title: Characterization of Two Distinct Nucleosome Remodeling and Deacetylase (NuRD) Complex Assemblies in Embryonic Stem Cells.
abstract: Pluripotency and self-renewal, the defining properties of embryonic stem cells, are brought about by transcriptional programs involving an intricate network of transcription factors and chromatin remodeling complexes. The Nucleosome Remodeling and Deacetylase (NuRD) complex plays a crucial and dynamic role in the regulation of stemness and differentiation. Several NuRD-associated factors have been reported but how they are organized has not been investigated in detail. Here, we have combined affinity purification and blue native polyacrylamide gel electrophoresis followed by protein identification by mass spectrometry and protein correlation profiling to characterize the topology of the NuRD complex. Our data show that in mouse embryonic stem cells the NuRD complex is present as two distinct assemblies of differing topology with different binding partners. Cell cycle regulator Cdk2ap1 and transcription factor Sall4 associate only with the higher mass NuRD assembly. We further establish that only isoform Sall4a, and not Sall4b, associates with NuRD. By contrast, Suz12, a component of the PRC2 Polycomb repressor complex, associates with the lower mass entity. In addition, we identify and validate a novel NuRD-associated protein, Wdr5, a regulatory subunit of the MLL histone methyltransferase complex, which associates with both NuRD entities. Bioinformatic analyses of published target gene sets of these chromatin binding proteins are in agreement with these structural observations. In summary, this study provides an interesting insight into mechanistic aspects of NuRD function in stem cell biology. The relevance of our work has broader implications because of the ubiquitous nature of the NuRD complex. The strategy described here can be more broadly applicable to investigate the topology of the multiple complexes an individual protein can participate in.
authors.affiliation: From the ‡Proteomic Mass Spectrometry, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton CB10 1SA, UK;
authors.forename: Daniel
authors.initials: D
authors.lastname: Bode
authors.affiliation: From the ‡Proteomic Mass Spectrometry, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton CB10 1SA, UK;
authors.forename: Lu
authors.initials: L
authors.lastname: Yu
authors.affiliation: §Stem Cell Engineering, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton CB10 1SA, UK.
authors.forename: Peri
authors.initials: P
authors.lastname: Tate
authors.affiliation: From the ‡Proteomic Mass Spectrometry, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton CB10 1SA, UK; mp3@sanger.ac.uk.
authors.forename: Mercedes
authors.identifier: http://orcid.org/0000-0002-3477-9695
authors.initials: M
authors.lastname: Pardo
authors.affiliation: From the ‡Proteomic Mass Spectrometry, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton CB10 1SA, UK;
authors.forename: Jyoti
authors.identifier: http://orcid.org/0000-0003-0881-5477
authors.initials: J
authors.lastname: Choudhary
mesh_terms: D000818:Animals; D042002:Chromatin Assembly and Disassembly; D004268:DNA-Binding Proteins / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D013058:Mass Spectrometry / Q000379:methods; D056245:Mi-2 Nucleosome Remodeling and Deacetylase Complex / Q000737:chemistry* / Q000302:isolation & purification*; D051379:Mice; D000066450:Mouse Embryonic Stem Cells / Q000378:metabolism*; D063347:Native Polyacrylamide Gel Electrophoresis / Q000379:methods; D009707:Nucleosomes / Q000378:metabolism*; D063151:Polycomb Repressive Complex 2 / Q000378:metabolism; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009707:Nucleosomes; D011506:Proteins; C509167:Sall4 protein, mouse; C494163:Suz12 protein, mouse; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C443118:Wdr5 protein, mouse; D063151:Polycomb Repressive Complex 2; D011494:Protein Kinases; C496849:Cdk2ap1 protein, mouse; D056245:Mi-2 Nucleosome Remodeling and Deacetylase Complex