MECHANISMS OF GENE REGULATION BY TR DURING DEVELOPMENT. Based on TR expression profiles and its molecular properties, we have previously proposed a dual function model for TR during frog development. That is, the heterodimers between TR and RXR (9-cis retinoic acid receptor) bind to target genes in vivo. 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 TH-inducible genes to initiate metamorphosis. Our studies in the last several years have provided molecular and genetic support for this model. Furthermore, we have revealed important roles of corepressor and coactivator complexes in TR action during metamorphosis. As these cofactor complexes can modify histone acetylation and methylation levels, we have been interested to determine if gene regulation by TR involves alterations in histone modifications, especially considering the fact that little is known on whether and how histone modifications change upon gene regulation by TR during development of any vertebrates. By using chromatin immunoprecipitation (ChIP) assay and the intestinal metamorphosis in Xenopus tropicalis as a model, we demonstrated for the first time in vivo during vertebrate development that liganded TR induces the removal of core histones at the promoter region and the recruitment of RNA polymerase. Furthermore, we observed changes in histone acetylation and methylation induced by liganded TR that suggest that some histone activation and repression marks for gene regulation during vertebrate development differ from those assigned based on correlations with mRNA levels in cell cultures, suggesting the importance of tissue- and developmental context in the roles of histone modifications in gene regulation. Our findings further provide important mechanistic insights on how chromatin remodeling affects developmental gene regulation in vivo. ANALYZING THE GENE EXPRESSION PROGRAMS UNDERLYING THE TEMPORAL TRANSFORMATIONS DURING METAMORPHOSIS. The complexity of metamorphic changes in different organs argues for the presence of different gene regulation programs regulated by TR. Knowledge on this systematic gene regulation will help to identify not only molecular markers but also important cellular pathways or critical genes for future mechanistic studies. Thus, we have begun to use the recently developed Xenopus laevis cDNA array to analyze genome-wide gene expression changes associated with TH-induced intestinal remodeling, a process that involves selective degeneration of the larval epithelium through apoptosis and de novo development of the adult epithelium. Our earlier analysis using total intestinal RNA revealed co-expressed genes involved in essential cell processes such as apoptosis and proliferation. Furthermore, we showed that most of the genes highly induced at metamorphic climax were also upregulated in the mouse intestine around birth, the postembryonic period resembling metamorphosis, supporting conservation in the underlying molecular pathways. More recently, we have carried out cDNA arrays by using isolated epithelium and non-epithelium tissues of the intestine at different stages during metamorphosis. Preliminary analysis suggested the existence of distinct epithelial and non-epithelial genes that are likely involved in the development of adult intestinal stem cells.
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