RESUMO
Human p66alpha and p66beta are two potent transcriptional repressors that interact with the methyl-CpG-binding domain proteins MBD2 and MBD3. An analysis of the molecular mechanisms mediating repression resulted in the identification of two major repression domains in p66alpha and one in p66beta. Both p66alpha and p66beta are SUMO-modified in vivo: p66alpha at two sites (Lys-30 and Lys-487) and p66beta at one site (Lys-33). Expression of SUMO1 enhanced the transcriptional repression activity of Gal-p66alpha and Gal-p66beta. Mutation of the SUMO modification sites or using a SUMO1 mutant or a dominant negative Ubc9 ligase resulted in a significant decrease of the transcriptional repression of p66alpha and p66beta. The Mi-2/NuRD components MBD3, RbAp46, RbAp48, and HDAC1 were found to bind to both p66alpha and p66beta in vivo. Most of the interactions were not affected by the SUMO site mutations in p66alpha or p66beta, with two exceptions. HDAC1 binding to p66alpha was lost in the case of a p66alphaK30R mutant, and RbAp46 binding was reduced in the case of a p66betaK33R mutant. These results suggest that interactions within the Mi-2/NuRD complex as well as optimal repression are mediated by SUMOylation.
Assuntos
Histona Desacetilases/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Animais , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Ilhas de CpG , Metilação de DNA , Histona Desacetilases/química , Histona Desacetilases/genética , Humanos , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase , Camundongos , Complexos Multiproteicos , Mutagênese Sítio-Dirigida , Células NIH 3T3 , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Transcrição GênicaRESUMO
The Mi-2/NuRD complex is a multi-subunit protein complex with enzymatic activities involving chromatin remodeling and histone deacetylation. Targeting of Mi-2/NuRD to methylated CpG sequences mediates gene repression. The function of p66alpha and of p66beta within the multiple subunits has not been addressed. Here, we analyzed the in vivo function and binding of both p66-paralogs. Both factors function in synergy, since knocking-down p66alpha affects the repressive function of p66beta and vice versa. Both proteins interact with MBD2 functionally and biochemically. Mutation of a single amino acid of p66alpha abolishes in vivo binding to MBD2 and interferes with MBD2-mediated repression. This loss of binding results in a diffuse nuclear localization in contrast to wild-type p66alpha that shows a speckled nuclear distribution. Furthermore, wild-type subnuclear distribution of p66alpha and p66beta depends on the presence of MBD2. Both proteins interact with the tails of all octamer histones in vitro, and acetylation of histone tails interferes with p66 binding. The conserved region 2 of p66alpha is required for histone tail interaction as well as for wild-type subnuclear distribution. These results suggest a two-interaction forward feedback binding mode, with a stable chromatin association only after deacetylation of the histones has occurred.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Histona Desacetilases/metabolismo , Histonas/metabolismo , Proteínas Repressoras/metabolismo , Acetilação , Animais , Linhagem Celular , Núcleo Celular/química , Proteínas de Ligação a DNA/genética , Inativação Gênica , Humanos , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase , Camundongos , Mutação , Estrutura Terciária de Proteína , Proteínas Repressoras/química , Proteínas Repressoras/genéticaRESUMO
Methyl-CpG-binding domain proteins (MBD) mediate functional responses of methylated DNA. MBD2 and MBD3 are components of the MeCP1 protein complex, which contains the Mi-2/NuRD complex and includes 66- and 68-kDa polypeptides. Here we identified two highly related 66-kDa proteins in a yeast two-hybrid screen with MBD2b. Based on the high degree of sequence conservation to the previously identified Xenopus p66 subunit of the Mi-2/NuRD complex, we termed these proteins hp66alpha and hp66beta. hp66alpha is the human orthologue of Xenopus p66, whereas hp66beta, previously identified as a component of the human MeCP1 complex, is a second member of a p66 gene family. Coprecipitation of hp66alpha and MBD2 demonstrates their in vivo association. Furthermore, confocal microscopy shows a nuclear colocalization of hp66alpha with hp66beta and MBD2 in a speckled pattern. hp66alpha is a potent transcriptional repressor reducing gene activity about 100-fold and is ubiquitously coexpressed with hp66beta in cell lines and in fetal and adult tissues. We demonstrate direct binding of both p66 family members to MBD2 as well as MBD3. Interestingly, hp66alpha, which binds with a higher affinity than hp66beta, interacts via two interaction domains in contrast to a single interaction domain present in hp66beta. These results demonstrate that two highly related mammalian p66 proteins display overlapping functions and are involved in methylation dependent transcriptional repression.