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Protein arginine methyltransferase 1 (PRMT1) acts as a transcription coactivator for nuclear receptors through histone H4 R3 methylation. The in vivo function of PRMT1 is largely unknown. Here we investigated the role of PRMT1 in thyroid hormone (T3) receptor (TR)-mediated transcription in vivo during vertebrate development. By using intestinal remodeling during T3-dependent Xenopus laevis metamorphosis for in vivo molecular analysis, we first showed that PRMT1 expression was upregulated during metamorphosis when both TR and T3 were present. We then demonstrated a role for 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 T3 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 T3 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 T3-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 in regulating the rate of metamorphosis.

A fascinating feature of thyroid hormone (T3) receptors (TR) is that they constitutively bind to promoter regions of T3-response genes, providing dual functions. In the presence of T3, TR activates T3-inducible genes, while unliganded TR represses these same genes. Although this dual function model is well demonstrated at the molecular level, few studies have addressed the presence or the role of unliganded TR-induced repression in physiological settings. Here, we analyze the role of unliganded TR in Xenopus laevis development. The total dependence of amphibian metamorphosis upon T3 provides us a valuable opportunity for studying TR function in vivo. First, we designed a dominant negative form of TR-binding corepressor N-CoR (dnN-CoR) consisting of its receptor interacting domain. We confirmed its dominant negative activity by showing that dnN-CoR competes away the binding of endogenous N-CoR to unliganded TR and relieves unliganded TR-induced gene repression in frog oocytes. Next, we overexpressed dnN-CoR in tadpoles through transgenesis and analyzed its effect on gene expression and development. Quantitative RT-PCR revealed significant derepression of T3-response genes in transgenic animals. In addition, transgenic tadpoles developed faster than wild type siblings, with an acceleration of as much as 7 days out of the 30-day experiment. These data thus provide in vivo evidence for the presence and a role of unliganded TR-induced gene repression in physiological settings and strongly support our earlier model that unliganded TR represses T3-response genes in premetamorphic tadpoles to regulate the progress of development.

Thyroid hormone receptors (TRs) can repress or activate target genes depending on the absence or presence of thyroid hormone (T3), respectively. This hormone-dependent gene regulation is mediated by the recruitment of corepressors in the absence of T3 and coactivators in its presence. Many TR-interacting coactivators have been characterized in vitro. Among them is coactivator-associated arginine methyltransferase 1 (CARM1), which methylates histone H3. We are interested in investigating the role of CARM1 in TR-mediated gene expression in vivo during postembryonic development by using T3-dependent frog metamorphosis as a model. We first cloned the Xenopus laevis CARM1 and obtained two alternative splicing forms, CARM1a and CARM1b. Both isoforms are expressed throughout metamorphosis, supporting a role for these isoforms during the process. To investigate whether Xenopus CARM1s participate in gene regulation by TRs, transcriptional analysis was conducted in Xenopus oocyte, where the effects of cofactors can be studied in the context of chromatin in vivo. Surprisingly, overexpression of CARM1b had little effect on TR-mediated transcription, whereas CARM1a enhanced gene activation by liganded TR. Chromatin immunoprecipitation assays showed that both endogenous CARM1a and overexpressed CARM1a and b were recruited to the promoter by liganded TR. However, the binding of liganded TR to the target promoter was reduced when CARM1b was overexpressed, accompanied by a slight reduction in histone methylation at the promoter. These results suggest that CARM1 may play a role in TR-mediated transcriptional regulation during frog development and that its function is regulated by alternative splicing.

Gene activation by the thyroid hormone (T3) receptor (TR) involves the recruitment of specific coactivator complexes to T3-responsive promoters. A large number of coactivators for TR have been isolated and characterized in vitro. However, their roles and functions in vivo during development have remained largely unknown. We have utilized metamorphosis in Xenopus laevis to study the role of these coactivators during post-embryonic development. Metamorphosis is totally dependent on the thyroid hormone, and TR mediates a vast majority, if not all, of the developmental effects of the hormone. We have previously shown that TR recruits the coactivator SRC3 (steroid receptor coactivator-3) and that coactivator recruitment is essential for metamorphosis. To determine whether SRCs are indeed required, we have analyzed the in vivo role of the histone acetyltransferase p300/CREB-binding protein (CBP), which was reported to be a component of the SRC.coactivator complexes. Chromatin immunoprecipitation revealed that p300 is recruited to T3-responsive promoters, implicating a role of p300 in TR function. Further, transgenic tadpoles overexpressing a dominant negative form of p300, F-dnp300, containing only the SRC-interacting domain, displayed arrested or delayed metamorphosis. Molecular analyses of the transgenic F-dnp300 animals showed that F-dnp300 was recruited by TR (displacing endogenous p300) and inhibited the expression of T3-responsive genes. Our results thus suggest that p300 and/or its related CBP is an essential component of the TR-signaling pathway in vivo and support the notion that p300/CBP and SRC proteins are part of the same coactivator complex in vivo during post-embryonic development.

The current review focuses on the molecular mechanisms and developmental roles of thyroid hormone receptors (TRs) in gene regulation and metamorphosis in Xenopus laevis and discusses implications for TR function in vertebrate development and diversity. Questions addressed are: (1) what are the molecular mechanisms of gene regulation by TR, (2) what are the developmental roles of TR in mediating the thyroid hormone (TH) signal, (3) what are the roles of the different TR isoforms, and (4) how do changes in these molecular and developmental mechanisms affect evolution? Even though detailed knowledge of molecular mechanisms of TR-mediated gene regulation is available from in vitro studies, relatively little is known about how TR functions in development in vivo. Studies on TR function during frog metamorphosis are leading the way toward bridging the gap between in vitro and in vivo studies. In particular, a dual function model for the role of TR in metamorphosis has been proposed and investigated. In this model, TRs repress genes allowing tadpole growth in the absence of TH during premetamorphosis and activate genes important for metamorphosis when TH is present. Despite the lack of metamorphosis in most other vertebrates, TR has important functions in development across vertebrates. The underlying molecular mechanisms of TR in gene regulation are conserved through evolution, so other mechanisms involving TH-target genes and TH tissue-sensitivity and dependence underlie differences in role of TR across vertebrates. Continued analysis of molecular and developmental roles of TR in X. laevis will provide the basis for understanding how TR functions in gene regulation in vivo across vertebrates and how TR is involved in the generation of evolutionary diversity.

The biological effects of thyroid hormone (T3) are mediated by the thyroid hormone receptor (TR). Amphibian metamorphosis is one of the most dramatic processes that are dependent on T3. T3 regulates a series of orchestrated developmental changes, which ultimately result in the conversion of an aquatic herbivorous tadpole to a terrestrial carnivorous frog. T3 is presumed to bind to TRs, which in turn recruit coactivators, leading to gene activation. The best-studied coactivators belong to the p160 or SRC family. Members of this family include SRC1/NCoA-1, SRC2/TIF2/GRIP1, and SRC3/pCIP/ACTR/AIB-1/RAC-3/TRAM-1. These SRCs interact directly with liganded TR and function as adapter molecules to recruit other coactivators such as p300/CBP. Here, we studied the expression patterns of these coactivators during various stages of development. Amongst the coactivators cloned in Xenopus laevis, SRC3 was found to be dramatically upregulated during natural and T3-induced metamorphosis, and SRC2 and p300 are expressed throughout postembryonic development with little change in their expression levels. These results support the view that these coactivators participate in gene regulation by TR during metamorphosis.