Thyroid Hormone Induces DNA Demethylation in Xenopus Tadpole Brain

Thyroid hormone (T-3) plays pivotal roles in vertebrate development, acting via nuclear T-3 receptors (TRs) that regulate gene transcription by promoting post-translational modifications to histones. Methylation of cytosine residues in deoxyribonucleic acid (DNA) also modulates gene transcription, and our recent finding of predominant DNA demethylation in the brain of Xenopus tadpoles at metamorphosis, a T-3-dependent developmental process, caused us to hypothesize that T-3 induces these changes in vivo. Treatment of premetamorphic tadpoles with T-3 for 24 or 48 hours increased immunoreactivity in several brain regions for the DNA demethylation intermediates 5-hydroxymethylcytosine (5-hmC) and 5-carboxylcytosine, and the methylcytosine dioxygenase ten-eleven translocation 3 (TET3). Thyroid hormone treatment induced locus-specific DNA demethylation in proximity to known T-3 response elements within the DNA methyltransferase 3a and Kruppel-like factor 9 genes, analyzed by 5-hmC immunoprecipitation and methylation sensitive restriction enzyme digest. Chromatin-immunoprecipitation (ChIP) assay showed that T-3 induced TET3 recruitment to these loci. Furthermore, the messenger ribonucleic acid for several genes encoding DNA demethylation enzymes were induced by T-3 in a time-dependent manner in tadpole brain. A TR ChIP-sequencing experiment identified putative TR binding sites at several of these genes, and we provide multiple lines of evidence to support that tet2 contains a bona fide T-3 response element. Our findings show that T-3 can promote DNA demethylation in developing tadpole brain, in part by promoting TET3 recruitment to discrete genomic regions, and by inducing genes that encode DNA demethylation enzymes.