Thyroid Hormone Induces DNA Demethylation in Developing Tadpole Brain

DNA demethylation and thyroid hormone;Molecular;Cellular and Developmental Biology;Science;Molecular, Cellular, and Developmental Biology

Raj, SamhithaDuan, Cunming ;

University of Michigan

关键词: DNA demethylation and thyroid hormone;    Molecular;    Cellular and Developmental Biology;    Science;    Molecular, Cellular, and Developmental Biology;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/146012/samraj_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Thyroid hormone (T3) plays important roles in vertebrate brain development. The actions of T3 are mediated by transcriptional regulation through the T3 receptors (TR) that serve as epigenetic switches to modify chromatin structure. The role of T3 in histone modifications is well studied, but virtually nothing is known about a potential role for T3in modulating DNA methylation, which together with histone modifications alterschromatin structure and gene transcription. Methylation of DNA is a key epigeneticmodification regulating gene transcription typically leading to repression; while, DNAdemethylation favors activation. I used Xenopus tadpole metamorphosis, a T3 -dependent postembryonic developmental process, to investigate a possible role for T3in the regulation of DNA methylation in brain development of a vertebrate.I investigated developmental and T3-dependent changes in mRNA levels ofgenes that code for DNA demethylation enzymes (tet2, tet3, idh1/2/3, gadd45α/β/γ andtdg) in the diencephalon of X. tropicalis brain. I found that the mRNAs for each of thesegenes increased during metamorphosis and reached a maximum at metamorphicclimax, and that tet2 and gadd45γ are direct T3 target genes. Usingimmunohistochemistry, I investigated the changes in distribution of ten eleventranslocase 3 (TET3), a methylated DNA-binding dioxygenase that catalyzes conversionof 5 methylcytosine (5-mC) to 5 hydroxymethylcytosine (5-hmC), and the active DNAdemethylation intermediates 5-hmC and 5 carboxy methylcytosine (5-caC)immunoreactivity (ir). I found that the TET3, 5-hmC and 5-caC ir increased around the thalamic nuclei and ventral hypothalamus of the tadpole brain, known to be highlyresponsive to actions of T3 during metamorphosis and reached a maximum atmetamorphic climax; TET3, 5-hmC and 5-caC ir could also be induced by treatingpremetamorphic tadpoles with exogenous T3.I used three independent assays to study locus specific changes in DNAmethylation at T3 response elements (TREs) of the known T3-regulated gene, DNAmethyltransferase 3a (dnmt3a). Using bisulfite sequencing, I discovered that one of theTREs within dnmt3a (TRE-A) underwent DNA demethylation during spontaneousmetamorphosis. Using immunoprecipitation for 5-hmC, I found that treatment ofpremetamorphic tadpoles with T3 increased 5-hmC around the dnmt3a TRE-A in thegenome of tadpole neural cells and that T3 treatment increased recruitment to chromatinof TET3 around dnmt3a TRE-A evidenced by chromatin immunoprecipitation assay. Ialso found that TET3 was recruited, to chromatin at regions of TREs of the T3 targetgenes trb, th/bzip, klf9 and gadd45γ.Taken together, my findings support that T3 induces DNA demethylation in the X.tropicalis tadpole brain and this is mediated in part, by direct T3-mediated regulation ofDNA demethylation genes, particularly tet2 and gadd45γ and also by direct T3-inducedDNA demethylation at TREs of known T3 response genes catalyzed by T3-dependentactive recruitment of TET3. To my knowledge, this is the first study to identify a role forT3 in modulating DNA methylation in the developing brain of a vertebrate and identifyinga clear positive correlation between T3, mRNA levels of DNA demethylation genes andDNA demethylation intermediates. It is also the first study to suggest that T3 could induce locus specific DNA demethylation through recruitment of TET3 to the sites ofDNA demethylation.