DEMONSTRATED THAT TR CONTROLS METMORPHIC TIMING AND RATE BUT NOT ESSENTIAL FOR METAMORPHOSIS. We developed a TALEN that could mutate TR gene in X. tropicalis with over 90% efficiency by injecting TALEN mRNAs into fertilized eggs, making it possible to analyze the role of TR in the resulting F0 animals. Using these F0 animals, we provided evidence to support a dual function model for TR during frog development: that is unliganded TR represses gene expression and prevent precocious metamorphosis while liganded TR activate target gene transcription and promote metamorphosis. More recently, we have used these F0 animals to generate animals with total knockout of TR gene. Surprisingly, we have observed that total knockout TR did not block metamorphosis, suggesting that the second TR gene, TR, was able to mediate the metamorphic effects of TH. While TR is not essential for metamorphosis, TR knockout accelerated animal development, with the knockout animals reaching the onset of metamorphosis earlier. On the other hand, they were resistant to exogenous TH treatment and had slower rate of natural metamorphosis. These findings are consistent with our earlier studies with the TR knockdown animals. Thus, our studies directly demonstrated a critical role of endogenous TR both in mediating the metamorphic effect of TH during metamorphosis and in preventing precocious initiation of metamorphosis when TH is absent. Additionally, our knockout animal studies also confirmed our initial novel observation on the growth of TR knockdown animals. That is TR knockout enhanced tadpole growth in premetamorphic tadpoles, in part by enhancing the expression of the two growth hormone genes in Xenopus. REVEALED THAT HISTONE METHYLTRANSFERASE DOT1L IS COACTIVATOR FOR TR AND HAVING AN ESSENTIAL ROLE IN PREMETAMORPHIC TADPOLE GROWTH. We have earlier identified Dot1L, the only HMT capable of methylating histone H3K79 in vitro, as a direct target gene of TH. Interestingly, the level of H3K79 methylation is strongly increased by TH at TR target genes, suggesting that Dot1L is upregulated by TR and in turn functions as a TR coactivator. Indeed, in cell culture assays and in the frog oocyte transcription system, we have shown that overexpression of Dot1L enhanced gene transcription by TR in the presence of TH. More importantly, transgenic overexpression of Dot1L in tadpoles also enhances gene activation of endogenous TR target genes by TH. To investigate the role of endogenous Dot1L, we generated a Dot1L-specific TALEN that was extremely efficient in mutating Dot1L when expressed in X. tropicalis fertilized eggs, creating animals with almost no Dot1L and little H3K79 methylation. We observed that Dot1L knockdown had no apparent effect on embryogenesis. On the other hand, it severely retarded tadpole growth and led to tadpole lethality before metamorphosis. Furthermore, the expression of endogenous TR target genes was also reduced. These findings suggest that Dot1L and H3K79 methylation plays an important role for tadpole growth and development prior to metamorphosis and that Dot1L functions at least in part as a coactivator for TR.
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