Distinct expression profiles of transcriptional coactivators for thyroid hormone receptors during Xenopus laevis metamorphosis

The biological effects of thyroid hormone (T3) are mediated by the thyroid hormone receptor (TR). Amphibian metamorphosis is one of the most dramatic processes that are dependent on T3. T3 regulates a series of orchestrated developmental changes, which ultimately result in the conversion of an aquatic herbivorous tadpole to a terrestrial carnivorous frog. T3 is presumed to bind to TRs, which in turn recruit coactivators, leading to gene activation. The best-studied coactivators belong to the p160 or SRC family. Members of this family include SRC1/NCoA-1, SRC2/TIF2/GRIP1, and SRC3/pCIP/ACTR/AIB-1/RAC-3/TRAM-1. These SRCs interact directly with liganded TR and function as adapter molecules to recruit other coactivators such as p300/CBP. Here, we studied the expression patterns of these coactivators during various stages of development. Amongst the coactivators cloned in Xenopus laevis, SRC3 was found to be dramatically upregulated during natural and T3-induced metamorphosis, and SRC2 and p300 are expressed throughout postembryonic development with little change in their expression levels. These results support the view that these coactivators participate in gene regulation by TR during metamorphosis.     

SRC-p300 coactivator complex is required for thyroid hormone-induced amphibian metamorphosis

Gene activation by the thyroid hormone (T3) receptor (TR) involves the recruitment of specific coactivator complexes to T3-responsive promoters. A large number of coactivators for TR have been isolated and characterized in vitro. However, their roles and functions in vivo during development have remained largely unknown. We have utilized metamorphosis in Xenopus laevis to study the role of these coactivators during post-embryonic development. Metamorphosis is totally dependent on the thyroid hormone, and TR mediates a vast majority, if not all, of the developmental effects of the hormone. We have previously shown that TR recruits the coactivator SRC3 (steroid receptor coactivator-3) and that coactivator recruitment is essential for metamorphosis. To determine whether SRCs are indeed required, we have analyzed the in vivo role of the histone acetyltransferase p300/CREB-binding protein (CBP), which was reported to be a component of the SRC.coactivator complexes. Chromatin immunoprecipitation revealed that p300 is recruited to T3-responsive promoters, implicating a role of p300 in TR function. Further, transgenic tadpoles overexpressing a dominant negative form of p300, F-dnp300, containing only the SRC-interacting domain, displayed arrested or delayed metamorphosis. Molecular analyses of the transgenic F-dnp300 animals showed that F-dnp300 was recruited by TR (displacing endogenous p300) and inhibited the expression of T3-responsive genes. Our results thus suggest that p300 and/or its related CBP is an essential component of the TR-signaling pathway in vivo and support the notion that p300/CBP and SRC proteins are part of the same coactivator complex in vivo during post-embryonic development.     

Tissue- and gene-specific recruitment of steroid receptor coactivator-3 by thyroid hormone receptor during development

Numerous coactivators that bind nuclear hormone receptors have been isolated and characterized in vitro. Relatively few studies have addressed the developmental roles of these cofactors in vivo. By using the total dependence of amphibian metamorphosis on thyroid hormone (T3) as a model, we have investigated the role of steroid receptor coactivator 3 (SRC3) in gene activation by thyroid hormone receptor (TR) in vivo. First, expression analysis showed that SRC3 was expressed in all tadpole organs analyzed. In addition, during natural as well as T3-induced metamorphosis, SRC3 was up-regulated in both the tail and intestine, two organs that undergo extensive transformations during metamorphosis and the focus of the current study. We then performed chromatin immunoprecipitation assays to investigate whether SRC3 is recruited to endogenous T3 target genes in vivo in developing tadpoles. Surprisingly, we found that SRC3 was recruited in a gene- and tissue-dependent manner to target genes by TR, both upon T3 treatment of premetamorphic tadpoles and during natural metamorphosis. In particular, in the tail, SRC3 was not recruited in a T3-dependent manner to the target TRbetaA promoter, suggesting either no recruitment or constitutive association. Finally, by using transgenic tadpoles expressing a dominant negative SRC3 (F-dnSRC3), we demonstrated that F-dnSRC3 was recruited in a T3-dependent manner in both the intestine and tail, blocking the recruitment of endogenous coactivators and histone acetylation. These results suggest that SRC3 is utilized in a gene- and tissue-specific manner by TR during development.     

Transcriptional regulation of the Xenopus laevis Stromelysin-3 gene by thyroid hormone is mediated by a DNA element in the first intron

The matrix metalloproteinase (MMP) stromelysin-3 (ST3) (MMP11) was first isolated as a breast cancer-associated gene and is expressed in diverse human carcinomas and various developmental processes involving apoptosis. The Xenopus laevis ST3 is highly up-regulated by thyroid hormone (T3) during amphibian metamorphosis, and its expression is spatially and temporally correlated with apoptosis in different tissues. Furthermore, it has been shown in vivo and in organ cultures to play a critical role in regulating T3-induced epithelial cell death during intestinal metamorphosis. Earlier studies suggest that ST3 is a direct T3 response gene, although a thyroid hormone response element (TRE) was not found in the initial analysis of the ST3 promoter. Here, we have identified a strong TRE consisting of two nearly perfect direct repeats of the consensus nuclear hormone receptor binding element AGGTCA separated by 4 bp in the first intron of the Xenopus ST3 gene. We show that the heterodimers of T3 receptor (TR) and 9-cis-retinoic acid receptor bind to the TRE both in vitro and in vivo in the context of chromatin. Furthermore, T3 induces strong activation of the promoter through the intronic TRE. Interestingly, although the unliganded TR/9-cis-retinoic acid receptor was able to recruit corepressors to the promoter, it had little repressive effect on the promoter in vivo. These results suggest that the intronic TRE mediates the inductive effect of T3 and that promoter context plays an important role in gene repression by unliganded TR.     

Acetylation of nuclear hormone receptor superfamily members: thyroid hormone causes acetylation of its own receptor by a mitogen-activated protein kinase-dependent mechanism

Because the androgen and estrogen nuclear hormone receptors are subject to acetylation, we speculated that the nuclear thyroid hormone receptor-beta1 (TRbeta1), another superfamily member, was also subject to this posttranslational modification. Treatment of 293T cells that contain TRbeta1(wt) with l-thyroxine (T4)(10(-7)M, total concentration) resulted in the accumulation of acetylated TR in nuclear fractions at 30-45 min and a decrease in signal by 60 min. A similar time course characterized recruitment by TR of p300, a coactivator protein with intrinsic transacetylase activity. Recruitment by the receptor of SRC-1, a TR coactivator that also acetylates nucleoproteins, was also demonstrated. Inhibition of the MAPK (ERK1/2) signal transduction cascade by PD 98059 blocked the acetylation of TR caused by T4. Tetraiodothyroacetic acid (tetrac) decreased T4-induced acetylation of TR. At 10(-7)M, 3,5,3'-triiodo-l-thyronine (T3) was comparably effective to T4 in causing acetylation of TR. We studied acetylation in TR that contained mutations in the DNA-binding domain (DBD) (residues 128-142) that are known to be relevant to recruitment of coactivators and to include the MAPK docking site. In response to T4 treatment, the K128A TR mutant transfected into CV-1 cells recruited p300, but not SRC-1, and was subject to acetylation. R132A complexed with SRC-1, but not p300; it was acetylated equally well in both the absence and presence of T4. S142E was acetylated in the absence and presence of T4 and bound SRC-1 under both conditions; this mutant was also capable of binding p300 in the presence of T4. There was no serine phosphorylation of TR in any of these mutants. We conclude that (1) TRbeta1, like AR and ER, is subject to acetylation; (2) the process of acetylation of TR requires thyroid hormone-directed MAPK activity, but not serine phosphorylation of TR by MAPK, suggesting that the contribution of MAPK is upstream in the activation of the acetylase; (3) the amino acid residue 128-142 region of the DBD of TR is important to thyroid hormone-associated recruitment of p300 and SRC-1; (4) acetylation of TR DBD mutants that is directed by T4 appears to be associated with recruitment of p300.