RESUMO
The transcription of type I collagen genes is tightly regulated, but few cis-acting elements have been identified that can modulate the levels of expression of these genes. Generation of transgenic mice harboring various segments of the mouse pro-alpha1(I) collagen promoter led us to suspect that a repressor element was located between -10.5 and -17 kilobase pairs. Stable and transient transfection experiments in ROS17/2.8 osteoblastic cells confirmed the existence of such a repressor element at about -14 kilobase pairs and showed that it consisted in an almost perfect three-time repeat of a 41-base pair sequence. This element, which we named COIN-1, contains three E2-boxes, and a point mutation in at least two of them completely abolished its repressor effect. In gel shift assays, COIN-1 bound a DNA-binding protein named delta EF1/ZEB-1, and mutations that abolished the repressor effect of COIN-1 also suppressed the binding of delta EF1. We also showed that the repressor effect of COIN-1 was not mediated by chromatin compaction. Furthermore, overexpression of delta EF1 in ROS17/2.8 osteoblastic cells enhanced the inhibitory effect of COIN-1 in a dose-dependent manner and repressed the expression of the pro-alpha 1(I) collagen gene. Thus, delta EF1 appears to repress the expression of the mouse pro-alpha 1(I) collagen gene, through its binding to COIN-1.
Assuntos
Colágeno Tipo I/genética , Inativação Gênica , Proteínas de Homeodomínio/metabolismo , Proteínas Nucleares/metabolismo , Osteoblastos/fisiologia , Regiões Promotoras Genéticas/fisiologia , Fatores de Transcrição , Animais , Sequência de Bases , Células Cultivadas , Cromatina/química , DNA/análise , Deleção de Genes , Genes Reporter , Genoma , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Peso Molecular , Conformação de Ácido Nucleico , Ratos , Transfecção , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
Type I collagen is the major component of many extracellular matrices, and its accumulation characterizes most fibrotic processes. It is synthesized by a small number of discrete cell types, including fibroblasts, osteoblasts and odontoblasts. Analysis of transgenic mice harbouring different segments of the promoters of the mouse pro-alpha1 (I) and pro-alpha2 (I) genes has led to the conclusion that this tissue-specific expression is controlled by different cis-acting elements which are responsible for the expression of type I collagen genes in different type I collagen-producing cells. Transacting factors which bind to these different tissue-specific elements are still unknown, but they probably act by modifying the chromatin structure. In fibroblastic cells, various soluble molecules can modulate the transcription of type I collagen genes. Analysis of the pro-alpha1 (I) and pro-alpha2 (I) proximal promoters has led to the identification of different cis-acting elements which can modulate the expression of reporter genes, in transfection experiments. Among these cis-acting elements, a sequence located between -378 and -183 bp in the human pro-alpha2 (I) promoter appears to mediate the transcriptional effects of transforming growth factor-beta. It binds a large multimeric complex which contains Sp1, as well as AP1 and other DNA-binding proteins which have not yet been identified.
