COLLAGEN GENE REGULATION IN DEVELOPMENT AND DISEASE

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Type I collagen, the most abundant protein in vertebrates, has diverse biological functions: it promotes cell migration, differentiation, and tissue morphogenesis during development, and, in the adult organism, provides tensile strength to connective tissues such as bone, tendons, and skin, and forms supporting framework of connective tissues in all major internal organs. Defects in the structure or synthesis of type I collagen are the cause of, or associated with, various acquired or genetic disorders which are characterized by symptoms such as brittle bones (osteogenesis imperfecta) or hyperflexible joints or skin, or by pathological fibrogenesis in diseases such as pulmonary fibrosis, liver cirrhosis, or atherosclerosis. A detailed knowledge of the synthesis and functions of type I collagen is therefore essential for understanding normal human development as well as disease processes. We are studying the developmental and tissue-specific transcriptional regulation of the gene encoding a1(I) collagen, the a1 subunit of type I collagen. Cis-regulatory DNA elements were previously identified in the 5'flanking region and first intron of the gene, and additional elements have now been found in its first exon and 3'flanking region. Most of these have not been characterized in much detail. In order to further elucidate their functions, we will analyze the trans-acting factors interacting with these elements by Dnase footprint and mobility shift assays, and determine their effect on the tissue-specific activity of the a1(I) promoter using reporter and minigene constructs, site- directed mutagenesis, and transection experiments. The functions of the various regulatory elements in the development activation of the a1(I) collagen gene will be analyzed using an in-vitro differentiation system of embryonal carcinoma cells, and their role in fibrotic diseases will be assessed in a model system for hepatic fibrosis. DNA methylation contributes to gene regulation in higher vertebrates, and several recent findings implicate a role of aberrant DNA methylation in various human diseases and cancer. The underlying mechanisms are only poorly understood. We have obtained evidence that DNA methylation may also be important for a1(I) collagen gene regulation. We will further address several possible direct and indirect mechanisms by which DNA methylation may function in regulating a1 (I) promoter activity using 5- aza-cytidine treatment and heterokaryon analyses, mobility shift assays with methylated factor binding sites, and transection experiments with methylated reporter gene constructs. Eukaryotic genomes are organized into chromatin loop domains which are attached at their ends to the nuclear matrix. Distal DNA elements and nuclear matrix attachment sites with regulatory functions have been identified. A chromatin structure analysis of the a1(I) collagen domain revealed distal DNase-hypersensitive sites in the 5-and 3- flanking regions of the gene, although it is not known whether they play a role in a1(I) gene regulation. We will determine the borders of the a1(I) chromatin domain and identifying remote regulatory elements using chromatin analyses. The function of distal elements will be assessed using minigene construct and transection experiments,and attachment sites to the nuclear matrix will be identified by matrix binding assays. The long-term goal of this work is a thorough knowledge of the cellular, biochemical, and molecular mechanisms involved in the regulation of type I collagen during normal development and differentiation and their derangement in pathological conditions.