COLLAGEN GENE REGULATION IN DEVELOPMENT AND DISEASE

I型胶原蛋白是脊椎动物中最丰富的蛋白质，具有多种多样的结构。 生物学功能：促进细胞迁移、分化， 在发育过程中的组织形态发生，并且，在成年生物体中， 为结缔组织如骨，腱， 和皮肤，并形成结缔组织的支撑框架， 主要的内部器官。 I型结构或合成缺陷 胶原蛋白是各种获得性或 遗传性疾病，其特征是症状， 骨（骨生成）或过度灵活的关节或皮肤，或通过 例如肺纤维化、肝纤维化 肝硬化或动脉粥样硬化。 详细了解合成和 因此，I型胶原蛋白的功能对于理解 正常的人类发育以及疾病过程。 我们正在研究发育和组织特异性转录 调节编码α 1（I）胶原蛋白的基因，α 1型胶原蛋白的α 1亚基 我胶原蛋白。 顺式调控DNA元件先前在 所述基因的5 '侧翼区和第一内含子，以及另外的 在其第一外显子和3 '端侧翼区发现了3个元件。 其中大多数尚未详细描述。 为了 进一步阐明其功能，我们将分析反式作用 通过DNA酶足迹和迁移率与这些元素相互作用的因素 偏移测定，并确定其对组织特异性活性的影响 使用报告基因和小基因构建体， 定向诱变和横切实验。 的职能 在a1（I）的发育激活中的各种调节元件 将使用体外分化系统分析胶原基因 胚胎癌细胞，以及它们在纤维化疾病中的作用， 在肝纤维化的模型系统中进行评估。 DNA甲基化有助于高等脊椎动物的基因调控， 最近的一些发现暗示了异常DNA甲基化在 各种人类疾病和癌症。 潜在的机制仅仅是 不太了解。 我们有证据表明DNA甲基化可能 对α 1（I）胶原基因调控也很重要。 我们将进一步 解决几种可能的直接和间接的机制， 甲基化可能在调节α 1（I）启动子活性中起作用， 氮杂胞苷处理和异核体分析，迁移率变动分析 与甲基化因子结合位点，和横断实验， 甲基化报告基因构建体。 真核基因组被组织成染色质环结构域， 其末端附着在核基质上。 远端DNA元件和 具有调节功能的核基质附着位点已经被 鉴定 α 1（I）胶原结构域的染色质结构分析 揭示了5-和3-侧翼的远端DNA酶超敏位点， 基因的区域，尽管尚不清楚它们是否发挥作用， 在a1（I）基因调控中。 我们将确定a1（I）的边界 染色质结构域和鉴定远程调控元件 染色质分析 将评估远端元件的功能 使用小基因构建体和横切实验，以及附着位点， 将通过基质结合测定来鉴定与核基质的结合。 这项工作的长期目标是彻底了解细胞， 生物化学和分子机制参与调节的类型 I型胶原在正常发育和分化过程中的作用及其 病理状态下的紊乱。

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.