RESUMO
TIF1 (transcriptional intermediary factor 1) proteins are encoded by an expanding family of developmental and physiological control genes that are conserved from flies to man. These proteins are characterized by an N-terminal RING-B box-coiled-coil (RBCC) motif and a C-terminal PHD finger/bromodomain unit, and have been implicated in epigenetic mechanisms of transcriptional repression involving histone modifiers and heterochromatin-binding proteins. We describe here the isolation and functional characterization of a fourth murine TIF1 gene, TIF1delta. The predicted TIF1delta protein displays all the structural hallmarks of a bona fide TIF1 family member and resembles the other TIF1s in that it can exert a deacetylase-dependent silencing effect when tethered to a promoter region. Moreover, like TIF1alpha and TIF1beta, TIF1delta can homodimerize and contains a PXVXL motif necessary and sufficient for HP1 (heterochromatin protein 1) binding. Although TIF1alpha and TIF1beta also bind nuclear receptors and Kruppel-associated boxes specifically and respectively, TIF1delta appears to lack nuclear receptor- and Kruppel-associated box binding activity. Furthermore, TIF1delta is unique among the TIF1 family proteins in that its expression is largely restricted to the testis and confined to haploid elongating spermatids, where it associates preferentially with HP1 isotype gamma (HP1gamma) and forms discrete foci dispersed within the centromeric chromocenter and the surrounding nucleoplasm. Collectively, these data are consistent with specific, nonredundant functions for the TIF1 family members in vivo and suggest a role for TIF1delta in heterochromatin-mediated gene silencing during postmeiotic phases of spermatogenesis.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas Nucleares/metabolismo , Isoformas de Proteínas/metabolismo , Espermátides/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Dimerização , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Isoformas de Proteínas/genética , Estrutura Terciária de Proteína , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Alinhamento de Sequência , Espermátides/citologia , Testículo/citologia , Testículo/metabolismo , Distribuição Tecidual , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
Haploinsufficiency of the NSD1 gene is a hallmark of Sotos syndrome, and rearrangements of this gene by translocation can cause acute myeloid leukemia. The NSD1 gene product is a SET-domain histone lysine methyltransferase that has previously been shown to interact with nuclear receptors. We describe here a novel NSD1-interacting protein, Nizp1, that contains a SCAN box, a KRAB-A domain, and four consensus C2H2-type zinc fingers preceded by a unique finger derivative, referred to herein as the C2HR motif. The C2HR motif functions to mediate protein-protein interaction with the cysteine-rich (C5HCH) domain of NSD1 in a Zn(II)-dependent fashion, and when tethered to RNA polymerase II promoters, represses transcription in an NSD1-dependent manner. Mutations of the cysteine or histidine residues in the C2HR motif abolish the interaction of Nizp1 with NSD1 and compromise the ability of Nizp1 to repress transcription. Interestingly, converting the C2HR motif into a canonical C2H2 zinc finger has a similar effect. Thus, Nizp1 contains a novel type of zinc finger motif that functions as a docking site for NSD1 and is more than just a degenerate evolutionary remnant of a C2H2 motif.
Assuntos
Proteínas de Transporte/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Nucleares/metabolismo , Dedos de Zinco/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Proteínas de Transporte/genética , Linhagem Celular , Sequência Conservada , DNA Complementar/genética , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/genética , Humanos , Técnicas In Vitro , Camundongos , Dados de Sequência Molecular , Mutação , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Homologia de Sequência de Aminoácidos , Síndrome , Técnicas do Sistema de Duplo-Híbrido , Dedos de Zinco/genéticaRESUMO
Mammalian heterochromatin protein 1 (HP1) alpha, HP1beta and HP1gamma are closely related non-histone chromosomal proteins that function in gene silencing, presumably by organizing higher order chromatin structures. Here, we show by co-immunoprecipitation that HP1alpha, but neither HP1beta nor HP1gamma, forms a complex with the BRG1 chromatin-remodeling factor in HeLa cells. In vitro, BRG1 interacts directly and preferentially with HP1alpha. The region conferring this preferential binding has been mapped to residues 106-180 of the HP1alpha C-terminal chromoshadow domain. Using site-directed mutagenesis, we have identified three amino acid residues I113, A114 and C133 in HP1alpha (K, P and S in HP1beta and HP1gamma) that are essential for the selective interaction of HP1alpha with BRG1. Interestingly, these residues were also shown to be critical for the silencing activity of HP1alpha. Taken together, these results demonstrate that mammalian HP1 proteins are biochemically distinct and suggest an entirely novel function for BRG1 in modulating HP1alpha-containing heterochromatic structures.
