Verification of microarray data by qRT-PCR. Primer sets for indicated genes were used for qRT-PCR analysis with independently isolated total RNA from control, T3-treated, CHX-treated, and T3 plus CHX-treated X. laevis tadpoles (n = 4 in each treatment group). A, three previously known T3 direct-regulated genes, one late T3-down-regulated gene (IFABP), and one non-regulated gene (HOXA1) were analyzed by TaqMan qRT-PCR with rpL8 (ribosomal protein L8) as the control gene (non-regulated). Note that IFABP was down-regulated by T3 alone but not by T3 plus CHX. B, four newly identified T3 direct response genes were analyzed by SYBR Green qRT-PCR with EF1α (elongation factor 1α) as the control gene (non-regulated). For all genes, the signal was normalized with the control gene and the mean of the control group was set to 1. Student's t test was carried out between pairs of control and T3-treated groups and pairs of CHX-treated and CHX plus T3-treated groups, and all pairwise comparisons had p values <0.05.

The binding of TR/RXR heterodimers to the TREs identified in the direct response genes. A, schematic diagram of the putative TREs in the cyclin J and REV1 promoter regions (−2000 to +2000 bp around the transcriptional start sites). The TREs are shown as up-side-down triangles with arrows indicating the orientation of each TRE. Each confirmed TRE is shown with a box drawn around it. B, the sequences of the putative TREs are shown with the positions (relative to the transcriptional start sites). The confirmed TREs are in bold letters. C and D, in vitro recognition of the putative TREs by TR/RXR heterodimers. The binding activity of cyclin J (CycJ) and REV1 TREs to in vitro translated TRα/RXRα heterodimers was analyzed by gel retardation assay to determine their ability to compete away the formation of the complex between the end-labeled X. laevis TRβ promoter TRE and in vitro translated TRα/RXRα. The competition was done with 4-, 20-, or 100-fold of indicated, unlabeled competitor TRE oligonucleotides: the TRβ TRE itself, mutated TRβ TRE (TRβ mTRE), or the putative TREs from the CycJ promoter region (C) or REV1 promoter region (D). The resulting binding mixture was analyzed on native polyacrylamide gels.

TR regulates the T3-direct response genes in vivo. A, TR/RXR heterodimer activates cyclin J, collagenase-3, and deiodinase-3 promoters in the presence of T3. The indicated promoter constructs for firefly luciferase were co-injected with the internal control plasmid phRG-TK driving the expression of Renilla luciferase into the nuclei of the oocytes with or without prior microinjection of mRNAs for TRα and RXRα (TR/RXR) into the cytoplasm. The oocytes were incubated at 18 °C overnight in the presence or absence of 100 nm T3 and then subjected to dual luciferase assays. The normalized firefly luciferase activities of the reporters were plotted. The bars represent the means ± S.E. of one independent experiment performed in quintuple (p < 0.05 was found for all samples); the experiment was performed three times with similar results. B, TR is bound to the TREs of the newly identified target genes in tadpoles. Chromatin was isolated from three whole premetamorphic tadpoles treated in either the presence or absence of 10 nm T3 for 48 h and used in the ChIP assay. Nine TRE regions in four T3-responsive promoters (cyclin J, REV1, collagenase-3, and deiodinase-3) and a control DNA region, REV1 exon 5, were analyzed for TR binding after immunoprecipitation with an anti-TR antibody (new PB-1) by using quantitative PCR with the eluted DNA. The data are represented as percentage of input chromatin. The bars represent mean ± S.E. from two to three replicate experiments performed. Statistically significant (p < 0.05) increases in TR binding in response to T3 at some TREs are marked by an asterisk. Note that all TRE regions have statistically significant enrichment (p value < 0.05) compared with REV1 exon 5, although the ChIP signal varied considerably among different TREs (see text for more details).

Venn diagrams showing that only a small fraction of genes are commonly regulated by T3 or T3 in the presence of CHX. A, 311 genes were up-regulated by T3 alone (T3 group compared with control group), whereas 188 were up-regulated by T3 in the presence of CHX (T3 plus CHX group compared with CHX group). Among them, 71 genes were commonly up-regulated by T3 or T3 plus CHX. B, 162 genes were down-regulated by T3 alone (T3 group compared with control group), whereas 249 were down-regulated by T3 in the presence of CHX (T3 plus CHX group compared with CHX group). Among them, 29 genes were commonly down-regulated by T3 or T3 plus CHX.

Conservation of the T3-regulation of newly identified genes in X. tropicalis. Selected up-regulated genes were analyzed by SYBR Green qRT-PCR in X. tropicalis tadpoles. Primers sets of four selected T3 direct response genes (same genes as in Fig. 3B) were used to analyze the expression of the genes in total RNA from control, T3-treated, CHX-treated, and T3 plus CHX-treated X. tropicalis tadpole samples (n = 4 in each treatment group). EF1α (elongation factor 1α) was also analyzed as the control gene (non-regulated). For all genes, the signal was normalized with the control gene and the mean of the control group was set to 1. Student's t test was carried out between pairs of control and T3-treated groups, and pairs of CHX-treated and CHX+T3-treated groups and all pairwise comparisons had p values <0.05.

A consensus TRE for Xenopus TR target genes. A, sequences of the 17 new TREs identified in X. tropicalis genes from this study along with the five known TREs for X. laevis genes (the bottom five). B, graphic representation of the consensus TRE generated from the 22 TREs above. The positions of residues in the TRE are shown on the horizontal axis, and the relative frequencies of the four bases at each position are shown in color as bits in the vertical axis. C, the frequencies of the four bases at each position in the 22 Xenopus TREs. The consensus TRE is shown at the bottom.