Abstract
The TET family of dioxygenases (TET1/2/3) can convert 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) and has been shown to be involved in active and passive DNA demethylation. Here, we demonstrate that altering TET dioxygenase levels within physiological range can affect DNA methylation dynamics of HEK293 cells. Overexpression of TET1 increased global 5hmC levels and was accompanied by mild DNA demethylation of promoters, gene bodies and CpG islands. Conversely, the simultaneous knockdown of TET1, TET2, and TET3 led to decreased global 5hmC levels and mild DNA hypermethylation of above-mentioned regions. The methylation changes observed in the overexpression and knockdown studies were mostly non-reciprocal and occurred with different preference depending on endogenous methylation and gene expression levels. Single-nucleotide 5hmC profiling performed on a genome-wide scale revealed that TET1 overexpression induced 5mC oxidation without a distribution bias among genetic elements and structures. Detailed analysis showed that this oxidation was related to endogenous 5hmC levels. In addition, our results support the notion that the effects of TET1 overexpression on gene expression are generally unrelated to its catalytic activity.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
We would like to thank Ludger Klein-Hitpass from the BioChip laboratory Essen and Walter Pulverer from the Austrian Institute of Technology for their kind help with the project. We would also like to thank Fabian Müller (Max Planck Institute for Informatics) and Zymo Research for their kind help. We thank Anjana Rao (La Jolla Institute for Allergy and Immunology) for providing the TET1 cDNA. Thanks also go to Hannah Demond for proofreading of the manuscript.
Supplemental Material
Supplemental data for this article can be accessed on the publisher's website.
Funding
This project is supported by the Deutsche Forschungsgemeinschaft (DFG) graduate research training program no. 1431: “Transcription, Chromatin Structure and DNA Repair in Development and Differentiation.” The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.