Tissue- and Gene-specific Recruitment of Steroid Receptor Coactivator-3 by Thyroid Hormone Receptor during Development
Journal of Biological Chemistry
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 (T 3 ) 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
... in all tadpole organs analyzed. In addition, during natural as well as T 3 -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 T 3 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 T 3 treatment of premetamorphic tadpoles and during natural metamorphosis. In particular, in the tail, SRC3 was not recruited in a T 3 -dependent manner to the target TR␤A 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 T 3 -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. Thyroid hormone (T 3 ) 1 affects diverse organ functions and metabolism in vertebrates (1-3) and plays critical roles in postembryonic organogenesis and tissue remodeling in vertebrates (1-6). The effects of T 3 are mediated by T 3 receptors (TRs), which are transcription factors belonging to the nuclear receptor superfamily (3, 7-11). TR forms a heterodimer with 9-cis-retinoic acid receptor (RXR) and binds to thyroid hormone response elements (TREs) of T 3 -responsive promoters to modulate transcription. TR/RXR heterodimers function to repress or activate target gene transcription in the absence or presence of T 3 , respectively, by recruiting corepressors or coactivators (3, 12-17). The best characterized coactivators for TR belong to the SRC or p160 family, comprising three homologous members, SRC1/ NCoA-1, SRC2/TIF2/GRIP1, and SRC3/pCIP/ACTR/AIB-1/ RAC-3/TRAM-1 (18 -26). These proteins share considerable structural homology and are evolutionarily related, being about 40% identical among each other, with extensive similarity at the N-terminal basic helix-loop-helix and PAS dimerization domain (27-29). The central region of SRC proteins contain three leucine rich, LXXLL (L, leucine; X, any amino acid) motifs, forming short amphipathic ␣-helices (19, 26, 30 -32) and constitute the receptor interaction domain. SRC proteins interact with nuclear receptors directly in a ligand-dependent manner and facilitate transcription via distinct activation domains, AD1 and AD2. AD1, which has two LXXLL motifs, can bind to the histone acetyltransferase CBP/p300 (21, 32, 33). SRCs themselves have also been reported to possess weak intrinsic histone acetyltransferase activity (21, 34). AD2 has been reported to interact with chromatin modifying enzymes including methylases such as coactivator-associated arginine methyltransferase-1 (CARM-1) and protein arginine methyltransferase-1 (PRMT-1) (35, 36) . Despite the enormous accumulation of molecular and biochemical information on coactivator-nuclear receptor interactions, the in vivo role of SRCs and their physiological significance in nuclear receptor-mediated developmental processes in vertebrates have remained essentially unexplored. Even when gene knock-out studies reveal that cofactor deficiency leads to specific development defects (37-42), the underlying molecular mechanisms are unknown, largely due to the fact that these cofactors are involved in transcriptional regulation by many diverse transcription factors and the difficulty to access and manipulate postembryonic development in mammals. Amphibian metamorphosis bears strong similarities to postembryonic development in mammals (2, 4, 5) and offers a unique opportunity to study the role of cofactors in nuclear receptor function in vertebrate development. A major advantage of this model is that all tissues/organs require T 3 despite undergoing vastly different transformations during metamorphosis (2, 43). These changes range from the development of adult organs de novo from undifferentiated stem cells to the regression of larval-specific organs such as the gills and tail and occur at developmentally distinct stages. All these changes are believed to be due to gene regulation by T 3 through TR (44) and can be easily manipulated by blocking the synthesis of endogenous T 3 or adding physiological concentrations of T 3 to the tadpole rearing water. We have shown earlier that the mRNAs of TR interacting cofactors, SRC2, SRC3, and p300, are expressed during meta-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.