HNF3-mediated chromatin remodeling: A role in liver gene regulation

描述(申请人提供)：在早期发育过程中，分化细胞必须激活以前没有表达的基因，这些基因是细胞类型指定和器官发生所必需的。为了启动这些基因，染色质必须被解压，以允许转录因子访问DNA模板。本研究的目的是研究染色质重塑的潜在机制。具体地说，我们将在肝脏发育的模型中研究肝脏富含转录因子HNF3在白蛋白增强子处的染色质开放。HNF3是一种开创性的转录因子，可以在体外不存在染色质重塑复合体的情况下与白蛋白增强子上紧密结合的染色质结合，并在体外打开紧密连接的染色质。为了确定这一现象的生理学相关性，我们计划使用F9畸胎癌细胞株在体内检测HNF3在染色质重塑中的作用。在维甲酸存在的情况下，这些细胞分化为内脏内胚层，并在分化过程中开启白蛋白基因的表达。因为我们对研究白蛋白增强子的激活很感兴趣，所以这些细胞将作为一个很好的模型来询问关于HNF3在染色质开放中的作用。为了确定染色质开放的变化，我们将使用DNase I、微球菌核酸酶(MNase)和染色质免疫沉淀(ChIP)分析，并比较染色质可及性与白蛋白基因上调的关系。通过突变白蛋白增强子上的HNF3结合位点，我们可以确定体内白蛋白增强子的染色质重塑是否需要HNF3结合。针对HNF3的短发夹状RNA(ShRNAs)的额外实验将确定HNF3的转录活性是否需要在白蛋白增强子上打开染色质。除了确定HNF3在白蛋白增强子重塑中的作用外，我们还将研究HNF3在激活其他肝脏特异性基因方面的潜在作用，包括转甲状腺激素和甲胎蛋白。通过充分了解正常发育过程中基因激活的机制，我们有望更好地理解这一过程中可能导致发育缺陷和癌症的异常。

DESCRIPTION (provided by applicant): During early development, differentiating cells must activate genes previously not expressed that are required for cell type specification and organogenesis. For these genes to be turned on, chromatin must be de-compacted to allow transcription factors access to the DNA template. The goal of this research is to study the underlying mechanisms involved in chromatin remodeling. Specifically, we will examine chromatin opening at the albumin enhancer by the liver enriched transcription factor, HNF3, in a model of liver development. HNF3 is a pioneer transcription factor that can bind to and open compacted chromatin at the albumin enhancer in vitro in the absence of chromatin remodeling complexes. In order to establish the physiological relevance of this phenomenon, we plan to examine the role of HNF3 in chromatin remodeling in vivo using the F9 teratocarcinoma cell line. These cells, in the presence of retinoic acid, differentiate into visceral endoderm and turn on albumin gene expression as they differentiate. Because we are interested in examining the activation of the albumin enhancer, these cells will serve as a good model to ask questions about the role of HNF3 in chromatin opening. In order to determine changes in chromatin opening, we will use DNase I, micrococcal nuclease (MNase) and chromatin immunoprecipitation (ChIP) assays and compare chromatin accessibility to albumin gene upregulation. By mutating HNF3 binding sites on the albumin enhancer, we can determine whether HNF3 binding is required for chromatin remodeling at the albumin enhancer in vivo. Additional experiments with short hairpin RNAs (shRNAs) against HNF3 will determine whether HNF3 transcriptional activity is required to open chromatin at the albumin enhancer. In addition to determining the role of HNF3 in albumin enhancer remodeling, we will also examine a potential role for HNF3 in the activation of other liver specific genes, including transthyretin and alpha-fetoprotein. By fully understanding the mechanisms involved in gene activation during normal development, we can hopefully better understand aberrations in this process that may lead to developmental defects and cancer.