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 majorities 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. 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. In premetamorphic tadpoles, they repress gene expression in the absence of TH to prevent metamorphosis, thus ensuring a proper tadpole growth period. When TH is present either from endogenous synthesis during development or exogenous addition to the raring water of premetamorphic tadpoles, TR/RXR heterodimers activate the TH-inducible genes to initiate metamorphosis. By using the sperm-mediated transgenic approach, we have previously shown that TR is both necessary and sufficient to mediate the metamorphic effects of TH. ? ? To investigate how TR differentially regulates different genes in various organs/tissues during metamorphosis, we have examined, by using chromatin immunoprecipitation (ChIP) assay, TR binding to promoters in vivo. We have analyzed two target genes with TH response elements (TRE) in their promoters, TR? and TH/bZIP (TH-responsive basic leucine zipper transcription factor). Using an antibody that recognizes both TR? and TR?, we found that TR binding to TR? promoter is constitutive. Surprisingly, TR binding to TH/bZIP promoter increases dramatically after TH treatment of premetamorphic tadpoles and during metamorphosis. Using an antibody specific to TR?, TR? binding increases at both promoters in response to TH, likely due to TH-induced increase in TR? expression. In vitro biochemical studies have revealed that TRs bind TH/bZIP TRE with 4-fold lower affinity than to TR? TRE. Our data, showing that only high affinity TR? TRE is occupied by limiting levels of TR during premetamorphosis and that lower affinity TH/bZIP TRE becomes occupied only when overall TR expression is higher during metamorphosis, provide the first in vivo evidence to suggest that one mechanism for tissue and gene-specific regulation of TR target gene expression is through tissue and developmental stage-dependent regulation of TR levels, likely a critical mechanism for coordinating development in different organs during postembryonic development.? ? 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. On the other hand, much less 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. ? ? Among corepressors, we have studied 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. We have shown previously that both are expressed and more importantly, bound to TH-response genes in premetamorphic tadpoles and thus bring to the promoters other components, such as TBLR1 (transducin beta-like protein 1-related protein), of the histone deacetylase-containing complexes. Furthermore, TH treatment of premetamorphic tadpoles leads to the release of TBLR1, together with N-CoR and/or SMRT, and increased histone acetylation and gene activation. These results argue that TBLR1-containing N-CoR/SMRT deacetylase complexes or related complexes are required for transcription repression by unliganded TR in tadpoles and that their release is one of the mechanisms by which TH response genes are activated during metamorphosis. ? ? On the activator side, we have used ChIP assay to show that Xenopus coactivator SRC3 is recruited in a gene- and tissue-dependent manner to target genes by TR. Furthermore, we have generated transgenic tadpoles expressing a dominant negative form of SRC3 (F-dnSRC3). Phenotypic and molecular analyses of the animals demonstrate that coactivator recruitment, aside from corepressor release, is required for TH function. Using a similar approach, we have now determined that the coactivator p300 is also recruited to TH target promoters by liganded TR. We have generated a dominant negative form of p300 that contains only the SRC-binding domain of p300, which is capable of inhibiting activation of a reporter gene by liganded TR in vivo. Through transgenesis, we show that this dominant negative p300 is also capable of inhibiting gene activation by TR in developing animals and thereby preventing metamorphosis, demonstrating a critical role of SRC-p300 complexes in TR-mediated metamorphosis. ? ? INVOLVEMENT OF MATRIX METALLOPROTEINASES DURING TH-INDUCED TISSUE REMODELING. We have previously identified several TH-response genes encoding matrix metalloproteinases (MMPs) during intestinal remodeling. Expression and organ culture studies have led us to propose that the MMP stomelysin-3 (ST3) is directly or indirectly involved in ECM (extracellular matrix) remodeling, which in turn influences cell behavior. Through transgenesis, we have shown that catalytically active ST3 is sufficient to induce apoptosis in the tadpole epithelium, accompanied by drastic remodeling of the basal lamina, or the ECM that separates the connective tissue and epithelium in the intestine. Toward understanding the mechanism by which ST3 affects tissue remodeling, we have isolated the 37 kd laminin receptor precursor (LR) as a likely substrate. LR binds to ST3 in vitro and can be cleaved by ST3 at two sites, distinct from where other MMPs cleave, in the extracellular domain between the transmembrane domain and laminin binding sequence, suggesting that LR cleavage by ST3 will alter cell-ECM interaction. We have also shown that LR is cleaved by ST3 in vivo. Interestingly, ST3 cleavage sites in LR are conserved in human LR. Furthermore, high levels of LR are known to be expressed in tumor cells, which are often surrounded by fibroblasts expressing ST3. Thus, LR may be a conserved substrate of ST3 and that its cleavage by ST3 may alter cell-ECM interactions, thus, playing a role in mediating the effects of ST3 on cell fate and behavior during development and pathogenesis. In addition to ST3, our recent expression studies suggest that gelatinase A (GelA) and membrane type MMP-1 (MT1-MMP) likely function together in ECM remodeling and that MT1-MMP also has GelA-independent roles during metamorphosis.
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