Liganded thyroid hormone receptor induces nucleosome removal and histone modifications to activate transcription during larval intestinal cell death and adult stem cell development

Abstract

Thyroid hormone (T(3)) plays an important role in regulating multiple cellular and metabolic processes, including cell proliferation, cell death, and energy metabolism, in vertebrates. Dysregulation of T(3) signaling results in developmental abnormalities, metabolic defects, and even cancer. We used T(3)-dependent Xenopus metamorphosis as a model to study how T(3) regulates transcription during vertebrate development. T(3) exerts its metamorphic effects through T(3) receptors (TR). TR recruits, in a T(3)-dependent manner, cofactor complexes that can carry out chromatin remodeling/histone modifications. Whether and how histone modifications change upon gene regulation by TR during vertebrate development is largely unknown. Here we analyzed histone modifications at T(3) target genes during intestinal metamorphosis, a process that involves essentially total apoptotic degeneration of the simple larval epithelium and de novo development of the adult epithelial stem cells, followed by their proliferation and differentiation into the complex adult epithelium. We demonstrated for the first time in vivo during vertebrate development that TR induces the removal of core histones at the promoter region and the recruitment of RNA polymerase. Furthermore, a number of histone activation and repression marks have been defined based on correlations with mRNA levels in cell cultures. Most but not all correlate with gene expression induced by liganded TR during development, suggesting that tissue and developmental context influences the roles of histone modifications in gene regulation. Our findings provide important mechanistic insights on how chromatin remodeling affects developmental gene regulation in vivo.

Fig. 1.

Developmental expression profiles and T₃ regulation of TRβ, TH/bZIP, and IFABP in the intestine during natural and T₃-induced metamorphosis of X. tropicalis. A and B, The expression of two known direct TR target genes TRβ and TH/bZIP, and an indirectly repressed gene IFABP were analyzed during natural (A) and T₃-induced (B) metamorphosis by qRT-PCR. EF1α was also analyzed as the control gene (nonregulated) and used to normalize the expression of the other genes. Note that the levels of TRβ and TH/bZIP mRNA were gradually up-regulated during natural development and reached a peak at stage 60 and 62, respectively (A). The expression of mRNA was then reduced by stage 66, the end of metamorphosis (A). On the other hand, the level of IFABP mRNA was down-regulated at stage 62, at the climax of metamorphosis. After 2 d of T₃ treatment of premetamorphic tadpoles at stage 54, the expression of TRβ and TH/bZIP mRNA were up-regulated, whereas that of IFABP was down-regulated (B), just like during natural metamorphosis. Error bars indicate sem. C, Schematics of the promoters of X. tropicalis TRβ and TH/bZIP genes showing the locations of the TRE relative to the transcription start site (+1). Note that the positions were based on those for the corresponding X. laevis genes (52, 68) because the exact start sites of the X. tropicalis genes have not been mapped but the TRE regions are conserved (7). The arrows indicate the relative locations of the PCR primers used for the analysis of the ChIP DNA.

Fig. 2.

TR binding to target genes leads to the recruitment of RNA Pol II and loss of core histones during T₃-induced metamorphosis. 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 (A), anti-Pol II (B), anti-H3 (C), anti-H2B (D), or anti-Id14 (extracellular protein, as a negative control) (E) antibody. The immunoprecipitated DNA was analyzed by qPCR for the presence of the TRE regions of the TRβ and TH/bZIP promoters. A region of exon 5 of the TRβ gene was analyzed as a negative control. The promoter region of the IFABP gene was also analyzed as a non-TR direct target gene control. Note that TR bound to both genes in the absence of T₃ in premetamorphic tadpoles. In the presence of T₃, TR binding to the TRE was increased, accompanied by the recruitment Pol II and reduction in histones at the TRE regions of both TH/bZIP and TRβ promoters. There was no TR binding to exon 5 in the presence or absence of T₃. However, increased Pol II was observed in the exon region due to increased transcription in the presence of T₃. Only background signals were observed with the anti-ID14 antibody. Error bars indicate sem (n = 3). Student's t test was carried out between pairs of control and T₃-treated groups. The one and two stars indicate pairs of samples with significant differences (P < 0.05 and P < 0.01, respectively).

Fig. 3.

Histone modifications associated with gene activation are increased at TR-target genes during T₃-induced metamorphosis. Premetamorphic tadpoles at stage 54 were treated with T₃ for 2 d. The intestine was isolated and subjected to a ChIP assay with anti-H3K4me3 (A), anti-H3K79me3 (B), or anti-H3R17me2a (C), anti-AcH3 (D), or anti-AcH4 antibody (E). ChIP signals were normalized with the ChIP signals of histone H3 in Fig. 2 for the corresponding promoter/exon regions. Error bars indicate sem (n = 3). A Student's t test was carried out between pairs of control and T₃-treated groups. The one and two stars indicate pairs of samples with significant differences (P < 0.05 and P < 0.01, respectively).

Fig. 4.

Of the two histone marks associated with gene silencing, H3K9me3 and H3K27me3, only H3K27me3 was reduced upon gene activation by T₃ during metamorphosis. Premetamorphic tadpoles at stage 54 were treated with T₃ for 2 d. The intestine was isolated and subjected to a ChIP assay with anti-H3K27me3 (A) or anti-H3K9me3 antibody (B). ChIP signals were normalized with the ChIP signals of histone H3 in Fig. 2 for the corresponding promoter/exon regions. Note that surprisingly, the amount of H3K9me3 was increased in the downstream transcribed region of the TRβ gene (exon 5) upon gene activation by T₃. Error bars indicate sem (n = 3). A Student's t test was carried out between pairs of control and T₃-treated groups. The one and two stars indicate pairs of samples with significant differences (P < 0.05 and P < 0.01, respectively).

Fig. 5.

TR binding to target genes increases during natural metamorphosis and is accompanied by increased recruitment of RNA Pol II and reduced core histones at the TRE. The intestine was isolated from tadpoles at premetamorphosis (stage 54), early metamorphic climax (stage 58), metamorphic climax (stage 60–62), and the end of metamorphosis (stage 66) and subjected to ChIP assay with the anti-TR (A), anti-Pol II (B), anti-H3 (C), anti-H2B (D), or anti-Id14 (extracellular protein, as a negative control) (E) antibody. The ChIP DNA was analyzed as in Fig. 2. Note that TR binding to the TRE was higher during metamorphosis (stages 58–62), accompanied by increased Pol II and reduced amounts of core histones at TRE of both TR-target promoters at stages 58–62. Only background signals were observed with the anti-ID14 antibody. Error bars indicate sem.

Fig. 6.

Histone modifications associated with gene activation are increased at TR-target genes during natural metamorphosis. The ChIP assay was performed as in Fig. 5 on intestines isolated from tadpoles at the indicated stages with anti-H3K4me3 (A), anti-H3K79me3 (B), anti-H3R17me2a (C), anti-AcH3 (D), or anti-AcH4 antibody (E). ChIP signals were normalized with the ChIP signals of histone H3 in Fig. 5 for the corresponding promoter/exon regions. Note that in general, these activation histone marks were increased at the two promoters during metamorphosis. Error bars indicate sem.

Fig. 7.

A and B, Of the two histone marks associated with gene silencing, H3K9me3 and H3K27me3, only K3K27me3 was reduced upon gene activation during natural metamorphosis. The ChIP assay was performed as in Fig. 5 on intestines isolated from tadpoles at the indicated stages during metamorphosis with anti-H3K27me3 (A) or anti-H3K9me3 antibody (B). ChIP signals were normalized with the ChIP signals of histone H3 in Fig. 5 for the corresponding promoter/exon regions. Note that surprisingly but in agreement with T₃-induced metamorphosis, the amount of H3K9me3 was increased in the downstream transcribed region of TRβ gene (exon 5) upon gene activation during natural metamorphosis (stages 58–62). Error bars indicate sem. C, A model for nucleosome removal and histone modifications during gene activation by liganded TR. In the absence of T₃, TR recruits corepressor complexes containing histone deacetylases, which results in a repressed chromatin state (dashed arrow) with many repression histone marks such as H3K27me3 (triangles). The presence of T₃ induces a conformational change in TR, leading to the recruitment of coactivator complexes containing chromatin remodeling and histone modification enzymes. This results in nucleosomal removal at the promoter region, a reduction in repression histone marks, an increase in activation histone marks such as H3K4me3, H3K7me3, R17me2a, AcH3, and AcH4 (circles), and gene activation (thick arrow).