CONTROL OF METAMOPRHIC TIMING BY UNLIGANDED TR. 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) 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. We 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. To investigate the function of unliganded TR in premetamorphic tadpoles, we designed a dominant negative form of the TR-binding corepressor N-CoR (nuclear receptor corepressor) (dnN-CoR), which contains only the receptor interacting domain of N-CoR. We overexpressed dnN-CoR under the control of a heat shock inducible promoter in tadpoles through transgenesis. We observed significant derepression of TH-response genes in transgenic animals. More importantly, transgenic tadpoles developed faster than wild type siblings, with an acceleration of as much as 7 days out of the 30-day experiment. By the end of the experiments, the animals reached early stages of metamorphosis. These data thus suggest that unliganded TR recruit corepressors to control metamorphic timing, as predicted by our model. ? ? 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. On the other hand, this analysis cannot determine whether the TH-response genes thus identified are directly or indirectly regulated by TRs. As directly regulated genes act earlier in the metamorphic process and are more likely important regulators of metamorphosis, we are currently using cDNA array analysis to identify such genes by using RNA from animals treated with TH in the presence or absence of protein synthesis inhibitors. By determining tissue specific direct target genes of TR, we should be able to gain insight into how TH controls tissue-specific changes during metamorphosis.? ? DEVELOPING XENOPUS TROPICALIS AS A MODEL FOR FUNCTIONAL STUDIES OF TR. While studies so far using Xenopus laevis as a model have led to a number of important in vivo findings on the function and mechanisms of TR, the lack of genomic sequence information, its tetraploid genome, and lengthy developmental cycle make it difficult for further analyses on TR functions in this species. On the other hand, the highly related species, Xenopus tropicalis, offers many advantages. Toward developing X. tropicalis for genome-wide and genetic studies of TR function, we have recently shown that TR/RXR expression correlates with organ transformations in X. tropicalis and that TR/RXR heterodimers are capable of repressing and activating gene expression in vivo in the absence and presence of TH, respectively. Furthermore, TRs are bound to endogenous target genes in X. tropicalis tadpoles. Our results thus support a role of TRs in mediating the metamorphic effects of TH in X. tropicalis. More importantly, the similarities in the expression and function between X. tropicalis and X. laevis TRs and RXRs as demonstrated by our study also pave the way to take advantages of existing morphological, molecular, and cellular knowledge of X. laevis development and the genetic and sequence superiority of X. tropicalis to dissect the molecular pathways governing tissue/organ-specific transformations during vertebrate 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 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 and SMRT (silencing mediator of retinoid and thyroid receptors) in gene repression by TR in premetamorphic tadpoles. Our studies with the dnN-CoR as summarized above have now demonstrated in vivo a role of corepressor complexes in the control of metamorphic timing. On the activator side, we have shown that Xenopus laevis SRC3 (steroid receptor coactivator 3)/p300-containing coactivator complexes or related complexes are required for gene regulation by liganded TR and for TH-dependent metamorphosis. These findings represent the first example whether specific coactivator complexes have been shown to play critical roles for developmental function of a nuclear receptor in vertebrates. ? ? The SRC/p300 complexes also contain methyltransferases CARM1 and PRMT1. Both have 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 CARM1 and PRMT1 and have shown that they are recruited by liganded TR to enhance transcriptional activation by TH. Currently, we are investigating the in vivo function of these methyltransferases during metamorphosis.? ? In addition to histone modifications involving complexes such as SRC-p300, transcriptional regulation by TR also involves chromatin remodeling. In particular, the BRG1-containing, ATP-dependent chromatin remodeling complexes have been implicated to play a role in gene regulation by nuclear receptors. To investigate their potential involvement in metamorphosis, we have now shown that the expression of BRG1, a chromatin-remodeling enzyme, is up-regulated at the climax of Xenopus laevis metamorphosis, while BAF57, a BRG1-binding protein in BRG1-containing chromatin remodeling complexes, is constitutively expressed during development. Consistently, TH-treatment of pre-metamorphic tadpoles led to up-regulation of the expression of BRG1 but not BAF57. Studies using a reconstituted TH-dependent Xenopus oocyte transcription system, where we can study TR function in the context of chromatin, revealed that BRG1 enhances the transcriptional activation by ligand-bound TRs in a dose-dependent manner, while a remodeling-defective BRG1 mutant inhibited the activation, suggesting that this process relies on chromatin remodeling. Further studies showed that BAF57 interacted with BRG1 in oocytes and enhanced gene activation by TR cooperatively with BRG1 in vivo. Chromatin immunoprecipitation revealed that BAF57 was recruited to the TR-regulated promoter in the presence of TR and TH. Together, these findings suggest a role of BRG1/BAF57-containing chromatin remodeling complexes in TR-regulated gene expression during metamorphosis.
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