RESUMO
We have characterized the promoter of the human gene coding for the apoferritin L subunit. Transient transfections of 5' and 3' deletion mutants indicate that the efficiency of the L promoter depends on both negative and positive cis-elements, located upstream and downstream of the transcription start point. DNaseI footprinting analysis of this DNA region revealed the presence of five protected segments. The most upstream one (element 1) corresponds to the negative cis-element and is recognized by factor(s) sharing a GC-sequence specificity. Three positive elements are in the region upstream of the start of transcription; a fifth positive cis-element (element 5) is localized in the first exon of the L gene.
Assuntos
Apoferritinas/genética , Regiões Promotoras Genéticas , Sequência de Bases , DNA/química , DNA/metabolismo , Pegada de DNA , Desoxirribonuclease I , Éxons , Deleção de Genes , Células HeLa , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , TransfecçãoRESUMO
In this paper, we examine the mechanisms that regulate the expression of the heavy (H) ferritin subunit in the colon carcinoma Caco-2 cell line allowed to differentiate spontaneously in vitro. The differentiation process of these cells in continuous culture is accompanied by an accumulation of the mRNA coding for the apoferritin H chain. The analysis of Caco-2 subclones stably transfected with an H-chain promoter-chloramphenicol acetyltransferase (CAT) construct revealed that the mRNA increase is paralleled by an enhanced transcription of the H gene, driven by the -100 to +4 region of the H promoter. The H gene transcriptional activation seems to be a specific feature of differentiated Caco-2 cells, since the activity of other promoters did not change upon differentiation. The -100 to +4 region of the H promoter binds a transcription factor called Bbf (B-box binding factor); electrophoretic-mobility-shift-assay analyses showed that the retarded complex due to Bbf-H promoter interaction is significantly increased in the differentiated cells. We propose that the activation of H-ferritin gene expression may be associated with the establishment of a differentiated phenotype in Caco-2 cells, and that the H-ferritin gene transcriptional up-regulation is accompanied by a modification in the activity of the transcription factor Bbf.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas de Drosophila , Ferritinas/genética , Fatores de Transcrição , Ativação Transcricional/fisiologia , Sequência de Bases , Células CACO-2 , Diferenciação Celular/fisiologia , Proteína A de Ligação a Elemento de Resposta do AMP Cíclico , Ferritinas/biossíntese , Regulação Neoplásica da Expressão Gênica , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Transcription of the H ferritin gene in vivo is stimulated by cAMP and repressed by the E1A oncoprotein. We report here the identification of the cis-element in the human promoter responsive to both cAMP- and E1A-mediated signals. This promoter region is included between positions -62 to -45 and binds a approximate 120-kDa transcription factor called Bbf. Bbf forms a complex in vivo with the coactivator molecules p300 and CBP. Recombinant E1A protein reduces the formation of these complexes. In vivo overexpression of p300 in HeLa cells reverses the E1A-mediated inhibition of the ferritin promoter transcription driven by Bbf. These data suggest the existence of a common mechanism for the cAMP activation and the E1A-mediated repression of H ferritin transcription.
Assuntos
Proteínas E1A de Adenovirus/fisiologia , AMP Cíclico/fisiologia , Ferritinas/genética , Transcrição Gênica , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Células HeLa , Humanos , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismoRESUMO
The frequency and distribution of angiotensin converting enzyme insertion/deletion (ACE I/D) polymorphism, and its association with other known risk factors for coronary atherosclerosis, has been studied, in a normal south Italian population. Subjects homozygous for deletion showed elevated fasting blood glucose levels when compared with subjects homozygous for insertion. The difference was consistent with an increased number of type 2 diabetics among the former group of subjects.
Assuntos
Glicemia/genética , Deleção de Genes , Peptidil Dipeptidase A/genética , Polimorfismo Genético/genética , Doença da Artéria Coronariana/genética , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de RiscoRESUMO
The human gene coding for the apoferritin H subunit belongs to a complex multigene family constituted by the expressed gene and by an undefined number of pseudogenes. We have used a strategy based on PCR to amplify specifically the H pseudogenes from a sample of human genomic DNA. With this approach, three new H pseudogenes have been cloned and characterized by DNA sequence analysis. In addition, we have identified a new type of pseudogene, the size of which (700 bp) is caused by multiple detection events in the putative coding region.