OGT prevents DNA demethylation and suppresses the expression of transposable elements in heterochromatin by restraining TET activity genome-wide

Background 

The storyline of this preprint revolves around four main characters:

• Transposable elements (TEs) – as the genomic loci of interest.
• DNA methylation – as the epigenetic modification that represses TE expression.
• The TET & DNMT1 enzymes – as the regulators of DNA methylation.
• The metabolic enzyme OGT – as the regulator of TET & DNMT1 levels and activity.

Transposable elements are dynamically expressed in pre-implantation mouse embryos in a stage-specific manner [1, 2]. Pluripotent stem cells, derived from the inner cell mass of the blastocyst, also express TEs [3]. Recently, TEs have been identified as the key regulator of cell fate decisions in preimplantation mouse embryos [4].

Repressive histone modifications and DNA methylation restrict the expression of TEs [5]. DNA methylation, primarily at the cytosine (C) residue, is carried out by DNA methyltransferases (DNMT1, DNM3A, DNM3B) to give rise to 5-methylcytosine (5mC). The demethylation is carried out by another enzyme family – The Ten-eleven translocation (TET1,2&3), which oxidise 5mC to 5-hydroxymethylcytosine (5hmC).

The central stage in this story is occupied by OGT (O-GlcNAc transferase), a metabolic enzyme that transfers N-acetylglucosamine from UDP-GlcNAc to a Ser/Thr residue of a protein. Inhibition of OGT leads to developmental arrest in mouse embryos [6]. In diverse model systems, OGT is known to interact with chromatin-modifying enzymes including the TET proteins. In this preprint, the authors delve into understanding the functional relevance of OGT-TET interaction in mouse embryonic stem cells (mESCs).

Key findings 

• The Interactome of OGT – Using immunoprecipitation followed by mass spectrometry, components of epigenetic regulatory complexes were found to interact with physiological levels of OGT in mESCs. These complexes include the NURD complex (MBD3, HDAC1, HDAC2), PR-DUB complex (ASXL1-BAP1), PRC2 complex (SUZ12, EED, 267 JARID2), MLL/COMPASS complex (HCF1), PROSER1, KAP1, ZFP57 and ZFP655, DNMT1, TET1, and TET2.

• OGT deletion affects DNA methylation – The Ogt iKO cells showed a decrease in DNMT, UHRF1, and TET protein levels. This was accompanied by a global reduction of DNA methylation in 10 kb windows across the entire genome and an increase in 5hmc (product of TET) in both euchromatin and heterochromatin. Interestingly, the disruption in TET-OGT interaction via a point mutation in TET1 phenocopied the decrease in global DNA methylation (5mC+5hmC). The gain in 5hmC, despite the reduction in TET1 and TET2 protein levels, was suggestive of two roles for OGT – (i) maintenance of TET1 and TET2 protein levels in wildtype mESCs and (ii) suppression of TET enzymatic activity.

• Change in Transposable element expression in Ogt-iKO mESCs – The decrease in 5mC on TE loci located in the heterochromatin led to their de-repression. MERVL, IAPs, and young L1 TF, GF, and A elements were the TE families that were most strikingly upregulated in the knock-out cell lines. The Chip-Seq for O-GlcNAc showed an enrichment of O-GlcNAc modification on the regulatory regions of transposable elements in wildtype. The authors speculate that spurious expression of TEs after Ogt deletion might be a consequence of loss of 5mC and/or loss of the O-GlcNAc modification in chromatin proteins binding TE regulatory regions.

• Gene expression changes in Ogt iKO cells  –  Ogt iKO mESCs showed upregulation of 2C-like genes, imprinted genes, and Interferon-Stimulated Genes (ISGs), whereas genes encoding the rapidly evolving KRAB ZFPs were downregulated.

Taken together, this preprint reveals a previously unknown function of OGT in regulating transposable elements and gene expression in mESCs by influencing TET levels and activity, and DNA methylation dynamics.

What I like about this preprint

During my PhD, I probed for the total proteome changes that occur in mESCs upon incubation with a-ketoglutarate and sodium-acetate for 24 hours. Interestingly, OGT, DNMT1, and ZFP57 protein levels changed significantly in both conditions. These were mere correlations though; this preprint has gone into understanding the functional consequence of the interaction between a metabolic enzyme and an epigenetic regulator in pluripotent stem cells. This is a very difficult thing to do, but the authors have used a series of high throughput and sensitive methods to uncover a mechanism explaining how OGT-TET acts together to modulate gene expression.

Questions to the authors  

1. Since OGT is a metabolic enzyme, did you observe any notable changes in the glucose metabolic profile of Ogt iKO mECSs after 4-OHT induction?
2. What happens to the core pluripotency transcriptional network (OCT4, SOX2, NANOG, KLF4) in cells deficient for OGT?
3. TE expression may be a stochastic alteration in gene expression or could lead to lineage priming. Do you see any differences in Ogt iKO cells in terms of (a) differentiation potential into three germ layers, (b) self-organizing properties, or (c) reprogramming efficiency to totipotent-like stage?

References: 

1. Zhang, W., et al., Zscan4c activates endogenous retrovirus MERVL and cleavage embryo genes.Nucleic acids research, 2019. 47(16): p. 8485-8501.
2. Oomen, M.E. and M.-E. Torres-Padilla, Jump-starting life: balancing transposable element co-option and genome integrity in the developing mammalian embryo.EMBO reports, 2024: p. 1-13.
3. Hackett, J.A., et al., Activation of lineage regulators and transposable elements across a pluripotent spectrum.Stem cell reports, 2017. 8(6): p. 1645-1658.
4. Sakashita, A., et al., Transcription of MERVL retrotransposons is required for preimplantation embryo development.Nature genetics, 2023. 55(3): p. 484-495.
5. Lu, X., Regulation of endogenous retroviruses in murine embryonic stem cells and early embryos.Journal of Molecular Cell Biology, 2023. 15(8): p. mjad052.
6. Chi, F., et al., Glycolysis-independent glucose metabolism distinguishes TE from ICM fate during mammalian embryogenesis.Developmental cell, 2020. 53(1): p. 9-26. e4.

 

doi: https://doi.org/10.1242/prelights.37128

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