Thyroid Hormone Induces DNA Demethylation in Developing Tadpole Brain

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 T3 in modulating DNA methylation, which together with histone modifications alters chromatin structure and gene transcription. Methylation of DNA is a key epigenetic modification regulating gene transcription typically leading to repression; while, DNA demethylation favors activation. I used Xenopus tadpole metamorphosis, a T3 - dependent postembryonic developmental process, to investigate a possible role for T3 in the regulation of DNA methylation in brain development of a vertebrate. I investigated developmental and T3-dependent changes in mRNA levels of genes that code for DNA demethylation enzymes (tet2, tet3, idh1/2/3, gadd45α/β/γ and tdg) in the diencephalon of X. tropicalis brain. I found that the mRNAs for each of these genes increased during metamorphosis and reached a maximum at metamorphic climax, and that tet2 and gadd45γ are direct T3 target genes. Using immunohistochemistry, I investigated the changes in distribution of ten eleven translocase 3 (TET3), a methylated DNA-binding dioxygenase that catalyzes conversion of 5 methylcytosine (5-mC) to 5 hydroxymethylcytosine (5-hmC), and the active DNA demethylation 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 highly responsive to actions of T3 during metamorphosis and reached a maximum at metamorphic climax; TET3, 5-hmC and 5-caC ir could also be induced by treating premetamorphic tadpoles with exogenous T3. I used three independent assays to study locus specific changes in DNA methylation at T3 response elements (TREs) of the known T3-regulated gene, DNA methyltransferase 3a (dnmt3a). Using bisulfite sequencing, I discovered that one of the TREs within dnmt3a (TRE-A) underwent DNA demethylation during spontaneous metamorphosis. Using immunoprecipitation for 5-hmC, I found that treatment of premetamorphic tadpoles with T3 increased 5-hmC around the dnmt3a TRE-A in the genome of tadpole neural cells and that T3 treatment increased recruitment to chromatin of TET3 around dnmt3a TRE-A evidenced by chromatin immunoprecipitation assay. I also found that TET3 was recruited, to chromatin at regions of TREs of the T3 target genes 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 of DNA demethylation genes, particularly tet2 and gadd45γ and also by direct T3-induced DNA demethylation at TREs of known T3 response genes catalyzed by T3-dependent active recruitment of TET3. To my knowledge, this is the first study to identify a role for T3 in modulating DNA methylation in the developing brain of a vertebrate and identifying a clear positive correlation between T3, mRNA levels of DNA demethylation genes and DNA 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 of DNA demethylation.PHDMolecular, Cellular, and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/146012/1/samraj_1.pd

DNA methylation dynamics underlie metamorphic gene regulation programs in Xenopus tadpole brain

International audienceMethylation of cytosine residues in DNA influences chromatin structure and gene transcription, and its regulation is crucial for brain development. There is mounting evidence that DNA methylation can be modulated by hormone signaling. We analyzed genome-wide changes in DNA methylation and their relationship to gene regulation in the brain of Xenopus tadpoles during metamorphosis, a thyroid hormone-dependent developmental process. We studied the region of the tadpole brain containing neurosecretory neurons that control pituitary hormone secretion, a region that is highly responsive to thyroid hormone action. Using Methylated DNA Capture sequencing (MethylCap-seq) we discovered a diverse landscape of DNA methylation across the tadpole neural cell genome, and pairwise stage comparisons identified several thousand differentially methylated regions (DMRs). During the pre-to pro-metamorphic period, the number of DMRs was lowest (1,163), with demethylation predominating. From pre-metamorphosis to metamorphic climax DMRs nearly doubled (2,204), with methylation predominating. The largest changes in DNA methylation were seen from metamorphic climax to the completion of metamorphosis (2960 DMRs), with 80% of the DMRs representing demethylation. Using RNA sequencing, we found negative correlations between differentially expressed genes and DMRs localized to gene bodies and regions upstream of transcription start sites. DNA demethylation at metamorphosis revealed by MethylCap-seq was corroborated by increased immunoreactivity for the DNA demethylation intermediates 5-hydroxymethylcytosine and 5-carboxymethylcytosine, and the methylcytosine dioxygenase ten eleven translocation 3 that catalyzes DNA demethylation. Our findings show that the genome of tadpole neural cells undergoes significant changes in DNA methylation during metamorphosis, and these changes likely influence chromatin architecture, and gene regulation programs occurring during this developmental period

AI-driven techniques for controlling the metal melting production: a review, processes, enabling technologies, solutions, and research challenges

Artificial Intelligence has left no stone unturned, and mechanical engineering is one of its biggest consumers. Such technological advancements in metal melting can help in process simplification, hazard reduction, human involvement reduction & lesser process time. Implementing the AI models in the melting technology will ultimately help various industries, i.e., Foundry, Architecture, Jewelry Industry, etc. This review extensively sheds light on Artificial Intelligence models implemented in metal melting processes or the metal melting aspect, alongside explaining additive manufacturing as a competitor to the current melting processes and its advances in metal melting and AI implementations

Deciphering the regulatory logic of an ancient, ultraconserved nuclear receptor enhancer module.

Cooperative, synergistic gene regulation by nuclear hormone receptors can increase sensitivity and amplify cellular responses to hormones. We investigated thyroid hormone (TH) and glucocorticoid (GC) synergy on the Krüppel-like factor 9 (Klf9) gene, which codes for a zinc finger transcription factor involved in development and homeostasis of diverse tissues. We identified regions of the Xenopus and mouse Klf9 genes 5-6 kb upstream of the transcription start sites that supported synergistic transactivation by TH plus GC. Within these regions, we found an orthologous sequence of approximately 180 bp that is highly conserved among tetrapods, but absent in other chordates, and possesses chromatin marks characteristic of an enhancer element. The Xenopus and mouse approximately 180-bp DNA element conferred synergistic transactivation by hormones in transient transfection assays, so we designate this the Klf9 synergy module (KSM). We identified binding sites within the mouse KSM for TH receptor, GC receptor, and nuclear factor κB. TH strongly increased recruitment of liganded GC receptor and serine 5 phosphorylated (initiating) RNA polymerase II to chromatin at the KSM, suggesting a mechanism for transcriptional synergy. The KSM is transcribed to generate long noncoding RNAs, which are also synergistically induced by combined hormone treatment, and the KSM interacts with the Klf9 promoter and a far upstream region through chromosomal looping. Our findings support that the KSM plays a central role in hormone regulation of vertebrate Klf9 genes, it evolved in the tetrapod lineage, and has been maintained by strong stabilizing selection. Mol Endocrinol 2015 Jun; 29(6):856-72