Gene Regulation And Function Of Thyroid Hormone

Child Health And Human Development

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Abstract

This laboratory is exploring molecular mechanisms in amphibian metamorphosis. The control of this developmental process by thyroid hormone (TH) offers a unique paradigm in which to study gene function in postembryonic organ development. During metamorphosis, different organs undergo vastly different changes. Some, like the tail, undergoes complete resorption, while others, such as the limb, are developed de novo. The majority of the larval organs persist through metamorphosis but are dramatically remodeled to function in a frog. For example, tadpole intestine in Xenopus laevis is a simple tubular structure consisting of primarily a single layer of primary epithelial cells. During metamorphosis, it is transformed into a multiply folded adult epithelium with elaborate connective tissue and muscles through specific cell death and selective cell proliferation and differentiation. The wealth of knowledge from past research and the ability to manipulate amphibian metamorphosis both in vivo by using transgenesis or hormone treatment of whole animals, and in vitro in organ cultures offer an excellent opportunity to 1) study the developmental function of thyroid hormone receptors (TRs) and the underlying mechanisms in vivo and 2) identify and functionally characterize genes which are critical for postembryonic organ development in vertebrates. ROLES OF COFACTORS IN GENE REGULATION BY TR. TR regulates gene transcription by recruiting cofactors to target genes. In the presence of TH, TR can bind to coactivators while the unliganded TR binds to corepressors. Many biochemical and molecular studies have been done on such cofactors. Despite of these, little is known about whether and how they participate in gene regulation by TR in different biological processes in vivo. Our focus is to investigate how TR utilizes different cofactors in the context of development in various organs. The frog metamorphosis is particularly well suited for such a purpose. Based on earlier studies, we have proposed a dual function model for TR during frog development. That is, the heterodimers between TR and RXR (9-cis retinoic acid receptor) activate gene expression during metamorphosis when TH is present, while in premetamorphic tadpoles, they repress gene expression to prevent metamorphosis, thus ensuring a tadpole growth period. To investigate how TR activates gene transcription during metamorphosis, we have begun to study p300 and SRC, which are among the best studied coactivators in vitro and in tissue culture cell studies. We have obtained the cDNA clones for Xenopus p300, SRC2, and SRC3, and have shown that they are expressed during metamorphosis. To investigate their involvement in gene regulator by TR, we utilized the frog oocyte system that we have established earlier for studying TR function in the context of chromatin, and demonstrated recently, by using chromatin immunoprecipitation (ChIP) assay, that TR can recruit SRC3 to a target promoter. Furthermore, we have generated a dominant negative form of SRC3 (dnSRC3) that can be recruited by TR, thus blocking the recruitment of wild type SRC and gene activation. Currently, we are carrying out similar studies on p300. More importantly, we are introducing the dnSRC3 into tadpoles through transgenesis to investigate whether SRC recruitment is important for gene regulation by TR in developing animals and for frog metamorphosis. To determine whether and how TR represses target genes in premetamorphic tadpoles when there is no TH, we have cloned the full or partial coding regions of corepressors N-CoR (nuclear receptor corepressor) and SMRT (silencing mediator of retinoid and thyroid receptors), which are the best studied corepressors that bind to TR in vitro. We have shown that both are expressed and more importantly, bound to TH-response genes in premetamorphic tadpoles. Their association with TH-response genes are released upon treatment of the tadpoles with TH, indicating that their binding to TH-response gene promoters is due to recruitment by unliganded TR. In agreement with the ability of these corepressors to form complexes containing histone deacetylases (HDACs), TH treatment leads to increase in local histone acetylation at the TH-response genes at least in the intestine and tail, arguing that histone acetylation is an important factor in gene regulation by TR. FUNCTION OF TR DURING DEVELOPMENT. Our dual function model for TR during frog development argues that transcriptional activation by TR is essential for frog metamorphosis. To test such a hypothesis, we have adapted sperm-mediated transgenic method to generate transgenic animals expressing a dominant negative TR (dnTR). Phenotypic analysis indicates that dnTR overexpression inhibits TH-induced metamorphosis. More importantly, we have shown that dnTR specifically blocks the expression of TH response genes. To determine the underlying mechanism by which TR regulates TH response genes in vivo, we used ChIP assay to show that dnTR is bound to target promoters, which led to retention of corepressors N-CoR and SMRT, and continued histone deacetylation even in the presence of TH. These results thus provide direct in vivo evidence for a molecular mechanism of altering gene expression by a dnTR. The correlation between dnTR-mediated gene repression and inhibition of metamorphosis also supports a key aspect of the dual function model for TR in development: during TH-induced metamorphosis, TR functions via the release of corepressors and promotion of histone acetylation to activate target gene expression, thereby inducing morphological changes. INVOLVEMENT OF MATRIX METALLOPROTEINASES DURING TH-INDUCED TISSUE REMODELING. In an effort to identify genes important for postembryonic development, we isolated many TH response genes during metamorphosis. Expression analyses and other studies have led us to focus on the TH-response genes encoding matrix metalloproteinases (MMPs) for functional investigations. MMPs are extracellular enzymes capable of digesting various ECM components. Our earlier studies have led us to propose that the MMP stomelysin-3 (ST3) is directly or indirectly involved in ECM remodeling, which in turn influences cell behavior. This has been supported by organ culture studies in vitro where we showed that ST3 function is important for TH-induced apoptosis of larval intestinal epithelial cells and the invasion of the proliferating adult epithelial cells into the connective tissue. To directly investigate the roles of MMPs in developing animals, we are employing the transgenic approach to express wild type and mutant MMPs in Xenopus embryos and tadpoles. By using a heat-shock inducible promoter to drive the expression of ST3 or an inactive mutant form of ST3, we generated transgenic tadpoles that will expression wild type or mutant ST3 upon heat shock. Heat shock at tadpole stages led to overexpression of wild type or mutant ST3 in all organs, although no visible morphological changes of the tadpoles were observed for up to 4 days. Analysis of the intestine showed that overexpression of wild type but not mutant ST3 caused premature apoptosis in the tadpole epithelium, consistent with our organ culture studies earlier. Furthermore, the apoptosis is accompanied by drastic remodeling of the basal lamina, or the ECM that separates the connective tissue and epithelium in the intestine. Together, our results suggest that ST3 directly or indirectly modifies the ECM, which in turn facilitate cell fate changes and tissue morphogenesis during metamorphosis.