FIG. 5.

The dnTR competes effectively with wild-type TRs for binding to endogenous-T₃-response genes in transgenic tadpoles. Wild-type tadpoles (wt, lanes 1 to 2) or transgenic tadpoles carrying the GFP-TR fusion gene dnTR (Tg, lanes 3) were treated with (lanes 2 to 3) or without (lanes 1) 5 nM T₃ for 1 day. Nuclei were isolated from intestine or tail and subjected to ChIP assay with the antibodies to indicated proteins for the binding of the TRs to the TRE regions of the endogenous TRβ and TH/bZip genes. Note that the ChIP assay with the GFP antibody clearly showed that the fusion protein is bound to the T₃ response genes in different organs of the transgenic animals. The ChIP assay with the TR antibody, which recognizes both TRα and TRβ, showed that TR is constitutively bound to both genes in different organs or whole animals (not shown) as expected (the weaker signal in the transgenic animals is likely due to possible interference of the immunoprecipitation by the GFP moiety in the fusion protein. Regardless of the exact cause, it does not affect the main conclusion that dnTR can compete with endogenous TR for binding to TREs). It is unclear why the T₃ treatment enhanced the binding of TR to the TH/bZip promoter in the intestines but not the tails. However, this has no impact on our conclusion regarding dnTR binding to endogenous promoters. The INPUT control shows the DNA level prior to immunoprecipitation with the antibodies.