RESUMO
The Angelman/Prader-Willi syndrome (AS/PWS) domain contains at least 8 imprinted genes regulated by a bipartite imprinting center (IC) associated with the SNRPN gene. One component of the IC, the PWS-IC, governs the paternal epigenotype and expression of paternal genes. The mechanisms by which imprinting and expression of paternal genes within the AS/PWS domain - such as MKRN3 and NDN - are regulated by the PWS-IC are unclear. The syntenic region in the mouse is organized and imprinted similarly to the human domain with the murine PWS-IC defined by a 6 kb interval within the Snrpn locus that includes the promoter. To identify regulatory elements that may mediate PWS-IC function, we mapped the location and allele-specificity of DNase I hypersensitive (DH) sites within the PWS-IC in brain cells, then identified transcription factor binding sites within a subset of these DH sites. Six major paternal-specific DH sites were detected in the Snrpn gene, five of which map within the 6 kb PWS-IC. We postulate these five DH sites represent functional components of the murine PWS-IC. Analysis of transcription factor binding within multiple DH sites detected nuclear respiratory factors (NRF's) and YY1 specifically on the paternal allele. NRF's and YY1 were also detected in the paternal promoter region of the murine Mrkn3 and Ndn genes. These results suggest that NRF's and YY1 may facilitate PWS-IC function and coordinately regulate expression of paternal genes. The presence of NRF's also suggests a link between transcriptional regulation within the AS/PWS domain and regulation of respiration. 3C analyses indicated Mkrn3 lies in close proximity to the PWS-IC on the paternal chromosome, evidence that the PWS-IC functions by allele-specific interaction with its distal target genes. This could occur by allele-specific co-localization of the PWS-IC and its target genes to transcription factories containing NRF's and YY1.
Assuntos
Síndrome de Angelman/genética , Regulação da Expressão Gênica , Fatores Nucleares Respiratórios/genética , Síndrome de Prader-Willi/genética , Elementos Reguladores de Transcrição , Fator de Transcrição YY1/genética , Proteínas Centrais de snRNP/genética , Alelos , Síndrome de Angelman/metabolismo , Síndrome de Angelman/patologia , Animais , Sequência de Bases , Sítios de Ligação , Desoxirribonuclease I/metabolismo , Loci Gênicos , Impressão Genômica , Humanos , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Fatores Nucleares Respiratórios/metabolismo , Síndrome de Prader-Willi/metabolismo , Síndrome de Prader-Willi/patologia , Ligação Proteica , Sintenia , Transcrição Gênica , Fator de Transcrição YY1/metabolismo , Proteínas Centrais de snRNP/metabolismoRESUMO
Repression and activation of gene transcription involves multiprotein complexes that modify chromatin structure. The integration of these complexes at regulatory sites can be assisted by co-factors that link them to DNA-bound transcriptional regulators. In humans, one such co-factor is the herpes simplex virus host-cell factor 1 (HCF-1), which is implicated in both activation and repression of transcription. We show here that disruption of the gene encoding the Drosophila melanogaster homolog of HCF-1, dHCF, leads to a pleiotropic phenotype involving lethality, sterility, small size, apoptosis, and morphological defects. In Drosophila, repressed and activated transcriptional states of cell fate-determining genes are maintained throughout development by Polycomb Group (PcG) and Trithorax Group (TrxG) genes, respectively. dHCF mutant flies display morphological phenotypes typical of TrxG mutants and dHCF interacts genetically with both PcG and TrxG genes. Thus, dHCF inactivation enhances the mutant phenotypes of the Pc PcG as well as brm and mor TrxG genes, suggesting that dHCF possesses Enhancer of TrxG and PcG (ETP) properties. Additionally, dHCF interacts with the previously established ETP gene skd. These pleiotropic phenotypes are consistent with broad roles for dHCF in both activation and repression of transcription during fly development.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Epigênese Genética , Alelos , Animais , Apoptose/genética , Tamanho Corporal/genética , Tamanho Celular , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes de Insetos/genética , Recombinação Homóloga/genética , Discos Imaginais/citologia , Discos Imaginais/metabolismo , Masculino , Mutação/genética , Oogênese/genética , Tamanho do Órgão , Fenótipo , Proteínas do Grupo Polycomb , Ligação Proteica , Proteínas Repressoras/metabolismo , Asas de Animais/citologia , Asas de Animais/metabolismoRESUMO
The imprinted SNRPN locus is a complex transcriptional unit that encodes the SNURF and SmN polypeptides as well as multiple non-coding RNAs. SNRPN is located within the Prader-Willi and Angelman syndrome (PWS/AS) region that contains multiple imprinted genes, which are coordinately regulated by a bipartite imprinting center (IC). The SNRPN 5' region co-localizes with the PWS-IC and contains two DNase I hypersensitive sites, DHS1 at the SNRPN promoter, and DHS2 within intron 1, exclusively on the paternally inherited chromosome. We have examined DHS1 and DHS2 to identify cis- and trans-acting regulatory elements within the endogenous SNRPN 5' region. Analysis of DHS1 by in vivo footprinting and chromatin immunoprecipitation identified allele-specific interaction with multiple regulatory proteins, including NRF-1, which regulates genes involved in mitochondrial and metabolic functions. DHS2 acted as an enhancer of the SNRPN promoter and contained a highly conserved region that showed allele-specific interaction with unphosphorylated RNA polymerase II, YY1, Sp1 and NRF-1, further suggesting a key role for NRF-1 in regulation of the SNRPN locus. We propose that one or more of the regulatory elements identified in this study may also contribute to PWS-IC function.