Targeted chromatin binding and histone acetylation in vivo by thyroid hormone receptor during amphibian development

Abstract

Amphibian metamorphosis is marked by dramatic, thyroid hormone (TH)-induced changes involving gene regulation by TH receptor (TR). It has been postulated that TR-mediated gene regulation involves chromatin remodeling. In the absence of ligand, TR can repress gene expression by recruiting a histone deacetylase complex, whereas liganded TR recruits a histone acetylase complex for gene activation. Earlier studies have led us to propose a dual function model for TR during development. In premetamorphic tadpoles, unliganded TR represses transcription involving histone deacetylation. During metamorphosis, endogenous TH allows TR to activate gene expression through histone acetylation. Here using chromatin immunoprecipitation assay, we directly demonstrate TR binding to TH response genes constitutively in vivo in premetamorphic tadpoles. We further show that TH treatment leads to histone deacetylase release from TH response gene promoters. Interestingly, in whole animals, changes in histone acetylation show little correlation with the expression of TH response genes. On the other hand, in the intestine and tail, where TH response genes are known to be up-regulated more dramatically by TH than in most other organs, we demonstrate that TH treatment induces gene activation and histone H4 acetylation. These data argue for a role of histone acetylation in transcriptional regulation by TRs during amphibian development in some tissues, whereas in others changes in histone acetylation levels may play no or only a minor role, supporting the existence of important alternative mechanisms in gene regulation by TR.

Figure 1

Up-regulation of TRα genes during late embryogenesis correlates with TR and RXR binding to TR target genes. (A) TRα mRNA expression increases between embryonic and tadpole stages. Total RNA was isolated from stage 20 embryos and stage 47 tadpoles and used for PCR analysis of TRα and TRβ mRNA levels. PCR products (10 μl) were electrophoresed on 2% agarose gels, and the gels were stained with ethidium bromide. The expression of ribosomal protein gene Rpl8 was used as an internal control. (B) TR and RXR binding to T₃ response gene promoters increases between embryonic and tadpole stages. Chromatin from stage 20 embryo and stage 47 tadpole nuclei was immunoprecipitated with antibodies against TR or RXR and analyzed by PCR for the presence of the fragments containing the TREs of the two T₃ response genes. Aliquots of the chromatin before immunoprecipitation were used directly for PCR as control (input). The figure represents one of three independent experiments with different batch of animals, all yielding same results.

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.

Figure 3

T₃ treatment leads to the release of histone deacetylase Rpd3 from TH response gene promoters. Stage 47 tadpole nuclei were isolated after 100 nM T₃ treatment for 24 h. Chromatin was immunoprecipitated with antibody against histone deacetylase Rpd3 and analyzed by PCR as in Fig. 2. The data represents one out of several independent experiments with identical result.

Figure 4

T₃ and TSA induce transcription of T₃ response genes without altering TR/RXR binding to TRE in premetamorphic tadpoles. (A) Differential effects of T₃ and TSA on gene expression. Stage 55 tadpoles were treated for 2 days with T₃ (10 nM) or TSA (100 nM). Total RNA was extracted from whole animals, intestine, or tail tissues and used for PCR analysis of TRβ and TH/bZIP expression. The expression of ribosomal protein gene Rpl8 was used as an internal control. Note that T₃ treatment increased mRNA levels of T₃ response genes in whole animals, intestine, and tail, whereas TSA treatment altered T₃ response gene expression only in intestine and tail. (B) TR/RXR binds to TREs in chromatin constitutively. Chromatin isolated from whole tadpoles, the intestine, or the tail of the T₃- or TSA-treated stage 55 tadpoles (A) was immunoprecipitated with antibodies against TR or RXR and analyzed by PCR as in Fig. 2. The figure represents one of two independent experiments with identical results.

Figure 5

T₃ treatment increases histone H4 acetylation specifically at the TRE regions of T₃ response genes in premetamorphic tadpole intestine and tail. (A) Organ-specific changes in histone acetylation by T₃ and TSA. Stage 55 tadpoles were treated for 2 days with T₃ (10 nM) or TSA (100 nM). Nuclei extract from whole tadpole, intestine, or tail were used for ChIP assay by using an antibody against acetylated histone H4. Aliquots of the chromatin before immunoprecipitation were used directly in PCR as a DNA control (input). (B) TSA but not T₃ increases overall histone acetylation level in the intestine. Nuclear proteins (20 μg) from intestine of the above animals were loaded on a Tris-glycine 18% acrylamide gel (Novex). Proteins were transferred to a poly(vinylidene difluoride) Immobilon-P membrane (Millipore), and rabbit anti-acetyl-lysine polyclonal antibody (Upstate Biotechnology) was used to analyze core histone acetylation states. (C) T₃ and TSA treatment has no effects on histone H4 acetylation in the transcribed region of TRβ gene far from the promoter in the intestine. ChIP assay was performed as in A for an internal region of TRβ gene instead of the promoter region. (D) Histone H4 acetylation levels at the promoter of IFABP gene, which is not a directly T₃ response gene, in the intestine, tail, and whole tadpole (WT). Note that histone acetylation of the promoter could be detected in the intestine or whole tadpoles but not in the tail, where the IFABP gene is not expressed. TSA but not T₃ treatment increased slightly histone H4 acetylation level at the promoter of IFABP gene in the intestine. All experiments were done at least twice.