RESUMO
The mouse beta-globin gene locus control region (LCR), located upstream of the beta-globin gene cluster, is essential for the activated transcription of genes in the cluster. The LCR contains multiple binding sites for transactivators, including Maf-recognition elements (MAREs). However, little is known about the specific proteins that bind to these sites or the time at which they bind during erythroid differentiation. We have performed chromatin immunoprecipitation experiments to determine the recruitment of the erythroid-specific transactivator p45 NF-E2/MafK (p18 NF-E2) heterodimer and small Maf proteins to various regions in the globin gene locus before and after the induction of murine erythroleukemia (MEL) cell differentiation. We report that, before induction, the LCR is occupied by small Maf proteins, and, on erythroid maturation, the NF-E2 complex is recruited to the LCR and the active globin promoters, even though the promoters do not contain MAREs. This differentiation-coupled recruitment of NF-E2 complex correlates with a greater than 100-fold increase in beta-major globin transcription, but is not associated with a significant change in locus-wide histone H3 acetylation. These findings suggest that the beta-globin gene locus exists in a constitutively open chromatin conformation before terminal differentiation, and we speculate that recruitment of NF-E2 complex to the LCR and active promoters may be a rate-limiting step in the activation of beta-globin gene expression.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Globinas/genética , Fatores de Transcrição/metabolismo , Acetilação , Animais , Proteínas de Bactérias/genética , Linhagem Celular , Fatores de Ligação de DNA Eritroide Específicos , Regulação da Expressão Gênica , Histonas/metabolismo , Leucemia Eritroblástica Aguda/genética , Leucemia Eritroblástica Aguda/metabolismo , Região de Controle de Locus Gênico , Fator de Transcrição MafK , Camundongos , Modelos Biológicos , Família Multigênica , Fator de Transcrição NF-E2 , Subunidade p45 do Fator de Transcrição NF-E2 , Regiões Promotoras Genéticas , Transcrição Gênica , Células Tumorais CultivadasRESUMO
In the early stage of Drosophila embryogenesis, DNA replication initiates at unspecified sites in the chromosome. In contrast, DNA replication initiates in specified regions in cultured cells. We investigated when and where the initiation regions are specified during embryogenesis and compared them with those observed in cultured cells by two-dimensional gel methods. In the DNA polymerase alpha gene (DNApolalpha) locus, where an initiation region, oriDalpha, had been identified in cultured Kc cells, repression of origin activity in the coding region was detected after formation of cellular blastoderms, and the range of the initiation region had become confined by 5 h after fertilization. During this work we identified other initiation regions between oriDalpha and the Drosophila E2F gene (dE2F) downstream of DNApolalpha. At least four initiation regions showing replication bubbles were identified in the 65-kb DNApolalpha-dE2F locus in 5-h embryos, but only two were observed in Kc cells. These results suggest that the specification levels of origin usage in 5-h embryos are in the intermediate state compared to those in more differentiated cells. Further, we found a spatial correlation between the active promoter regions for dE2F and the active initiation zones of replication. In 5-h embryos, two known transcripts differing in their first exons were expressed, and two regions close to the respective promoter regions for both transcripts functioned as replication origins. In Kc cells, only one transcript was expressed and functional replication origins were observed only in the region including the promoter region for this transcript.
Assuntos
DNA Polimerase Dirigida por DNA/genética , Proteínas de Drosophila , Drosophila melanogaster/genética , Genes de Insetos , Origem de Replicação , Fatores de Transcrição/genética , Animais , Sequência de Bases , Replicação do DNA , Drosophila melanogaster/embriologia , Fator de Transcrição E2F2 , Eletroforese em Gel Bidimensional , Feminino , Dados de Sequência MolecularRESUMO
The Escherichia coli gene recA is essential for homologous recombination and DNA repair, and homologs have been identified in eukaryotes. A basidiomycete, Coprinus cinereus, which has many advantages for the study of meiosis, was recently reported to have a homolog of one of these, RAD51. In the yeast Saccharomyces, mutations in the RAD51 gene cause defects in both somatic and meiotic cells. Based on this finding, we screened for a meiosis-specific homolog of recA, equivalent to Lilium LIM15 or Saccharomyces DMC1, in C. cinereus, and isolated a clone containing a 1.2-kb DNA fragment from a cDNA library constructed with Coprinus poly(A)+ RNA isolated from cells undergoing meiosis. The predicted amino acid sequence was 52% identical to the putative gene product of the lily cDNA clone LIM15 and 61% identical to Saccharomyces DMC1, and showed limited sequence similarity to the products of RAD52, 55, and 57. The synchrony of meiosis in Coprinus provides an ideal system for the investigation of differential gene expression in relation to meiosis and fruiting body development. Northern analysis indicated that Coprinus LIM15/DMC1 was expressed at meiotic prophase within 8 h after the onset of karyogamy, suggesting that the gene functions mostly at the stage at which the homologous chromosomes pair, but may not be essential at the point at which they recombine. The gene is not expressed in somatic cells.
Assuntos
Proteínas de Ciclo Celular , Cromossomos Fúngicos , Coprinus/genética , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Meiose/genética , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Southern Blotting , Primers do DNA , DNA Fúngico , Dados de Sequência Molecular , RNA Mensageiro/genética , Proteínas de Saccharomyces cerevisiae , Homologia de Sequência de AminoácidosRESUMO
Mammalian E2F transcription factors comprise a family of proteins encoded by distinct genes which function in the form of heterodimers with DP proteins. In Drosophila melanogaster, only a single E2F-related transcription factor, dE2F, has been reported. We have now identified and characterized a cDNA encoding another E2F family member in Drosophila, termed dE2F2. The predicted amino acid sequence shares 38.8% identity with dE2F, including the QKRRIYDITNVLEGI motif which is highly conserved in mammalian E2F family members and dE2F. The 18 amino acids, located in the carboxy-terminal region of the mammalian E2F family, sufficient for binding to pRb are also conserved in dE2F2. Band mobility shift analyses with glutathione S-transferase fusion proteins revealed dE2F2 binding to E2F-recognition sites to be dependent on the presence of dDP protein, in apparent contrast to dE2F. Furthermore, cotransfection experiments in Kc cells demonstrated dE2F2 repression of the PCNA gene promoter activity, while dE2F caused activation, the target site for the repression being identical to the dE2F-recognition site.
Assuntos
Proteínas de Transporte , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Proteínas de Drosophila , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Transativadores , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , DNA Complementar , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Fatores de Transcrição E2F , Fator de Transcrição E2F2 , Embrião não Mamífero/metabolismo , Genes de Insetos , Larva , Mamíferos , Dados de Sequência Molecular , Proteína 1 de Ligação ao Retinoblastoma , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
Two mRNA species were observed for the Drosophila E2F (dE2F) gene, differing with regard to the first exons (exon 1-a and exon 1-b), which were expressed differently during development. A single transcription initiation site for mRNA containing exon 1-b was mapped by primer extension analysis and numbered +1. We found three tandemly aligned sequences, similar to the DNA replication-related element (DRE; 5'-TATCGATA), which is commonly required for transcription of genes related to DNA replication and cell proliferation, in the region upstream of this site. Band mobility shift analyses using oligonucleotides containing the DRE-related sequences with or without various base substitutions revealed that two out of three DRE-related sequences are especially important for binding to the DRE-binding factor (DREF). On footprinting analysis with Kc cell nuclear extracts and a glutathione S-transferase fusion protein with the N-terminal fragment (1-125 amino acid residues) of DREF, all three DRE-related sequences were found to be protected. Transient luciferase expression assays in Kc cells demonstrated that the region containing the three DRE-related sequences is required for high promoter activity. We have established transgenic lines of Drosophila in which ectopic expression of DREF was targeted to the eye imaginal disc cells. Overexpression of DREF in eye imaginal disc cells enhanced the promoter activity of dE2F. The obtained results indicate that the DRE/DREF system activates transcription of the dE2F gene.
Assuntos
Proteínas de Transporte , Proteínas de Ciclo Celular , Proteínas de Drosophila , Drosophila/embriologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Transativadores , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Animais , Animais Geneticamente Modificados , Sequência de Bases , Sítios de Ligação/genética , Pegada de DNA , Proteínas de Ligação a DNA/análise , Fatores de Transcrição E2F , Éxons/genética , Olho/crescimento & desenvolvimento , Imuno-Histoquímica , Óperon Lac/genética , Dados de Sequência Molecular , Morfogênese/genética , Regiões Promotoras Genéticas/genética , RNA Mensageiro/metabolismo , Proteína 1 de Ligação ao Retinoblastoma , Deleção de Sequência/genética , Transcrição Gênica/genética , Ativação Transcricional/genéticaRESUMO
A DNA polymerase from cauliflower (Brassica oleracea var. botrytis) inflorescence has been purified to near homogeneity through five successive column chromatographies, and temporally designated cauliflower polymerase 1. Cauliflower polymerase 1 is a monopolypeptide with a molecular mass of 100 kDa. The enzyme efficiently uses synthetic DNA homopolymers and moderately activated DNA and a synthetic RNA homopolymer as template-primers. The enzyme is strongly sensitive to dideoxythymidine triphosphate and N-ethylmaleimide, but it is insensitive to aphidicolin. It was stimulated with 250 mM KCl. Its mode of DNA synthesis is high-processive with or without proliferating-cell nuclear antigen. A 3'-->5' exonuclease activity is associated with cauliflower polymerase 1. The enzyme is clearly different from cauliflower mitochondrial polymerase and does not resemble the four different types of wheat DNA polymerase, designated wheat DNA polymerases A, B, CI and CII. In the present paper the role of the enzyme in plant DNA synthesis is discussed.
Assuntos
Brassica/enzimologia , DNA Polimerase Dirigida por DNA/química , Cátions/farmacologia , Inibidores Enzimáticos/farmacologia , Exonucleases/metabolismo , Peso Molecular , Proteínas de Plantas/química , Especificidade por SubstratoRESUMO
A 135kDa DNA polymerase alpha lacking primase activity has been purified to near homogeneity from Coprinus meiotic tissues. The activity of the DNA polymerase was sensitive to aphidicolin and N-ethylmaleimide, but was insensitive to dideoxythymidine triphosphate. DNA synthesis was proceeded with a low processivity. Neither activity nor processivity were affected by PCNA in the presence or absence of KCI. Monovalent cation inhibited its activity. These biochemical properties are almost identical to those of Coprinus DNA polymerase alpha -primase complex. However, the 135kDa DNA polymerase did not use activated DNA as a template-primer, inconsistent with Coprinus DNA polymerase alpha-primase complex. The 135kDa DNA polymerase was purified from the tissues at meiotic pro-metaphase I, suggesting that the alpha- DNA polymerase-primase complex dissociates as the meiotic cell cycle progresses and only the catalytic subunit remains at this stage.