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（在小鼠肝脏染色质中与ChIP-SEQ结合）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，核受体介导的功能转录单位组装。关于这一应用，本挑战主题指出：“.全基因组响应元件占有率分析的应用具有快速和全面地揭示基因调控的新机制的潜力。"...肝脏中相当大比例的代谢和响应性基因调控是通过核受体（NR）超家族的成员发生的，特别是那些由含RXR β的异源二聚体对调控的，然而我们只知道基因组中一小部分RXR β结合位点。一个基因的基因结合位点的评估是不可行的，而最近的技术进步强烈表明，我们目前的偏见研究结合位点附近的转录起始位点（TSS）大大低估了实际的结合，发生在活细胞。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，> 50%的体内结合发生在> 10 kb的TSS，并且尚未被探索。当前技术可以克服的主要未满足的挑战，以及本提案中具体解决的问题是：1。鉴定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.）使RXR（与染色质的结合与基因表达相关;和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2个目标来实现：目标1 RXR的ChIP-SEQ（在小鼠肝脏体内）：我们假设小鼠肝脏中ChIP-SEQ的整体描绘将识别所有RXR（染色质中的结合位点。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发现。我们还将探讨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。我们将使用来自可获得的肝细胞选择性DBD缺陷RXR的肝脏（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调节基因具有信息性和不可预测的作用，表明参与RXR在基因调节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 RXR的验证（体内结合和IL-1诱导炎症后的结合：我们假设Aim 1的发现需要针对选定的基因和区域进行详细验证，以确认全球发现。关注男性和女性肝脏之间存在差异的基因和区域，以及wt和hs-？DBD-RXR（，将为选择确认提供合理的框架。方法学将包括蛋白质印迹、ChIP qPCR、通过rtPCR进行RNA定量、通过ChIP探索转录复合物的其他成员以及凝胶移位。我们将利用以前的目标的发现，以及我们实验室正在进行的炎症中肝胆转运蛋白基因表达改变的描述，来探索和关联染色质响应IL-1信号传导的结果。小鼠肝脏中RXR β结合区域的全基因组定位将为学术界和制药界正在进行的识别肝脏中所有RXR β调控基因的研究提供一个框架。目前的治疗重点是探索NR配体作为各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，研究的hs-？DBD-RXR α提供了了解基本NR功能的独特窗口，并且可能提供关于RXR α的哪些结构域用于不同转录复合物的新见解。 公共卫生相关性：对于大多数肝脏疾病的有效疗法存在迫切需求，因为肝脏疾病影响超过10%的美国成年人口，并且是第九大死亡原因。其中更有吸引力的候选药物是通过改变主基因调节剂的活性来改变肝功能的药物，该提案旨在完全绘制主肝脏基因调节剂RXR结合和发挥作用的DNA中的所有数千个位点。这些知识预计将迅速加速合理设计和验证的治疗肝脏疾病的药物发现。

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.