RESUMO
Actin and nuclear myosin 1c (NM1) cooperate in RNA polymerase I (pol I) transcription. NM1 is also part of a multiprotein assembly, B-WICH, which is involved in transcription. This assembly contains the chromatin remodeling complex WICH with its subunits WSTF and SNF2h. We report here that NM1 binds SNF2h with enhanced affinity upon impairment of the actin-binding function. ChIP analysis revealed that NM1, SNF2h, and actin gene occupancies are cell cycle-dependent and require intact motor function. At the onset of cell division, when transcription is temporarily blocked, B-WICH is disassembled due to WSTF phosphorylation, to be reassembled on the active gene at exit from mitosis. NM1 gene knockdown and motor function inhibition, or stable expression of NM1 mutants that do not interact with actin or chromatin, overall repressed rRNA synthesis by stalling pol I at the gene promoter, led to chromatin alterations by changing the state of H3K9 acetylation at gene promoter, and delayed cell cycle progression. These results suggest a unique structural role for NM1 in which the interaction with SNF2h stabilizes B-WICH at the gene promoter and facilitates recruitment of the HAT PCAF. This leads to a permissive chromatin structure required for transcription activation.
Assuntos
Actinas , Pontos de Checagem do Ciclo Celular , Montagem e Desmontagem da Cromatina/genética , Miosina Tipo I , RNA Ribossômico , Acetilação , Actinas/genética , Actinas/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Núcleo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Células HEK293 , Células HeLa , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Miosina Tipo I/genética , Miosina Tipo I/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , RNA Polimerase I/metabolismo , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , 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.
Assuntos
Adenosina Trifosfatases/metabolismo , Montagem e Desmontagem da Cromatina/genética , Proteínas Cromossômicas não Histona/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Miosina Tipo I/metabolismo , Proteínas Nucleares/metabolismo , RNA Polimerase I/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Núcleo Celular/química , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Miosina Tipo I/química , Miosina Tipo I/genética , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Ligação Proteica/genética , RNA Polimerase I/biossíntese , RNA Polimerase I/química , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Actin and myosin have been individually implicated in different aspects of gene expression. Here, we show in vivo evidence for a specific nucleolar actin-myosin complex physically associated with both the RNA polymerase I holoenzyme and ribosomal genes. We find that this specific actin-myosin complex is functionally coupled to elongating ribosomal RNA transcripts in living cells. From these observations, we conclude that an actin-based myosin motor is associated with transcribing ribosomal genes in the cell nucleus. These results correlate with an involvement of both actin and myosin in regulating mRNA synthesis and suggest that actin-myosin motors may provide a general mechanism to facilitate elongation of RNA transcripts during transcription of both ribosomal genes and protein-coding genes.