RESUMO
Embryonic stem cell (ESC) self-renewal and pluripotency is maintained by an external signaling pathways and intrinsic regulatory networks involving ESC-specific transcriptional complexes (mainly formed by OCT3/4, Sox2 and Nanog proteins), the Polycomb repressive complex 2 (PRC2) and DNA methylation [1-8]. Among these, Nanog represents the more ESC specific factor and its repression correlates with the loss of pluripotency and ESC differentiation [9-11]. During ESC early differentiation, many development-associated genes become upregulated and although, in general, much is known about the pluripotency self-renewal circuitry, the molecular events that lead ESCs to exit from pluripotency and begin differentiation are largely unknown. Snai1 is one the most early induced genes during ESC differentiation in vitro and in vivo [12,13]. Here we show that Snai1 is able to directly repress several stemness-associated genes including Nanog. We use a ESC stable-line expressing a inducible Snai1 protein. We here show microarray analysis of embryonic stem cells (ESC) expressing Snail-ER at various time points of induction with 4-OH. Data were deposited in Gene Expression Omnibus (GEO) datasets under reference GSE57854 and here: http://epigenetics.hugef-research.org/data.php.
RESUMO
The nuclear factor of activated T-cells (NFAT) family transcription factors play a key role in the control of cytokine gene expression in T-cells. Although initially identified in T-cells, recent data have unveiled unanticipated roles for NFATs in the development, proliferation, and differentiation of other tissues. Here we report the identification, cDNA cloning, and functional characterization of a new isoform of NFAT1 highly expressed in mouse brain. This isoform, which we named NFAT1-D, is identical to NFAT1 throughout the N-terminal regulatory domain and the portion of the Rel domain which includes the minimal region required for specific binding to DNA and interaction with AP-1. The homology stops sharply upstream of the 3'-boundary of the Rel homology domain and is followed by a short unique C-terminal region. NFAT1-D was expressed at high levels in all brain districts and was found as a constitutively active transcription complex. Transfection of a NFAT/luciferase reporter in the neuronal cell line PC12, which also expresses NFAT1-D, showed that these cells expressed a constitutive NFAT activity that was enhanced after nerve growth factor-induced differentiation but was resistant to the immunosuppressant cyclosporin A. NFAT1-D was, however, inducibly activated in a cyclosporin A-sensitive manner when expressed in T-cells, suggesting that the activity of NFAT proteins might be controlled by their specific cellular context.
Assuntos
Encéfalo/metabolismo , Proteínas de Ligação a DNA/química , Proteínas Nucleares , Fatores de Transcrição/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Complexo CD3/metabolismo , Diferenciação Celular , Divisão Celular , Linhagem Celular , Núcleo Celular/metabolismo , Clonagem Molecular , Ciclosporina/farmacologia , DNA Complementar/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Inibidores Enzimáticos/farmacologia , Genes Reporter , Glutationa Transferase/metabolismo , Humanos , Immunoblotting , Células Jurkat , Luciferases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Microscopia Confocal , Dados de Sequência Molecular , Fatores de Transcrição NFATC , Fator de Crescimento Neural/farmacologia , Neurônios/metabolismo , Células PC12 , Testes de Precipitina , Isoformas de Proteínas , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/metabolismo , Fatores de Tempo , Distribuição Tecidual , Fator de Transcrição AP-1/química , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Transcrição Gênica , TransfecçãoRESUMO
The utrophin gene codes for a large cytoskeletal protein closely related to dystrophin which, in the absence of dystrophin, can functionally substitute it. Utrophin is transcribed by two independently regulated promoters about 50 kb apart. The upstream promoter is TATA-less and contains a functional GABP binding site which, in muscle, restricts the promoter activity to post-synaptic nuclei. Transient transfections analysis of mutant promoters in rhabdomyosarcoma cells showed that the upstream promoter contains three functional GC elements that are recognised by Sp1 and Sp3 factors in vitro. Co-transfections of the promoter with Sp1, Sp3 and GABP factors in Drosophila SL2 Schneider cells, which lack of endogenous Sp factors, demonstrated that both Sp1 and Sp3 are positive regulators of the utrophin promoter and that they activate transcription synergistically with GABP. Consistent with this result, we observed physical interaction of both Sp factors with the GABPalpha subunit in vitro. Functional domain interaction analysis of Sp1 and Sp3 revealed that both factors interact with GABPalpha through their DNA binding zinc finger domain. The modulation and correct interaction between Sp1, Sp3 and GABP in muscle cells may be critical for the regulation of the utrophin promoter, and provide new targets for therapies of Duchenne muscular dystrophy.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Membrana/metabolismo , Fator de Transcrição Sp1/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas do Citoesqueleto/genética , Pegada de DNA , Análise Mutacional de DNA , Primers do DNA/química , Drosophila melanogaster , Fator de Transcrição de Proteínas de Ligação GA , Expressão Gênica , Vetores Genéticos , Glutationa Transferase/metabolismo , Humanos , Proteínas de Membrana/genética , Dados de Sequência Molecular , Músculos/patologia , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Rabdomiossarcoma/genética , Fator de Transcrição Sp3 , Transfecção , Células Tumorais Cultivadas , Utrofina , Dedos de Zinco/genéticaRESUMO
The utrophin gene codes for a large cytoskeletal protein closely related to dystrophin. Its transcription is driven by a TATA-less promoter. Here we analyzed 40 kilobases of the 5' end region of the utrophin gene searching for new utrophin regulatory elements in muscle cells. By transient transfection of utrophin genomic fragments in front of a reporter gene, we identified a new enhancer that maps downstream of the transcription start site within the second intron and co-localizes with a DNase I-hypersensitive site. By deletion analysis it was mapped to a sequence of 128 base pairs that retains the whole activity. Linker scanning mutagenesis showed that most of the enhancer sequence is essential for its transcriptional activity. Binding analysis with nuclear cell extracts demonstrated that the enhancer regulatory elements, identified by mutagenesis, are protected from DNase I digestion. Because utrophin can functionally substitute dystrophin, the identification and characterization of new regulatory elements provide new targets for possible therapies of Duchenne muscular dystrophy aiming at the up-regulation of the utrophin expression in muscle cells.
Assuntos
Proteínas do Citoesqueleto/genética , Elementos Facilitadores Genéticos/genética , Proteínas de Membrana/genética , Transcrição Gênica , Animais , Sequência de Bases , Linhagem Celular , Mapeamento Cromossômico , Proteínas do Citoesqueleto/metabolismo , Regulação da Expressão Gênica , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Distrofias Musculares/genética , Mutagênese Sítio-Dirigida , Transfecção , UtrofinaRESUMO
The dystrophin gene transcription is up-regulated during muscle cell differentiation. Its expression in muscle cells is induced by the binding of the positive regulators serum response factor and dystrophin promoter bending factor (DPBF) on a regulatory CArG element present on the promoter. Here we show that the dystrophin CArG box is also recognized by the zinc finger nuclear factor YY1. Transient transfection experiments show that YY1 negatively regulates dystrophin transcription in C2C12 muscle cells. On the dystrophin CArG element YY1 competes with the structural factor DPBF. We further show that YY1 and DPBF binding to the CArG element induce opposite DNA bends suggesting that their binding induces alternative promoter structures. Along with C2C12 myotube formation YY1 is reduced and we observed that YY1, but not DPBF, is a substrate of m-calpain, a protease that is up-regulated in muscle cell differentiation. Thus, high levels of YY1 in non-differentiated muscle cells down-regulate the dystrophin promoter, at least in part, by interfering with the spatial organization of the promoter.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Distrofina/genética , Regulação da Expressão Gênica , Músculos/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Sequência de Bases , Calpaína/metabolismo , Linhagem Celular , Primers do DNA , Distrofina/metabolismo , Fatores de Ligação de DNA Eritroide Específicos , Hidrólise , Músculos/citologia , Ligação Proteica , Transcrição Gênica , Fator de Transcrição YY1RESUMO
The minimal muscle-specific dystrophin promoter contains the consensus sequence CC(A/T)6GG, or the CArG element, which can be found in serum-inducible or muscle-specific promoters. The serum response factor (SRF), which mediates the transcriptional activation of the c-fos gene in response to serum stimulation, can bind to different CArG box elements, suggesting that it could be involved in muscle-constitutive transcription. Here we show that SRF binds to the dystrophin promoter and regulates its muscle-specific transcription. In transient transfections, an altered-binding-specificity SRF mutant restores the muscle-constitutive transcription of a dystrophin promoter with a mutation in its CArG box element. The muscle-constitutive transcription of the dystrophin promoter also requires the sequence GAAACC immediately downstream of the CArG box. This sequence is recognized by a novel DNA bending factor which was named dystrophin promoter-bending factor (DPBF). Mutations of the CArG flanking sequence abolish both DPBF binding and the promoter activity in muscle cells. Its replacement with a p62/ternary complex factor binding site changes the promoter specificity from muscle constitutive to serum responsive. These results show that, on the dystrophin promoter, the transcriptional activation induced by SRF requires the DNA bending induced by DPBF. The bending, next to the CArG box, could promote interactions between SRF and other proteins in the transcriptional complex.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/química , Distrofina/genética , Proteínas Nucleares/metabolismo , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas/genética , Células 3T3 , Animais , Sequência de Bases , Extratos Celulares , Linhagem Celular , Sequência Consenso , DNA/metabolismo , Regulação da Expressão Gênica , Genes fos/genética , Humanos , Camundongos , Dados de Sequência Molecular , Músculos/citologia , Músculos/fisiologia , Fator de Resposta Sérica , Transcrição Gênica , TransfecçãoRESUMO
We report here three related patients with a duplication of exons 19-41 of the dystrophin gene, having dissimilar clinical phenotype and dystrophin immunohistochemistry. Two brothers aged six and three years had myalgia, proximal muscular weakness and hypertrophic calves, with 10- 20-fold increase of serum creatine kinase. Muscle biopsy showed dystrophic changes and reduced, patchy binding of dystrophin. The clinical and laboratory findings were consistent with a diagnosis of Becker muscular dystrophy with early onset. Their 14-year-old cousin had only mild hyperCKemia. His muscle biopsy was normal with only mild reduction of dystrophin immunostaining. At follow-up, he is still without symptoms and signs at age 19. All three patients had the same gene duplication and an increased dystrophin size of 507 kDa. Expression of the dystrophin-associated glycoproteins adhalin, alpha-dystroglycan, and beta-dystroglycan were normal in the three patients. An intrafamilial variability in patients carrying a partial duplication of the dystrophin gene may be related to a quantitative difference in mRNA.
Assuntos
Distrofina/genética , Família Multigênica/genética , Distrofias Musculares/genética , Southern Blotting , Western Blotting , Criança , Pré-Escolar , DNA/biossíntese , Distrofina/análise , Distrofina/metabolismo , Éxons , Imunofluorescência , Humanos , Imuno-Histoquímica , Masculino , Músculo Esquelético/patologia , Distrofias Musculares/metabolismo , Distrofias Musculares/patologia , FenótipoRESUMO
The genomic organization of most of the human dystrophin gene has not been defined at single-exon level, owing to its enormous size (2300 kb). By taking advantage of a YAC-based restriction map of the gene previously constructed, we have localized individual dystrophin exons from 42 to 79 along the central and 3' regions of the gene. These data elucidate the general organization of this large portion of the gene (1250 kb) and, in particular, characterize the genomic region most frequently involved in deletion mutations responsible for Duchenne and Becker muscular dystrophies.
Assuntos
Distrofina/genética , Mapeamento por Restrição , Deleção de Genes , Genoma , HumanosRESUMO
Starting from a group of 265 Italian patients affected with Duchenne or Becker muscular dystrophy a screening for duplications in the dystrophin gene was performed on 112 cases in which no deletions had previously been detected. The 21 intragenic duplications detected account for 7.9% of the total. Among these, one duplication including exons from 3 to 43 is the largest reported so far. Data from this study were combined with those from the literature and breakpoint distribution by intron was analysed. In general breakpoints occur mostly in the proximal third of the gene, in particular in intron 7. However, both the frequency of duplications and the distribution of breakpoints by intron are different in the Japanese sample compared with the other groups of patients. The role of geographical differentiation of intron sequences by genetic drift and of transposon-like sequences in explaining these differences is discussed.
Assuntos
Distrofina/genética , Íntrons , Família Multigênica , Distrofias Musculares/genética , Canadá , Geografia , Humanos , Itália , Japão , Países BaixosRESUMO
We have analysed the sequence organization of the pseudoautosomal region at the telomeres of the long arms of the human sex chromosomes and shown that it is 320 kb long. A LINE sequence is present on both the X and Y chromosomes immediately adjacent to the breakpoint in homology suggesting that the homology arose as a result of an ectopic recombination event mediated by LINE sequences originally present in non-homologous stretches of X and Y chromosomal DNA. This led to the translocation of sequences from the X chromosome telomere onto the Y chromosome and created a new pseudoautosomal region.