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，TH在儿童和成人生活中都是代谢、生长和体重的重要调节因子。TH通过其同源核受体，甲状腺激素受体异构体（TRs）的信号传导介导其作用。TRs通过与协同调节蛋白（根据TH的存在而变化）的相互作用，积极或消极地调节基因表达。基于对体外和细胞系的大量研究，人们推测，TR对基因表达的影响是由目标基因调控区域中TR应答元件（TREs）的存在所决定的。然而，这在体内从未在肝等TH作用的关键靶组织中显示出来，因为目前针对TR的抗体加上靶组织中TR的表达量使得体内染色质免疫沉淀（ChIP）非常困难。为了解决这个问题并确定TR在体内的结合位点，我们将开发一种肽标记的TR21异构体，该异构体可以通过生物素连接酶BirA在体内进行生物素化。然后用链亲和素在ChIP中沉淀生物素化的TR21异构体，回收的DNA进行深度平行测序（ChIP-seq）以确定目标问题的TR细胞。本研究的第一个目的是通过腺病毒将标记的tr21亚型传递到表达BirA的TR2敲除小鼠的肝脏中，然后通过ChIP-seq确定肝脏中的TR细胞。在第二个目标中，生物素化肽将被敲入内源性TR21基因组位点，从而允许开发小鼠模型，只要BirA表达，ChIP-seq就可以在任何组织中进行。总之，这些目标的完成将为理解体内的TR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。

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.