CHROMATIN REMODELING DURING LIVER DEVELOPMENT

这项提案的主要目的是审查下列程序 发育调节转录的顺序结合 因子组织组成增强子的顺式作用序列 将血清白蛋白基因分为三个翻译阶段的序列 包含基本增强因子的核小体样颗粒 肝脏染色质中的结合部位。这将使用三个 不同的方法。首先，将白蛋白增强剂组装成 肝脏发育过程中一种活性较高的核蛋白复合体 之后将比较白蛋白的核小体时相 肝脏染色质在连续发育阶段的增强剂。这些 观察结果将与最近完成的体内DMS相结合 鉴定转录因子的保护性分析 负责激活白蛋白增强剂。第二，有能力 纯化的转录因子单独和组合结合到和 白蛋白增强子体外重组的扰动核小体 将确定片段和核心组蛋白，以便确定 转录因子指定核小体的机制 白蛋白增强剂的结构。最后，具体发挥的作用 转录因子在白蛋白染色质重构中的作用 增强子将通过研究活性和染色质进行评估 含有白蛋白增强子突变的转基因的结构 转基因肝脏发育过程中增强子结合位点的选择 老鼠。这些研究应该会加深我们对所起作用的理解 染色质结构在转录调控中的作用 洞察监管因素决定正常的机制 肝脏生长发育。

The primary aim of this proposal is to examine the processes whereby the sequential binding of developmentally regulated transcription factors organizes the cis-acting sequences which make up the enhancer of the serum albumin gene into a series of three translationally phased nucleosome-like particles encompassing the essential enhancer factor binding sites in liver chromatin. This will be accomplished using three different approaches. First, the assembly of the albumin enhancer into an active higher-order nucleoprotein complex during liver development will be followed by comparison of nucleosome phasing at the albumin enhancer in liver chromatin at successive developmental stages. These observations will be combined with recently completed in vivo DMS protection analyses which have identified the transcription factors responsible for activating the albumin enhancer. Second, the ability of purified transcription factors alone and in combination to bind to and perturb nucleosomes reconstituted in vitro from albumin enhancer fragments and core histones will be determined, in order to determine the mechanisms whereby transcription factors specify the nucleosome structure of the albumin enhancer. Finally, the role played by specific transcription factors in the chromatin remodeling of the albumin enhancer will be assessed by studying the activity and chromatin structure of albumin enhancer transgenes containing mutations in selected enhancer binding sites during liver development in transgenic mice. These studies should further our understanding of the role played by chromatin architecture in transcriptional regulation as well as lend insight into the mechanisms whereby regulatory factors dictate normal liver growth and development.