Molecular mechanisms of Tet1 and Tet2 mediated DNA demethylation
Final Report Abstract
The aim of the project was to dissect the molecular mechanism which TET enzymes and cellular machinery use for guiding demethylation activity to relevant genomic locations and to understand how demethylation activity is modulated by vitamins. In the first part of the project, we aimed to understand how the activity of TET enzymes is controlled by vitamin C and A. Vitamin C was shown to promote the catalytic activity of TET enzymes and enhance the process of reprogramming to pluripotency. However, the molecular mechanism behind the observed effects was not understood. We have used a combination of biochemical and cellular assays aided with epigenomics to dissect this process. We discovered that ascorbate (vitamin C)-mediated enhancement of TET catalytic activity is due to preservation Fe2+ abundance through reduction of Fe3+ to Fe2+ both in vitro and in stem cells. Vitamin A, on the other hand, upregulates the expression of TET enzymes in the cells while showing no direct effect on the catalytic proficiency of the enzymes. Cosupplementation of vitamin A and vitamin C to cell culture media, revealed an additive effect providing optimal demethylation activity in cells leading to enhanced efficiency of epiblast cell de-differentiation to pluripotency. In the second part of the project, we have identified that the catalytic domains of mammalian TET enzymes possess a very strong (>250 fold) intrinsic sequence preference that targets the enzymes to a subset of CG sites embedded in a specific sequence context. Ultimately, we discovered that E-box sites (CACGTG), which are also binding sites for the iconic c-Myc and Jun/Fos immediate early TF, are the most preferred substrate site for mammalian TETs. The least preferred TET substrates are CG sequences embedded in GCGC flanks which are also bound by methylation dependent TFs, like Oct4. We observed this dramatic preference with purified recombinant enzymes in vitro, as well as in TET- triple knock-out embryonic stem cells inducibly expressing TET enzymes (in cellulo) and in vivo by analysing the loss of methylation signatures in cell systems where TET activity is critical for the observed cell state transition. We have used X-ray crystallography to elucidate the molecular principle governing this striking sequence preference. Comparison of the obtained high-resolution X-ray structures of human TET2 catalytic domain bound to the best and worst DNA substrate, allowed us to discover that the structural principle guiding TET preference. These obtained results are highly significant to understanding of TET biology and in particular how TET enzymes impact the cellular epigenetic and transcriptional programmes. In the course of this project a very large amount of nucleic acid samples had to be purified and handled. Because the classical column-based extraction protocols are not easily automated and are very costly when used at scale, we have developed in house magnetic bead-based protocols for highthroughput RNA/DNA isolations. These protocols allowed us to process hundreds and thousands of samples with a tight budget and limited workforce. We have offered these open-access protocols to the wider scientific community (http://bomb.bio) and benefited the multiple laboratories across the world. Last but not least, due to the scalability of the BOMB protocols, these methods and protocols are currently successfully used in multiple countries (New Zealand, UK, Columbia, Finland and many other) for automated COVID19 testing, allowing high-throughput, magnetic isolation of COVID19 RNA and downstream analysis. Overall, in this project we uncovered fundamental principles behind TET biology which bring new light and understanding of the role of TET proteins in cell state transition and homeostasis and will benefit wider scientific community and in particular developmental biologists, cancer biologists, epigeneticist.
Publications
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(2016) Retinol and ascorbate drive erasure of epigenetic memory and enhance reprogramming to naive pluripotency by complementary mechanisms. Proceedings of the National Academy of Sciences 113 (43), 12202-12207
TA Hore, F von Meyenn, M Ravichandran, Martin Bachman, Gabriella Ficz, David Oxley, Fátima Santos, Shankar Balasubramanian, TP Jurkowski, W Reik
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(2018) Target specificity of mammalian DNA methylation and demethylation machinery. Organic & biomolecular chemistry 16 (9), 1419-1435
M Ravichandran, RZ Jurkowska, TP Jurkowski
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(2019) Bio-On-Magnetic-Beads (BOMB): Open platform for high-throughput nucleic acid extraction and manipulation. PLoS biology 17 (1), e3000107
P Oberacker, P Stepper, DM Bond, S Höhn, J Focken, V Meyer, L Schelle, VJ Sugrue, G-J Jeunen, T Moser, SR Hore, F von Meyenn, K Hipp, TA Hore, TP Jurkowski
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(2019) Establishment, Erasure and Synthetic Reprogramming of DNA Methylation in Mammalian Cells. Book chapter in “The DNA, RNA, and Histone Methylomes”, 1-26
RZ Jurkowska, TP Jurkowski
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(2019) Non-invasive detection of DNA methylation states in carcinoma and pluripotent stem cells using Raman microspectroscopy and imaging. Scientific reports 9 (1), 1-13
R Daum, EM Brauchle, DAC Berrio, TP Jurkowski, K Schenke-Layland
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(2020) Technologies and Applications for the Assessment of 5-Hydroxymethylcytosine. Epigenetics Methods - book chapter
TP Jurkowski
