Figure 5.

TRs are bound to target genes during T₃-induced X. tropicalis metamorphosis. A, RT-PCR analysis shows that T₃ induces the expression of X. tropicalis TH/bZIP gene. The RNA samples in Fig. 2 were analyzed for the expression of TH/bZIP. Again, the expression of Ef1α gene was analyzed in the same PCR as an internal control. Note that X. tropicalis TH/bZIP was induced by T₃ ubiquitously, just like the X. laevis TH/bZIP gene. The experiment was repeated twice with similar results. B, The TRE regions of the T₃-target genes TRβ and TH/bZIP are conserved between X. laevis and X. tropicalis. The sequences flanking the TRE regions used for quantitative PCR analysis of ChIP DNA from the intestine were aligned between the X. tropicalis TRβ (genomic scaffold_26:1899211–1899280) and X. laevis TRβ (38) (top panel) and between X. tropicalis TH/bZIP (genomic scaffold_448:1011455–1011524) and X. laevis TH/bZIP (37) (bottom panel), respectively. Note that there are 96 and 100% identities for TRβ and TH/bZIP genes, respectively. The TREs are shown in bold letters and the primers/probe for qPCR analysis are also indicated. C, ChIP assays shows that TRs are bound to endogenous target genes TRβ and TH/bZIP in the intestine of X. tropicalis. Tadpoles at stage 54 were treated with or without T₃ for 2 d, and the intestine was isolated for ChIP assay with the anti-TR (new PB), anti-TR (PB), or anti-Id14 (extracellular protein, as a negative control) antibody. The precipitated DNA was analyzed by quantitative PCR for the presence of the TRE regions of the TRβ and TH/bZIP promoters. A region of exon 5 of TRβ gene was also analyzed as a negative control. Note that TR was bound to both promoters in the intestine of stage 54 tadpoles, and T₃ treatment led to enhanced binding, whereas only background signal was detected for exon 5.