Defining the thyroid hormone receptor Cistrome in vivo

描述（由申请方提供）：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是童年和成年生活中代谢、生长和体重的关键调节剂。TH通过其同源核受体（甲状腺激素受体亚型（TRs））的信号传导介导其作用。TRs通过与辅调节蛋白的相互作用，根据TH的存在而变化，从而正向或负向调节基因表达。基于在体外和细胞系中的重要工作，已经推测TR对基因表达的影响是由靶基因的调控区中TR应答元件（TREs）的存在决定的。然而，这从未在体内TH作用的关键靶组织如肝脏中显示，因为目前针对TR的抗体与靶组织中表达的TR的量偶联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结合位点在体内，我们将开发一个肽标记的TR 21亚型，可以在体内生物素连接酶BirA生物素化。然后，生物素化的TR 21同种型可以通过链霉亲和素在ChIP中沉淀，并且回收的DNA进行深度平行测序（ChIP-seq）以确定靶组织中的TR顺式组。在该提议的第一个目的中，将标记的TR 21同种型递送至TR 2敲除小鼠的肝脏，所述TR 2敲除小鼠通过腺病毒表达BirA，然后通过ChIP-seq来定义肝脏中的TR顺式组。在第二个目标中，生物素化肽将被敲入内源性TR 21基因组位点，以开发小鼠模型，只要BirA表达，就可以在任何组织中进行ChIP-seq。总之，这些目标的完成将为了解体内TR作用提供一个新的平台，并允许更深入地了解TH如何调节体内关键的转录途径。

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.