RESUMO
Class I histone deacetylase complexes play essential roles in many nuclear processes. Whilst they contain a common catalytic subunit, they have diverse modes of action determined by associated factors in the distinct complexes. The deacetylase module from the NuRD complex contains three protein domains that control the recruitment of chromatin to the deacetylase enzyme, HDAC1/2. Using biochemical approaches and cryo-electron microscopy, we have determined how three chromatin-binding domains (MTA1-BAH, MBD2/3 and RBBP4/7) are assembled in relation to the core complex so as to facilitate interaction of the complex with the genome. We observe a striking arrangement of the BAH domains suggesting a potential mechanism for binding to di-nucleosomes. We also find that the WD40 domains from RBBP4 are linked to the core with surprising flexibility that is likely important for chromatin engagement. A single MBD2 protein binds asymmetrically to the dimerisation interface of the complex. This symmetry mismatch explains the stoichiometry of the complex. Finally, our structures suggest how the holo-NuRD might assemble on a di-nucleosome substrate.
Assuntos
Cromatina/genética , Proteínas de Ligação a DNA/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Proteínas Repressoras/genética , Proteína 4 de Ligação ao Retinoblastoma/genética , Transativadores/genética , Sequência de Aminoácidos/genética , Microscopia Crioeletrônica , Proteínas de Ligação a DNA/ultraestrutura , Histona Desacetilase 1/genética , Histona Desacetilase 1/ultraestrutura , Histona Desacetilases/genética , Histona Desacetilases/ultraestrutura , Humanos , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/ultraestrutura , Nucleossomos/genética , Nucleossomos/ultraestrutura , Ligação Proteica/genética , Domínios Proteicos/genética , Proteínas Repressoras/ultraestrutura , Proteína 4 de Ligação ao Retinoblastoma/ultraestrutura , Transativadores/ultraestruturaRESUMO
The histone deacetylase (HDAC) enzymes have emerged as an important class of molecular targets in cancer therapy, with five inhibitors in clinical use. Recently, it has been shown that a lack of selectivity between the 11 Zn-dependent HDAC isoforms may lead to unwanted side-effects. In this paper, we show that piano stool Ru complexes can act as HDAC inhibitors, and variation in the capping arene leads to differences in HDAC isoform selectivity.
Assuntos
Proliferação de Células/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Neoplasias/tratamento farmacológico , Compostos de Rutênio/farmacologia , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Células HeLa , Histona Desacetilase 1/antagonistas & inibidores , Histona Desacetilase 1/genética , Histona Desacetilase 1/ultraestrutura , Desacetilase 6 de Histona/antagonistas & inibidores , Desacetilase 6 de Histona/genética , Desacetilase 6 de Histona/ultraestrutura , Inibidores de Histona Desacetilases/química , Humanos , Neoplasias/genética , Conformação Proteica/efeitos dos fármacos , Isoformas de Proteínas/genética , Rutênio/química , Rutênio/farmacologia , Compostos de Rutênio/químicaRESUMO
The constitutively nuclear histone deacetylases (HDACs) 1, 2, and 3 erase acetyl marks on acetyllysine residues, alter the landscape of histone modifications, and modulate chromatin structure and dynamics and thereby crucially regulate gene transcription in higher eukaryotes. Nuclear HDACs exist as at least six giant multiprotein complexes whose nonenzymatic subunits confer genome targeting specificity for these enzymes. The deacetylase activity of HDACs has been shown previously to be enhanced by inositol phosphates, which also bridge the catalytic domain in protein-protein interactions with SANT (Swi3, Ada2, N-Cor, and TFIIIB) domains in all HDAC complexes except those that contain the Sin3 transcriptional corepressors. Here, using purified recombinant proteins, coimmunoprecipitation and HDAC assays, and pulldown and NMR experiments, we show that HDAC1/2 deacetylase activity in one of the most ancient and evolutionarily conserved Sin3L/Rpd3L complexes is inducibly up-regulated by inositol phosphates but involves interactions with a zinc finger motif in the Sin3-associated protein 30 (SAP30) subunit that is structurally unrelated to SANT domains, indicating convergent evolution at the functional level. This implies that this mode of regulation has evolved independently multiple times and provides an evolutionary advantage. We also found that constitutive association with another core subunit, Rb-binding protein 4 chromatin-binding factor (RBBP4), further enhances deacetylase activity, implying both inducible and constitutive regulatory mechanisms within the same HDAC complex. Our results indicate that inositol phosphates stimulate HDAC activity and that the SAP30 zinc finger motif performs roles similar to that of the unrelated SANT domain in promoting the SAP30-HDAC1 interaction and enhancing HDAC activity.
