COLLAGEN GENE REGULATION IN DEVELOPMENT AND DISEASE

发育和疾病中的胶原蛋白基因调控

• 批准号: 2413977
• 负责人: MICHAEL BREINDL
• 金额: $ 16.93万
• 依托单位: SAN DIEGO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-25 至 1998-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2413977
• 关键词: DNA footprinting; DNA methylation; chromatin; collagen; connective tissue development; gel mobility shift assay; gene expression; genetic regulatory element; genetic transcription; human genetic material tag; introns; laboratory mouse; nucleic acid sequence; protein biosynthesis; protein structure function; site directed mutagenesis; tissue /cell culture; transcription factor; transfection

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.

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 生物学功能：它促进细胞迁移，分化和 在发育过程中以及成人生物体中的组织形态发生， 为结缔组织提供拉伸强度，例如骨骼，肌腱， 和皮肤，并形成所有在所有人中的结缔组织的支撑框架 主要内部器官。 I型的结构或合成中的缺陷 胶原蛋白是各种获得或与之相关的原因或 以脆性为特征的遗传疾病 骨骼（成骨不完美）或过度柔滑的关节或皮肤，或者 疾病中的病理纤维发生，例如肺纤维化，肝脏 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至关重要 正常的人类发展以及疾病过程。 我们正在研究发育和组织特异性的转录 调节编码A1（I）胶原蛋白的基因，类型的A1亚基 我胶原蛋白。 以前在 5'Flanking区域和基因的第一个内含子，以及其他 现在已经在其第一个外显子和3'Franking地区发现了元素。 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DNase足迹和流动性与这些元素相互作用的因素 转移测定，并确定其对组织特异性活性的影响 使用记者和微型构建体的A1（i）启动子 定向诱变和横切实验。 功能 A1（i）开发激活中的各种调节元素 胶原蛋白基因将使用体外分化系统进行分析 胚胎癌细胞及其在纤维化疾病中的作用将 在用于肝纤维化的模型系统中进行评估。 DNA甲基化有助于较高脊椎动物的基因调节，并且 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。 基本机制仅是 理解不佳。 我们已经获得了DNA甲基化的证据 对于A1（i）胶原基因调节也很重要。 我们将进一步 解决DNA的几种可能的直接和间接机制 甲基化可能在使用5-- AZA-胞苷治疗和异质分析，移动性转移测定法 与甲基化因子结合位点，以及与 甲基化的报告基因构建体。 真核基因组被组织成染色质环状结构域 附着在核矩阵上。 远端DNA元素和 具有调节功能的核基质附件位点已经 确定。 A1（i）胶原蛋白结构域的染色质结构分析 在5和3侧面显示的 该基因的区域，尽管尚不清楚它们是否扮演角色 在A1（i）基因调节中。 我们将确定A1（i）的边界 染色质结构域并使用 染色质分析。 将评估远端元素的功能 使用微型构建和横切实验以及附件位点 核基质将通过基质结合测定确定。 这项工作的长期目标是对细胞的彻底了解， 与类型调节有关的生化和分子机制 我在正常发展和差异化期间胶原蛋白及其 病理条件下的危险。