Genome-Wide Analysis of RXR-alpha Binding In Mouse Liver Chromatin with ChIP-SEQ

摘要：本提案将解决挑战领域08（基因组学）的核心项目，特别针对挑战主题08- dk -107，核受体介导的功能性转录单元组装。关于这一应用，本挑战主题指出：“……全基因组响应元件占用分析的应用有可能快速、全面地揭示基因调控的新机制。肝脏中相当大比例的代谢和应答性基因调控是通过核受体（NR）超家族成员发生的，特别是那些由含有RXR的异源二聚体对调控的基因，但我们只知道基因组中RXR结合位点的一小部分。对结合位点进行逐个基因的评估是不可行的，而最近的技术进步强烈表明，我们目前倾向于研究转录起始位点（TSS）附近的结合位点，这大大低估了活细胞中发生的实际结合。在迄今为止进行的大多数全基因组分析中，对于NR家族成员和其他转录因子，bb50 %的体内结合发生在距离TSS bb10 kb处，尚未被探索。当前技术可以克服的主要挑战是：1.)确定RXR的基因组结合位点的全局阵列（在雄性和雌性小鼠肝脏中）；2.)将RXR（与染色质结合）与基因表达相关联；3.)确定RXR的其他功能域（除了dna结合外，驱动其参与染色质结合和基因表达）的作用。这些目标将通过以下两个目标来实现：目标1小鼠肝脏体内RXR的ChIP-SEQ：我们假设小鼠肝脏中ChIP-SEQ的全局描述将识别染色质中所有RXR结合位点。根据与Pol II结合的相关性，我们将确定RXR（结合）与肝脏基因表达相关的区域，并比较雄性和雌性小鼠的发现。我们还将探索RXR的dna结合域（DBD）以外的区域参与小鼠肝脏基因调控的机制。我们将使用一种独特的肝细胞选择性dbd缺陷RXR（小鼠）的肝脏进行ChIP-SEQ。DBD - RXR()。初步数据表明，这种内部截断的RXR对先前报道的RXR调控基因具有信息性和不可预测的影响，表明参与RXR在基因调控中的有效性的其他结构域和相互作用具有意想不到的作用。目的2：体内和IL-1诱导炎症后RXR结合的验证：我们假设Aim1的发现需要在选择的基因和区域进行详细验证，以确认整体发现。研究重点是男性和女性肝脏之间的基因和区域差异，以及wt和hs-？DBD- RXR将为选择验证提供合理的框架。方法将包括western blotting、ChIP qPCR、rtPCR的RNA定量、ChIP对转录复合物其他成员的探索和凝胶转移。我们将利用先前的研究结果，以及我们实验室对炎症中肝胆转运蛋白基因表达变化的持续描述，探索染色质对IL-1信号的反应并将其关联起来。小鼠肝脏中RXR结合区域的全基因组图谱将为学术界和制药企业快速启动正在进行的肝脏中所有RXR调控基因的鉴定研究提供一个框架。目前的治疗重点是探索NR配体作为多种肝脏疾病的治疗药物，这些数据对于寻找治疗靶点、避免毒性和筛选可能的候选分子至关重要。此外，对hs-？DBD- RXR（）为研究NR的基本功能提供了一个独特的窗口，并可能为研究RXR的哪些结构域在不同的转录复合体中被利用提供新的见解。

DESCRIPTION (provided by applicant): Genome-wide analysis of RXR( binding in mouse liver chromatin with ChIP-SEQ Abstract: This proposal will address core items in Challenge Area 08 (Genomics), specifically targeting Challenge Topic 08-DK-107, Nuclear receptor mediated assembly of functional transcription units. In relation to this application, this Challenge Topic states: "... The application of genome-wide analyses of response element occupancy has the potential to rapidly and comprehensively reveal novel mechanisms of gene regulation."... A substantial proportion of metabolic and responsive gene regulation in the liver occurs via members of the Nuclear Receptor (NR) superfamily, specifically those regulated by RXR(-containing heterodimer pairs, yet we only know a small fraction of RXR( binding sites in the genome. A gene-by-gene assessment of binding sites is not feasible, while recent technological advances strongly indicate that our current bias towards studying binding sites near the transcription start sites (TSS) vastly underestimates the actual binding that occurs in living cells. In most genome-wide analyses performed to date, for both NR family members and other transcription factors, > 50% of in vivo binding takes place > 10 kb from the TSS, and has not yet been explored. The major unmet Challenges that are now surmountable with current technology, and specifically addressed in this proposal, are: 1.) to identify the global array of genomic binding sites for RXR( in both male and female mouse liver; 2.) correlate RXR( binding to chromatin with gene expression; and 3.) identify roles for other functional domains of RXR( apart from DNA-binding, that drive its participation in chromatin binding and gene expression. These goals will be addressed via the following 2 Aims: Aim 1 ChIP-SEQ of RXR( in mouse livers vivo: We hypothesize that global delineation of ChIP-SEQ in mouse liver will identify all RXR( binding sites in chromatin. With correlation to Pol II binding, we will identify those regions where RXR( binding is linked to liver gene expression, and compare the findings in male and female mice. We will also explore the mechanisms where regions other than the DNA-binding Domain (DBD) of RXR( participate in gene regulation in mouse liver. We will perform ChIP-SEQ using livers from a uniquely-available hepatocyte-selective DBD-deficient RXR( mice (hs-? DBD- RXR(). Preliminary data indicate informative and non-predictable effects of this internally-truncated RXR( on previously reported RXR(-regulated genes, indicating unexpected roles for other domains and interactions that participate in RXR('s effectiveness in gene regulation. Aim 2 Validation of RXR( binding in vivo and after IL-1¿ induced inflammation: We hypothesize that the findings of Aim1 will need to be validated in detail for select genes and regions in order to confirm the global findings. A focus upon genes and regions that show discrepancies between male and female livers, and wt and hs-?DBD- RXR(, will provide the rational framework for select validations. Methodologies will include western blotting, ChIP qPCR, RNA quantitation by rtPCR, exploration by ChIP of other members of the transcription complexes, and gel shifts. We will utilize the findings from previous Aims, and our lab's ongoing delineation of alterations in hepatobiliary transporter gene expression in inflammation, to explore and correlate the consequences in chromatin in response to IL-1¿ signaling. Genome-wide mapping of RXR( binding regions in mouse liver will provide a framework to jumpstart ongoing investigations into identification of all RXR(-regulated genes in liver in both academia and pharma. With current therapeutic emphases on exploring NR ligands as therapeutics for a variety of liver diseases, these data are critical for finding therapeutic targets, avoiding toxicities, and screening likely candidate molecules. In addition, studies of the hs-?DBD- RXR(, provide a unique window into basic NR functioning, and are likely to provide novel insights into which domains of RXR( are utilized in different transcription complexes. PUBLIC HEALTH RELEVANCE: There is a dire need for effective therapeutics for most liver diseases, since liver disease affects over 10% of the adult US population, and is the 9th leading cause of death. Among the more attractive candidates are drugs that modify liver function through changing activities of master gene regulators, and this proposal aims to fully map all the thousands of sites in the DNA where the master liver gene regulator RXR( binds and functions. Such knowledge is expected to rapidly accelerate rationally-designed and validated drug discovery for liver disease.