Steroid-receptor coactivator complexes in thyroid hormone-regulation of Xenopus metamorphosis

Abstract

Anuran metamorphosis is perhaps the most drastic developmental change regulated by thyroid hormone (T3) in vertebrate. It mimics the postembryonic development in mammals when many organs/tissues mature into adult forms and plasma T3 level peaks. T3 functions by regulating target gene transcription through T3 receptors (TRs), which can recruit corepressor or coactivator complexes to target genes in the absence or presence of T3, respectively. By using molecular and genetic approaches, we and others have investigated the role of corepressor or coactivator complexes in TR function during the development of two highly related anuran species, the pseudo-tetraploid Xenopus laevis and diploid Xenopus tropicalis. Here we will review some of these studies that demonstrate a critical role of coactivator complexes, particularly those containing steroid receptor coactivator (SRC) 3, in regulating metamorphic rate and ensuring the completion of metamorphosis.

Keywords: Chromatin remodeling; Histone modification; Intestine; Stem cell; Thyroid hormone receptor; Xenopus laevis; Xenopus tropicalis.

Fig. 1

T3 and postembryonic development. (A) Plasma T3 levels peak during postembryonic development in human and anuran metamorphosis. Postembryonic development in mammals refers to the perinatal period when many organs mature into their adult forms, i.e., about 4 months before to several months after birth in human (Tata, 1993). This period corresponds to metamorphosis in anurans such as Xenopus laevis (Leloup & Buscaglia, 1977; Nieuwkoop & Faber, 1965). (B) Xenopus metamorphosis involves three major types of transformations: de novo development of adult organs such as limbs, complete degeneration of larval organs such as the tail, and remodeling of organs that are present in both tadpoles and frogs such as the intestine. Note that at least some T3-induced apoptosis occurs in these examples of all three types. EP, epithelium; CT, connective tissue; MU, muscle.

Fig. 2

Dual function of TR during Xenopus metamorphosis. TR can repress and activate T3-inducible genes in the absence and presence of T3, respectively. The presence of little or no T3 in premetamorphic tadpoles enables most or all TRs remaining in the unliganded state and TR/RXR heterodimers recruit HDAC-containing corepressors complexes to target genes. This in turn reduces the levels of activation histone marks and increases the levels of repression histone marks, resulting in gene repression. With rising levels of T3 during metamorphosis, TRs become T3-bound and TR/RXR heterodimers recruit coactivator complexes. This then causes chromatin remodeling, including the loss of 2–3 nucleosomes around the TRE and histone modifications, particularly an increase in activation histone marks and a decrease in repression histone marks, and eventually transcriptional activation. N-CoR, nuclear corepressor; HDAC, histone deacetylase; SRC3, steroid receptor coactivator 3; p300, a histone acetyltransferase; PRMT1, protein arginine methyltransferase 1.

Fig. 3

Transgenic overexpression of a Flag-tagged dominant negative steroid receptor coactivator 3 (F-dnSRC3) inhibits metamorphosis. (A) A schematic representation of the full-length SRC3. bHLH/PAS, basic helix–loop–helix and PAS dimerization domains; RID, receptor interaction domain; CID, CBP/p300 interaction domain. The LXXLL motifs present in the protein are numbered from i to vi. A glutamine (Q)-rich region is present toward the C-terminal end of the protein. The Flag-tagged dominant negative form (F-dnSRC3) used in transgenesis is shown below the full length SRC3. It covers aa 600 to aa 751, which comprise the LXXLL motifs i to iii, forming the RID and fused to an N-terminal peptide containing the Flag tag and nuclear localization sequences. (B) Transgenic expression of F-dnSRC3 does not affect T3-induced release of corepressor SMRT but inhibits the recruitment of endogenous wild type SRC3 to the T3-regulated TH/bZIP promoter, thus reducing histone acetylation. Wild type (WT) and transgenic (Tg) animals at stage 54 were treated with 10 nM T3 for 2 days. Intestinal nuclei were isolated and ChIP assays performed by using anti-SRC3 (for endogenous wild type SRC3), anti-acetylated histone H4 (AcH4), anti-SMRT (for endogenous corepressor SMRT), and anti-Flag (for transgenic F-dnSRC3) antibodies. The TRE region of the T3-response gene TH/bZIP was analyzed by real time PCR after immunoprecipitation with the indicated antibodies. Note that F-dnSRC3 was recruited to the promoter upon T3-treatment, competing against the recruitment of endogenous SRC3 recruitment and reducing local AcH4 level in the transgenic animals. (C) F-dnSRC3 inhibits metamorphosis. Wild type (a) and transgenic Xenopus laevis tadpoles (b) at stage 54 after treatment with 5 nM T3 for 3 days, with the resorption of gills and development of the Meckel’s cartilage (leading to protrusion of the jaw) in the wild type but not transgenic tadpole after T3 treatment. (c) A transgenic tadpole which failed to undergo tail resorption during natural development and eventually died. See Paul, Fu, et al. (2005) for more details.

Fig. 4

SRC3 knockout reduces histone H4 acetylation and inhibits T3-induced metamorphosis. (A)/(B) Western blots showing that homozygous SRC3 knockout tadpoles have no detectable SRC3 protein and reduced histone H4 acetylation. Total protein was isolated from whole body of wild type (WT), heterozygous (SRC3^+/−), and compound heterozygous (functionally homozygous, SRC3^−/−) SRC3 mutant tadpoles at stage 46 and subjected to Western blot analyses with antibodies against SRC3, acetyl-histone H4, or histone H4 (A). The signal intensity of the bands was measured by image j and presented as the mean ± standard deviation of n = 3 (B). Statistical analysis: ANOVA one-way followed by Tukey analysis. *P <0.05. (C) SRC3 knockout reduces gene activation by T3 in the intestine. The intestine was isolated from wild type (WT), SRC3^+/− and SRC3^−/− tadpoles at stage 54 with or without 18 h T3 treatment before RNA isolation and qRT-PCR analysis of the indicated TR target genes. **P < 0.01 for T3 treated vs. WT intestine. (D) SRC knockout inhibits T3-induced limb metamorphosis. Wild type (WT) and SRC3 knockout (SRC3^−/−) tadpoles at stage 54 were treated with 10 nM T3 for 3 days at 25°C and hindlimb region was photographed. The lower panels were the same as the top panels except the added white line marking the approximate boundary of the outer limb epithelium and/or the boundaries of the digits (white arrowheads indicate some of the digits of the wild type animal treated with T3). (E) Adult intestinal epithelium development is inhibited by SRC3 knockout. Cross-sections of the intestine in WT and SRC3^−/− tadpoles as in (D) were stained with methyl green-pyronin Y (staining DNA blue and RNA red). Note that the adult epithelium folds began to form after 3 days of T3 treatment in WT but not SRC3^−/− tadpoles. See Tanizaki, Bao, et al. (2021) for more details.