Dataset Information

Thyroid Hormone Induces DNA Demethylation in Xenopus Tadpole Brain.

ABSTRACT: Thyroid hormone (T3) plays pivotal roles in vertebrate development, acting via nuclear T3 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 T3-dependent developmental process, caused us to hypothesize that T3 induces these changes in vivo. Treatment of premetamorphic tadpoles with T3 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 T3 response elements within the DNA methyltransferase 3a and Krüppel-like factor 9 genes, analyzed by 5-hmC immunoprecipitation and methylation sensitive restriction enzyme digest. Chromatin-immunoprecipitation (ChIP) assay showed that T3 induced TET3 recruitment to these loci. Furthermore, the messenger ribonucleic acid for several genes encoding DNA demethylation enzymes were induced by T3 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 T3 response element. Our findings show that T3 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.

SUBMITTER: Raj S

PROVIDER: S-EPMC7947600 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Methylation 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.

Project description:The Xenopus laevis experimental system has provided significant insight into the development and plasticity of neural circuits. Xenopus neuroscience research would be enhanced by additional tools to study neural circuit structure and function. Rabies viruses are powerful tools to label and manipulate neural circuits and have been widely used to study mesoscale connectomics. Whether rabies virus can be used to transduce neurons and express transgenes in Xenopus has not been systematically investigated. Glycoprotein-deleted rabies virus transduces neurons at the axon terminal and retrogradely labels their cell bodies. We show that glycoprotein-deleted rabies virus infects local and projection neurons in the Xenopus tadpole when directly injected into brain tissue. Pseudotyping glycoprotein-deleted rabies with EnvA restricts infection to cells with exogenous expression of the EnvA receptor, TVA. EnvA pseudotyped virus specifically infects tadpole neurons with promoter-driven expression of TVA, demonstrating its utility to label targeted neuronal populations. Neuronal cell types are defined by a combination of features including anatomical location, expression of genetic markers, axon projection sites, morphology, and physiological properties. We show that driving TVA expression in one hemisphere and injecting EnvA pseudotyped virus into the contralateral hemisphere, retrogradely labels neurons defined by cell body location and axon projection site. Using this approach, rabies can be used to identify cell types in Xenopus brain and simultaneously to express transgenes which enable monitoring or manipulation of neuronal activity. This makes rabies a valuable tool to study the structure and function of neural circuits in Xenopus.Significance StatementStudies in Xenopus have contributed a great deal to our understanding of brain circuit development and plasticity, regeneration, and hormonal regulation of behavior and metamorphosis. Here, we show that recombinant rabies virus transduces neurons in the Xenopus tadpole, enlarging the toolbox that can be applied to studying Xenopus brain. Rabies can be used for retrograde labeling and expression of a broad range of transgenes including fluorescent proteins for anatomical tracing and studying neuronal morphology, voltage or calcium indicators to visualize neuronal activity, and photo- or chemosensitive channels to control neuronal activity. The versatility of these tools enables diverse experiments to analyze and manipulate Xenopus brain structure and function, including mesoscale connectivity.

Dataset Information

Thyroid Hormone Induces DNA Demethylation in Xenopus Tadpole Brain.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets