BUCHHOLZ, D.R.*; PAUL, B.P.; SHI, Y.-B.; Laboratory of Gene Regulation, National Institute of Child Health and Human Development: Gene-specific changes in promoter occupancy by thyroid hormone receptor during frog metamorphosis: Implications for developmental gene regulation

In all vertebrates, thyroid hormones (TH) affect post-embryonic developmental processes. The role of TH receptor (TR) in mediating the TH signal during development is complex as evidenced by divergent phenotypes in mice lacking TH compared to TR knockout mice. We have proposed a dual function model for the role of TR during development based on studies of frog metamorphosis, where TR functions as a transcriptional repressor or activator during premetamorphosis and metamorphic climax, respectively. Here, we examined an important assumption of this dual function model using the chromatin immunoprecipitation (ChIP) assay, namely constitutive TR binding to promoters in vivo throughout development. We examined two TH direct response genes with TH response elements (TRE) in their promoters, TR? itself and TH/bZIP (TH-responsive basic leucine zipper transcription factor). Using an antibody that recognizes both TR? and TR? in the ChIP assay, we found that TR binding to the TR? promoter is indeed constitutive, whereas TR binding to the TH/bZIP promoter increases 5- to 10-fold from near background levels after TH treatment of premetamorphic tadpoles and at metamorphic climax. Using an antibody specific to TR?, TR? binding increases at both TR? and TH/bZIP promoters in response to TH. In-vitro biochemical studies showed that TRs bind the TH/bZIP TRE with 4-fold lower affinity than to the TR? TRE. Our data, suggesting that only high affinity TR? TREs are occupied by limiting levels of TR during premetamorphosis and that lower affinity TH/bZIP TRE becomes occupied only when overall TR expression is higher during metamorphosis, have broad implications for developmental gene regulation.