RESUMO
DNA helicases are known to play important roles in the maintenance of genome integrity including the replication of trinucleotide repeats in the cells. Here, we report the HFM1 gene, which encodes the putative human DNA helicase. The HFM1 gene comprises 39 exons mapping to human chromosome 1p22.2. The HFM1 cDNA encompasses 4931 nucleotides with a single open reading frame (ORF) of 1435 amino acid residues encoding a predicted 172 kDa protein (hHFM1). The deduced protein sequence shares similar domain and motif structures to those of Mer3, a DNA helicase of Saccharomyces cerevisiae; seven consecutive motifs conserved among the DEXH-box type of DNA/RNA helicases at the N-terminal and a single putative zinc finger motif at the C-terminal regions of the protein. Further, the HFM1 transcript is preferentially expressed in testis and ovary. Collectively, hHFM1 is the evolutionally conserved putative human DNA helicase, which may function as a modulator for genome integrity in germ-line tissues.
Assuntos
DNA Helicases/genética , Células Germinativas/enzimologia , Sequência de Aminoácidos , Sequência de Bases , DNA Helicases/metabolismo , DNA Complementar/metabolismo , Éxons , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Dedos de ZincoRESUMO
Huntington's disease (HD) is a neurodegenerative disease caused by a CAG repeat expansion in exon 1 of the HD gene, and the expression level of either normal or mutant huntingtin is implicated in the pathogenesis of HD. However, a molecular base of the HD gene transcription has not been elucidated as yet. In this study, we identified two proteins, HDBP1 and HDBP2, which bind to the promoter region for the HD gene using a yeast one-hybrid system. Amino acid sequence analysis of the proteins deduced the presence of nuclear localization signal, nuclear export signal, zinc finger, serine/proline-rich region, and highly conserved C-terminal region. In vitro DNA binding assay indicated that the C-terminal conserved regions of the proteins were responsible for binding to the unique promoter DNA sequences of the HD gene. The DNA sequence protected from DNase I digestion was a 7-bp consensus sequence (GCCGGCG), which resides in triplicate at intervals of 13 bp within and proximal to the 20-bp direct repeat sequences of the HD promoter region. The mutation of 7-bp consensus sequence abolishes the HD promoter function in a neuronal cell line (IMR32). In human cultured cells, ectopically expressed green fluorescent protein-fused HDBP1 and HDBP2 localized in the cytoplasm, but both proteins totally shift from cytoplasm to nucleus by the treatment with an inhibitor of the nuclear export, leptomycin B, and mutagenesis of the putative nuclear export signals. Taken together, HDBP1 and HDBP2 are novel transcription factors shuttling between nucleus and cytoplasm and bind to the specific GCCGGCG, which is an essential cis-element for HD gene expression in neuronal cells.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fatores de Transcrição/fisiologia , Sequência de Aminoácidos , Northern Blotting , Linhagem Celular , Sequência Conservada , Citoplasma/metabolismo , DNA Complementar/metabolismo , Proteínas de Ligação a DNA/genética , Desoxirribonuclease I/metabolismo , Ácidos Graxos Insaturados/farmacologia , Biblioteca Gênica , Genes Reporter , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Proteína Huntingtina , Luciferases/metabolismo , Proteínas Luminescentes/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Sinais de Localização Nuclear , Plasmídeos/metabolismo , Prolina/química , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Serina/química , Fatores de Transcrição/genética , Transcrição Gênica , Técnicas do Sistema de Duplo-Híbrido , Dedos de ZincoRESUMO
Neuronal apoptosis inhibitory protein (NAIP) has been shown to inhibit apoptosis in vitro and in vivo with an expression which is regulated in a variety of cells and tissues and may be modulated by a variety of external stimuli. To understand the molecular basis of the transcriptional regulation of the NAIP gene, we have analyzed the 5'-flanking region and transcription of the human NAIP gene. The functional promoter and silencer elements were identified by luciferase reporter constructs in transient transfection experiments using four different human cells. Although the location of the functional elements were shared among the different cells used, the activities for the NAIP promoter varied. Further, cell type-specific protein binding activities were observed by an electrophoretic mobility shift assay (EMSA). EMSA analysis with specific antibodies and DNA sequence analysis identified the POU domain transcription factor Brn-2 as a candidate transcriptional regulator of the NAIP gene. The DNA sequence of the promoter region of the PsiNAIP gene, a copy gene for NAIP, was nearly identical to that of the NAIP gene, indicating a common regulatory mechanism for transcription of the NAIP and PsiNAIP genes. Indeed, the transcript of the PsiNAIP gene was identified. These results provided the first evidence for the functional promoter and candidate transcriptional factor for the NAIP gene and transcription of the PsiNAIP gene.
