Metamorphic T3-response genes have specific co-regulator requirements

Abstract

Thyroid hormone receptors (TRs) have several regulatory functions in vertebrates. In the absence of thyroid hormone (T3; tri-iodothyronine), apo-TRs associate with co-repressors to repress transcription, whereas in the presence of T3, holo-TRs engage transcriptional coactivators. Although many studies have addressed the molecular mechanisms of T3 action, it is not known how specific physiological responses arise. We used T3-dependent amphibian metamorphosis to analyse how TRs interact with particular co-regulators to differentially regulate gene expression during development. Using chromatin immuno-precipitation to study tissue from pre-metamorphic tadpoles, we found that TRs are physically associated with T3-responsive promoters, whether or not T3 is present. Addition of T3 results in histone H4 acetylation specifically on T3-response genes. Most importantly, we show that individual T3-response genes have distinct co-regulator requirements, the T3-dependent co-repressor-to-coactivator switch being gene-specific for both co-regulator categories.

Figure 1

Effects of T₃ on transcription and DNA binding by thyroid hormone receptor at T₃-response genes in pre-metamorphic stage NF55 tadpole tail. (A) T₃ induces transcription of T₃-response genes. Tadpoles were treated for 48 h with 10 nM T₃. Total RNA was extracted from tail tissue and used for RT–PCR (PCR after reverse transcription) analysis of thyroid hormone receptor b (TRb), TH/bZIP (a basic leucine-zipper TH-response gene), MyoD, intestinal fatty acid binding protein (IFABP), elongation factor 1a (EF1a) and ribosomal protein L8 (Rpl8) expression. The internal control was Rpl8. The results were also quantified by phosphoimager scanning. The average values ± s.e.m. of three independent experiments are expressed as multiples of induction, where 1 is equal to expression in the absence of T₃ (control level). For each sample, densitometry readings were normalized against the value for Rpl8 RNA (except for the Rpl8 data, which were not normalized). Statistical significance as compared with untreated animals is indicated as NS (not significant), ^* (p < 0.05) or ^*** (p < 0.001). (B) T₃ does not affect TR binding to T₃ response elements. Chromatin isolated from tails of T₃-treated tadpoles (10 nM T₃ for 48 h) was immunoprecipitated (IP) with antibodies against TR and analysed by PCR. Aliquots of the chromatin taken before immunoprecipitation were used directly for PCR as a control (input). For TRb promoters, we distinguished two sequences containing T₃REs (sequence 1 at position +266 and sequence 2 at positions −800 to −500). All experiments were carried out at least three times. T₃, thyroid hormone (triiodothyronine).

Figure 2

T₃ treatment increases histone H4 acetylation specifically at the T₃-response elements of T₃-response genes in pre-metamorphic tadpoles. Chromatin isolated from tail or intestine of T₃-treated stage NF55 tadpoles (treated with 10 nM T₃ for 48 h) was immunoprecipitated (IP) with antibodies against acetylated histone H4 (AcH4) and analysed by PCR, as described for Fig. 1. Each experiment was carried out at least twice. EF1a, elongation factor 1a; IFABP, intestinal fatty acid binding protein; T₃, thyroid hormone (triiodothyronine); TH/bZIP, a basic leucine-zipper Th-response gene; TRb, thyroid hormone receptor b.

Figure 3

Effects of T₃ on Rpd3 and NCoR co-repressor expression and recruitment on T₃-response elements of T₃-response genes in pre–metamorphic tadpoles. (A) Rpd3 and NCoR protein levels in tail nuclei are not affected by T₃ treatment. Western blot analysis of protein extracts from the tail nuclei of tadpoles treated with 10 nM T₃ for 48 h. (B) Chromatin isolated from tails of T₃-treated tadpoles (treated with 10 nM T₃ for 48 h) was immunoprecipitated (IP) with antibodies against Rpd3 or NCoR and analysed by PCR, as described for Fig. 1. Pre-immune serum (Pre-I) was used as a control for antibody specificity. The data represent one of several independent experiments with identical results. EF1a, elongation factor 1a; IFABP, intestinal fatty acid binding protein; NCoR, nuclear co-repressor; T₃, thyroid hormone (triiodothyronine); TH/bZIP, a basic leucine-zipper Th-response gene; TRb, thyroid hormone receptor b.

Figure 4

Effects of T₃ on steroid receptor coactivator 3 and p300 coactivator expression and recruitment on T₃-response elements of T₃-response genes in pre-metamorphic tadpoles. (A) Steroid receptor coactivator 3 (SRC3) and p300 protein levels in tail nuclei are not affected by T₃ treatment. Western blot analysis of protein extracts fron tail nuclei of tadpoles treated with 10 nM T₃ for 48 h. (B) Chromatin isolated from tails of T₃-treated tadpoles (treated with 10 nM T₃ for 48 h) was immunoprecipitated (IP) with antibodies against SRC3 or p300 and analysed by PCR, as described for Fig. 1. Pre-immune serum (Pre-I) was used as a control for antibody specificity. All experiments were carried out at least three times. EF1a, elongation factor 1a; IFABP, intestinal fatty acid binding protein; T₃, thyroid hormone (triiodothyronine); TH/bZIP, a basic leucine-zipper Th-response gene; TRb, thyroid hormone receptor b.