Figure 2.
Stage-dependent effects of T3 and TSA on transcription, DNA binding by TRs, and histone H4 acetylation at T3 response genes. (A) Premetamorphic tadpoles but not embryos are competent to respond to T3 treatment. Stage 20 embryos and stage 47 tadpoles were treated with 100 nM T3 or 100 nM TSA for 24 h. Total RNA was extracted from whole animals and used for PCR analysis of TRα, TRβ, and TH/bZIP expression. The expression of ribosomal protein gene Rpl8 was used as an internal control. Note that only TRβ and TH/bZIP genes are direct TH response genes, and they were induced only in tadpoles but not in embryos, which had little TR. (B) TR binding to TREs of T3 response genes was not affected by T3 or TSA treatment. Chromatin from animals treated as in A was immunoprecipitated with antibody against TR and analyzed by PCR for the presence of immunoprecipitated TRE-containing fragments. Aliquots of the chromatin before immunoprecipitation were used directly for PCR as control (input). (C) Histone H4 acetylation levels of the chromatin containing the TREs of the TH response genes are up-regulated between embryos and tadpoles by TSA but not T3 treatment. Chromatin isolated as above was immunoprecipitated with antibody against acetylated histone H4 and analyzed by PCR as above. As the same chromatin samples were used for TR and H4 ChIP assays, the input control was the same as shown in B. Note it is unclear why the intensity in the + T3 lane at stage 47 was slightly lower than the control, although the input intensity was also lower in the + T3 lane (see B). However, as we observed little correlation of histone acetylation with gene expression in whole animals, the result did not affect our conclusions. The figure represents one of two independent experiments with identical results.