Molecular mechanism of thyroid hormone receptor function during metamorphosis

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

Investigated the function of endogenous TR genes by using knockout animals: By using the TALEN technology, we have previously generated X. tropicalis animals lacking any functional TR and observed that TR knockout animals are surprisingly able to complete metamorphosis at a similar age as the wild typing siblings. Carefully analyses during development, however, revealed multiple roles of TR during Xenopus development, including preventing precocious initiation of metamorphosis when TH is absent, regulating the rate of metamorphic progression, and coordinating temporal regulation of metamorphosis in different tissues/organs. We have now also generated TR heterozygous knockout animals and begun to analyze the role of TR in Xenopus development. In addition, global RNA sequencing (RNA-seq) and chromatin immunoprecipitation (ChIP)-sequencing (ChIP-seq) are being carried out on wild type and TR-knockout animal tissues to determine the molecular pathways regulated by different TRs in different tissues during metamorphosis. Demonstrated that histone methyltransferase Dot1L is a coactivator for thyroid hormone receptor during Xenopus development: Histone modifications are associated with transcriptional regulation by diverse transcription factors. Genome-wide correlation studies have revealed that histone activation marks and repression marks, associated with activated and repressed gene expression, respectively. Among the histone activation marks is histone H3 K79 methylation, which is carried out by only a single methyltransferase, Dot1L. Interestingly, our ChIP-on-chip analysis above revealed that Dot1L is regulated by TH at the transcriptional level and our previously studies have shown that H3K79 methylation levels are induced at TH target genes during natural and TH-induced metamorphosis. These suggest that TH induces Dot1L expression and Dot1L in turn functions as a TR coactivator to promote vertebrate development. We have now shown that in co-transfection studies or in the reconstituted frog oocyte in vivo transcription system, overexpression of Dot1L enhances gene activation by TR in the presence of TH. Furthermore, making use of the ability to carry out transgenesis in Xenopus laevis and gene knockdown in Xenopus tropicalis, we have demonstrated that endogenous Dot1L is critical for TH-induced activation of endogenous TR target genes while transgenic Dot1L enhances endogenous TR function in premetamorphic tadpoles in the presence of TH. Our studies thus provide, for the first time, complementary gain- and loss-of functional evidence in vivo for a cofactor, Dot1L, in gene activation by TR during vertebrate development.