ROLES OF COFACTORS IN GENE REGULATION BY TR. TR 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 shown that TR is both necessary and sufficient for the metamorphic effects of TH. Thus metamorphosis provides the first example where TR is shown to mediate directly and sufficiently the developmental effects of TH. Furthermore, we have shown that TR regulates metamorphic timing by recruiting corepressor complexes to target genes in premetamorphic tadpoles to prevent precocious metamorphosis. During metamorphosis, TR needs to recruit coactivator complexes containing SRC3 (steroid receptor coactivator 3)/p300 to target genes for their activation and metamorphosis. These findings represent the first example whether specific cofactor complexes have been shown to play critical roles for developmental function of a nuclear receptor in vertebrates. The SRC/p300 complexes also contain the methyltransferase PRMT1, which has been implicated in TR function in mammalian cell culture studies. Thus, to further investigate the role and mechanisms of the SRC/p300 complexes in development, we have cloned and characterized Xenopus laevis PRMT1. By using intestinal remodeling during Xenopus laevis metamorphosis for in vivo molecular analysis, we showed that PRMT1 expression was upregulated during metamorphosis when both TR and TH were present. We then demonstrated a role of PRMT1 in TR-mediated transcription by showing that PRMT1 enhanced transcriptional activation by liganded TR in the frog oocyte transcription system and was recruited to the TH response element (TRE) of the target promoter in the oocyte as well as to endogenous TREs during frog metamorphosis. Surprisingly, we found that PRMT1 was only transiently recruited to the TREs in the target during metamorphosis and observed no PRMT1 recruitment to TREs at the climax of intestinal remodeling when both PRMT1 and TH were at peak levels. Mechanistically, we showed that overexpression of PRMT1 enhanced TR binding to TREs both in the frog oocyte model system and during metamorphosis. More importantly, transgenic overexpression of PRMT1 enhanced gene activation in vivo and accelerated both natural and TH-induced metamorphosis. These results thus indicate that PRMT1 functions transiently as a coactivator in TR-mediated transcription by enhancing TR-TRE binding and further suggest that PRMT1 has tissue specific roles to regulate the rate of metamorphosis. ANALYZING THE GENE EXPRESSION PROGRAMS UNDERLYING THE TEMPORAL AND TISSUE-DEPENDENT 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. Our initial analysis of animals treated with TH for different number of days have provided a molecular description of the gene regulation pathways associated with different metamorphic processes in the intestine. The success of this study also prompted us to ask whether we could use metamorphosis coupled with cDNA array analysis as a model to study whether endocrine disrupting compounds (EDCs) can affect vertebrate development via the TH signaling pathway. EDCs are exogenous substances that alter function(s) of the endocrine system and consequently cause adverse health effects in an intact organism, or its progeny, or (sub) populations. As TH plays a central role in vertebrate development, growth, and metabolism, the effects of EDCs on TH signaling will undoubtedly pose a threat to human and wildlife health. However, the lack of a suitable in vivo model to study EDCs effects on TR function in vertebrate development impedes our understanding on whether and how persistent exposure to these bioaccumulative compounds affects human health. As a test case, we analyzed the effect of bisphenol A (BPA), on Xenopus metamorphosis. BPA, a chemical widely used to manufacture plastics, is estrogenic and capable of disrupting sex differentiation. However, recent in vitro studies have shown that BPA can also antagonize TH activation of TRs. The difficulty in studying uterus-enclosed mammalian embryos has hampered the analysis on the direct effects of BPA during vertebrate development. We studied the effect of BPA on TH-dependent metamorphosis at both morphological and molecular levels. After 4 days of exposure, BPA inhibited TH-induced intestinal remodeling in premetamorphic Xenopus laevis tadpoles. Importantly, microarray analysis revealed that BPA antagonized the regulation of most TH-response genes, thereby explaining the inhibitory effect of BPA on metamorphosis. Surprisingly, most of the genes affected by BPA in the presence of TH were TH-response genes, suggesting that BPA predominantly affected TH-signaling pathways during metamorphosis. Our finding that this endocrine disruptor, well known for its estrogenic activity in vitro, functions to inhibit TH-pathways to affect vertebrate development in vivo thus not only provides a mechanism for the likely deleterious effects of BPA on human development but also demonstrates the importance of studying endocrine-disruption in a developmental context in vivo.
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