Dual functions of thyroid hormone receptors in vertebrate development: the roles of histone-modifying cofactor complexes

Abstract

Thyroid hormone (TH) receptor (TR) plays critical roles in vertebrate development. Transcription studies have shown that TR activates or represses TH-inducible genes by recruiting coactivators or corepressors in the presence or absence of TH, respectively. However, the developmental roles of these TR cofactors remain largely unexplored. Frog metamorphosis is totally dependent on TH and mimics the postembryonic period in mammalian development during which TH levels are also high. We have previously proposed a dual function model for TR in the development of the anuran Xenopus laevis. That is, unliganded TR recruits corepressors to TH-inducible genes in premetamorphic tadpoles to repress these genes and prevent premature metamorphic changes and subsequently, when TH becomes available, liganded TR recruits coactivators to activate these same genes, leading to metamorphosis. Over the years, we and others have used molecular and genetic approaches to demonstrate the importance of the dual functions of TR in Xenopus laevis. In particular, unliganded TR has been shown to recruit histone deacetylase-containing corepressor complexes in premetamorphic tadpoles to control metamorphic timing. In contrast, metamorphosis requires TH-bound TR to recruit coactivator complexes containing histone acetyltransferases and methyltransferases to activate transcription. Furthermore, the concentrations of coactivators appear to regulate the rate of metamorphic progression. Studies in mammals also suggest that the dual function model for TR is conserved across vertebrates.

FIG. 1.

(A) Mechanisms of transcriptional regulation by thyroid hormone (TH) receptor (TR). For TH-inducible genes, TR heterodimerized with 9-cis-retinoic acid receptors (RXR) constitutively binds the TH response elements (TREs) in their promoters or enhancers. In the absence of TH, TR binds corepressor complexes, such as those containing histone deacetylase HDAC3 and the highly related protein N-CoR (nuclear corepressor) or SMRT (silencing mediator of retinoid and thyroid hormone receptors) to inhibit transcription from the promoters through deacetylation of lysine residues of histone H3 and H4 to induce a “closed” chromatin state, as suggested by the folding of histone tails (red beaded structure) on to the DNA helix because of the charge–charge interaction between the positively charged histone tails and negatively charged DNA. The binding by TH induces a conformational change in TR, leading to the binding of coactivator complexes, such as those containing the related coactivators SRC 1, 2, or 3, which in turn binds p300 and PRMT1. SRCs and p300 have histone acetyltransferase (HAT) activity to acetylate histones H3 and H4, and PRMT1 can methylate histone H4. Histone acetylation is believed to facilitate the formation of an “open” chromatin state, as diagramed by the unfolding of histone tails (red beaded structure) away from the DNA helix due to the neutralization of the positive charges on the histone tails by acetylation. There are other corepressors and coactivators that can participate in this process, including those involved in chromatin disruption upon transcriptional activation by TR (27,28,37). For simplicity, these are not shown here. (B) A dual function model of TR in frog development. TH-inducible genes are expressed at the basal levels during embryogenesis when the levels of TH and TRs are low. After tadpole feeding begins at stage 45, the increased expression of TRα and RXRs, e.g., RXRα as shown in the figure, leads to the formation of TR-RXR heterodimers that bind to the TREs in the target genes and recruit corepressor complexes to repress gene expression. After stage 55, the synthesis of endogenous TH leads to the binding of TH to TR, causing the release of the corepressor complexes and recruitment of coactivator complexes to the promoters. This results in the activation of the genes and initiates metamorphosis.

FIG. 2.

TH-dependent recruitment of coactivator SRC3 and histone acetylation at TH-responsive promoters, TRβA and TH/bZIP, in the intestine of tadpoles during natural metamorphosis. (A) Schematic diagrams showing the location of the transcription start site (thick arrow) for the two TH-response genes and the locations of the TREs and the primers (thin arrows) used to analyze the DNA in the ChIP assay (–112). (B) ChIP assays on intestinal samples from premetamorphic (stage 54, when TH level is low) and metamorphosing (stage 62, when endogenous TH is at the peak level) tadpoles. The Input represents DNA prior to immunoprecipitation with antibodies against TR, SRC3, or acetylated histone H4 (AcH4) for the ChIP assay. Note that TR binds to the TREs at both stages while SRC3 recruitment is high when TH level is high at stage 62, accompanied by increased histone acetylation (65).

FIG. 3.

Expression of a TH-independent, constitutively active, dominant positive TR in transgenic tadpoles initiates metamorphosis. (A) Premetamorphic wild type tadpoles and sibling tadpoles transgenic for the dominant positive TR under the control of a heat shock–inducible promoter were reared together in methimazole to block endogenous TH synthesis and were heat-shocked daily for 8 days or treated with 5 nM 3,5,3′-triiodothyronine (T₃) for 3 days. Note that expression of the dominant positive TR in transgenic animals upon heat shock treatment induced the same metamorphic changes, e.g., the gills (white brackets) and the hind limbs (white arrow heads), in the absence of T₃, as those induced by T₃ in wild-type tadpoles. (B) The dominant positive TR regulates gene expression in the animal intestine just like TH treatment in wild type tadpoles. Premetamorphic wild-type and dominant positive TR transgenic tadpoles were heat shocked for 4 days or treated with 5 nM T₃ for 3 days. RT-PCR analysis was carried out on RNA isolated from the intestine for the expression of TH-response genes with the TH-independent gene rpl8 as an internal control. The genes analyzed were TRβ and TH/bZIP, ubiquitously expressed, direct TH response genes; stromelysin-3 (ST3), a fibroblast-specific, direct TH-response gene; sonic hedgehog (xhh), an intestinal epithelium-specific, direct TH-response gene; and bone morphogenic protein (BMP)-4, a fibroblast-specific, late upregulated gene. See Buchholz et al. (76) for more details.

FIG. 3.

Expression of a TH-independent, constitutively active, dominant positive TR in transgenic tadpoles initiates metamorphosis. (A) Premetamorphic wild type tadpoles and sibling tadpoles transgenic for the dominant positive TR under the control of a heat shock–inducible promoter were reared together in methimazole to block endogenous TH synthesis and were heat-shocked daily for 8 days or treated with 5 nM 3,5,3′-triiodothyronine (T₃) for 3 days. Note that expression of the dominant positive TR in transgenic animals upon heat shock treatment induced the same metamorphic changes, e.g., the gills (white brackets) and the hind limbs (white arrow heads), in the absence of T₃, as those induced by T₃ in wild-type tadpoles. (B) The dominant positive TR regulates gene expression in the animal intestine just like TH treatment in wild type tadpoles. Premetamorphic wild-type and dominant positive TR transgenic tadpoles were heat shocked for 4 days or treated with 5 nM T₃ for 3 days. RT-PCR analysis was carried out on RNA isolated from the intestine for the expression of TH-response genes with the TH-independent gene rpl8 as an internal control. The genes analyzed were TRβ and TH/bZIP, ubiquitously expressed, direct TH response genes; stromelysin-3 (ST3), a fibroblast-specific, direct TH-response gene; sonic hedgehog (xhh), an intestinal epithelium-specific, direct TH-response gene; and bone morphogenic protein (BMP)-4, a fibroblast-specific, late upregulated gene. See Buchholz et al. (76) for more details.

FIG. 4.

Transgenic, constitutive expression of a Flag-tagged dominant negative coactivator SRC3, containing the receptor interaction domain of Xenopus laevis SRC3, inhibits natural and TH-induced metamorphosis by competing for recruitment to endogenous target genes. (A) Transgenic dominant negative SRC3 competes with endogenous wild-type SRC3 for binding to liganded TR at TH-regulated promoters in the intestine, leading to reduced histone acetylation, but without an effect on TH-induced corepressor release. Premetamorphic wild type (WT) and transgenic (Tg) animals were treated with 10 nM T₃ for 2 days. Intestinal nuclei were isolated and ChIP assays performed using anti-SRC3 (for endogenous wild-type SRC3), anti-acetylated histone H4 (AcH4), anti-SMRT (for endogenous corepressor SMRT), and anti-Flag (for the dominant negative SRC3) antibodies. The TRE regions of TH/bZIP and TRβA promoters in the ChIP DNA were analyzed by real-time PCR. Note the reduced or absence of endogenous SRC3 recruitment and the corresponding histone acetylation in TH-treated transgenic tadpoles compared to wild type ones. On the other hand, as expected, there was no difference in the release of the corepressor SMRT upon TH treatment. (B) Overexpression of the dominant negative SRC3 inhibits TH-induced metamorphosis. Premetamorphic wild-type and transgenic tadpoles at stage 54 were treated with 5 nM T₃ for 3 days. The characteristic TH-induced changes such as resorption of gills (bracketed) and the morphogenesis of the hind limbs (arrows) were induced in wild-type animals treated with TH. These changes were all inhibited in transgenic animals. (C) Transgenic tadpoles overexpressing the dominant negative SRC3 fail to complete metamorphosis. Left: a wild type tadpole completed metamorphosis about 2 months after fertilization. Right, two transgenic siblings retained their tail and some gills (the top one) even at 4 months of age. See Paul et al. (64) for more details.

FIG. 4.

Transgenic, constitutive expression of a Flag-tagged dominant negative coactivator SRC3, containing the receptor interaction domain of Xenopus laevis SRC3, inhibits natural and TH-induced metamorphosis by competing for recruitment to endogenous target genes. (A) Transgenic dominant negative SRC3 competes with endogenous wild-type SRC3 for binding to liganded TR at TH-regulated promoters in the intestine, leading to reduced histone acetylation, but without an effect on TH-induced corepressor release. Premetamorphic wild type (WT) and transgenic (Tg) animals were treated with 10 nM T₃ for 2 days. Intestinal nuclei were isolated and ChIP assays performed using anti-SRC3 (for endogenous wild-type SRC3), anti-acetylated histone H4 (AcH4), anti-SMRT (for endogenous corepressor SMRT), and anti-Flag (for the dominant negative SRC3) antibodies. The TRE regions of TH/bZIP and TRβA promoters in the ChIP DNA were analyzed by real-time PCR. Note the reduced or absence of endogenous SRC3 recruitment and the corresponding histone acetylation in TH-treated transgenic tadpoles compared to wild type ones. On the other hand, as expected, there was no difference in the release of the corepressor SMRT upon TH treatment. (B) Overexpression of the dominant negative SRC3 inhibits TH-induced metamorphosis. Premetamorphic wild-type and transgenic tadpoles at stage 54 were treated with 5 nM T₃ for 3 days. The characteristic TH-induced changes such as resorption of gills (bracketed) and the morphogenesis of the hind limbs (arrows) were induced in wild-type animals treated with TH. These changes were all inhibited in transgenic animals. (C) Transgenic tadpoles overexpressing the dominant negative SRC3 fail to complete metamorphosis. Left: a wild type tadpole completed metamorphosis about 2 months after fertilization. Right, two transgenic siblings retained their tail and some gills (the top one) even at 4 months of age. See Paul et al. (64) for more details.

FIG. 5.

Cofactors regulate developmental rate and timing. (A) Transgenic tadpoles (Tg) overexpressing PRMT1 under the control of a heat shock-inducible promoter develop to more advanced metamorphic stages than wild-type siblings (Wt) when treated with TH. Wt and Tg animals were heat-shocked for 30 minutes twice daily starting at stage 56. Three days after the first heat-shock treatment, all the animals were treated with 2 nM T₃ and continuously subjected to heat shock treatment daily. The Wt and Tg animals were kept in the same container and analyzed morphologically every 3 days throughout the 15-day experiment. The average developmental stages were plotted. See Matsuda et al. (57) for more details. (B) Tg tadpoles expressing a dominant negative N-CoR under the control of a heat shock-inducible promoter initiate metamorphosis earlier than Wt siblings. Wt and Tg sibling tadpoles were heat-shocked every day starting from stage 46, the onset of tadpole feeding. Developmental stages of the tadpoles were examined every 5 days for 30 days. The average developmental stages were plotted. Note that at the end of the 30-day experiments, Tg animals had initiated metamorphosis (passed stage 55) while Wt ones were one stage behind. It would take Wt animals about 7 more days to begin metamorphosis. See for Sato et al. (93) more details. *p < 0.05 when Tg animals were compared to the Wt ones.