Inhibition of DNMT1 methyltransferase activity via glucose-regulated O-GlcNAcylation alters the epigenome
- City of Hope, United States
- Translational Genomics Research Institute, United States
Abstract
The DNA methyltransferase activity of DNMT1 is vital for genomic maintenance of DNA methylation. We report here that DNMT1 function is regulated by O-GlcNAcylation, a protein modification that is sensitive to glucose levels, and that elevated O-GlcNAcylation of DNMT1 from high glucose environment leads to alterations to the epigenome. Using mass spectrometry and complementary alanine mutation experiments, we identified S878 as the major residue that is O-GlcNAcylated on human DNMT1. Functional studies in human and mouse cells further revealed that O-GlcNAcylation of DNMT1-S878 results in an inhibition of methyltransferase activity, resulting in a general loss of DNA methylation that preferentially occurs at partially methylated domains (PMDs). This loss of methylation corresponds with an increase in DNA damage and apoptosis. These results establish O-GlcNAcylation of DNMT1 as a mechanism through which the epigenome is regulated by glucose metabolism and implicates a role for glycosylation of DNMT1 in metabolic diseases characterized by hyperglycemia.
Data availability
PromethION sequencing data have been deposited in the NCBI Gene Expression Omnibus (GEO) and Sequence Read Archive (SRA) under accession no. GSE201470. Mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD043031.
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Inhibition of DNMT1 methyltransferase activity via glucose-regulated O-GlcNAcylation alters the epigenomeNCBI Gene Expression Omnibus, GSE201470.
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Whole-genome analysis of the methylome and hydroxymethylome in normal and malignant lung and liverNCBI Gene Expression Omnibus, GSE70091.
Article and author information
Author details
Funding
National Institutes of Health (R01DK112041)
- Dustin E Schones
National Institutes of Health (R01CA220693)
- Dustin E Schones
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: All animal experiments conducted have been approved by the Institutional Animal Care and Use Committees at City of Hope. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols (#17010).
Copyright
© 2023, Shin et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Inhibition of DNMT1 methyltransferase activity via glucose-regulated O-GlcNAcylation alters the epigenome
eLife 12:e85595.
https://doi.org/10.7554/eLife.85595
Further reading
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- Chromosomes and Gene Expression
- Genetics and Genomics
Among the major classes of RNAs in the cell, tRNAs remain the most difficult to characterize via deep sequencing approaches, as tRNA structure and nucleotide modifications can each interfere with cDNA synthesis by commonly-used reverse transcriptases (RTs). Here, we benchmark a recently-developed RNA cloning protocol, termed Ordered Two-Template Relay (OTTR), to characterize intact tRNAs and tRNA fragments in budding yeast and in mouse tissues. We show that OTTR successfully captures both full-length tRNAs and tRNA fragments in budding yeast and in mouse reproductive tissues without any prior enzymatic treatment, and that tRNA cloning efficiency can be further enhanced via AlkB-mediated demethylation of modified nucleotides. As with other recent tRNA cloning protocols, we find that a subset of nucleotide modifications leave misincorporation signatures in OTTR datasets, enabling their detection without any additional protocol steps. Focusing on tRNA cleavage products, we compare OTTR with several standard small RNA-Seq protocols, finding that OTTR provides the most accurate picture of tRNA fragment levels by comparison to "ground truth" Northern blots. Applying this protocol to mature mouse spermatozoa, our data dramatically alter our understanding of the small RNA cargo of mature mammalian sperm, revealing a far more complex population of tRNA fragments - including both 5′ and 3′ tRNA halves derived from the majority of tRNAs – than previously appreciated. Taken together, our data confirm the superior performance of OTTR to commercial protocols in analysis of tRNA fragments, and force a reappraisal of potential epigenetic functions of the sperm small RNA payload.
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- Chromosomes and Gene Expression
O-GlcNAcylation is the reversible post-translational addition of β-N-acetylglucosamine to serine and threonine residues of nuclear and cytoplasmic proteins. It plays an important role in several cellular processes through the modification of thousands of protein substrates. O-GlcNAcylation in humans is mediated by a single essential enzyme, O-GlcNAc transferase (OGT). OGT, together with the sole O-GlcNAcase OGA, form an intricate feedback loop to maintain O-GlcNAc homeostasis in response to changes in cellular O-GlcNAc using a dynamic mechanism involving nuclear retention of its fourth intron. However, the molecular mechanism of this dynamic regulation remains unclear. Using an O-GlcNAc responsive GFP reporter cell line, we identify SFSWAP, a poorly characterized splicing factor, as a trans-acting factor regulating OGT intron detention. We show that SFSWAP is a global regulator of retained intron splicing and exon skipping that primarily acts as a negative regulator of splicing. In contrast, knockdown of SFSWAP leads to reduced inclusion of a ‘decoy exon’ present in the OGT retained intron which may mediate its role in OGT intron detention. Global analysis of decoy exon inclusion in SFSWAP and UPF1 double knockdown cells indicate altered patterns of decoy exon usage. Together, these data indicate a role for SFSWAP as a global negative regulator of pre-mRNA splicing and positive regulator of intron retention.
