RESUMO
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.
Assuntos
Regulação da Expressão Gênica/genética , Histona Desacetilases/metabolismo , Nucleossomos/metabolismo , Humanos , Modelos MolecularesRESUMO
Chromatin structure and function is regulated by reader proteins recognizing histone modifications and/or histone variants. We recently identified that PWWP2A tightly binds to H2A.Z-containing nucleosomes and is involved in mitotic progression and cranial-facial development. Here, using in vitro assays, we show that distinct domains of PWWP2A mediate binding to free linker DNA as well as H3K36me3 nucleosomes. In vivo, PWWP2A strongly recognizes H2A.Z-containing regulatory regions and weakly binds H3K36me3-containing gene bodies. Further, PWWP2A binds to an MTA1-specific subcomplex of the NuRD complex (M1HR), which consists solely of MTA1, HDAC1, and RBBP4/7, and excludes CHD, GATAD2 and MBD proteins. Depletion of PWWP2A leads to an increase of acetylation levels on H3K27 as well as H2A.Z, presumably by impaired chromatin recruitment of M1HR. Thus, this study identifies PWWP2A as a complex chromatin-binding protein that serves to direct the deacetylase complex M1HR to H2A.Z-containing chromatin, thereby promoting changes in histone acetylation levels.
Assuntos
Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Histona Desacetilases/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Proteínas Repressoras/metabolismo , Acetilação , Animais , Proteínas Cromossômicas não Histona/genética , Células HEK293 , Histona Desacetilases/genética , Histonas/genética , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilação , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Camundongos , Nucleossomos/metabolismo , RNA Interferente Pequeno , Proteínas Repressoras/genética , TransativadoresRESUMO
The nucleosome remodelling and deacetylase (NuRD) complex is essential for the development of complex animals. NuRD has roles in regulating gene expression and repairing damaged DNA. The complex comprises at least six proteins with two or more paralogues of each protein routinely identified when the complex is purified from cell extracts. To understand the structure and function of NuRD, a map of direct subunit interactions is needed. Dozens of published studies have attempted to define direct inter-subunit connectivities. We propose that conclusions reported in many such studies are in fact ambiguous for one of several reasons. First, the expression of many NuRD subunits in bacteria is unlikely to lead to folded, active protein. Second, interaction studies carried out in cells that contain endogenous NuRD complex can lead to false positives through bridging of target proteins by endogenous components. Combining existing information on NuRD structure with a protocol designed to minimize false positives, we report a conservative and robust interaction map for the NuRD complex. We also suggest a 3D model of the complex that brings together the existing data on the complex. The issues and strategies discussed herein are also applicable to the analysis of a wide range of multi-subunit complexes. ENZYMES: Micrococcal nuclease (MNase), EC 3.1.31.1; histone deacetylase (HDAC), EC 3.5.1.98.
Assuntos
Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/química , Nucleossomos/química , Mapeamento de Interação de Proteínas/métodos , Animais , Artefatos , Western Blotting , Escherichia coli , Células HEK293 , Células HeLa , Histona Desacetilase 1/química , Humanos , Camundongos , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , Subunidades Proteicas , Coelhos , Proteínas Recombinantes de Fusão/química , ReticulócitosRESUMO
Breast cancer treatment has experienced several advancements in the past few decades with the discovery of specific predictive and prognostic biomarkers that make possible the application of individualized therapies. In addition to traditional prognostic factors of breast carcinoma, molecular biomarkers have played a significant role in tumor prediction and treatment. The most frequent genetic alterations of breast cancer are gained along chromosome 1q, 8q, 17q, 20q, and 11q and losses along 8p, 13q, 16q, 18q, and 11q. Interestingly, many of these chromosomal fragments harbor known proto oncogenes or tumor suppressor genes such as BRCA1, BRCA2, p53, HER2-neu, cyclin D1, and cyclin E, which are briefly described in this review.