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.
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.
|Ramadoss, Preeti; Abraham, Brian J; Tsai, Linus et al. (2014) Novel mechanism of positive versus negative regulation by thyroid hormone receptor *1 (TR*1) identified by genome-wide profiling of binding sites in mouse liver. J Biol Chem 289:1313-28|