RESUMO
Fragile X syndrome (FXS) is a leading genetic cause of intellectual disability and autism. FXS results from the loss of function of fragile X mental retardation protein (FMRP), which represses translation of target transcripts. Most of the well-characterized target transcripts of FMRP are synaptic proteins, yet targeting these proteins has not provided effective treatments. We examined a group of FMRP targets that encode transcriptional regulators, particularly chromatin-associated proteins. Loss of FMRP in mice results in widespread changes in chromatin regulation and aberrant gene expression. To determine if targeting epigenetic factors could reverse phenotypes associated with the disorder, we focused on Brd4, a BET protein and chromatin reader targeted by FMRP. Inhibition of Brd4 function alleviated many of the phenotypes associated with FXS. We conclude that loss of FMRP results in significant epigenetic misregulation and that targeting transcription via epigenetic regulators like Brd4 may provide new treatments for FXS.
Assuntos
Azepinas/farmacologia , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Síndrome do Cromossomo X Frágil/tratamento farmacológico , Síndrome do Cromossomo X Frágil/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Triazóis/farmacologia , Animais , Células Cultivadas , Epigênese Genética , Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Histonas/metabolismo , Camundongos , Camundongos Knockout , Naftiridinas/farmacologia , Neurônios/metabolismo , Fenazinas , Transcrição GênicaRESUMO
Developing neurons undergo a progression of morphological and gene expression changes as they transition from neuronal progenitors to mature neurons. Here we used RNA-seq and H3K4me3 and H3K27me3 ChIP-seq to analyze how chromatin modifications control gene expression in a specific type of CNS neuron: the mouse cerebellar granule cell (GC). We found that in proliferating GC progenitors (GCPs), H3K4me3/H3K27me3 bivalency is common at neuronal genes and undergoes dynamic changes that correlate with gene expression during migration and circuit formation. Expressing a fluorescent sensor for bivalent domains revealed subnuclear bivalent foci in proliferating GCPs. Inhibiting H3K27 methyltransferases EZH1 and EZH2 in vitro and in organotypic cerebellar slices dramatically altered the expression of bivalent genes, induced the down-regulation of migration-related genes and up-regulation of synaptic genes, inhibited glial-guided migration, and accelerated terminal differentiation. Thus, histone bivalency is required to regulate the timing of the progression from progenitor cells to mature neurons.
Assuntos
Epigênese Genética , Histonas , Animais , Camundongos , Histonas/metabolismo , Ativação Transcricional , Diferenciação Celular/genéticaRESUMO
Eight types of short-chain Lys acylations have recently been identified on histones: propionylation, butyrylation, 2-hydroxyisobutyrylation, succinylation, malonylation, glutarylation, crotonylation and ß-hydroxybutyrylation. Emerging evidence suggests that these histone modifications affect gene expression and are structurally and functionally different from the widely studied histone Lys acetylation. In this Review, we discuss the regulation of non-acetyl histone acylation by enzymatic and metabolic mechanisms, the acylation 'reader' proteins that mediate the effects of different acylations and their physiological functions, which include signal-dependent gene activation, spermatogenesis, tissue injury and metabolic stress. We propose a model to explain our present understanding of how differential histone acylation is regulated by the metabolism of the different acyl-CoA forms, which in turn modulates the regulation of gene expression.
Assuntos
Regulação da Expressão Gênica , Histonas/química , Histonas/metabolismo , Acetilcoenzima A/metabolismo , Acil Coenzima A/metabolismo , Acilação , Animais , Ácidos Graxos Voláteis/metabolismo , Histonas/genética , Humanos , Lisina/metabolismo , Masculino , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Espermatogênese , Estresse FisiológicoRESUMO
Acquired chromosomal instability and copy number alterations are hallmarks of cancer. Enzymes capable of promoting site-specific copy number changes have yet to be identified. Here, we demonstrate that H3K9/36me3 lysine demethylase KDM4A/JMJD2A overexpression leads to localized copy gain of 1q12, 1q21, and Xq13.1 without global chromosome instability. KDM4A-amplified tumors have increased copy gains for these same regions. 1q12h copy gain occurs within a single cell cycle, requires S phase, and is not stable but is regenerated each cell division. Sites with increased copy number are rereplicated and have increased KDM4A, MCM, and DNA polymerase occupancy. Suv39h1/KMT1A or HP1γ overexpression suppresses the copy gain, whereas H3K9/K36 methylation interference promotes gain. Our results demonstrate that overexpression of a chromatin modifier results in site-specific copy gains. This begins to establish how copy number changes could originate during tumorigenesis and demonstrates that transient overexpression of specific chromatin modulators could promote these events.
Assuntos
Replicação do DNA , Dosagem de Genes , Histona Desmetilases com o Domínio Jumonji/metabolismo , Neoplasias/genética , Cromatina/metabolismo , Cromossomos Humanos Par 1 , Instabilidade Genômica , Células HEK293 , Humanos , Histona Desmetilases com o Domínio Jumonji/química , Histona Desmetilases com o Domínio Jumonji/genética , Metilação , Neoplasias/metabolismo , Estrutura Terciária de Proteína , Fase SRESUMO
Polycomb repressive complex 2 (PRC2) regulates gene expression during lineage specification through trimethylation of lysine 27 on histone H3 (H3K27me3). In Drosophila, polycomb binding sites are dynamic chromatin regions enriched with the histone variant H3.3. Here, we show that, in mouse embryonic stem cells (ESCs), H3.3 is required for proper establishment of H3K27me3 at the promoters of developmentally regulated genes. Upon H3.3 depletion, these promoters show reduced nucleosome turnover measured by deposition of de novo synthesized histones and reduced PRC2 occupancy. Further, we show H3.3-dependent interaction of PRC2 with the histone chaperone, Hira, and that Hira localization to chromatin requires H3.3. Our data demonstrate the importance of H3.3 in maintaining a chromatin landscape in ESCs that is important for proper gene regulation during differentiation. Moreover, our findings support the emerging notion that H3.3 has multiple functions in distinct genomic locations that are not always correlated with an "active" chromatin state.
Assuntos
Células-Tronco Embrionárias/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Cromatina/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Células-Tronco Embrionárias/citologia , Chaperonas de Histonas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Fatores de Transcrição/metabolismo , Regulação para CimaRESUMO
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.
Assuntos
Transformação Celular Neoplásica/genética , Cromatina/química , Cromatina/genética , Histonas/deficiência , Histonas/genética , Linfoma/genética , Linfoma/patologia , Alelos , Animais , Linfócitos B/metabolismo , Linfócitos B/patologia , Autorrenovação Celular , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/genética , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Genes Supressores de Tumor , Centro Germinativo/patologia , Histonas/metabolismo , Humanos , Linfoma/metabolismo , Camundongos , Mutação , Células-Tronco/metabolismo , Células-Tronco/patologiaRESUMO
Eukaryotic chromosomes contain compartments of various functions, which are marked by and enriched with specific histone modifications. However, the molecular mechanisms by which these histone marks function in chromosome compartmentalization are poorly understood. Constitutive heterochromatin is a largely silent chromosome compartment characterized in part by H3K9me2 and 3. Here, we show that heterochromatin protein 1 (HP1), an H3K9me2 and 3 "reader," interacts with SUV39H1, an H3K9me2 and 3 "writer," and with TRIM28, an abundant HP1 scaffolding protein, to form complexes with increased multivalent engagement of H3K9me2 and 3-modified chromatin. H3K9me2 and 3-marked nucleosomal arrays and associated complexes undergo phase separation to form macromolecule-enriched liquid droplets. The droplets are reminiscent of heterochromatin as they are highly dense chromatin-containing structures that are resistant to DNase and exclude the general transcription factor TFIIB. Our data suggest a general mechanism by which histone marks regulate chromosome compartmentalization by promoting phase separation.
Assuntos
Montagem e Desmontagem da Cromatina , Heterocromatina/metabolismo , Histonas/metabolismo , Gotículas Lipídicas/metabolismo , Nucleossomos/metabolismo , Processamento de Proteína Pós-Traducional , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Células HEK293 , Heterocromatina/genética , Humanos , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Complexos Multiproteicos , Nucleossomos/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Tempo , Proteína 28 com Motivo Tripartido/genética , Proteína 28 com Motivo Tripartido/metabolismoRESUMO
Two recent studies mapped nucleosomes across the yeast and human genomes, teasing apart the relative contributions of DNA sequence and chromatin remodelers to nucleosome organization. These data suggest two emerging models: chromatin remodelers position nucleosomes around transcriptional start sites in yeast, and a few "locked" nucleosomes may serve as barriers from which nucleosome arrays emanate in human genomes.
Assuntos
Montagem e Desmontagem da Cromatina , Genoma Fúngico , Genoma Humano , Nucleossomos/metabolismo , Trifosfato de Adenosina/metabolismo , Humanos , Modelos Genéticos , Regiões Promotoras GenéticasRESUMO
Little is known about how combinations of histone marks are interpreted at the level of nucleosomes. The second PHD finger of human BPTF is known to specifically recognize histone H3 when methylated on lysine 4 (H3K4me2/3). Here, we examine how additional heterotypic modifications influence BPTF binding. Using peptide surrogates, three acetyllysine ligands are indentified for a PHD-adjacent bromodomain in BPTF via systematic screening and biophysical characterization. Although the bromodomain displays limited discrimination among the three possible acetyllysines at the peptide level, marked selectivity is observed for only one of these sites, H4K16ac, in combination with H3K4me3 at the mononucleosome level. In support, these two histone marks constitute a unique trans-histone modification pattern that unambiguously resides within a single nucleosomal unit in human cells, and this module colocalizes with these marks in the genome. Together, our data call attention to nucleosomal patterning of covalent marks in dictating critical chromatin associations.
Assuntos
Antígenos Nucleares/metabolismo , Histonas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Nucleossomos/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Animais , Núcleo Celular/metabolismo , Cromatina/química , Cromatina/metabolismo , Código das Histonas , Histonas/química , Humanos , Modelos Moleculares , Nucleossomos/química , Peptídeos/química , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , XenopusRESUMO
Modifications of histone proteins have essential roles in normal development and human disease. Recognition of modified histones by 'reader' proteins is a key mechanism that mediates the function of histone modifications, but how the dysregulation of these readers might contribute to disease remains poorly understood. We previously identified the ENL protein as a reader of histone acetylation via its YEATS domain, linking it to the expression of cancer-driving genes in acute leukaemia1. Recurrent hotspot mutations have been found in the ENL YEATS domain in Wilms tumour2,3, the most common type of paediatric kidney cancer. Here we show, using human and mouse cells, that these mutations impair cell-fate regulation by conferring gain-of-function in chromatin recruitment and transcriptional control. ENL mutants induce gene-expression changes that favour a premalignant cell fate, and, in an assay for nephrogenesis using murine cells, result in undifferentiated structures resembling those observed in human Wilms tumour. Mechanistically, although bound to largely similar genomic loci as the wild-type protein, ENL mutants exhibit increased occupancy at a subset of targets, leading to a marked increase in the recruitment and activity of transcription elongation machinery that enforces active transcription from target loci. Furthermore, ectopically expressed ENL mutants exhibit greater self-association and form discrete and dynamic nuclear puncta that are characteristic of biomolecular hubs consisting of local high concentrations of regulatory factors. Such mutation-driven ENL self-association is functionally linked to enhanced chromatin occupancy and gene activation. Collectively, our findings show that hotspot mutations in a chromatin-reader domain drive self-reinforced recruitment, derailing normal cell-fate control during development and leading to an oncogenic outcome.
Assuntos
Linhagem da Célula , Cromatina/genética , Proteínas de Ligação a DNA/metabolismo , Mutação com Ganho de Função , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Células HEK293 , Humanos , Camundongos , Néfrons/metabolismo , Néfrons/patologia , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Complex organisms can rapidly induce select genes in response to diverse environmental cues. This regulation occurs in the context of large genomes condensed by histone proteins into chromatin. The sensing of pathogens by macrophages engages conserved signalling pathways and transcription factors to coordinate the induction of inflammatory genes1-3. Enriched integration of histone H3.3, the ancestral histone H3 variant, is a general feature of dynamically regulated chromatin and transcription4-7. However, how chromatin is regulated at induced genes, and what features of H3.3 might enable rapid and high-level transcription, are unknown. The amino terminus of H3.3 contains a unique serine residue (Ser31) that is absent in 'canonical' H3.1 and H3.2. Here we show that this residue, H3.3S31, is phosphorylated (H3.3S31ph) in a stimulation-dependent manner along rapidly induced genes in mouse macrophages. This selective mark of stimulation-responsive genes directly engages the histone methyltransferase SETD2, a component of the active transcription machinery, and 'ejects' the elongation corepressor ZMYND118,9. We propose that features of H3.3 at stimulation-induced genes, including H3.3S31ph, provide preferential access to the transcription apparatus. Our results indicate dedicated mechanisms that enable rapid transcription involving the histone variant H3.3, its phosphorylation, and both the recruitment and the ejection of chromatin regulators.
Assuntos
Histonas/química , Histonas/metabolismo , Transcrição Gênica , Regulação para Cima/genética , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Quinase I-kappa B/química , Quinase I-kappa B/metabolismo , Macrófagos/metabolismo , Masculino , Metilação , Camundongos , Modelos Moleculares , FosforilaçãoRESUMO
The MLL1 gene is a frequent target for recurrent chromosomal translocations, resulting in transformation of hematopoietic precursors into leukemia stem cells. Here, we report on structure-function studies that elucidate molecular events in MLL1 binding of histone H3K4me3/2 marks and recruitment of the cyclophilin CyP33. CyP33 contains a PPIase and a RRM domain and regulates MLL1 function through HDAC recruitment. We find that the PPIase domain of CyP33 regulates the conformation of MLL1 through proline isomerization within the PHD3-Bromo linker, thereby disrupting the PHD3-Bromo interface and facilitating binding of the MLL1-PHD3 domain to the CyP33-RRM domain. H3K4me3/2 and CyP33-RRM target different surfaces of MLL1-PHD3 and can bind simultaneously to form a ternary complex. Furthermore, the MLL1-CyP33 interaction is required for repression of HOXA9 and HOXC8 genes in vivo. Our results highlight the role of PHD3-Bromo cassette as a regulatory platform, orchestrating MLL1 binding of H3K4me3/2 marks and cyclophilin-mediated repression through HDAC recruitment.
Assuntos
Ciclofilinas/metabolismo , Histona Desacetilases/metabolismo , Proteína de Leucina Linfoide-Mieloide/química , Sequência de Aminoácidos , Linhagem Celular , Cristalografia por Raios X , Histona-Lisina N-Metiltransferase , Histonas/metabolismo , Humanos , Metilação , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Prolina/química , Domínios e Motivos de Interação entre ProteínasRESUMO
The incorporation of histone H3 variants has been implicated in the epigenetic memory of cellular state. Using genome editing with zinc-finger nucleases to tag endogenous H3.3, we report genome-wide profiles of H3 variants in mammalian embryonic stem cells and neuronal precursor cells. Genome-wide patterns of H3.3 are dependent on amino acid sequence and change with cellular differentiation at developmentally regulated loci. The H3.3 chaperone Hira is required for H3.3 enrichment at active and repressed genes. Strikingly, Hira is not essential for localization of H3.3 at telomeres and many transcription factor binding sites. Immunoaffinity purification and mass spectrometry reveal that the proteins Atrx and Daxx associate with H3.3 in a Hira-independent manner. Atrx is required for Hira-independent localization of H3.3 at telomeres and for the repression of telomeric RNA. Our data demonstrate that multiple and distinct factors are responsible for H3.3 localization at specific genomic locations in mammalian cells.
Assuntos
Histonas/análise , Telômero/química , Animais , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células-Tronco Embrionárias/metabolismo , Genoma , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Histonas/genética , Histonas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Telômero/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Sítio de Iniciação de TranscriçãoRESUMO
Mutations in epigenetic pathways are common oncogenic drivers. Histones, the fundamental substrates for chromatin-modifying and remodelling enzymes, are mutated in tumours including gliomas, sarcomas, head and neck cancers, and carcinosarcomas. Classical 'oncohistone' mutations occur in the N-terminal tail of histone H3 and affect the function of polycomb repressor complexes 1 and 2 (PRC1 and PRC2). However, the prevalence and function of histone mutations in other tumour contexts is unknown. Here we show that somatic histone mutations occur in approximately 4% (at a conservative estimate) of diverse tumour types and in crucial regions of histone proteins. Mutations occur in all four core histones, in both the N-terminal tails and globular histone fold domains, and at or near residues that contain important post-translational modifications. Many globular domain mutations are homologous to yeast mutants that abrogate the need for SWI/SNF function, occur in the key regulatory 'acidic patch' of histones H2A and H2B, or are predicted to disrupt the H2B-H4 interface. The histone mutation dataset and the hypotheses presented here on the effect of the mutations on important chromatin functions should serve as a resource and starting point for the chromatin and cancer biology fields in exploring an expanding role of histone mutations in cancer.
Assuntos
Transformação Celular Neoplásica/genética , Histonas/genética , Mutação/genética , Neoplasias/genética , Histonas/química , Histonas/metabolismo , Humanos , Lisina/genética , Lisina/metabolismo , Metilação , Neoplasias/patologia , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/metabolismo , Domínios Proteicos/genética , Processamento de Proteína Pós-TraducionalRESUMO
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.
Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , DNA Intergênico/metabolismo , Histonas/metabolismo , Animais , Linhagem Celular , DNA Metiltransferase 3A , Estudo de Associação Genômica Ampla , Transtornos do Crescimento/genética , Transtornos do Crescimento/fisiopatologia , Humanos , Camundongos , Ligação Proteica , Domínios Proteicos , Transporte Proteico , Síndrome de Sotos/genética , Síndrome de Sotos/fisiopatologiaRESUMO
Information encoded in DNA is interpreted, modified, and propagated as chromatin. The diversity of inputs encountered by eukaryotic genomes demands a matching capacity for transcriptional outcomes provided by the combinatorial and dynamic nature of epigenetic processes. Advances in genome editing, visualization technology, and genome-wide analyses have revealed unprecedented complexity of chromatin pathways, offering explanations to long-standing questions and presenting new challenges. Here, we review recent findings, exemplified by the emerging understanding of crossregulatory interactions within chromatin, and emphasize the pathologic outcomes of epigenetic misregulation in cancer.
Assuntos
Transformação Celular Neoplásica/genética , Montagem e Desmontagem da Cromatina , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Mutação , Neoplasias/genética , Oncogenes , Animais , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Metilação de DNA , Histonas/metabolismo , Humanos , Metilação , Modelos Moleculares , Neoplasias/metabolismo , Neoplasias/patologia , Conformação de Ácido Nucleico , Fosforilação , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Transcrição GênicaRESUMO
The inflammatory response requires coordinated activation of both transcription factors and chromatin to induce transcription for defense against pathogens and environmental insults. We sought to elucidate the connections between inflammatory signaling pathways and chromatin through genomic footprinting of kinase activity and unbiased identification of prominent histone phosphorylation events. We identified H3 serine 28 phosphorylation (H3S28ph) as the principal stimulation-dependent histone modification and observed its enrichment at induced genes in mouse macrophages stimulated with bacterial lipopolysaccharide. Using pharmacological and genetic approaches, we identified mitogen- and stress-activated protein kinases (MSKs) as primary mediators of H3S28ph in macrophages. Cell-free transcription assays demonstrated that H3S28ph directly promotes p300/CBP-dependent transcription. Further, MSKs can activate both signal-responsive transcription factors and the chromatin template with additive effects on transcription. Specific inhibition of MSKs in macrophages selectively reduced transcription of stimulation-induced genes. Our results suggest that MSKs incorporate upstream signaling inputs and control multiple downstream regulators of inducible transcription.
Assuntos
Proteínas de Ciclo Celular/genética , Cromatina/química , Histonas/genética , Mitose , Modelos Estatísticos , Fatores de Transcrição/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Cromatina/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Retroalimentação Fisiológica , Células HeLa , Histonas/metabolismo , Humanos , Cinética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Imagem Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteína Vermelha FluorescenteRESUMO
Recognition of histone covalent modifications by chromatin-binding protein modules ("readers") constitutes a major mechanism for epigenetic regulation, typified by bromodomains that bind acetyllysine. Non-acetyl histone lysine acylations (e.g., crotonylation, butyrylation, propionylation) have been recently identified, but readers that prefer these acylations have not been characterized. Here we report that the AF9 YEATS domain displays selectively higher binding affinity for crotonyllysine over acetyllysine. Structural studies revealed an extended aromatic sandwiching cage with crotonyl specificity arising from π-aromatic and hydrophobic interactions between crotonyl and aromatic rings. These features are conserved among the YEATS, but not the bromodomains. Using a cell-based model, we showed that AF9 co-localizes with crotonylated histone H3 and positively regulates gene expression in a YEATS domain-dependent manner. Our studies define the evolutionarily conserved YEATS domain as a family of crotonyllysine readers and specifically demonstrate that the YEATS domain of AF9 directly links histone crotonylation to active transcription.