COLLAGEN GENE REGULATION IN DEVELOPMENT AND DISEASE

I型胶原是脊椎动物中含量最丰富的蛋白质，具有多种 生物学功能：促进细胞迁移、分化和 发育过程中的组织形态发生，在成年生物体中， 为结缔组织提供抗拉强度，如骨、肌腱、 和皮肤，形成结缔组织的支撑骨架 主要内脏器官。第I类结构或合成中的缺陷 胶原蛋白是各种获得性或与之相关的疾病的原因 以脆性等症状为特征的遗传性疾病 骨骼(成骨不全)或高度灵活的关节或皮肤，或通过 肺纤维化、肝纤维化等疾病的病理性纤维化 肝硬变，或动脉粥样硬化。关于合成的详细知识和 因此，I型胶原的功能对于理解 人类的正常发育以及疾病的过程。 我们正在研究发育和组织特异性转录 A1(I)型胶原编码基因的调控 我的胶原蛋白。顺式调控的DNA元件之前在 基因的5‘侧翼区和第一内含子，以及附加的 现在已经在它的第一个外显子和3‘侧翼区域发现了元素。 其中大多数还没有得到太多细节的描述。为了 进一步阐明它们的功能，我们将分析它们的反式作用 通过DNA酶足迹和流动性与这些元素相互作用的因素 改变检测方法，并确定它们对组织特异性活性的影响 在使用报告基因和微基因结构的A1(I)启动子中， 定向诱变和横切实验。的功能 A1的发育激活过程中的各种调节元件(I) 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 在肝纤维化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，并且 最近的几项发现表明，DNA甲基化异常在 人类的各种疾病和癌症。潜在的机制只是 人们对此知之甚少。我们已经获得证据表明DNA甲基化可能 对A1(I)胶原基因的调控也很重要。我们将进一步 阐述DNA通过几种可能的直接和间接机制 甲基化可能通过5-甲基化调节A1(I)启动子活性 氮杂胞苷治疗和异核体分析、迁移率变化分析 与甲基化因子结合部位，和横切实验与 甲基化的报告基因结构。 真核生物基因组被组织成染色质环域，这些区域是 它们的两端附着在核矩阵上。远端DNA元件和 具有调节功能的核基质连接位点已经被 已确认身份。A1(I)胶原区的染色质结构分析 在5-侧翼和3-侧翼发现远端DNA酶敏感部位 基因的区域，尽管还不知道它们是否起作用 在A1(I)基因调控中。我们将确定A1(I)的边界 染色质结构域和识别远程调控元件 染色质分析。将评估远端元件的功能 使用微型基因构建和横切实验，以及附着点 与核基质的结合将通过基质结合试验进行鉴定。 这项工作的长期目标是彻底了解细胞， 参与类型调控的生化和分子机制 I型胶原在正常发育和分化中的作用及其机制 病理状态下的精神错乱。

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.