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1.
Cell ; 183(3): 802-817.e24, 2020 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-33053319

RESUMO

Mammalian SWI/SNF complexes are ATP-dependent chromatin remodeling complexes that regulate genomic architecture. Here, we present a structural model of the endogenously purified human canonical BAF complex bound to the nucleosome, generated using cryoelectron microscopy (cryo-EM), cross-linking mass spectrometry, and homology modeling. BAF complexes bilaterally engage the nucleosome H2A/H2B acidic patch regions through the SMARCB1 C-terminal α-helix and the SMARCA4/2 C-terminal SnAc/post-SnAc regions, with disease-associated mutations in either causing attenuated chromatin remodeling activities. Further, we define changes in BAF complex architecture upon nucleosome engagement and compare the structural model of endogenous BAF to those of related SWI/SNF-family complexes. Finally, we assign and experimentally interrogate cancer-associated hot-spot mutations localizing within the endogenous human BAF complex, identifying those that disrupt BAF subunit-subunit and subunit-nucleosome interfaces in the nucleosome-bound conformation. Taken together, this integrative structural approach provides important biophysical foundations for understanding the mechanisms of BAF complex function in normal and disease states.


Assuntos
Doença , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Montagem e Desmontagem da Cromatina , Microscopia Crioeletrônica , DNA Helicases/química , DNA Helicases/genética , DNA Helicases/metabolismo , Doença/genética , Humanos , Mutação de Sentido Incorreto/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Ligação Proteica , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/metabolismo , Homologia Estrutural de Proteína , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
2.
Cell ; 179(6): 1342-1356.e23, 2019 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-31759698

RESUMO

Mammalian switch/sucrose non-fermentable (mSWI/SNF) complexes are multi-component machines that remodel chromatin architecture. Dissection of the subunit- and domain-specific contributions to complex activities is needed to advance mechanistic understanding. Here, we examine the molecular, structural, and genome-wide regulatory consequences of recurrent, single-residue mutations in the putative coiled-coil C-terminal domain (CTD) of the SMARCB1 (BAF47) subunit, which cause the intellectual disability disorder Coffin-Siris syndrome (CSS), and are recurrently found in cancers. We find that the SMARCB1 CTD contains a basic α helix that binds directly to the nucleosome acidic patch and that all CSS-associated mutations disrupt this binding. Furthermore, these mutations abrogate mSWI/SNF-mediated nucleosome remodeling activity and enhancer DNA accessibility without changes in genome-wide complex localization. Finally, heterozygous CSS-associated SMARCB1 mutations result in dominant gene regulatory and morphologic changes during iPSC-neuronal differentiation. These studies unmask an evolutionarily conserved structural role for the SMARCB1 CTD that is perturbed in human disease.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Proteínas Cromossômicas não Histona/metabolismo , Mutação/genética , Nucleossomos/metabolismo , Proteína SMARCB1/genética , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Elementos Facilitadores Genéticos/genética , Feminino , Genoma Humano , Células HEK293 , Células HeLa , Heterozigoto , Humanos , Masculino , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ligação Proteica , Domínios Proteicos , Proteína SMARCB1/química , Proteína SMARCB1/metabolismo
3.
Cell ; 175(5): 1272-1288.e20, 2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30343899

RESUMO

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.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Fatores de Transcrição/metabolismo , Animais , Cromatina/química , Proteínas Cromossômicas não Histona/análise , Proteínas Cromossômicas não Histona/genética , Drosophila/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Espectrometria de Massas , Mutagênese , Subunidades Proteicas/análise , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Fatores de Transcrição/análise , Fatores de Transcrição/genética
4.
Mol Cell ; 71(4): 554-566.e7, 2018 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-30078722

RESUMO

Chromosomal rearrangements resulting in the fusion of TMPRSS2, an androgen-regulated gene, and the ETS family transcription factor ERG occur in over half of prostate cancers. However, the mechanism by which ERG promotes oncogenic gene expression and proliferation remains incompletely understood. Here, we identify a binding interaction between ERG and the mammalian SWI/SNF (BAF) ATP-dependent chromatin remodeling complex, which is conserved among other oncogenic ETS factors, including ETV1, ETV4, and ETV5. We find that ERG drives genome-wide retargeting of BAF complexes in a manner dependent on binding of ERG to the ETS DNA motif. Moreover, ERG requires intact BAF complexes for chromatin occupancy and BAF complex ATPase activity for target gene regulation. In a prostate organoid model, BAF complexes are required for ERG-mediated basal-to-luminal transition, a hallmark of ERG activity in prostate cancer. These observations suggest a fundamental interdependence between ETS transcription factors and BAF chromatin remodeling complexes in cancer.


Assuntos
Carcinogênese/genética , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Neoplasias da Próstata/genética , Serina Endopeptidases/genética , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células , Cromatina/química , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos Transgênicos , Proteínas Nucleares/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Organoides/metabolismo , Organoides/patologia , Próstata/metabolismo , Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-ets , Serina Endopeptidases/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulador Transcricional ERG/genética , Regulador Transcricional ERG/metabolismo
5.
Nat Genet ; 54(6): 861-873, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35681054

RESUMO

Mammalian SWI/SNF (mSWI/SNF) ATP-dependent chromatin remodeling complexes establish and maintain chromatin accessibility and gene expression, and are frequently perturbed in cancer. Clear cell meningioma (CCM), an aggressive tumor of the central nervous system, is uniformly driven by loss of SMARCE1, an integral subunit of the mSWI/SNF core. Here, we identify a structural role for SMARCE1 in selectively stabilizing the canonical BAF (cBAF) complex core-ATPase module interaction. In CCM, cBAF complexes fail to stabilize on chromatin, reducing enhancer accessibility, and residual core module components increase the formation of BRD9-containing non-canonical BAF (ncBAF) complexes. Combined attenuation of cBAF function and increased ncBAF complex activity generates the CCM-specific gene expression signature, which is distinct from that of NF2-mutated meningiomas. Importantly, SMARCE1-deficient cells exhibit heightened sensitivity to small-molecule inhibition of ncBAF complexes. These data inform the function of a previously elusive SWI/SNF subunit and suggest potential therapeutic approaches for intractable SMARCE1-deficient CCM tumors.


Assuntos
Neoplasias Meníngeas , Meningioma , Animais , Cromatina , Montagem e Desmontagem da Cromatina/genética , Mamíferos/genética , Neoplasias Meníngeas/genética , Meningioma/genética , Fatores de Transcrição/metabolismo
6.
Nat Struct Mol Biol ; 27(9): 836-845, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32747783

RESUMO

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.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Histonas/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Sarcoma Sinovial/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitinas/metabolismo , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/química , Células HEK293 , Humanos , Modelos Moleculares , Proteínas de Neoplasias/química , Nucleossomos/metabolismo , Nucleossomos/patologia , Proteínas de Fusão Oncogênica/química , Conformação Proteica , Proteínas Proto-Oncogênicas/química , Proteínas Repressoras/química , Sarcoma Sinovial/patologia , Fatores de Transcrição/química , Ubiquitinação
7.
Nat Genet ; 51(4): 618-626, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30858614

RESUMO

Perturbations to mammalian switch/sucrose non-fermentable (mSWI/SNF) chromatin remodeling complexes have been widely implicated as driving events in cancer1. One such perturbation is the dual loss of the SMARCA4 and SMARCA2 ATPase subunits in small cell carcinoma of the ovary, hypercalcemic type (SCCOHT)2-5, SMARCA4-deficient thoracic sarcomas6 and dedifferentiated endometrial carcinomas7. However, the consequences of dual ATPase subunit loss on mSWI/SNF complex subunit composition, chromatin targeting, DNA accessibility and gene expression remain unknown. Here we identify an ATPase module of subunits that is required for functional specification of the Brahma-related gene-associated factor (BAF) and polybromo-associated BAF (PBAF) mSWI/SNF family subcomplexes. Using SMARCA4/2 ATPase mutant variants, we define the catalytic activity-dependent and catalytic activity-independent contributions of the ATPase module to the targeting of BAF and PBAF complexes on chromatin genome-wide. Finally, by linking distinct mSWI/SNF complex target sites to tumor-suppressive gene expression programs, we clarify the transcriptional consequences of SMARCA4/2 dual loss in SCCOHT.


Assuntos
Adenosina Trifosfatases/genética , Proteínas Cromossômicas não Histona/genética , Mamíferos/genética , Fatores de Transcrição/genética , Animais , Catálise , Cromatina/genética , Montagem e Desmontagem da Cromatina/genética , Genômica/métodos
8.
Curr Opin Genet Dev ; 42: 56-67, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28391084

RESUMO

Mammalian SWI/SNF (BAF) chromatin remodeling complexes orchestrate a diverse set of chromatin alterations which impact transcriptional output. Recent whole-exome sequencing efforts have revealed that the genes encoding subunits of mSWI/SNF complexes are mutated in over 20% of cancers, spanning a wide range of tissue types. The majority of mutations result in loss of subunit protein expression, implicating mSWI/SNF subunits as tumor suppressors. mSWI/SNF-deficient cancers remain a therapeutic challenge, owing to a lack of potent and selective agents which target complexes or unique pathway dependencies generated by mSWI/SNF subunit perturbations. Here, we review the current landscape of mechanistic insights and emerging therapeutic opportunities for human malignancies driven by mSWI/SNF complex perturbation.


Assuntos
Proteínas Cromossômicas não Histona/genética , Neoplasias/genética , Fatores de Transcrição/genética , Animais , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Humanos , Mamíferos , Mutação , Neoplasias/terapia , Fatores de Transcrição/antagonistas & inibidores
10.
ACS Chem Biol ; 10(3): 667-74, 2015 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-25397901

RESUMO

The DOT1L lysine methyltransferase has emerged as a validated therapeutic target in MLL-rearranged (MLLr) acute leukemias. Although S-adenosylmethionine competitive inhibitors have demonstrated pharmacological proof-of-principle in MLLr-leukemia, these compounds require further optimization to improve cellular potency and pharmacokinetic stability. Limiting DOT1L inhibitor discovery and ligand optimization have been complex biochemical methods often using radionucleotides and cellular methods requiring prolonged culture. We therefore developed a new suite of assay technologies that allows comparative assessment of chemical tools for DOT1L in a miniaturized format. Coupling these assays with structural information, we developed new insights into DOT1L ligand binding and identified several functionalized probes with increased cellular potency (IC50 values ∼10 nM) and excellent selectivity for DOT1L. Together these assay technologies define a platform capability for discovery and optimization of small-molecule DOT1L inhibitors.


Assuntos
Antineoplásicos/síntese química , Inibidores Enzimáticos/síntese química , Regulação Neoplásica da Expressão Gênica , Ensaios de Triagem em Larga Escala , Histonas/antagonistas & inibidores , Metiltransferases/antagonistas & inibidores , Adenosina/análogos & derivados , Adenosina/síntese química , Adenosina/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Histona-Lisina N-Metiltransferase , Histonas/genética , Histonas/metabolismo , Humanos , Ligantes , Metilação , Metiltransferases/química , Metiltransferases/genética , Metiltransferases/metabolismo , Modelos Moleculares , Compostos de Fenilureia/síntese química , Compostos de Fenilureia/farmacologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade
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