Defining the thyroid hormone receptor Cistrome in vivo

描述(由申请人提供)：甲状腺激素(TH)对小鼠模型和人类的正常发育至关重要。此外，在儿童和成人生活中，TH都是新陈代谢、生长和体重的重要调节器。TH通过其同源核受体--甲状腺激素受体亚型(TRs)传递信号来调节其效应。TRs通过与协同调节蛋白的相互作用，对基因的表达进行正向或负向调控。基于在体外和细胞系中的大量工作，人们推测，受体对基因表达的影响是由靶基因调控区域中存在的受体反应元件(TRE)决定的。然而，在肝脏等TH作用的关键靶组织中从未显示出这一点，因为目前针对TRR的抗体加上靶组织中表达的TRR的数量使得在体内进行染色质免疫共沉淀(ChIP)变得非常困难。为了解决这一问题并在体内确定TRR结合部位，我们将开发一种多肽标记的TR21亚型，它可以在体内被生物素连接酶BIRA生物素化。然后，生物素化的TR21亚型可以被链霉亲和素沉淀在芯片中，回收的DNA经过深度平行测序(CHIP-SEQ)来确定靶问题中的TR21亚型。在这项提议的第一个目标中，标记的-TR21亚型将通过腺病毒传递给表达BIRA的TR2基因敲除小鼠的肝脏，然后是芯片序列，以确定肝脏中的TRR环路。在第二个目标中，生物素化多肽将被敲打到内源性TR21基因组位置，以便开发出一种小鼠模型，在该模型中，只要表达了Bira，就可以在任何组织中执行ChIP-SEQ。总之，这些目标的完成将为理解体内的TRR作用提供一个新的平台，并允许更深入地了解TH如何调节体内的关键转录途径。 公共卫生相关性：甲状腺激素是人类发育、生长和新陈代谢的关键调节器。虽然关于甲状腺激素是如何通过其靶标受体--甲状腺激素受体亚型(TRs)发出信号的已知很多，但TH和TRR的直接基因靶标还没有得到很好的描述。通过在体内确定受体的直接基因靶点，我们将对TH信号的机制有更深入的了解。这将有助于更好地了解TH如何调节发育、生长和新陈代谢，这些都是调节人类健康的关键过程。

DESCRIPTION (provided by applicant): Thyroid hormone (TH) is critical for normal development in murine models and humans. In addition, TH is a critical regulator of metabolism, growth and body weight in both childhood and adult life. TH mediates its effects by signaling through its cognate nuclear receptors, the thyroid hormone receptor isoforms (TRs). The TRs, through their interactions with coregulators proteins that vary depending upon the presence of TH, regulate gene expression positively or negatively. Based on significant work in vitro and in cell lines, it has been presumed that the effects of the TR on gene expression are dictated by the presence of TR response elements (TREs) in the regulatory regions of targeted genes. However, this has never been shown in vivo in key target tissues of TH action such as the liver, because current antibodies directed against the TR coupled with the amount of expressed TR in target tissues has made in vivo chromatin imunoprecipitation (ChIP) very difficult. To attack this issue and define TR binding sites in vivo, we will develop a peptide-tagged TR21 isoform that can be biotinylated in vivo by the biotin ligase BirA. The biotinylated TR21 isoform can then be precipitated in ChIP by streptavidin and the recovered DNA subjected to deep parallel sequencing (ChIP-seq) to define the TR cistrome in target issues. In the first aim of this proposal the tagged-TR21 isoform will be delivered to livers of TR2 knockout mice that express BirA by adenovirus followed by ChIP-seq to define the TR cistrome in liver. In the second aim, the biotinylation peptide will be knocked in to the endogenous TR21 genomic locus to allow for the development of a mouse model where ChIP-seq can be performed in any tissue as long as BirA is expressed. Together, completion of these Aims will provide a new platform for understanding TR action in vivo and allow for greater insight into how TH regulates key transcriptional pathways in vivo. PUBLIC HEALTH RELEVANCE: Thyroid hormone is a critical regulator of development, growth and metabolism in humans. Though much is known about how thyroid hormone signals through its target receptors, the thyroid hormone receptor isoforms (TRs), the direct gene targets of the TH and the TR are not well-described. By identifying the direct gene targets of the TR in vivo we will gain great insight into the mechanism of TH signaling. This will allow for a better understanding of how TH regulates development, growth and metabolism which are key processes in regulating human health.