RESUMO
Transcription factors of the nuclear factor 1 (NFI) family regulate normal brain development in vertebrates. However, multiple splice variants of four NFI isoforms exist, and their biological functions have yet to be elucidated. Here, we cloned and analyzed human NFI-X3, a novel splice variant of the nfix gene, which contains a unique transcriptional activation (TA) domain completely conserved in primates. In contrast to previously cloned NFI-X1, overexpression of NFI-X3 potently activates NFI reporters, including glial fibrillary acidic protein (GFAP) reporter, in astrocytes and glioma cells. The GAL4 fusion protein containing the TA domain of NFI-X3 strongly activates the GAL4 reporter, whereas the TA domain of NFI-X1 is ineffective. The expression of NFI-X3 is dramatically up-regulated during the differentiation of neural progenitors to astrocytes and precedes the expression of astrocyte markers, such as GFAP and SPARCL1 (Secreted Protein, Acidic and Rich in Cysteines-like 1). Overexpression of NFI-X3 dramatically up-regulates GFAP and SPARCL1 expression in glioma cells, whereas the knockdown of NFI-X3 diminishes the expression of both GFAP and SPARCL1 in astrocytes. Although activation of astrocyte-specific genes involves DNA demethylation and subsequent increase of histone acetylation, NFI-X3 activates GFAP expression, in part, by inducing alterations in the nucleosome architecture that lead to the increased recruitment of RNA polymerase II.
Assuntos
Processamento Alternativo/fisiologia , Astrócitos/citologia , Astrócitos/fisiologia , Fatores de Transcrição NFI/genética , Sequência de Aminoácidos , Animais , Proteínas de Ligação ao Cálcio/genética , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Sequência Conservada , Células-Tronco Embrionárias/citologia , Proteínas da Matriz Extracelular/genética , Fibroblastos/citologia , Marcadores Genéticos , Proteína Glial Fibrilar Ácida/genética , Glioblastoma , Células HEK293 , Humanos , Mamíferos , Camundongos , Dados de Sequência Molecular , Fatores de Transcrição NFI/química , Fatores de Transcrição NFI/metabolismo , Regiões Promotoras Genéticas/fisiologia , Estrutura Terciária de Proteína , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Ativação Transcricional/fisiologiaRESUMO
BACKGROUND: The neural cell adhesion molecule L1 plays a crucial role in development and plasticity of the nervous system. Neural cells thus require precise control of L1 expression. RESULTS: We identified a full binding site for nuclear factor I (NFI) transcription factors in the regulatory region of the mouse L1 gene. Electrophoretic mobility shift assay (EMSA) showed binding of nuclear factor I-A (NFI-A) to this site. Moreover, for a brain-specific isoform of NFI-A (NFI-A bs), we confirmed the interaction in vivo using chromatin immunoprecipitation (ChIP). Reporter gene assays showed that in neuroblastoma cells, overexpression of NFI-A bs repressed L1 expression threefold. CONCLUSION: Our findings suggest that NFI-A, in particular its brain-specific isoform, represses L1 gene expression, and might act as a second silencer of L1 in addition to the neural restrictive silencer factor (NRSF).
Assuntos
Regulação para Baixo , Fatores de Transcrição NFI/metabolismo , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Encéfalo/metabolismo , Linhagem Celular , Cricetinae , Íntrons , Camundongos , Dados de Sequência Molecular , Fatores de Transcrição NFI/química , Fatores de Transcrição NFI/genética , Molécula L1 de Adesão de Célula Nervosa/genética , Neuroblastoma/genética , Neuroblastoma/metabolismo , Especificidade de Órgãos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoAssuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Mutação de Sentido Incorreto , Fatores de Transcrição NFI/genética , Proteínas de Neoplasias/genética , Mutação Puntual , Policitemia Vera/genética , Deleção de Sequência , Substituição de Aminoácidos , Sequência de Bases , Cromossomos Humanos Par 1/genética , Erros de Diagnóstico , Progressão da Doença , Feminino , Genes Supressores de Tumor , Humanos , Janus Quinase 2/genética , Leucemia Monocítica Aguda/genética , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Fatores de Transcrição NFI/química , Proteínas de Neoplasias/química , Policitemia Vera/diagnóstico , Estrutura Terciária de Proteína/genética , Trombocitemia Essencial/diagnósticoRESUMO
The transcription factor nuclear factor I (NFI) has been shown previously both in vivo and in vitro to be involved in the cooperative regulation of whey acidic protein (WAP) gene transcription along with the glucocorticoid receptor and STAT5. In addition, one of the specific NFI isoforms, NFI-B2, was demonstrated in transient co-transfection experiments in JEG cells, which lack endogenous NFI, to be preferentially involved in the cooperative regulation of WAP gene expression. A comparison of the DNA-binding specificities of the different NFI isoforms only partially explained their differential ability to activate the WAP gene transcription. Here, we analyzed the transactivation regions of two NFI isoforms by making chimeric proteins between the NFI-A and B isoforms. Though, their DNA-binding specificities were not altered as compared to the corresponding wild-type transcription factors, the C-terminal region of the NFI-B isoform was shown to preferentially activate WAP gene transcription in cooperation with GR and STAT5 in transient co-transfection assays in JEG-3 cells. Furthermore, determination of serine and threonine-specific glycosylation (O-linked N-acetylglucosamine) of the C-terminus of the NFI-B isoform suggested that the secondary modification by O-GlcNAc might play a role in the cooperative regulation of WAP gene transcription by NFI-B2 and STAT5.