RESUMO
macroH2A (mH2A) is an unusual histone variant consisting of a histone H2A-like domain fused to a large nonhistone region. In this work, we show that histone mH2A represses p300- and Gal4-VP16-dependent polymerase II transcription, and we have dissected the mechanism by which this repression is realized. The repressive effect of mH2A is observed at the level of initiation but not at elongation of transcription, and mH2A interferes with p300-dependent histone acetylation. The nonhistone region of mH2A is responsible for both the repression of initiation of transcription and the inhibition of histone acetylation. In addition, the presence of this domain of mH2A within the nucleosome is able to block nucleosome remodeling and sliding of the histone octamer to neighboring DNA segments by the remodelers SWI/SNF and ACF. These data unambiguously identify mH2A as a strong transcriptional repressor and show that the repressive effect of mH2A is realized on at least two different transcription activation chromatin-dependent pathways: histone acetylation and nucleosome remodeling.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Histona Acetiltransferases/antagonistas & inibidores , Histonas/metabolismo , Nucleossomos/metabolismo , Proteínas Repressoras/metabolismo , Transativadores/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Transcrição Gênica , Acetilação , Animais , Proteínas de Ciclo Celular/metabolismo , DNA Polimerase II/metabolismo , Regulação para Baixo , Histona Acetiltransferases/metabolismo , Proteínas Nucleares/metabolismo , Nucleossomos/química , Estrutura Terciária de Proteína , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Xenopus laevis , Fatores de Transcrição de p300-CBPRESUMO
The histone variant H2A.Bbd appeared to be associated with active chromatin, but how it functions is unknown. We have dissected the properties of nucleosome containing H2A.Bbd. Atomic force microscopy (AFM) and electron cryo-microscopy (cryo-EM) showed that the H2A.Bbd histone octamer organizes only approximately 130 bp of DNA, suggesting that 10 bp of each end of nucleosomal DNA are released from the octamer. In agreement with this, the entry/exit angle of the nucleosomal DNA ends formed an angle close to 180 degrees and the physico-chemical analysis pointed to a lower stability of the variant particle. Reconstitution of nucleosomes with swapped-tail mutants demonstrated that the N-terminus of H2A.Bbd has no impact on the nucleosome properties. AFM, cryo-EM and chromatin remodeling experiments showed that the overall structure and stability of the particle, but not its property to interfere with the SWI/SNF induced remodeling, were determined to a considerable extent by the H2A.Bbd docking domain. These data show that the whole H2A.Bbd histone fold domain is responsible for the unusual properties of the H2A.Bbd nucleosome.
Assuntos
Histonas/química , Histonas/metabolismo , Nucleossomos/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Microscopia Crioeletrônica , DNA/química , DNA/metabolismo , Variação Genética , Histonas/genética , Histonas/ultraestrutura , Técnicas In Vitro , Microscopia de Força Atômica , Dados de Sequência Molecular , Mutação , Dobramento de Proteína , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/ultraestrutura , Xenopus laevisRESUMO
A histone variant H2ABbd was recently identified, but its function is totally unknown. Here we have studied the structural and functional properties of nucleosome and nucleosomal arrays reconstituted with this histone variant. We show that H2ABbd can replace the conventional H2A in the nucleosome, but this replacement results in alterations of the nucleosomal structure. The remodeling complexes SWI/SNF and ACF are unable to mobilize the variant H2ABbd nucleosome. However, SWI/SNF was able to increase restriction enzyme access to the variant nucleosome and assist the transfer of variant H2ABbd-H2B dimer to a tetrameric histone H3-H4 particle. In addition, the p300- and Gal4-VP16-activated transcription appeared to be more efficient for H2ABbd nucleosomal arrays than for conventional H2A arrays. The intriguing mechanisms by which H2ABbd affects both nucleosome remodeling and transcription are discussed.