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 postembryonic development. The role of the TH receptor (TR) in mediating the TH signal is complex as evidenced by divergent phenotypes in mice lacking TH compared with TR knock-out mice. We have proposed a dual function model for TR during development based on studies of frog metamorphosis. Here we examined an important assumption of this dual function model by using the chromatin immunoprecipitation assay, namely constitutive TR binding to promoters in vivo. We examined two target genes with TH-response elements (TRE) in their promoters, TR beta itself and TH/bZIP (TH-responsive basic leucine zipper transcription factor). By using an antibody that recognizes both TR alpha and TR beta, we found that TR binding to the TR beta promoter is indeed constitutive. Most surprisingly, TR binding to the TH/bZIP promoter increases dramatically after TH treatment of premetamorphic tadpoles and during metamorphosis. By using an antibody specific to TR beta, TR beta binding increases at both promoters in response to TH. In vitro biochemical studies showed that TRs bind TH/bZIP TRE with 4-fold lower affinity than to TR beta TRE. Our data show that only high affinity TR beta TRE is occupied by limiting levels of TR during premetamorphosis and that lower affinity TH/bZIP TRE becomes occupied only when overall the TR expression is higher during metamorphosis. These data provide the first in vivo evidence to suggest that one mechanism for tissue- and gene-specific regulation of TR target gene expression is through tissue and developmental stage-dependent regulation of TR levels, likely a critical mechanism for coordinating development in different organs during postembryonic development.