RESUMEN
Mammalian SWI/SNF (mSWI/SNF) ATP-dependent chromatin remodeling complexes are multi-subunit molecular machines that play vital roles in regulating genomic architecture and are frequently disrupted in human cancer and developmental disorders. To date, the modular organization and pathways of assembly of these chromatin regulators remain unknown, presenting a major barrier to structural and functional determination. Here, we elucidate the architecture and assembly pathway across three classes of mSWI/SNF complexes-canonical BRG1/BRM-associated factor (BAF), polybromo-associated BAF (PBAF), and newly defined ncBAF complexes-and define the requirement of each subunit for complex formation and stability. Using affinity purification of endogenous complexes from mammalian and Drosophila cells coupled with cross-linking mass spectrometry (CX-MS) and mutagenesis, we uncover three distinct and evolutionarily conserved modules, their organization, and the temporal incorporation of these modules into each complete mSWI/SNF complex class. Finally, we map human disease-associated mutations within subunits and modules, defining specific topological regions that are affected upon subunit perturbation.
Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Factores de Transcripción/metabolismo , Animales , Cromatina/química , Proteínas Cromosómicas no Histona/análisis , Proteínas Cromosómicas no Histona/genética , Drosophila/metabolismo , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Espectrometría de Masas , Mutagénesis , Subunidades de Proteína/análisis , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Factores de Transcripción/análisis , Factores de Transcripción/genéticaRESUMEN
Mammalian SWI/SNF (mSWI/SNF or BAF) ATP-dependent chromatin remodeling complexes play critical roles in governing genomic architecture and gene expression and are frequently perturbed in human cancers. Transcription factors (TFs), including fusion oncoproteins, can bind to BAF complex surfaces to direct chromatin targeting and accessibility, often activating oncogenic gene loci. Here, we demonstrate that the FUS::DDIT3 fusion oncoprotein hallmark to myxoid liposarcoma (MLPS) inhibits BAF complex-mediated remodeling of adipogenic enhancer sites via sequestration of the adipogenic TF, CEBPB, from the genome. In mesenchymal stem cells, small-molecule inhibition of BAF complex ATPase activity attenuates adipogenesis via failure of BAF-mediated DNA accessibility and gene activation at CEBPB target sites. BAF chromatin occupancy and gene expression profiles of FUS::DDIT3-expressing cell lines and primary tumors exhibit similarity to SMARCB1-deficient tumor types. These data present a mechanism by which a fusion oncoprotein generates a BAF complex loss-of-function phenotype, independent of deleterious subunit mutations.
Asunto(s)
Liposarcoma Mixoide , Animales , Línea Celular Tumoral , Cromatina/genética , Liposarcoma Mixoide/genética , Liposarcoma Mixoide/metabolismo , Liposarcoma Mixoide/patología , Mamíferos/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) play a critical role in development and normal physiology. Alterations in 5-mC and 5-hmC patterns are common events in hematopoietic neoplasms. In this review, we begin by emphasizing the importance of 5-mC, 5-hmC, and their enzymatic modifiers in hematological malignancies. Then, we discuss the functions of 5-mC and 5-hmC at distinct genic contexts, including promoter regions, gene bodies, intron-exon boundaries, alternative promoters, and intragenic microRNAs. Recent advances in technology have allowed for the study of 5-mC and 5-hmC independently and specifically permitting distinction between the bases that show them to have transcriptional effects that vary by their location relative to gene structure. We extend these observations to their functions at enhancers and transcription factor binding sites. We discuss dietary influences on 5-mC and 5-hmC levels and summarize the literature on the effects of folate and vitamin C on 5-mC and 5-hmC, respectively. Finally, we discuss how these new themes in the functions of 5-mC and 5-hmC will likely influence the broader research field of epigenetics.
Asunto(s)
5-Metilcitosina/metabolismo , Citosina/análogos & derivados , Neoplasias Hematológicas/metabolismo , Animales , Ácido Ascórbico/metabolismo , Citosina/metabolismo , Metilación de ADN , Dieta , Ácido Fólico/metabolismo , Neoplasias Hematológicas/genética , Humanos , MicroARNs/genética , Regiones Promotoras Genéticas/genética , Relación Estructura-Actividad , Factores de Transcripción/genéticaRESUMEN
Epigenetic alterations play a central role in the control of normal and malignant blood cell development. We demonstrate here that expression of a truncated DNA methyltransferase 3B isoform DNMT3B7, which has been shown to alter cellular epigenetic patterns, decreases the overall number of hematopoietic stem and progenitor cells (HSPCs), and markedly diminishes blood cell reconstitution within the female hormonal microenvironment. Gene expression profiling of HSPCs isolated from DNMT3B7 transgenic embryos identified Apolipoprotein E (Apoe) as overexpressed. The CpG island controlling Apoe expression had lower levels of modified cytosines in DNMT3B7 transgenic HSPCs, corresponding with the observed increase in gene expression. Furthermore, we observed that spleens and bone marrows of female mice transplanted with DNMT3B7 transgenic HSPCs express very high levels of Apoe. Finally, the introduction of Apoe-overexpressing HSPCs into male recipients decreased bone marrow engraftment, recapitulating our original observations in female recipients. Our work reveals a dynamic interplay between the intrinsic epigenetic changes in HSPCs and extrinsic endocrine factors acting on these cells to regulate the efficiency of HSPC engraftment and reconstitution. We have identified a novel mechanism by which gender-specific hormones modulate HSPC function, which could serve as a target for augmenting hematopoiesis in cases with limited HSC functionality.
Asunto(s)
Apolipoproteínas E/biosíntesis , Islas de CpG/fisiología , Epigénesis Genética/fisiología , Hematopoyesis/fisiología , Caracteres Sexuales , Animales , Apolipoproteínas E/genética , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Femenino , Masculino , Ratones , Ratones NoqueadosRESUMEN
Cbx7 is one of five mammalian orthologs of the Drosophila Polycomb. Cbx7 recognizes methylated lysine residues on the histone H3 tail and contributes to gene silencing in the context of the Polycomb repressive complex 1 (PRC1). However, our knowledge of Cbx7 post-translational modifications remains limited. Through combined biochemical and mass spectrometry approaches, we report a novel phosphorylation site on mouse Cbx7 at residue Thr-118 (Cbx7T118ph), near the highly conserved Polycomb box. The generation of a site-specific antibody to Cbx7T118ph demonstrates that Cbx7 is phosphorylated via MAPK signaling. Furthermore, we find Cbx7T118 phosphorylation in murine mammary carcinoma cells, which can be blocked by MEK inhibitors. Upon EGF stimulation, Cbx7 interacts robustly with other members of PRC1. To test the role of Cbx7T118 phosphorylation in gene silencing, we employed a RAS-induced senescence model system. We demonstrate that Cbx7T118 phosphorylation moderately enhances repression of its target gene p16. In summary, we have identified and characterized a novel MAPK-mediated phosphorylation site on Cbx7 and propose that mitogen signaling to the chromatin template regulates PRC1 function.
Asunto(s)
Sistema de Señalización de MAP Quinasas , Complejo Represivo Polycomb 1/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Senescencia Celular , Cromatina/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Silenciador del Gen , Células HEK293 , Humanos , Ratones , Datos de Secuencia Molecular , Mutación , Fosforilación , Complejo Represivo Polycomb 1/química , Complejo Represivo Polycomb 1/genética , Unión Proteica , Estructura Terciaria de Proteína , RatasRESUMEN
Interactions between chromatin-associated proteins and the histone landscape play major roles in dictating genome topology and gene expression. Cancer-specific fusion oncoproteins, which display unique chromatin localization patterns, often lack classical DNA-binding domains, presenting challenges in identifying mechanisms governing their site-specific chromatin targeting and function. Here we identify a minimal region of the human SS18-SSX fusion oncoprotein (the hallmark driver of synovial sarcoma) that mediates a direct interaction between the mSWI/SNF complex and the nucleosome acidic patch. This binding results in altered mSWI/SNF composition and nucleosome engagement, driving cancer-specific mSWI/SNF complex targeting and gene expression. Furthermore, the C-terminal region of SSX confers preferential affinity to repressed, H2AK119Ub-marked nucleosomes, underlying the selective targeting to polycomb-marked genomic regions and synovial sarcoma-specific dependency on PRC1 function. Together, our results describe a functional interplay between a key nucleosome binding hub and a histone modification that underlies the disease-specific recruitment of a major chromatin remodeling complex.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Histonas/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Sarcoma Sinovial/metabolismo , Factores de Transcripción/metabolismo , Ubiquitinas/metabolismo , Línea Celular Tumoral , Proteínas Cromosómicas no Histona/química , Células HEK293 , Humanos , Modelos Moleculares , Proteínas de Neoplasias/química , Nucleosomas/metabolismo , Nucleosomas/patología , Proteínas de Fusión Oncogénica/química , Conformación Proteica , Proteínas Proto-Oncogénicas/química , Proteínas Represoras/química , Sarcoma Sinovial/patología , Factores de Transcripción/química , UbiquitinaciónRESUMEN
Mammalian SWI/SNF chromatin remodelling complexes exist in three distinct, final-form assemblies: canonical BAF (cBAF), PBAF and a newly characterized non-canonical complex (ncBAF). However, their complex-specific targeting on chromatin, functions and roles in disease remain largely undefined. Here, we comprehensively mapped complex assemblies on chromatin and found that ncBAF complexes uniquely localize to CTCF sites and promoters. We identified ncBAF subunits as synthetic lethal targets specific to synovial sarcoma and malignant rhabdoid tumours, which both exhibit cBAF complex (SMARCB1 subunit) perturbation. Chemical and biological depletion of the ncBAF subunit, BRD9, rapidly attenuates synovial sarcoma and malignant rhabdoid tumour cell proliferation. Importantly, in cBAF-perturbed cancers, ncBAF complexes maintain gene expression at retained CTCF-promoter sites and function in a manner distinct from fusion oncoprotein-bound complexes. Together, these findings unmask the unique targeting and functional roles of ncBAF complexes and present new cancer-specific therapeutic targets.