RESUMEN
The nucleosome remodeling and deacetylase (NuRD) complex is essential for metazoan development but has been refractory to biochemical analysis. We present an integrated analysis of the native mammalian NuRD complex, combining quantitative mass spectrometry, cross-linking, protein biochemistry, and electron microscopy to define the architecture of the complex. NuRD is built from a 2:2:4 (MTA, HDAC, and RBBP) deacetylase module and a 1:1:1 (MBD, GATAD2, and Chromodomain-Helicase-DNA-binding [CHD]) remodeling module, and the complex displays considerable structural dynamics. The enigmatic GATAD2 controls the asymmetry of the complex and directly recruits the CHD remodeler. The MTA-MBD interaction acts as a point of functional switching, with the transcriptional regulator PWWP2A competing with MBD for binding to the MTA-HDAC-RBBP subcomplex. Overall, our data address the long-running controversy over NuRD stoichiometry, provide imaging of the mammalian NuRD complex, and establish the biochemical mechanism by which PWWP2A can regulate NuRD composition.
Asunto(s)
Regulación de la Expresión Génica/genética , Histona Desacetilasas/metabolismo , Nucleosomas/metabolismo , Humanos , Modelos MolecularesRESUMEN
BRCA1 is a tumor suppressor found to be mutated in hereditary breast and ovarian cancer and plays key roles in the maintenance of genomic stability by homologous recombination repair. It is recruited to damaged chromatin as a component of the BRCA1-A deubiquitinase, which cleaves K63-linked ubiquitin chains attached to histone H2A and H2AX. BRCA1-A contributes to checkpoint regulation, repair pathway choice, and HR repair efficiency through molecular mechanisms that remain largely obscure. The structure of an active core complex comprising two Abraxas/BRCC36/BRCC45/MERIT40 tetramers determined by negative-stain electron microscopy (EM) reveals a distorted V-shape architecture in which a dimer of Abraxas/BRCC36 heterodimers sits at the base, with BRCC45/Merit40 pairs occupying each arm. The location and ubiquitin-binding activity of BRCC45 suggest that it may provide accessory interactions with nucleosome-linked ubiquitin chains that contribute to their efficient processing. Our data also suggest how ataxia telangiectasia mutated (ATM)-dependent BRCA1 dimerization may stabilize self-association of the entire BRCA1-A complex.
Asunto(s)
Proteína BRCA1/química , Proteínas Portadoras/química , Enzimas Desubicuitinizantes/química , Histonas/química , Complejos Multiproteicos/química , Proteínas de la Ataxia Telangiectasia Mutada/química , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteína BRCA1/genética , Proteína BRCA1/ultraestructura , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proteínas Portadoras/genética , Proteínas Portadoras/ultraestructura , Cromatina/química , Cromatina/genética , Daño del ADN/genética , Reparación del ADN/genética , Enzimas Desubicuitinizantes/genética , Enzimas Desubicuitinizantes/ultraestructura , Inestabilidad Genómica , Histonas/genética , Humanos , Complejos Multiproteicos/genética , Complejos Multiproteicos/ultraestructura , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Reparación del ADN por Recombinación/genética , Ubiquitina/genéticaRESUMEN
Chromatin remodeling enzymes act to dynamically regulate gene accessibility. In many cases, these enzymes function as large multicomponent complexes that in general comprise a central ATP-dependent Snf2 family helicase that is decorated with a variable number of regulatory subunits. The nucleosome remodeling and deacetylase (NuRD) complex, which is essential for normal development in higher organisms, is one such macromolecular machine. The NuRD complex comprises â¼10 subunits, including the histone deacetylases 1 and 2 (HDAC1 and HDAC2), and is defined by the presence of a CHD family remodeling enzyme, most commonly CHD4 (chromodomain helicase DNA-binding protein 4). The existing paradigm holds that CHD4 acts as the central hub upon which the complex is built. We show here that this paradigm does not, in fact, hold and that CHD4 is a peripheral component of the NuRD complex. A complex lacking CHD4 that has HDAC activity can exist as a stable species. The addition of recombinant CHD4 to this nucleosome deacetylase complex reconstitutes a NuRD complex with nucleosome remodeling activity. These data contribute to our understanding of the architecture of the NuRD complex.
Asunto(s)
Autoantígenos/metabolismo , ADN Helicasas/metabolismo , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Nucleosomas/metabolismo , Animales , Autoantígenos/genética , Línea Celular , ADN Helicasas/genética , Histona Desacetilasa 1/genética , Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 2/genética , Histona Desacetilasa 2/metabolismo , Humanos , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/genética , Ratones , Nucleosomas/genéticaRESUMEN
We have developed an approach for directly isolating an intact multi-protein chromatin remodeling complex from mammalian cell extracts using synthetic peptide affinity reagent 4. FOG1(1-15), a short peptide sequence known to target subunits of the nucleosome remodeling and deacetylase (NuRD) complex, was joined via a 35-atom hydrophilic linker to the StreptagII peptide. Loading this peptide onto Streptactin beads enabled capture of the intact NuRD complex from MEL cell nuclear extract. Gentle biotin elution yielded the desired intact complex free of significant contaminants and in a form that was catalytically competent in a nucleosome remodeling assay. The efficiency of 4 in isolating the NuRD complex was comparable to other reported methods utilising recombinantly produced GST-FOG1(1-45).
Asunto(s)
Marcadores de Afinidad , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/aislamiento & purificación , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Péptidos/química , Secuencia de Aminoácidos , Animales , Catálisis , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Ratones , Datos de Secuencia Molecular , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Células Tumorales CultivadasRESUMEN
Recent data demonstrate that small synthetic compounds specifically targeting bromodomain proteins can modulate the expression of cancer-related or inflammatory genes. Although these studies have focused on the ability of bromodomains to recognize acetylated histones, it is increasingly becoming clear that histone-like modifications exist on other important proteins, such as transcription factors. However, our understanding of the molecular mechanisms through which these modifications modulate protein function is far from complete. The transcription factor GATA1 can be acetylated at lysine residues adjacent to the zinc finger domains, and this acetylation is essential for the normal chromatin occupancy of GATA1. We have recently identified the bromodomain-containing protein Brd3 as a cofactor that interacts with acetylated GATA1 and shown that this interaction is essential for the targeting of GATA1 to chromatin. Here we describe the structural basis for this interaction. Our data reveal for the first time the molecular details of an interaction between a transcription factor bearing multiple acetylation modifications and its cognate recognition module. We also show that this interaction can be inhibited by an acetyllysine mimic, highlighting the importance of further increasing the specificity of compounds that target bromodomain and extraterminal (BET) bromodomains in order to fully realize their therapeutic potential.