Gene Regulation /Function By Thyroid Hormone During Tiss

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 an organ of a multiply folded adult epithelium surrounded by 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. FUNCTION OF TR DURING DEVELOPMENT. 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 proper tadpole growth period. Such a model argues that transcriptional activation by TR is essential for frog metamorphosis. To test such a hypothesis, we previously adapted the sperm-mediated transgenic method to generate transgenic animals expressing a dominant negative TR (dnTR). This allowed us to show that gene activation by TR is necessary for metamorphosis. To determine whether gene activation by TR is sufficient for TH-dependent metamorphosis, we developed a dominant positive mutant TR (dpTR). Transgenic expression of dpTR under the control of a heat shock-inducible promoter in premetamorphic tadpoles led to precocious metamorphic transformations. Molecular analyses showed that dpTR induced metamorphosis by specifically binding to known T3-target genes, leading to increased local histone acetylation and gene activation, similar to T3-bound TR during natural metamorphosis. Thus, the metamorphic role of T3 is predominantly, if not exclusively, through genomic action of the hormone. They further provide the first example where TR is shown to mediate directly and sufficiently these developmental effects of T3 in individual organs by regulating target gene expression. 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. We have chosen for our studies several cofactors that are likely involved in development based on earlier in vitro work. Among the coactivators, we have began to study p300 and SRC. We have shown that Xenopus p300, SRC2, and SRC3 are expressed during metamorphosis. We are employing chromatin immunoprecipitation (ChIP) assay to analyze the recruitment of both SRC3 and p300 to endogenous TH-response genes during development. Furthermore, we have obtained preliminary evidence to show that transgenic tadpoles expressing dnSRC3 are resistant to TH-induced as welll as natural metamorphosis. Among the corepressors, we have been studying the role of N-CoR (nuclear receptor corepressor) and SMRT (silencing mediator of retinoid and thyroid receptors) in gene repression by TR in premetamorphic tadpoles. Our earlier and recent data showed that they are involved in gene repression by unliganded TR in vivo. Both N-CoR and SMRT are known to exist in large complexes in mammals. We previously showed that the frog TBLR1 (transducin beta-like protein 1-related protein) is expressed in the tadpoles and forms complexes with both N-CoR and SMRT, at least in the oocyte. We have now shown that a dominant negative N-CoR that contains only the TBLR1-interacting domain was able to block TBLR1 interaction with TR and derepress the promoter in the frog oocyte. Furthermore, TBLR1 is associated with endogenous TH-target promoters in premetamorphic tadpoles and this association is reduced or eliminated upon treatment of the tadpoles with TH or during natural metamorphosis, suggesting that the release of TBLR1-containing corepressor complexes is one of the mechanisms by which TH response genes are activated during metamorphosis. REGULATION AND FUNCTION OF TH RESPONSE GENES DURING TH-INDUCED TISSUE REMODELING. To identify genes important for postembryonic development, we isolated many TH response genes during metamorphosis. Of particular interests among them are those encoding matrix metalloproteinases (MMPs). MMPs are extracellular enzymes capable of digesting various ECM components. Our earlier studies have led us to propose that the MMP stromelysin-3 (ST3) is directly or indirectly involved in ECM remodeling, which in turn influences cell behavior in the remodeling intestine. To directly investigate its roles in vivo, we have generated transgenic tadpoles expressing wild type ST3 or a catalytically inactive mutant under the control of a heat-shock inducible promoter. Heat shock at tadpole stages led to overexpression of wild type or mutant ST3 in all organs. While no visible morphological changes were observed for up to a few weeks, analysis of the intestine showed that overexpression of wild type but not mutant ST3 caused premature apoptosis in the tadpole epithelium, accompanied by drastic remodeling of the basal lamina, the ECM that separates the connective tissue and epithelium in the intestine. Thus, our results suggest that ST3 directly or indirectly modifies the ECM, which in turn facilitate cell fate changes and tissue morphogenesis during metamorphosis. To determine how ST3 affects tissue remodeling, we have begun to isolate and characterizing ST3 substrates. As a first step, we investigated the ability of Xenopus laevis ST3 to cleave an in vitro substrate of mammalian ST3, the alpha 1-proteinase inhibitor (PI). We analyzed the ability of Xenopus laevis ST3 catalytic domain to cleave frog PI. Surprisingly, we found the ST3 failed to recognize the site in Xenopus PI equivalent to the major cleavage site in human PI by mammalian ST3. Sequence and mutagenic analyses revealed that multiple substitutions at P2-P3? positions between human and Xenopus PI contributed to the inability of Xenopus PI to be cleaved by ST3. Our studies showed that (A)(G/A)(A)(M)(F/A)(L) (P3-P3?) as a preferred cleavage site for ST3. We further demonstrated that mutations in the cleavage sites affected cleavage by ST3 differently from several other MMPs. These findings, together with earlier reports on ST3, showed that ST3 has distinct substrate specificities compared to other MMPs. Our results further suggest that PI is unlikely to be a physiological substrate for ST3, at least with regard to evolutionarily conserved developmental processes.