XB-ART-35883Mech Dev July 1, 2007; 124 (6): 476-88.
A role of unliganded thyroid hormone receptor in postembryonic development in Xenopus laevis.
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.
PubMed ID: 17482434
PMC ID: PMC1973152
Article link: Mech Dev
Genes referenced: hdac3 hoxa1 mmp11 myc ncor1 rpl8 shh
GO keywords: metamorphosis
Antibodies: Ncor1 Ab1
Article Images: [+] show captions
|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. 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.|