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

描述(由申请人提供)：RXR的全基因组分析(结合在小鼠肝脏染色质中的芯片序列)摘要：这项建议将解决挑战领域08(基因组学)中的核心项目，特别针对挑战主题08-DK-107，核受体介导的功能转录单位的组装。关于这一应用，这一挑战主题指出：“...对反应元件占有率的全基因组分析的应用有可能迅速和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反应基因调控是通过核受体(NR)超家族成员进行的，特别是那些受RXR(-包含异源二聚体)调控的家族，但我们只知道基因组中一小部分RXR(结合位点)。逐个基因地评估结合位点是不可行的，而最近的技术进步强烈表明，我们目前对转录起始位点(TSS)附近结合位点的研究偏见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，&gt；50%的体内结合发生在TSS的&gt；10kb处，尚未被探索。目前利用现有技术可以克服的主要未满足挑战，并在本提案中具体涉及：1.)鉴定RXR(在雄性和雌性小鼠肝脏中)的基因组结合位点的全局阵列；RXR(与染色质结合与基因表达相关；3.)确定RXR的其他功能结构域的作用(除了DNA结合，驱动其参与染色质结合和基因表达。这些目标将通过以下两个目标来实现：目标1 RXR的CHIP-SEQ(在小鼠肝脏中：我们假设在小鼠肝脏中CHIP-SEQ的全球描绘将识别染色质中的所有RXR(结合位点)。通过与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研究结果。我们还将探索RXR的DNA结合结构域(DBD)以外的区域参与小鼠肝脏基因调控的机制。我们将使用唯一可用的肝细胞选择性DBD缺陷RXR(小鼠(hs-？DBD-RXR()。初步数据表明，这种内部截短的RXR(对先前报道的RXR(调控基因)具有信息性和不可预测性的影响，表明参与RXR(‘S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验证RXR(体内结合和IL-1诱导炎症后的结合)：我们假设Aim1的发现需要对选定的基因和区域进行详细验证才能证实全球的发现。关注显示男性和女性肝脏之间差异的基因和区域，以及wt和hs-？DBD-RXR()，将为选择验证提供合理的框架。方法学将包括蛋白质印迹、芯片定量聚合酶链式反应、RT-PCR定量RNA、芯片对转录复合体其他成员的探索以及凝胶移位。我们将利用先前AIMS的发现，以及我们实验室正在描绘的炎症中肝胆转运蛋白基因表达的变化，来探索和关联IL-1信号在染色质中的后果。对小鼠肝脏中RXR(结合区)的全基因组作图将提供一个框架，以启动学术界和医药界正在进行的识别肝脏中所有RXR(受RXR)调控基因的研究。由于目前的治疗重点是探索NR配体作为各种肝病的治疗药物，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()的研究为了解基本的NR功能提供了一个独特的窗口，并可能为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。 公共卫生相关性：大多数肝病急需有效的治疗方法，因为肝病影响着美国10%以上的成年人口，是第九大主要死亡原因。其中更具吸引力的候选药物是通过改变主基因调节器的活动来改变肝功能的药物，这项提案旨在完整地绘制DNA中主肝脏基因调节器RXR(结合和功能)的所有数千个位点。这些知识有望迅速加速合理设计和验证的肝病药物发现。

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.