Inhibition of DNMT1 methyltransferase activity via glucose-regulated O-GlcNAcylation alters the epigenome

  1. Heon Shin
  2. Amy Leung
  3. Kevin R Costello
  4. Parijat Senapati
  5. Hiroyuki Kato
  6. Roger E Moore
  7. Michael Lee
  8. Dimitri Lin
  9. Xiaofang Tang
  10. Patrick Pirrotte
  11. Zhen Bouman Chen
  12. Dustin E Schones

  • Curated by eLife

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    eLife assessment

    This study explores the regulatory function of O-GlcNAcylation on DNA methyltransferase 1 and identifies serine 878 as the main target. This study is of interest to those in epigenetics and metabolism. The significance is important and the strength of the evidence is convincing.

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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.

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  1. Version published to 10.7554/elife.85595 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    In this study, Shin and colleagues investigate the role of the posttranslational modification of the DNA methyltransferase by covalent linkage of the N-Acetylglucosamine (O-GlcNAc).

    The authors present compelling evidence showing that a prolonged high fat/sucrose diet causes global protein O-GlcNAcylation in the liver and DNMT1 is among the proteins that increase their O-GlcNAc level. This result is significant because of the paucity of in vivo data addressing the interplay between metabolism and protein O-GlcNAcylation. The paper also shows that DNMT1's O-GlcNAcylation level correlated to the extracellular glucose levels in other cell types.

    Using mass spectrometry, the authors identify S878 as the main site for O-GlcNAcylation. It is noteworthy that the mapping was performed with hyper-O-GlcNAcylated cells and may be different in a physiological situation. To investigate how O-GlcNAcylation of S878 of DNMT1 impacts its activity and ultimately DNA methylation patterns, Shin and colleagues mostly use a cellular model of hyper O-GlcNAcylation induced by the combination of high glucose and a chemical inhibitor of OGA (the only enzyme responsible for O-GlcNAc removal). The data shows that increased O-GlcNAcylation resulting from the combination of high glucose and OGA inhibition causes a reduction of DNMT1 activity and local loss of DNA methylation specifically at partially methylated domains.

    This study brings completely new knowledge on the regulatory function of glycosylation of DNMT1 and its impact on its methyl-transferase activity and downstream genomic methylation. Furthermore, the manuscript introduces new data on the interplay between cellular metabolism and O-GlcNAcylation on DNMT1 and other proteins. The experiments are well-controlled, and their interpretation is sound. This study should be of special interest to the fields of fundamental and environmental epigenetics, as well as metabolism.

    The main limitation of the study is the convolution of the functional experiments where the perturbation is a combination of high glucose and chemical inhibition of OGA. The relative contribution of the two variables is partially addressed in Figure 3-figure supplement 1B which shows that high glucose increases DNMT1 activity (Hep3B cells) while Figure 3D shows that high glucose when combined with OGA inhibitor decreases DNMT1 activity (Hep3B cells). As discussed, the data suggest that high-glucose and OGA inhibition may have an antagonistic effect on DNMT1 activity. An experiment of treatment of the cells with the OGA inhibitor in physiological glucose conditions would address this gap of knowledge.

    We thank the reviewer for the suggestion. The physiological glucose levels are between 5 to 7 mM, and 25mM is in hyperglycemic range, which corresponds to severe diabetes. The new Figure 1A shows TMG treatment with physiological glucose conditions. We have included new WB data of 5mM glucose, 5mM glucose + TMG, 25mM glucose, and 25mM glucose + TMG (Figure 1A).

    To understand the impact of the environment (in this study: extracellular glucose level) on the epigenome, one should keep in mind the variation of cytosine methylation patterns between individuals and over time. A recent large-scale profiling of DNA methylation of 137 individuals shows a near absence of individual variation between replicates of the same cell type, suggesting that genomic methylation patterns are largely insensitive to the environment (https://doi.org/10.1038/s41586-022-05580-6).

    Comparative methylomes of healthy and diabetic individuals are needed to examine the medical significance of the findings presented here. It is possible that the modulation of DNMT1 activity by O-GlcNAc modification is relevant for a specific cell type or developmental stage that remains to be discovered.

    We thank the reviewer for the suggestion. While the present study is focused on the functional impact of glucose concentrations on O-GlcNAcylation of DNMT1, the extension of this work to diabetic individuals is a goal for a follow up project.

    Reviewer #2 (Public Review):

    I've read the manuscript by Shin et al with great interest. The authors describe the identification of O-GlcNAcylation of DNMT1 and the impact this modification has on the maintenance activity of DNMT1 genome-wide and that modification of S878 leads to enzyme inhibition. The manuscript is written in a clear and understandable way making it easy for the reader to understand the logic as well as the steps of the experimental approach.

    The authors identify O-GlcNAcylation of DNMT1 in a number of different cell lines by combining inhibition studies and WB and further on they identify the modification sites with LC/MS, predictions, and mutational studies. I really like the experimental approach, which while being straightforward (albeit technically challenging), is powerful and well-controlled in this case to unequivocally prove the modification of DNMT1 and identify the site. However, mutation of the two identified modification sites does not remove all the O-GlcNAcylation signal associated with DNMT1, thus possibly not all the possible sites were identified. While this is not a criticism of this manuscript, it would be interesting to know what other sites are modified and the enzymatic/biological effects associated.

    We completely agree with the reviewer. As the O-GlcNAc band was also detected in double mutated DNMT1 (Figure 2D), it is expected that undetected O-GlcNAcylated sites will exist. This is a limitation of current MS analysis and is known to be difficult to detect in the case of modified sites located at both 5’- and 3’- ends of the protein or around the site cut by endoprotease such as trypsin. In follow up work we plan to detect more diverse O-GlcNAc modified sites using more types of endoproteases and observe changes in the phenotype of various cells accordingly.

    Also, the authors isolate the modified DNMT1 from cells using immunoprecipitation, which is indeed useful to study the changes in catalytic activity but does not provide any information if the cellular localisation of modified DNMT1 changes.

    We apologize for this oversight. We have added a DNMT1 localization assay via immunofluorescence (IF) in the revised manuscript (Figure 3—figure supplement 3). We found no difference in DNMT1 localization between wild type and S878A mutants.

    Subsequently, the authors checked the impact of high glucose diet on the genome-wide DNA methylation patterns. The observed effects (Fig 4A) are very strong, almost as strong as observed with Aza treatment and therefore I wonder if LINE/IAP or other elements are getting activated (as observed with genome-wide demethylation with Aza).

    We thank the reviewer for the suggestion. Changes in methylation of LINE-1 by hyperglycemia condition are displayed in Figure 4—figure supplement 4. In the case of LINE-1, DNA methylation is lost globally in hyperglycemia conditions. While beyond the scope of this study, a more thorough examination of the impact of the observed loss of methylation under high glucose conditions is of interest.

    Do the authors see any changes in cell phenotype, slower/faster proliferation, or increased apoptosis due to the activation of mobile elements (not only ROS)?

    This is also a very interesting idea. We plan on further investigating this as part of a follow up study.

    Another point is that the S878A mutant seems not to be able to fully maintain the DNA methylation (Fig 4A). Does O-GlcNAcylation recruit any additional interactors? Given that the authors immunoprecipitated DNMT1 and use it for activity assay, it is possible, that the modification attracts an additional protein factor that could in turn inhibit DNMT1 activity (as observed). Therefore, the observed kinetic effect could be indirect, while still interesting and important, the mechanism of inhibition would be different.

    We thank the reviewer for the great suggestions. According to Figure 4A, in the case of mutated DNMT1, a slight methylation loss appears to occur in both conditions. There could be for a number of reasons. It may be due to interacting proteins or it may be caused by some damage of DNMT1 itself. A further investigation of this is planned as a follow up project.

    DNA methylation clock can be used to estimate the biological age of a tissue/cells. While not directly in the line of the manuscript, I was wondering if the DNA methylation changes in the high glucose diet would affect the methylation sites used for the DNAme clock. Meaning, would the cells/tissue epigenetically age faster when in high glucose media, and if the Ala mutant could provide resistance to that?

    We thank the reviewer for the interesting suggestion. We believe this is beyond the scope of this manuscript, but we'll consider this with interest in the future.

    In discussion, the authors write that this is the first investigation of O-GlcNAcylation in relation to DNA methylation, while this is true for DNMTs, TET enzymes, that oxidise 5mC and trigger active DNA demethylation have been shown before to also be modified.

    We have toned down the language throughout the revised manuscript. This is the first investigation into the maintenance of DNA methylation. Although there is a great deal of evidence regarding the important regulatory role of O-GlcNAcylation in gene regulation, a direct link with maintenance of DNA methylation has not previously been established.

    A nice and rigorous study, with important observations and connections to biological effects. It would be nice to prove that the effects are direct and not associated with other factors that could be recruited by the modification and impact the activity of DNMT1. I find it a bit surprising that phosphorylation of the target serine does not impact DNMT1 activity as well.

    We thank the reviewer for the positive comments and agree that there are many interesting avenues to follow up on this.

    Reviewer #3 (Public Review):

    The authors investigate the potential effect of OGlcNacylation on the activity of the DNA methyltransferase DNMT1.

    Some results that are convincingly obtained include:

    • There is more overall OGlcNacylation when Glucose concentration in the culture medium or the feed is high;
    • DNMT1 is OGlcNacylated, and more so in high glucose or on rich chow;
    • The position S878 can be OGlcNacylated;
    • The activity of transfected DNMT1 is decreased in high glucose conditions. This effect is lessened when S878 is mutated to A or D.

    Some results that are suggested but not fully backed by experimental data include:

    • This process happens to the endogenous protein under physiologically relevant conditions;

    We agree that we could not completely rule out endogenous DNMT1 in our experiments. We have adjusted the language in the revised manuscript to acknowledge this. However, we confirmed the change in activity of recombinant DNMT1 (Figure 3D), and also demonstrated the change in activity under physiological conditions (normal physiological glucose level vs hyperglycemic range) in Figure 3—figure supplement 1B. This is a result that directly shows that the activity of DNMT1 changes under physiological conditions. In addition, DNA hypomethylation due to high glucose has been previously reported, already (Kandilya et al., 2020; Lan et al., 2016). Our results suggest a possible mechanism for this.

    Kandilya, D., Shyamasundar, S., Singh, D.K., Banik, A., Hande, M.P., Stunkel, W., Chong, Y.S., and Dheen, S.T. (2020). High glucose alters the DNA methylation pattern of neurodevelopment associated genes in human neural progenitor cells in vitro. Sci Rep 10, 15676.

    Lan, C.C., Huang, S.M., Wu, C.S., Wu, C.H., and Chen, G.S. (2016). High-glucose environment increased thrombospondin-1 expression in keratinocytes via DNA hypomethylation. Transl Res 169, 91-101 e101-103.

    • This process is responsible for changes in DNA methylation, leading to changes in gene expression, leading to increased ROS and increased apoptosis.

    We confirmed that ROS levels increased under high glucose conditions through DCFH-DA fluorescence experiments (Figure 5A). In addition, γH2A.X fluorescence experiments showed that DNA damage was increased under high glucose conditions (Fig. 5B). On the other hand, in the case of the S878A mutant, DNA damage was reduced under hyperglycemic conditions compared to wild type DNMT1 despite an increase in ROS levels (Fig. 5B). Moreover, we verified that the DNA damage did not come from oxidative stress through 8-OHdG analysis (Figure 5—figure supplement 4). Therefore, DNA oxidative stress is suppressed by DNMT1 due to the increase of ROS under high glucose conditions. However, the reduction of DNA methylation by O-GlcNAcylation of DNMT1 induces apoptosis due to oxidative stress.

    Studying the connection between cellular metabolism and epigenetic phenomena is interesting. However, I feel that the article falls short of its aims because of the limits of the experimental system, some missing controls, and some data overinterpretation.

    We hope the reviewer finds our revised manuscript more suitable.

  3. eLife

    eLife assessment

    This study explores the regulatory function of O-GlcNAcylation on DNA methyltransferase 1 and identifies serine 878 as the main target. This study is of interest to those in epigenetics and metabolism. The significance is important and the strength of the evidence is convincing.

  4. eLife

    Reviewer #1 (Public Review):

    In this study, Shin and colleagues investigate the role of the posttranslational modification of the DNA methyltransferase by covalent linkage of the N-Acetylglucosamine (O-GlcNAc).

    The authors present compelling evidence showing that a prolonged high fat/sucrose diet causes global protein O-GlcNAcylation in the liver and DNMT1 is among the proteins that increase their O-GlcNAc level. This result is significant because of the paucity of in vivo data addressing the interplay between metabolism and protein O-GlcNAcylation. The paper also shows that DNMT1's O-GlcNAcylation level correlated to the extracellular glucose levels in other cell types.

    Using mass spectrometry, the authors identify S878 as the main site for O-GlcNAcylation. It is noteworthy that the mapping was performed with hyper-O-GlcNAcylated cells and may be different in a physiological situation. To investigate how O-GlcNAcylation of S878 of DNMT1 impacts its activity and ultimately DNA methylation patterns, Shin and colleagues mostly use a cellular model of hyper O-GlcNAcylation induced by the combination of high glucose and a chemical inhibitor of OGA (the only enzyme responsible for O-GlcNAc removal). The data shows that increased O-GlcNAcylation resulting from the combination of high glucose and OGA inhibition causes a reduction of DNMT1 activity and local loss of DNA methylation specifically at partially methylated domains.

    This study brings completely new knowledge on the regulatory function of glycosylation of DNMT1 and its impact on its methyl-transferase activity and downstream genomic methylation. Furthermore, the manuscript introduces new data on the interplay between cellular metabolism and O-GlcNAcylation on DNMT1 and other proteins. The experiments are well-controlled, and their interpretation is sound. This study should be of special interest to the fields of fundamental and environmental epigenetics, as well as metabolism.

    The main limitation of the study is the convolution of the functional experiments where the perturbation is a combination of high glucose and chemical inhibition of OGA. The relative contribution of the two variables is partially addressed in Figure 3-figure supplement 1B which shows that high glucose increases DNMT1 activity (Hep3B cells) while Figure 3D shows that high glucose when combined with OGA inhibitor decreases DNMT1 activity (Hep3B cells). As discussed, the data suggest that high-glucose and OGA inhibition may have an antagonistic effect on DNMT1 activity. An experiment of treatment of the cells with the OGA inhibitor in physiological glucose conditions would address this gap of knowledge.

    To understand the impact of the environment (in this study: extracellular glucose level) on the epigenome, one should keep in mind the variation of cytosine methylation patterns between individuals and over time. A recent large-scale profiling of DNA methylation of 137 individuals shows a near absence of individual variation between replicates of the same cell type, suggesting that genomic methylation patterns are largely insensitive to the environment (https://doi.org/10.1038/s41586-022-05580-6).

    Comparative methylomes of healthy and diabetic individuals are needed to examine the medical significance of the findings presented here. It is possible that the modulation of DNMT1 activity by O-GlcNAc modification is relevant for a specific cell type or developmental stage that remains to be discovered.

  5. eLife

    Reviewer #2 (Public Review):

    I've read the manuscript by Shin et al with great interest. The authors describe the identification of O-GlcNAcylation of DNMT1 and the impact this modification has on the maintenance activity of DNMT1 genome-wide and that modification of S878 leads to enzyme inhibition.
    The manuscript is written in a clear and understandable way making it easy for the reader to understand the logic as well as the steps of the experimental approach.

    The authors identify O-GlcNAcylation of DNMT1 in a number of different cell lines by combining inhibition studies and WB and further on they identify the modification sites with LC/MS, predictions, and mutational studies. I really like the experimental approach, which while being straightforward (albeit technically challenging), is powerful and well-controlled in this case to unequivocally prove the modification of DNMT1 and identify the site. However, mutation of the two identified modification sites does not remove all the O-GlcNAcylation signal associated with DNMT1, thus possibly not all the possible sites were identified. While this is not a criticism of this manuscript, it would be interesting to know what other sites are modified and the enzymatic/biological effects associated.

    Also, the authors isolate the modified DNMT1 from cells using immunoprecipitation, which is indeed useful to study the changes in catalytic activity but does not provide any information if the cellular localisation of modified DNMT1 changes. Subsequently, the authors checked the impact of high glucose diet on the genome-wide DNA methylation patterns. The observed effects (Fig 4A) are very strong, almost as strong as observed with Aza treatment and therefore I wonder if LINE/IAP or other elements are getting activated (as observed with genome-wide demethylation with Aza). Do the authors see any changes in cell phenotype, slower/faster proliferation, or increased apoptosis due to the activation of mobile elements (not only ROS)? Another point is that the S878A mutant seems not to be able to fully maintain the DNA methylation (Fig 4A). Does O-GlcNAcylation recruit any additional interactors? Given that the authors immunoprecipitated DNMT1 and use it for activity assay, it is possible, that the modification attracts an additional protein factor that could in turn inhibit DNMT1 activity (as observed). Therefore, the observed kinetic effect could be indirect, while still interesting and important, the mechanism of inhibition would be different.

    DNA methylation clock can be used to estimate the biological age of a tissue/cells. While not directly in the line of the manuscript, I was wondering if the DNA methylation changes in the high glucose diet would affect the methylation sites used for the DNAme clock. Meaning, would the cells/tissue epigenetically age faster when in high glucose media, and if the Ala mutant could provide resistance to that?

    In discussion, the authors write that this is the first investigation of O-GlcNAcylation in relation to DNA methylation, while this is true for DNMTs, TET enzymes, that oxidise 5mC and trigger active DNA demethylation have been shown before to also be modified.

    A nice and rigorous study, with important observations and connections to biological effects. It would be nice to prove that the effects are direct and not associated with other factors that could be recruited by the modification and impact the activity of DNMT1. I find it a bit surprising that phosphorylation of the target serine does not impact DNMT1 activity as well.

  6. eLife

    Reviewer #3 (Public Review):

    The authors investigate the potential effect of OGlcNacylation on the activity of the DNA methyltransferase DNMT1.

    Some results that are convincingly obtained include:
    - There is more overall OGlcNacylation when Glucose concentration in the culture medium or the feed is high;
    - DNMT1 is OGlcNacylated, and more so in high glucose or on rich chow;
    - The position S878 can be OGlcNacylated;
    - The activity of transfected DNMT1 is decreased in high glucose conditions. This effect is lessened when S878 is mutated to A or D.

    Some results that are suggested but not fully backed by experimental data include:
    - This process happens to the endogenous protein under physiologically relevant conditions;
    - This process is responsible for changes in DNA methylation, leading to changes in gene expression, leading to increased ROS and increased apoptosis.

    Studying the connection between cellular metabolism and epigenetic phenomena is interesting. However, I feel that the article falls short of its aims because of the limits of the experimental system, some missing controls, and some data overinterpretation.

  7. Version published to 10.1101/2022.05.11.491514v6 on bioRxiv
  8. Version published to 10.1101/2022.05.11.491514v5 on bioRxiv
  9. Version published to 10.1101/2022.05.11.491514v4 on bioRxiv
  10. Version published to 10.1101/2022.05.11.491514v3 on bioRxiv
  11. Version published to 10.1101/2022.05.11.491514v2 on bioRxiv
  12. Version published to 10.1101/2022.05.11.491514v1 on bioRxiv