Sato Y et al. (2007), A role of unliganded thyroid hormone receptor i...

XB-ART-35883

Mech Dev 2007 Jul 01;1246:476-88. doi: 10.1016/j.mod.2007.03.006.

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A role of unliganded thyroid hormone receptor in postembryonic development in Xenopus laevis.

Sato Y , Buchholz DR , Paul BD , Shi YB .

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A fascinating feature of thyroid hormone (T3) receptors (TR) is that they constitutively bind to promoter regions of T3-response genes, providing dual functions. In the presence of T3, TR activates T3-inducible genes, while unliganded TR represses these same genes. Although this dual function model is well demonstrated at the molecular level, few studies have addressed the presence or the role of unliganded TR-induced repression in physiological settings. Here, we analyze the role of unliganded TR in Xenopus laevis development. The total dependence of amphibian metamorphosis upon T3 provides us a valuable opportunity for studying TR function in vivo. First, we designed a dominant negative form of TR-binding corepressor N-CoR (dnN-CoR) consisting of its receptor interacting domain. We confirmed its dominant negative activity by showing that dnN-CoR competes away the binding of endogenous N-CoR to unliganded TR and relieves unliganded TR-induced gene repression in frog oocytes. Next, we overexpressed dnN-CoR in tadpoles through transgenesis and analyzed its effect on gene expression and development. Quantitative RT-PCR revealed significant derepression of T3-response genes in transgenic animals. In addition, transgenic tadpoles developed faster than wild type siblings, with an acceleration of as much as 7 days out of the 30-day experiment. These data thus provide in vivo evidence for the presence and a role of unliganded TR-induced gene repression in physiological settings and strongly support our earlier model that unliganded TR represses T3-response genes in premetamorphic tadpoles to regulate the progress of development.

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Species referenced: Xenopus laevis
Genes referenced: hdac3 hoxa1 mmp11 myc ncor1 rpl8 shh
GO keywords: metamorphosis [+]
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	Fig. 1. Schematic representation of dominant negative constructs of Xenopus laevis N-CoR. X. laevis N-CoR is a corepressor protein composed of 2498 amino acids. N-CoR can interact with nuclear hormone receptors including TR via the receptor interacting domain (ID) near the C-terminus. The repressor interacting domains (RDs) located in the N-terminal part are required for the recruitment of other corepressors such as TBL1/TBLR1 and HDAC3. The dominant negative forms, dnN-CoRs, used in this study are shown below. The myc-ID monomer comprises the ID (amino acids 1988–2349) fused to an N-terminal peptide containing myc tag and nuclear localizing sequences (NLS). The other dnN-CoR, myc-ID dimer, consists of two direct repeats of the ID separated by a linker sequence.
	Fig. 2. The dnN-CoRs derepress a T3-repsonse promoter by binding to TR and blocking endogenous N-CoR binding in the reconstituted frog oocyte system. (a) The dimeric dnN-CoR relieves reporter gene repression by unliganded TR more effectively than the monomeric dnN-CoR. The mRNAs for FLAG-TRα/RXR and myc-tagged dnN-CoRs (myc-ID dimer or monomer) were injected into the cytoplasm of the frog oocytes. The firefly luciferase reporter vector together with the control Renilla luciferase plasmid was then injected into the nucleus. After overnight incubation with or without T3, the oocytes were lysed and assayed for luciferase activities. The ratio of firefly luciferase activity to Renilla luciferase activity was determined as a measure of the reporter gene transcription level. The result from each group was expressed as a percentage of the basal transcription level that was obtained from the oocytes without TRα/RXR mRNA injection. The experiment was repeated 8 times and the combined results are presented here. The same oocyte samples used in luciferase assay were subjected to Western blotting with anti-myc and anti-FLAG antibodies. Representative results were shown in the lower panels, confirming the expression of dnN-CoRs and FLAG-TRα. Note that both dnN-CoRs relieved the unliganded TR-induced repression, but the derepression was more potent with the myc-ID dimer (left graph, ∗p < 0.05). Neither dnN-CoR affected the liganded-TR induced reporter gene activation (right graph). (b) myc-ID dimer interacts with unliganded TRα in frog oocytes. The mRNAs for FLAG-TRα/RXR and myc-ID dimer were injected into the cytoplasm of oocytes as indicated. After overnight incubation with or without 100 nM T3, the oocytes were lysed and subjected to immunoprecipitation (IP) with anti-FLAG antibody against TR. Pre-IP lysates and IP samples were immunoblotted with anti-FLAG and anti-myc antibodies. Note that myc-ID dimer was co-immunoprecipitated with FLAG-TRα in the sample without T3 treatment (lane 3) but not in the T3-treated sample (lane 4). (c) myc-ID dimer competes with the endogenous N-CoR for binding to unliganded TR. The mRNAs for FLAG-TRα/RXR and myc-ID dimer were injected into the cytoplasm of oocytes as indicated. After overnight incubation with or without 100 nM T3, the oocyte lysates were subjected to IP with anti-FLAG antibody. Pre-IP lysates and IP samples were immunoblotted with anti-FLAG, anti-N-CoR, and anti-myc antibodies. Similar to myc-ID dimer, endogenous N-CoR was co-immunoprecipitated with FLAG-TRα only in the sample without T3 treatment (compare lane 2 and 3 in the second panel). The myc-ID dimer overexpression reduced the amount of endogenous N-CoR co-immunoprecipitated with FLAG-TRα (compare lane 2 and 4 in the second panel). The experiments in Fig. 2 were repeated at least 3 times with similar results.
	Fig. 3. Transgenic analysis of the effects of overexpressing myc-ID dimer on animal development. (a) Schematic representation of the construct used for transgenesis. The heat shock-inducible promoter drives the expression of the myc-ID dimer transgene. The construct also harbors GFP driven by γ-crystallin promoter as a marker to identify transgenic animals. (b) A transgenic (Tg) and wild type (WT) Xenopus laevis tadpole at stage 46. The presence of GFP in the eye (arrows) indicates the presence of the transgene, myc-ID dimer, in the Tg tadpole. The arrowheads indicate the auto-fluorescence, likely due to the yolk remaining in the tadpoles. (c) Experimental scheme using myc-ID dimer transgenic tadpoles. WT and Tg tadpoles at stage 46 (about 10 days old, shortly after feeding begins at stage 45) were heat shocked for 1 h at 33–34 °C on days 1–30. Some of the tadpoles were also treated with 1 nM of T3 for the first 5 days. The developmental stages of the tadpoles were examined every 5 days. Note that the experiment ended after 30 days, when the tadpoles reached stage 55 and plasma thyroid hormones (T3, 3,5,3′-triiodo-l-thyronine, and T4, 3,5,3′,5′-tetraiodo-l-thyronine) become detectable, which would lead to the dissociation of both dnN-CoR and endogenous corepressors from TR.
	Fig. 4. Heat shock induces the expression of the myc-ID dimer transgene. (a) The expression of the myc-ID dimer mRNA is induced in the transgenic tadpoles after heat shock. Transgenic tadpoles were identified by the GFP in the eyes at the end of the treatments. Total RNAs were isolated from individual wild type (WT) and transgenic (Tg) animals before (0) and after 1, 3, 5, 10 days of heat shock with or without T3 treatment. The cDNAs reverse-transcribed from the total RNAs were subjected to PCR using primers specific for the myc-ID dimer or ribosomal protein L8 (rpL8) (Shi and Liang, 1994) as an internal control. Shown here is representative of three independent experiments with similar results. Each band corresponds to one WT or Tg animal heat-shocked for indicated number of days. (b) Immunoprecipitation (IP) analysis confirms the expression of myc-ID dimer protein in the transgenic animals after heat shock treatment. The protein lysates from WT and Tg animals after 30 days of heat shock were immunoprecipitated with anti-myc antibody. IP samples were immunoblotted with anti-myc antibody (upper panel). Pre-IP samples were blotted with β-actin antibody (lower panel) as a loading control.
	Fig. 5. A low level of T3 (1 nM) can activate T3-inducible genes and metamorphosis in early premetamorphic tadpoles. (a) T3 activates various T3-response genes in early premetamorphic tadpoles. Wild type tadpoles at stage 46 were reared in the water with or without 1 nM T3. Total RNA was isolated from individual animals after 0, 1, 3, and 5 days. Each group had four individual animals. The cDNAs reverse-transcribed from the total RNAs were subjected to quantitative PCR. T3-response genes TRβ, stromelysin-3 (ST3), T3-responsive basic leucine zipper transcription factor (TH/bZip), and sonic hedgehog (xhh), were examined. A retinoic acid-response gene (HoxA1) was analyzed as a negative control. The expression level of each gene was normalized to that of rpL8 and expressed in arbitrary units. Note that expression of T3-response genes was significantly upregulated by T3 treatment (∗p < 0.05), whereas HoxA1 was unaffected. The xhh gene is an organ-specific T3-response gene and thus the upregulation was not very dramatic when analyzed in whole animals (Stolow and Shi, 1995). Similar results were obtained from another experiment with an independent batch of animals. (b) T3 accelerates hind limb development in premetamorphic tadpoles. A wild type tadpole reared in (1 nM) T3-containing water for 5 days had larger hind limbs (arrowhead) compared to a control tadpole without T3 treatment.
	Fig. 6. Expression of myc-ID dimer derepresses T3-response genes in premetamorphic tadpoles. Wild type (WT) and myc-ID dimer transgenic (Tg) tadpoles at stage 46 were heat shocked for 1 h per day. Total RNA was isolated from individual animals before (day 0) and after 1 and 5 days of heat shock treatment. Each group had four individuals. The cDNAs reverse-transcribed from the total RNAs were subjected to qPCR analysis as in Fig. 5. White bars represent expression levels in WT animals and black bars represent those in Tg tadpoles. Note that expression of T3-response genes (TRβ, ST3, TH/bZip, and xhh) in Tg animals tended to be higher than WT counterparts at most of time points (∗p < 0.05), whereas such tendency was not seen in HoxA1 (slightly lower at one time point), a retinoic acid-response gene. Similar results were obtained from another experiment with an independent batch of animals.
	Fig. 7. Transgenic expression of myc-ID dimer accelerates tadpole development. (a) Wild type (WT) and myc-ID dimer transgenic (Tg) sibling tadpoles were heat shocked every day starting from stage 46. Developmental stages of 10 WT and 10 Tg tadpoles were examined every 5 days. The average developmental stages of the animals at different time points were plotted (also see Table 2). Note that stages of Tg animals were significantly more advanced after 20 days of heat shock (∗p < 0.05). At the end of the experiments, the transgenic animals accelerated their development by about 1 stage compared to the wild type siblings. Similar acceleration was observed in another experiment with an independent batch of animals. (b) Heat shocked Tg tadpoles develop to more advanced stages. Representative WT and Tg tadpoles are shown after 25 days of heat shock treatment. The right panel is a magnified image of the boxed area in the left panel, showing that limb buds of a Tg animal were larger and more advanced (around early stage 53) than those of a WT sibling (around early stage 52) (arrowheads).

References [+] :

Amano, Thyroid hormone regulation of a transcriptional coactivator in Xenopus laevis: implication for a role in postembryonic tissue remodeling. 2002, Pubmed, Xenbase