proteica
16.
Res Sq ; 2023 Jul 28.
Artículo en Inglés | MEDLINE | ID: mdl-37546815

RESUMEN

Tardigrades are remarkable in their ability to survive extreme environments. The damage suppressor (Dsup) protein is thought responsible for their extreme resistance to reactive oxygen species (ROS) generated by irradiation. Here we show that expression of Ramazzottius varieornatus Dsup in Saccharomyces cerevisiae reduces oxidative DNA damage and extends the lifespan of budding yeast exposed to chronic oxidative genotoxicity. This protection from ROS requires either the Dsup HMGN-like domain or sequences C-terminal to same. Dsup associates with no apparent bias across the yeast genome, using multiple modes of nucleosome binding; the HMGN-like region interacts with both the H2A/H2B acidic patch and H3/H4 histone tails, while the C-terminal region binds DNA. These findings give precedent for engineering an organism by physically shielding its genome to promote survival and longevity in the face of oxidative damage.

17.
bioRxiv ; 2023 Feb 23.
Artículo en Inglés | MEDLINE | ID: mdl-36865140

RESUMEN

The maintenance of gene expression patterns during metazoan development is achieved by the actions of Polycomb group (PcG) complexes. An essential modification marking silenced genes is monoubiquitination of histone H2A lysine 119 (H2AK119Ub) deposited by the E3 ubiquitin ligase activity of the non-canonical Polycomb Repressive Complex 1. The Polycomb Repressive Deubiquitinase (PR-DUB) complex cleaves monoubiquitin from histone H2A lysine 119 (H2AK119Ub) to restrict focal H2AK119Ub at Polycomb target sites and to protect active genes from aberrant silencing. BAP1 and ASXL1, subunits that form active PR-DUB, are among the most frequently mutated epigenetic factors in human cancers, underscoring their biological importance. How PR-DUB achieves specificity for H2AK119Ub to regulate Polycomb silencing is unknown, and the mechanisms of most of the mutations in BAP1 and ASXL1 found in cancer have not been established. Here we determine a cryo-EM structure of human BAP1 bound to the ASXL1 DEUBAD domain in complex with a H2AK119Ub nucleosome. Our structural, biochemical, and cellular data reveal the molecular interactions of BAP1 and ASXL1 with histones and DNA that are critical for remodeling the nucleosome and thus establishing specificity for H2AK119Ub. These results further provide a molecular explanation for how >50 mutations in BAP1 and ASXL1 found in cancer can dysregulate H2AK119Ub deubiquitination, providing new insight into understanding cancer etiology. One Sentence Summary: We reveal the molecular mechanism of nucleosomal H2AK119Ub deubiquitination by human BAP1/ASXL1.

18.
Cell Stem Cell ; 30(11): 1503-1519.e8, 2023 11 02.
Artículo en Inglés | MEDLINE | ID: mdl-37863054

RESUMEN

Somatic mutations accumulate in all cells with age and can confer a selective advantage, leading to clonal expansion over time. In hematopoietic cells, mutations in a subset of genes regulating DNA repair or epigenetics frequently lead to clonal hematopoiesis (CH). Here, we describe the context and mechanisms that lead to enrichment of hematopoietic stem cells (HSCs) with mutations in SRCAP, which encodes a chromatin remodeler that also influences DNA repair. We show that SRCAP mutations confer a selective advantage in human cells and in mice upon treatment with the anthracycline-class chemotherapeutic doxorubicin and bone marrow transplantation. Furthermore, Srcap mutations lead to a lymphoid-biased expansion, driven by loss of SRCAP-regulated H2A.Z deposition and increased DNA repair. Altogether, we demonstrate that SRCAP operates at the intersection of multiple pathways in stem and progenitor cells, offering a new perspective on the functional impact of genetic variants that promote stem cell competition in the hematopoietic system.


Asunto(s)
Hematopoyesis Clonal , Hematopoyesis , Animales , Humanos , Ratones , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Reparación del ADN/genética , Epigénesis Genética , Hematopoyesis/genética , Mutación/genética
19.
Sci Adv ; 9(32): eadg9832, 2023 08 09.
Artículo en Inglés | MEDLINE | ID: mdl-37556531

RESUMEN

Histone H2A lysine 119 (H2AK119Ub) is monoubiquitinated by Polycomb repressive complex 1 and deubiquitinated by Polycomb repressive deubiquitinase complex (PR-DUB). PR-DUB cleaves H2AK119Ub to restrict focal H2AK119Ub at Polycomb target sites and to protect active genes from aberrant silencing. The PR-DUB subunits (BAP1 and ASXL1) are among the most frequently mutated epigenetic factors in human cancers. How PR-DUB establishes specificity for H2AK119Ub over other nucleosomal ubiquitination sites and how disease-associated mutations of the enzyme affect activity are unclear. Here, we determine a cryo-EM structure of human BAP1 and the ASXL1 DEUBAD in complex with a H2AK119Ub nucleosome. Our structural, biochemical, and cellular data reveal the molecular interactions of BAP1 and ASXL1 with histones and DNA that are critical for restructuring the nucleosome and thus establishing specificity for H2AK119Ub. These results further provide a molecular explanation for how >50 mutations in BAP1 and ASXL1 found in cancer can dysregulate H2AK119Ub deubiquitination, providing insight into understanding cancer etiology.


Asunto(s)
Proteínas de Drosophila , Neoplasias , Humanos , Histonas/genética , Nucleosomas , Lisina , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismo , Proteínas del Grupo Polycomb/genética , Proteínas de Drosophila/genética , Neoplasias/genética , Proteínas Represoras/genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo
20.
Elife ; 122023 05 19.
Artículo en Inglés | MEDLINE | ID: mdl-37204295

RESUMEN

In nucleosomes, histone N-terminal tails exist in dynamic equilibrium between free/accessible and collapsed/DNA-bound states. The latter state is expected to impact histone N-termini availability to the epigenetic machinery. Notably, H3 tail acetylation (e.g. K9ac, K14ac, K18ac) is linked to increased H3K4me3 engagement by the BPTF PHD finger, but it is unknown if this mechanism has a broader extension. Here, we show that H3 tail acetylation promotes nucleosomal accessibility to other H3K4 methyl readers, and importantly, extends to H3K4 writers, notably methyltransferase MLL1. This regulation is not observed on peptide substrates yet occurs on the cis H3 tail, as determined with fully-defined heterotypic nucleosomes. In vivo, H3 tail acetylation is directly and dynamically coupled with cis H3K4 methylation levels. Together, these observations reveal an acetylation 'chromatin switch' on the H3 tail that modulates read-write accessibility in nucleosomes and resolves the long-standing question of why H3K4me3 levels are coupled with H3 acetylation.


Asunto(s)
Cromatina , Histonas , Histonas/metabolismo , Nucleosomas , Metilación , Acetilación
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