RESUMO
The ATP-dependent chromatin remodelling complexes SWI/SNF alter the chromatin structure in transcriptional regulation. Several classes of mammalian SWI/SNF complex have been isolated biochemically, distinguished by a few specific subunits, such as the BAF-specific BAF250A, BAF250B and BRM, and the PBAF-specific BAF180. We have determined the complex compositions using low stringency immunoprecipitation (IP) and shown that the pattern of subunit interactions was more diverse than previously defined classes had predicted. The subunit association at five gene promoters that depend on the SWI/SNF activity varied and the sequential chromatin immunoprecipitations revealed that different class-specific subunits occupied the promoters at the same time. The low-stringency IP showed that the BAF-specific BAF250A and BAF250B and the PBAF-specific BAF180 co-exist in a subset of SWI/SNF complexes, and fractionation of nuclear extract on size-exclusion chromatography demonstrated that sub-complexes with unorthodox subunit compositions were present in the cell. We propose a model in which the constellations of SWI/SNF complexes are "tailored" for each specific chromatin target and depend on the local chromatin environment to which complexes and sub-complexes are recruited.
Assuntos
Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Mamíferos/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Animais , Fracionamento Celular , Imunoprecipitação da Cromatina , Cromatografia em Gel , Proteínas Cromossômicas não Histona/isolamento & purificação , Proteínas de Ligação a DNA , Células HeLa , Humanos , Imunoprecipitação , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Especificidade de Órgãos/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Subunidades Proteicas/metabolismo , Sais , Fatores de Transcrição/isolamento & purificaçãoRESUMO
The WSTF (Williams syndrome transcription factor) protein is involved in vitamin D-mediated transcription and replication as a component of two distinct ATP-dependent chromatin remodeling complexes, WINAC and WICH, respectively. We show here that the WICH complex (WSTF-SNF2h) interacts with several nuclear proteins as follows: Sf3b155/SAP155, RNA helicase II/Gualpha, Myb-binding protein 1a, CSB, the proto-oncogene Dek, and nuclear myosin 1 in a large 3-MDa assembly, B-WICH, during active transcription. B-WICH also contains RNAs, 45 S rRNA, 5 S rRNA, 7SL RNA, and traces of the U2 small nuclear RNA. The core proteins, WSTF, SNF2h, and nuclear myosin 1, are associated with the RNA polymerase III genes 5 S rRNA genes and 7SL, and post-transcriptional silencing of WSTF reduces the levels of these transcripts. Our results show that a WSTF-SNF2h assembly is involved in RNA polymerase III transcription, and we suggest that WSTF-SNF2h-NM1 forms a platform in transcription while providing chromatin remodeling.
Assuntos
Adenosina Trifosfatases/química , Cromatina/química , Proteínas Cromossômicas não Histona/química , Fatores de Transcrição/química , Núcleo Celular/metabolismo , Cromatina/metabolismo , Células HeLa , Humanos , Imunoprecipitação , Modelos Biológicos , Miosinas/química , Ligação Proteica , Proto-Oncogene Mas , RNA/química , RNA Polimerase III/metabolismo , Frações Subcelulares , Transcrição GênicaRESUMO
Nuclear actin and myosin 1 (NM1) are key regulators of gene transcription. Here, we show by biochemical fractionation of nuclear extracts, protein-protein interaction studies and chromatin immunoprecipitation assays that NM1 is part of a multiprotein complex that contains WICH, a chromatin remodelling complex containing WSTF (Williams syndrome transcription factor) and SNF2h. NM1, WSTF and SNF2h were found to be associated with RNA polymerase I (Pol I) and ribosomal RNA genes (rDNA). RNA interference-mediated knockdown of NM1 and WSTF reduced pre-rRNA synthesis in vivo, and antibodies to WSTF inhibited Pol I transcription on pre-assembled chromatin templates but not on naked DNA. The results indicate that NM1 cooperates with WICH to facilitate transcription on chromatin.