DNMT3B PWWP mutations cause hypermethylation of heterochromatin

  1. Francesca Taglini
  2. Ioannis Kafetzopoulos
  3. Willow Rolls
  4. Kamila Irena Musialik
  5. Heng Yang Lee
  6. Yujie Zhang
  7. Mattia Marenda
  8. Lyndsay Kerr
  9. Hannah Finan
  10. Cristina Rubio-Ramon
  11. Philippe Gautier
  12. Hannah Wapenaar
  13. Dhananjay Kumar
  14. Hazel Davidson-Smith
  15. Jimi Wills
  16. Laura C Murphy
  17. Ann Wheeler
  18. Marcus D Wilson
  19. Duncan Sproul

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Abstract

The correct establishment of DNA methylation patterns is vital for mammalian development and is achieved by the de novo DNA methyltransferases DNMT3A and DNMT3B. DNMT3B localises to H3K36me3 at actively transcribing gene bodies via its PWWP domain. It also functions at heterochromatin through an unknown recruitment mechanism. Here, we find that knockout of DNMT3B causes loss of methylation predominantly at H3K9me3-marked heterochromatin and that DNMT3B PWWP domain mutations or deletion result in striking increases of methylation in H3K9me3-marked heterochromatin. Removal of the N-terminal region of DNMT3B affects its ability to methylate H3K9me3-marked regions. This region of DNMT3B directly interacts with HP1α and facilitates the bridging of DNMT3B with H3K9me3-marked nucleosomes in vitro. Our results suggest that DNMT3B is recruited to H3K9me3-marked heterochromatin in a PWWP-independent manner that is facilitated by the protein’s N-terminal region through an interaction with a key heterochromatin protein. More generally, we suggest that DNMT3B plays a role in DNA methylation homeostasis at heterochromatin, a process which is disrupted in cancer, aging and Immunodeficiency, Centromeric Instability and Facial Anomalies (ICF) syndrome.

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  1. Version published to 10.1038/s44319-024-00061-5
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    Reply to the reviewers

    General comments:

    We thank the reviewers for recognizing the importance of our work and for their supportive and insightful comments.

    Our planned revisions focus on addressing all the comments and especially in further elucidating the molecular mechanism underpinning our observations, their consequences for cell phenotypes and reproducing our observations in an additional cell line. Our revision plan is backed up in many cases by preliminary data.

    Our submitted manuscript demonstrated that DNMT3B’s recruitment to H3K9me3-marked heterochromatin was mediated by the N-terminal region of DNMT3B. Data generated since submission suggest that DNMT3B binds indirectly to H3K9me3 nucleosomes through an interaction mediated by a putative HP1 motif in its N-terminal region.

    Specifically, we have found that DNMT3B can pull down HP1a and H3K9me3 from cell extracts and that this interaction is abrogated when we remove the N-terminal region of DNMT3B (revision plan, figure 1a). Using purified proteins in vitro, we have shown binding of DNMT3B to HP1a that is dependent on the presence of DNMT3B’s N-terminus suggesting that the interaction with HP1a is direct and that this mediates DNMT3B’s recruitment to H3K9me3 (revision plan,* figure 1b*). Alphafold multimer modelling identified that DNMT3B's N-terminus binds the interface of a HP1 dimeric chromoshadow domain through a putative HP1 motif. Two point mutations in this motif ablate DNMT3B’s interaction with HP1a in vitro (revision plan, *figure 1b *- DNMT3B L166S I168N).

    We propose to further characterize DNMT3B’s interaction with HP1a in vitro and determine the significance of these observations in cells by microscopy in a revised manuscript. Together with the other proposed experiments and analyses, we believe the extra detail regarding the molecular mechanisms through which DNMT3B is recruited to H3K9me3 heterochromatin will help address the reviewer’s comments.

    Point by point response:

    We have reproduced the reviewer’s comments in their entirety and highlighted them in blue italics.

    Reviewer #1 (Evidence, reproducibility and clarity (Required)): Summary:

    This paper by Francesca Taglini, Duncan Sproul, and their coworkers, examines the mechanisms of DNA methylation in a human cancer cell line. They use the human colorectal cancer line HCT116, which has been very widely used to look at epigenetics in cancer, and to dissect the contribution of different proteins and chromatin marks to DNA methylation.

    The authors focus on the role of the de novo methyltransferase DNMT3B. It has been shown in ES cells in 2015 that its PWWP domain directs it to H3K36me3, typically found in gene bodies. More recently, the authors showed similar conclusions in colorectal cancer (Masalmeh Nat Comm 2021). Here they examine, more specifically, the role of the PWWP. The conclusions are described below.

    Major comments:

    • *1-I feel that this paper has several messages that are somewhat muddled. The main message, as expressed in the title and in the model, is that the PWWP domain of DNMT3B actively drags the protein to H3K36me3-marked regions. Inactivation of this domain by a point mutation, or removal of the Nter altogether, causes DNMT3B to relocate to other genomic regions that are H3K9me3-rich, and that see their DNA methylation increase in the mutant conditions. This first message is clear.

    We thank the reviewer for their positive comments on our observations. However, we note that our results suggest that removal of the N-terminal region has a different effect to point mutations in the PWWP domain. The data we present suggest that the N-terminus facilitates recruitment to H3K9me3 regions.

    The second message has to do with ICF. A mutant form of DNMT3B bearing a mutation found in ICF, S270P, is actually unstable and, therefore, does not go to H3K9me3 regions. I feel that here the authors go on a tangent that distracts from message #1. This could be moved to the supp data. At any rate, HCT116 are not a good model for ICF. In addition, a previous paper has looked at the S270P mutant, and it did not seem unstable in their hands (Park J Mol Med 2008, PMID: 18762900). So I really feel the authors do not do themselves a favor with this ICF angle.

    While we agree with the reviewer that HCT116 cells as a cancer cell line are not a good model for ICF1 syndrome, our observation that S270P destabilizes DNMT3B is important to consider in the context of this disease. In addition, the S270P mutant was reported to abrogate the interaction between DNMT3B and H3K36me3 (Baubec et al 2015 Nature PMID: 25607372) making it important to compare it to the other mutations we examine. In our revised version of the manuscript, we propose to move these data to the supplementary materials and add a statement to the discussion noting the caveat that HCT116 cells are likely not to model many aspects of ICF1.

    With regard to the differences between our results and that of Park et al, we note that stability of the S270P mutant was not assessed in that study whereas we directly assess stability in vitro and in cells. We propose to add discussion of this previous study to the revised manuscript.

    2-I feel that some major confounders exist that endanger the conclusions of the work. The most worrisome one, in my opinion, is the amount of WT or mutant DNMT3B in the cells. It is clear in figure 4C that the WT rescue construct is expressed much more than the W263A mutant (around 3 or 4 times more). Unless I am mistaken, we are never shown how the level of exogenous rescue protein compares to the level of DNMT3B in WT cells. This bothers me a lot. If the level is too low, we may have partial rescue. If it is too high, we might have artifactual effects of all that DNMT3B. I would also like to see the absolute DNA methylation values (determined by WGBS) compared to the value found in WT. From figure S1A, it looks like WT is aroun 80% methylation, and 3BKO is around 77% or so. I wonder if the rescue lines may actually have more methylation than WT?

    The rescue cell lines do express DNMT3B to a greater level than observed endogenously. In our manuscript we controlled for this effect by generating the knock-in W263A cells and, as reported in the manuscript, we observe similar effects to the rescue cells (manuscript, figure 2d) suggesting that our observations are not driven by the overexpression.

    We also expressed ectopic DNMT3B from a weaker promoter (EF1a) in DNMT3B KO cells but did not include these data in the submitted manuscript. We have previously shown that this promoter expresses DNMT3B at lower levels than the CAG promoter used in the submitted manuscript (Masalmeh et al 2021 Nature Communications PMID: 33514701). Bisulfite PCR of representative non-repetitive loci within heterochromatic H3K9me3 domains show that we observe similar gains of methylation with DNMT3BW263A (revision plan, figure 2).

    Revision plan figure 2. Expression of DNMT3BW236A from a weaker promoter leads to increased DNA methylation at selected H3K9me3 loci. Barplot of mean methylation by BS-PCR at H3K9me3 loci alongside the H3K4me3-marked BRCA2 promoter in DNMT3B mutant cells were DNMT3B is expressed from the EF1 promoter. P-values are from two-sided Wilcoxon rank sum tests.

    To reinforce that our conclusions are not solely a result of the level of DNMT3B expression, we propose to include these data in the revised manuscript.

    The reviewer is also correct that by WGBS, the rescue cell lines have higher levels of overall DNA methylation than HCT116 cells. We will note this in revised manuscript and include HCT116 cells in a revised version of Figure S1e.

    3-I guess the unarticulated assumption is that the gain of DNA methylation seen at H3K9me3 region upon expression of a mutant DNMT3B is due to DNMT3B itself. But we do not know this for sure, unless the authors test a double mutant (PWWP inactive, no catalytic activity). I am not necessarily asking that they do it, but minimally they should mention this caveat.

    The hypothesis that the gains in DNA methylation at H3K9me3 loci result from the direct catalytic activity of DNMT3B is supported by our observation that a catalytic dead DNMT3B does not remethylate heterochromatin (manuscript, figures 1d and e). However, we acknowledge that we have not formally shown that the additional DNA methylation seen with DNMT3BW263A are a direct result of its catalytic activity. We will conduct an analysis of the effect of catalytically dead DNMT3BW263A on DNA methylation at Satellite II and selected H3K9me3 loci and include this in the revised manuscript.

    4-I am confused as to why the authors look at different genomic regions in different figures. In figure 1 we are looking at a portion of the "left" arm of chr 16. But in figure 2B, we now look at a portion of the "right" arm of the same chromosome, which has a large 8-Mb block of H3K9me3, and is surprisingly lowly methylated in the 3BKO. This seems quite odd, and I wonder if there is a possible artifact, for instance mapping bias, deletion, or amplification in HCT116. Showing the coverage along with the methylation values would eliminate some of these concerns.

    By choosing different regions of the genome for different figures, we intended to reassure the reader that our results were not specific to any one region of the genome. In the revised manuscript, we propose to display a consistent genomic region between these figures.

    With regard to the low levels of DNA methylation in H3K9me3 domains in DNMT3B KO cells, H3K9me3 domains are partially methylated domains which have reduced methylation in HCT116 cells (see page 5 of the manuscript):

    … we found that hidden Markov model defined H3K9me3 domains significantly overlapped with extended domains of overall reduced methylation termed partially methylated domains (PMDs) defined in our HCT116 WGBS (Jaccard=0.575,p=1.07x10-6, Fisher’s test).

    These domains lose further DNA methylation in DNMT3B KO cells leading to the low methylation level noted by the reviewer. The methylation percentages calculated from WGBS are based on the ratio of methylated to total reads. Thus, a lack of coverage generates errors from division by zero rather than the low values observed in this domain in DNMT3B KO cells.

    We include a modified version of figure 2b from the manuscript below. This includes coverage for the 3 cell lines (revision plan, figure 3). Although WGBS coverage is slightly reduced in H3K9me3 domains, reads are still present and overall coverage equal between different cell lines.

    While we could potentially include the coverage tracks in revised versions of figures, we note that doing so for multiple cell lines would make these figures extensively cluttered and it would likely be difficult to observe the differences in DNA methylation in these figure panels due to shrinkage of the other tracks.

    Minor comments:

    1-The WGBS coverage is not very high, around 2.5X on average, occasionally 2X. I don't believe this affects the findings, as the authors look at large H3K9me3 regions. But the info in table S2 was hard to find and it is important. I would make it more accessible.

    In the revised manuscript we will specify the mean coverage in the text to ensure this is clearer.

    2-It would be nice to have a drawing showing exactly what part of the Nter was removed.

    We will add this in the figure in the revised manuscript.

    3-some figures could be clearer. I was not always sure when we were looking at a CRISPR mutant clone (W263A) versus a piggyBac rescue.

    In the revised manuscript we will clarify in the figure labels to ensure it is clear which data were generated using CRISPR clones.

    4-unless I am mistaken, all the ChIP-seq data (H3K9me3, H3K36me3 etc) come from WT cells. It is not 100% certain that they remain the same in the 3BKO, is it? This should be discussed.

    We performed ChIP-seq on both HCT116 and 3BKO cell lines and used ChIP-seq data from the 3BKO cell line for the rescue experiments where DNMT3Bs were expressed in 3BKO cells. We will ensure this is clearer in the revised version.

    Reviewer #1 (Significance (Required)):

    Strengths:

    The experiments are for the most part well done and well interpreted (save for the limitations mentioned above). The techniques are appropriate and well mastered by the team. The paper is well written, the figures are nice. The authors know the field well, which translates into a good intro and a good discussion. The bioinformatics are convincing.

    Limitations:

    All the work is done in a single cancer cell line. One might assume the conclusions will hold in other systems, but there is no certainty at this point.

    We acknowledge this limitation. To demonstrate that our results are applicable beyond HCT116 cells, we will include analysis of experiments on an independent cell line in the revised manuscript.

    HCT116 are not the best model system to study ICF, which mostly affects lymphocytes

    At present, I feel that the biological relevance of the findings is fairly unclear. The authors report what happens when DNMT3B has no functional PWWP domain. I am convinced by their conclusions, but what do they tell us, biologically? Are there, for instance, mutant forms of DNMT3B expressed in disease that have a mutant PWWP? Are there isoforms expressed during development or in certain cell types that do not have a PWWP? In these cell types, does the distribution of DNA methylation agree with what the authors predict?

    As stated in response to point 1, although we acknowledge the limitations of HCT116 cells as a model of ICF, we believe are finding that the S270P mutation results in unstable DNMT3B are still important to consider for ICF syndrome.

    We are not aware of reports of mutations affecting the residues of DNMT3B’s PWWP domain we have studied. Our preliminary analysis suggests that although mutations in DNMT3B’s PWWP domain are frequent, residues in the aromatic cage such as W263 and D266 are absent from the gnomAD catalogue (Karczewski et al 2020 Nature, PMID: 32461654). This suggests that they are incompatible with healthy human development.

    A number of different DNMT3B splice isoforms have been reported. These include DDNMT3B4 which lacks the PWWP domain and a portion of the N-terminal region (Wang et al 2006 International Journal of Oncology, PMID: 16773201). DDNMT3B4 is proposed to be expressed in non-small cell lung cancer (Wang et al 2006 Cancer Research, PMID: 16951144).

    We will include analysis of gnomAD and discussion of these points in the revised manuscript.

    In its present state, I feel the appeal of the findings is towards a semi-specialized audience, that is interested in aberrant DNA methylation in cancer and other diseases. This is not a small audience, by the way.

    We thank the reviewer for their comments and the suggestion that our findings are of interest to a cross-section of researchers.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    Note, we have added numbers to the comments made by reviewer 2 to aid cross-referencing.

    In this manuscript, Taglini et al., describe an increased activity of DNMT3B at H3K9me3-marked regions in HCT cells. They first identify that DNA methylation at K9me3-marked regions is strongly reduced in absence of DNMT3B. Next, the authors re-express DNMT3B and DNMT3B mutant variants in the DNMT3B-KO HCT cells and assess DNA methylation by WGBS where they identify a strong preference for re-methylation of K9me3 sites. Based on genome-wide binding maps for DNMT3B, including the mutant variants, they address how the localization of DNMT3B relates to the observed changes in methylation.

    Major points:

    • The authors show increased reduction of mCG at H3K9me3 (and K27me3) sites in absence of DNMT3B. This is based on correlating delta %mCG with histone modifications in 2kb bins. I find this approach to not fully support the major claim.* First, the correlation coefficients are very small -0.124 for K9me3 and -0.175 for K27me3, and just marginally better compared to, for example, K36me3 that does not seem to have any influence on mCG according to Sup Fig S1b. While I agree that mCG seems more reduced at K9me3 in absence of DNMT3B (e.g. in Fig 1a), is there a better way to visualize the global effect? The delta mCG Boxplots based on bins are not ideal (this applies to many figures using the same approach in the current manuscript).

    Our choice to examine the global effects using correlations in windows across the genome was motivated by similar previous analyses in other studies (for example: Baubec et al. 2015 Nature PMID 25607372, Weinberg at al 2021 Nature PMID:33986537, Neri et al 2017 Nature PMID: 28225755). These global analyses result in modest correlation coefficients because the vast majority of genomic windows are negative for a given mark. For this reason, we included specific analyses of H3K36me3, H3K9me3 and H3K27me3 domains in the manuscript (eg manuscript figure 1b, c and d) which reinforce the conclusions drawn from our global analyses.

    However, we acknowledge that while our data support a specific activity at H3K9me3 marked heterochromatin, these are not the only changes in DNMT3B KO cells as DNMTs are promiscuous enzymes that are localized to multiple genomic regions. We will add discussion of this point to the revised manuscript.

    *2. *Second, the calculation based on delta mCpG does not allow to see how much methylation was initially there. For example, S1b shows a median decrease of ~ 10% in K9me3 and ~7-8% in H3K4me3. What does this mean given that the starting methylation for both marks is completely different?

    Following this point, the authors mention that mCG is already low at K9me3 domains in HCT cells (compared to other sites in the genome). I am curious if this may influence the accelerated loss of methylation in absence of DNMT3B? Any comments on this?

    The observation that there is a greater loss at H3K9me3 domains than H3K27me3-only domains which also have low DNA methylation levels in HCT116 argue that the losses are not solely driven by the lower initial level of methylation in H3K9me3 domains. Our analyses later in the manuscript also support a specific activity at H3K9me3. In addition, we propose to reinforce this point through further data on exploring how DNMT3B interacts with HP1a (see general comments, revision plan figure 1).

    However, we acknowledge the possibility that part of the loss seen at H3K9me3 domains in DNMT3B KO cells could be in part a result of their low initial level of methylation. In the revised manuscript we propose to include discussion of this possibility.

    3. One issue is the lack of correlation in DNMT3B binding to H3K9me3 sites in WT cells (Fig 3). How does this explain the requirement for DNMT3B for maintenance of methylation at H3K9me3? While some of the tested mutants show some weak increase at K9me3 sites, these are not comparable to the strong binding preferences observed at K36me3 for the wt or delta N- term version.

    Using ChIP-seq we cannot say that DNMT3BWT does not bind at H3K9me3, only that it binds here to a lower level than at K36me3-marked loci. The normalized DNMT3BWT signal at H3K9me3 domains is higher than the background signal from DNMT3B KO cells (manuscript figure 3d) supporting the hypothesis that DNMT3BWT localizes to H3K9me3. This hypothesis is also supported by the observation that the correlation between DNMT3BDN and H3K9me3 is reduced compared to that of DNMT3BWT (manuscript figure 6c compared to figure 3c).

    There are several reasons why the apparent enrichment of DNMT3B at H3K9me3 may appear weaker than at H3K36me3 by ChIP-seq. Previous work has also suggested that formaldehyde crosslinking fails to capture transient interactions in cells (Schmiedeberg et al. 2009 PLoS One PMID: 19247482). H3K9me3-marked heterochromatin is also resistant to sonication (Becker et al. 2017 Molecular Cell PMID: 29272703) and this could further affect our ability to detect DNMT3B in these regions using ChIP-seq. Our new data also suggest that DNMT3B binds to H3K9me3 indirectly through HP1a (see general comments, revision plan figure 1) and this may also lead to weaker ChIP-seq enrichment at H3K9me3 compared to the direct interaction with H3K36me3 through DNMT3B’s PWWP domain.

    We propose to add discussion of these issues to the revised manuscript.

    4. Following the above comment, what about other methyltransferases in HCT cells? Could DNMT1 or DNMT3A function be altered in absence of DNMT3B, and the observed methylation changes could be indirectly influenced by DNMT3B? The authors could create a DNMT-TKO HCT cell line and re-introduce DNMT3B in this background and measure methylation to exclude that DNMT1 or DNT3A could have an influence. In this case, only H3K9me3 should gain DNA methylation.

    As discussed in response to reviewer 1 (point 3), we propose to examine the changes in DNA methylation upon expression of catalytically dead DNMT3BW263A to further strengthen the evidence that DNMT3BW263A is directly responsible for the increased DNA methylation at H3K9me3-marked loci.

    5. DNMT3B lacking N-terminal shows reduced K9me3 methylation & some localization by imaging. While the presented experiments show some support for this conclusion, I suggest to re-introduce a W263A mutant lacking the N-terminal part and measure changes in DNA methylation at H3K9. This should help to test the requirement for the N-terminal regions and further indicate which protein part (PWWP or N-term) is more important in regulating the balance between K9me3 and K36me3.

    We have performed this experiment and the data are shown in manuscript figure S6c and d. The results of these experiments show that DNMT3BΔN+W263A cells showed less methylation at H3K9me3 loci than DNMT3BW263A cells, supporting a role for the N-terminus in recruiting DNMT3B to H3K9me3-marked heterochromatin. In the revised version, we will ensure that these data are more clearly indicated.

    In the first paragraph of the discussion, the authors state: "Our results demonstrate that DNMT3B is recruited to and methylates heterochromatin in a PWWP- independent manner that is facilitated by its N-terminal region." Same statement is found in the abstract. This contradicts the ChIP-seq results that do not indicate a recruitment of DNMT3B to heterochromatin, and the N- terminal deletions are not fully supporting a role in this targeting since there is no localization to K9me3 to begin with. While changes in methylation are observed, it remains to be determined if this is indeed through direct DNMT3B delocalization or indirectly through influencing the remaining DNMTs.

    As discussed above, there are several potential reasons why DNMT3B ChIP-seq signal at H3K9me3 is weak (reviewer 2, point 3). The additional experiments we propose to include in the revised manuscript could reinforce this statement by clarifying whether DNMT3B is directly responsible for methylating H3K9me3-marked regions (reviewer 1, point 3) and by delineating the role of the putative HP1a motif in DNMT3B’s N-terminal region (general comments, revision plan figure 1).

    Reviewer #2 (Significance (Required)):

    Advance: detailed analysis of DNMT3B mutants in relation to K9me3. Builds up on previous studies. Audience: specialised audience

    We thank the reviewer for their insights.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    • *In this work, Taglini et al. examine how the de novo DNA methyltransferase DNMT3B localizes to constitutive heterochromatin marked by the repressive histone modification H3K9me3. The authors utilize a previously generated DNMT3B KO colorectal carcinoma cell line, HCT116 to study recruitment and activity of DNMT3B at constitutive H3K9me3 heterochromatin. The authors noted preferential decrease of DNA methylation (DNAme) at regions of the genome marked with H3K9me3 in DNMT3B KOs. The authors then rescued the deficiency through overexpression of WT and catalytic dead DNMT3A/B and confirmed that DNA methylation increase at H3K9me3+ region in the WT DNMT3B, but not catalytically inactive mutant nor DNMT3A. To examine which protein domains may be mediating DNMT3B's recruitment to H3K9me3 regions, the authors designed a series of mutants, primarily focusing on the PWWP domain which normally recognizes H3K36me3. In the PWWP mutants, DNMT3B binding to the genome is altered, showing depletion at some H3K36me3-marked regions and gain at H3K9me3 heterochromatin, which coincides with DNAme increase at satellites. In contrast, the clinically relevant ICF1 mutation S270P, shows DNMT3B protein destabilization and no such loss of DNAme at heterochromatin. Finally, the authors truncate the N-terminal portion of DNMT3B, and saw that this region of the protein is necessary for heterochromatin localization and subsequent DNAme of H3K9me3+ regions.

    The experiments are well done with extensive controls, and the results are interesting and convincing. The structure of the manuscript could be improved for clarity and flow - for example, the PWWP mutations and truncations should be mentioned and compared together. I also found the section on ICF1 mutant to be out-of-place.

    As described above (reviewer 1, point 1), we propose to move these data to the supplementary materials in the revised manuscript.

    More emphasis should be placed on the N- terminal mutant as this region seems to be critical to heterochromatin recruitment, and this may address whether the interaction to H3K9me3 is direct or indirect.

    As described above (general comments), the revised manuscript will include experiments clarifying the nature of DNMT3B’s interaction with H3K9me3. Our preliminary data support that it is an indirect interaction mediated through HP1a (revision plan figure 1).

    Finally, while the epigenetic crosstalk is well-examined in this work, I would strongly urge the authors to add RNA-seq data to determine the transcriptional consequence of such chromatin disruptions (e.g. are repetitive sequences up-regulated in DNMT3B KOs?).

    As suggested by the reviewer, we propose to generate and analyse RNA-seq data in the revised manuscript to understand the impact of DNMT3B on transcriptional programs.

    Comments

    1. A potential caveat to the study is the use of a single cell line - colorectal cancer cell HCT116 - to draw major conclusions on the function of DNMT3B. It is worth noting the Baubec et al. study examining DNMT3B recruitment to H3K36me3 was mainly performed in murine embryonic stem cells (mESCs). It would greatly strengthen the study if the authors could perform similar type of data analysis on an independent DNMT3B KO cell line. For example, does DNMT3B localize to H3K9me3 regions in WT mESCs?

    As described above in response to reviewer 1, we will include analysis in an additional cell line in the revised manuscript to demonstrate that our results are generalizable beyond HCT116 cells.

    2. Did the PWWP mutant W263A show the expected loss of DNAme at H3K36me3-marked regions? In other words, was there evidence of DNAme redistribution in loss at H3K36me3+ regions and inappropriate gain at H3K9me3+ regions? Please perform intersection analysis of DMRs with other epigenomic marks (e.g. H3K27me3, H3K36me3, CpG shores) in the PWWP mutants.

    Our analysis of DNMT3B KO cells (manuscript figure s1d) show that losses of DNA methylation in these cells are not correlated with H3K36me3 in gene bodies suggesting that DNMT3A and DNMT1 are sufficient to compensate in maintaining their methylation in DNMT3B KO cells. To clarify this point for the DNMT3BW263A knock in clones, in the revised manuscript we will directly examine whether these cells show loss of methylation at H3K36me3 marked gene bodies in a similar analysis and add discussion of these results.

    The study would also be strengthen greatly with the in addition of biochemical studies to confirm direct loss of binding, and possibly gain of H3K9me3 binding, in the DNMT3B PWWP mutants.

    As detailed above (general comments, revision plan figure 1), our data suggest that DNMT3B interacts indirectly with H3K9me3 through an HP1 motif in its N-terminal region. We will undertake further biochemical studies on this interaction which will be included in the revised manuscript. Specifically we will focus on using EMSAs with synthetic nucleosomes to clarify the degree to which the HP1a interaction is responsible for binding of DNMT3B to H3K9me3 modified nucleosomes.

    We also propose to undertake in vitro biochemical characterization of the effect of DNMT3B PWWP mutations on interaction with H3K36me3 using synthetic nucleosomes. However, we note that in the manuscript we have shown similar effects using two independent point mutations that are predicted to affect H3K36me3 binding (W263A and D266A) and deletion of the entire PWWP domain.

    3. Examining the tracks in Figure 3A,B, the PWWP mutants showed almost indiscriminate increase across the genome, and not specifically to H3K9me3-marked regions. Would ask the authors to speculate as to why the ChIP-seq of DNMT3B mutants do not recapitulate the heterochromatin co-localization shown by immunofluorescence.

    As discussed in response to reviewer 2 (point 3) we believe that the weak DNMT3B ChIP-seq signal at H3K9me3 loci is likely due to the nature of the interaction that DNMT3B has with chromatin in these regions. We will add discussion of these points to the revised manuscript.

    4. It's a shame that the ICF1 mutation S270P was not characterized to the same extent as the PWWP mutants. Would consider adding WGBS for this clinically relevant mutation.

    We have shown that this mutant does not produce stable protein in vitro or in our cells and we observe little difference in DNA methylation at selected loci. As WGBS is expensive, we believe that carrying out this experiment is not an efficient use of limited research resources.

    5. Figure 7 - please draw in the ICF1 and the N-terminal mutations in the model figure. Also provide legends.

    We will modify the manuscript to include these details in the revised manuscript.

    Reviewer #3 (Significance (Required)):

    This is an intersting study on a timely subject. It will be of interest to multiple fields from epigenetics to development and cancer. My expertise is in cancer, epigenetics, development.

    We thank the reviewer for highlighting the broad interest of our study.

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    Referee #3

    Evidence, reproducibility and clarity

    In this work, Taglini et al. examine how the de novo DNA methyltransferase DNMT3B localizes to constitutive heterochromatin marked by the repressive histone modification H3K9me3. The authors utilize a previously generated DNMT3B KO colorectal carcinoma cell line, HCT116 to study recruitment and activity of DNMT3B at constitutive H3K9me3 heterochromatin. The authors noted preferential decrease of DNA methylation (DNAme) at regions of the genome marked with H3K9me3 in DNMT3B KOs. The authors then rescued the deficiency through overexpression of WT and catalytic dead DNMT3A/B and confirmed that DNA methylation increase at H3K9me3+ region in the WT DNMT3B, but not catalytically inactive mutant nor DNMT3A. To examine which protein domains may be mediating DNMT3B's recruitment to H3K9me3 regions, the authors designed a series of mutants, primarily focusing on the PWWP domain which normally recognizes H3K36me3. In the PWWP mutants, DNMT3B binding to the genome is altered, showing depletion at some H3K36me3-marked regions and gain at H3K9me3 heterochromatin, which coincides with DNAme increase at satellites. In contrast, the clinically relevant ICF1 mutation S270P, shows DNMT3B protein destabilization and no such loss of DNAme at heterochromatin. Finally, the authors truncate the N-terminal portion of DNMT3B, and saw that this region of the protein is necessary for heterochromatin localization and subsequent DNAme of H3K9me3+ regions.

    The experiments are well done with extensive controls, and the results are interesting and convincing. The structure of the manuscript could be improved for clarity and flow - for example, the PWWP mutations and truncations should be mentioned and compared together. I also found the section on ICF1 mutant to be out-of-place. More emphasis should be placed on the N-terminal mutant as this region seems to be critical to heterochromatin recruitment, and this may address whether the interaction to H3K9me3 is direct or indirect. Finally, while the epigenetic crosstalk is well-examined in this work, I would strongly urge the authors to add RNA-seq data to determine the transcriptional consequence of such chromatin disruptions (e.g. are repetitive sequences up-regulated in DNMT3B KOs?).

    Comments

    1. A potential caveat to the study is the use of a single cell line - colorectal cancer cell HCT116 - to draw major conclusions on the function of DNMT3B. It is worth noting the Baubec et al. study examining DNMT3B recruitment to H3K36me3 was mainly performed in murine embryonic stem cells (mESCs). It would greatly strengthen the study if the authors could perform similar type of data analysis on an independent DNMT3B KO cell line. For example, does DNMT3B localize to H3K9me3 regions in WT mESCs?
    2. Did the PWWP mutant W263A show the expected loss of DNAme at H3K36me3-marked regions? In other words, was there evidence of DNAme redistribution in loss at H3K36me3+ regions and inappropriate gain at H3K9me3+ regions? Please perform intersection analysis of DMRs with other epigenomic marks (e.g. H3K27me3, H3K36me3, CpG shores) in the PWWP mutants. The study would also be strengthen greatly with the in addition of biochemical studies to confirm direct loss of binding, and possibly gain of H3K9me3 binding, in the DNMT3B PWWP mutants.
    3. Examining the tracks in Figure 3A,B, the PWWP mutants showed almost indiscriminate increase across the genome, and not specifically to H3K9me3-marked regions. Would ask the authors to speculate as to why the ChIP-seq of DNMT3B mutants do not recapitulate the heterochromatin co-localization shown by immunofluorescence.
    4. It's a shame that the ICF1 mutation S270P was not characterized to the same extent as the PWWP mutants. Would consider adding WGBS for this clinically relevant mutation.
    5. Figure 7 - please draw in the ICF1 and the N-terminal mutations in the model figure. Also provide legends.

    Significance

    This is an intersting study on a timely subject. It will be of interest to multiple fields from epigenetics to development and cancer.

    My expertise is in cancer, epigenetics, development.

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    Referee #2

    Evidence, reproducibility and clarity

    In this manuscript, Taglini et al., describe an increased activity of DNMT3B at H3K9me3-marked regions in HCT cells. They first identify that DNA methylation at K9me3-marked regions is strongly reduced in absence of DNMT3B. Next, the authors re-express DNMT3B and DNMT3B mutant variants in the DNMT3B-KO HCT cells and assess DNA methylation by WGBS where they identify a strong preference for re-methylation of K9me3 sites. Based on genome-wide binding maps for DNMT3B, including the mutant variants, they address how the localization of DNMT3B relates to the observed changes in methylation.

    Major points:

    The authors show increased reduction of mCG at H3K9me3 (and K27me3) sites in absence of DNMT3B. This is based on correlating delta %mCG with histone modifications in 2kb bins. I find this approach to not fully support the major claim.

    First, the correlation coefficients are very small -0.124 for K9me3 and -0.175 for K27me3, and just marginally better compared to, for example, K36me3 that does not seem to have any influence on mCG according to Sup Fig S1b. While I agree that mCG seems more reduced at K9me3 in absence of DNMT3B (e.g. in Fig 1a), is there a better way to visualize the global effect? The delta mCG Boxplots based on bins are not ideal (this applies to many figures using the same approach in the current manuscript).

    Second, the calculation based on delta mCpG does not allow to see how much methylation was initially there. For example, S1b shows a median decrease of ~ 10% in K9me3 and ~7-8% in H3K4me3. What does this mean given that the starting methylation for both marks is completely different?

    Following this point, the authors mention that mCG is already low at K9me3 domains in HCT cells (compared to other sites in the genome). I am curious if this may influence the accelerated loss of methylation in absence of DNMT3B? Any comments on this?

    One issue is the lack of correlation in DNMT3B binding to H3K9me3 sites in WT cells (Fig 3). How does this explain the requirement for DNMT3B for maintenance of methylation at H3K9me3? While some of the tested mutants show some weak increase at K9me3 sites, these are not comparable to the strong binding preferences observed at K36me3 for the wt or delta N-term version.

    Following the above comment, what about other methyltransferases in HCT cells? Could DNMT1 or DNMT3A function be altered in absence of DNMT3B, and the observed methylation changes could be indirectly influenced by DNMT3B? The authors could create a DNMT-TKO HCT cell line and re-introduce DNMT3B in this background and measure methylation to exclude that DNMT1 or DNT3A could have an influence. In this case, only H3K9me3 should gain DNA methylation.

    DNMT3B lacking N-terminal shows reduced K9me3 methylation & some localization by imaging. While the presented experiments show some support for this conclusion, I suggest to re-introduce a W263A mutant lacking the N-terminal part and measure changes in DNA methylation at H3K9. This should help to test the requirement for the N-terminal regions and further indicate which protein part (PWWP or N-term) is more important in regulating the balance between K9me3 and K36me3.

    In the first paragraph of the discussion, the authors state: "Our results demonstrate that DNMT3B is recruited to and methylates heterochromatin in a PWWP- independent manner that is facilitated by its N-terminal region." Same statement is found in the abstract. This contradicts the ChIP-seq results that do not indicate a recruitment of DNMT3B to heterochromatin, and the N-terminal deletions are not fully supporting a role in this targeting since there is no localization to K9me3 to begin with. While changes in methylation are observed, it remains to be determined if this is indeed through direct DNMT3B delocalization or indirectly through influencing the remaining DNMTs.

    Significance

    Advance: detailed analysis of DNMT3B mutants in relation to K9me3. Builds up on previous studies.

    Audience: specialised audience

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    This paper by Francesca Taglini, Duncan Sproul, and their coworkers, examines the mechanisms of DNA methylation in a human cancer cell line. They use the human colorectal cancer line HCT116, which has been very widely used to look at epigenetics in cancer, and to dissect the contribution of different proteins and chromatin marks to DNA methylation.

    The authors focus on the role of the de novo methyltransferase DNMT3B. It has been shown in ES cells in 2015 that its PWWP domain directs it to H3K36me3, typically found in gene bodies. More recently, the authors showed similar conclusions in colorectal cancer (Masalmeh Nat Comm 2021). Here they examine, more specifically, the role of the PWWP. The conclusions are described below.

    Major comments:

    1. I feel that this paper has several messages that are somewhat muddled. The main message, as expressed in the title and in the model, is that the PWWP domain of DNMT3B actively drags the protein to H3K36me3-marked regions. Inactivation of this domain by a point mutation, or removal of the Nter altogether, causes DNMT3B to relocate to other genomic regions that are H3K9me3-rich, and that see their DNA methylation increase in the mutant conditions. This first message is clear.

    The second message has to do with ICF. A mutant form of DNMT3B bearing a mutation found in ICF, S270P, is actually unstable and, therefore, does not go to H3K9me3 regions. I feel that here the authors go on a tangent that distracts from message #1. This could be moved to the supp data. At any rate, HCT116 are not a good model for ICF. In addition, a previous paper has looked at the S270P mutant, and it did not seem unstable in their hands (Park J Mol Med 2008, PMID: 18762900). So I really feel the authors do not do themselves a favor with this ICF angle.

    1. I feel that some major confounders exist that endanger the conclusions of the work. The most worrisome one, in my opinion, is the amount of WT or mutant DNMT3B in the cells. It is clear in figure 4C that the WT rescue construct is expressed much more than the W263A mutant (around 3 or 4 times more). Unless I am mistaken, we are never shown how the level of exogenous rescue protein compares to the level of DNMT3B in WT cells. This bothers me a lot. If the level is too low, we may have partial rescue. If it is too high, we might have artifactual effects of all that DNMT3B. I would also like to see the absolute DNA methylation values (determined by WGBS) compared to the value found in WT. From figure S1A, it looks like WT is aroun 80% methylation, and 3BKO is around 77% or so. I wonder if the rescue lines may actually have more methylation than WT?
    2. I guess the unarticulated assumption is that the gain of DNA methylation seen at H3K9me3 region upon expression of a mutant DNMT3B is due to DNMT3B itself. But we do not know this for sure, unless the authors test a double mutant (PWWP inactive, no catalytic activity). I am not necessarily asking that they do it, but minimally they should mention this caveat.
    3. I am confused as to why the authors look at different genomic regions in different figures. In figure 1 we are looking at a portion of the "left" arm of chr 16. But in figure 2B, we now look at a portion of the "right" arm of the same chromosome, which has a large 8-Mb block of H3K9me3, and is surprisingly lowly methylated in the 3BKO. This seems quite odd, and I wonder if there is a possible artifact, for instance mapping bias, deletion, or amplification in HCT116. Showing the coverage along with the methylation values would eliminate some of these concerns.

    Minor comments:

    1. The WGBS coverage is not very high, around 2.5X on average, occasionally 2X. I don't believe this affects the findings, as the authors look at large H3K9me3 regions. But the info in table S2 was hard to find and it is important. I would make it more accessible.
    2. It would be nice to have a drawing showing exactly what part of the Nter was removed.
    3. some figures could be clearer. I was not always sure when we were looking at a CRISPR mutant clone (W263A) versus a piggyBac rescue.
    4. unless I am mistaken, all the ChIP-seq data (H3K9me3, H3K36me3 etc) come from WT cells. It is not 100% certain that they remain the same in the 3BKO, is it? This should be discussed.

    Significance

    Strengths:

    The experiments are for the most part well done and well interpreted (save for the limitations mentioned above). The techniques are appropriate and well mastered by the team. The paper is well written, the figures are nice. The authors know the field well, which translates into a good intro and a good discussion. The bioinformatics are convincing.

    Limitations:

    All the work is done in a single cancer cell line. One might assume the conclusions will hold in other systems, but there is no certainty at this point.

    HCT116 are not the best model system to study ICF, which mostly affects lymphocytes

    At present, I feel that the biological relevance of the findings is fairly unclear. The authors report what happens when DNMT3B has no functional PWWP domain. I am convinced by their conclusions, but what do they tell us, biologically? Are there, for instance, mutant forms of DNMT3B expressed in disease that have a mutant PWWP? Are there isoforms expressed during development or in certain cell types that do not have a PWWP? In these cell types, does the distribution of DNA methylation agree with what the authors predict?

    In its present state, I feel the appeal of the findings is towards a semi-specialized audience, that is interested in aberrant DNA methylation in cancer and other diseases. This is not a small audience, by the way.

  6. Version published to 10.1101/2022.12.19.521050v2 on bioRxiv
  7. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/7529495.

    In this manuscript, Taglini and colleagues explore the role of de novo DNA methyltransferase DNMT3B in human heterochromatin methylation. Using a human colorectal cancer cell line lacking DNMT3B, the authors demonstrate a specific loss of DNA methylation over pericentromeric DNA and constitutive heterochromatin, which are normally characterized by intermediate levels of DNA methylation and high H3K9 methylation. This model is consistent with previous studies that link DNMT3B loss of function mutations with pericentromeric DNA demethylation, genome instability and ICF-1 syndrome.

    The authors then reconstituted DNMT3B-less cells with DNMT3B variants that carry a series of clinically relevant mutations, including the previously described S270P mutation in the PWWP domain, which was hypothesized to be the domain required for DNMT3B targeting to pericentromeric DNA. The authors also generated DNMT3B variants carrying PWWP domain mutations at paralogous amino acids to those that cause Heyn-Sproul-Jackson Syndrome (HESJAS) syndrome when mutated in DNMT3A (W263A and D266A) which result in methylation of DNA methylation valleys normally marked by H3K27me3. Unexpectedly, using two types of protein stability assays and Western blotting, the S270P mutation was shown to result in protein destabilization, conferring a loss of function mutation. Further characterization of pericentromeric DNA in revealed that S270P leads to loss of DNMT3B at heterochromatin hypomethylated pericentromeric DNA, likely due to protein activity loss. On the other hand, WGBS, southern blotting and bisulphite-PCR of W263A and D266A mutants show that both lead to an increase in pericentromeric and heterochromatin DNA methylation. Thus, while DNMT3B normally targets transcribed gene bodies and heterochromatin, mutations that alter PWWP function shift the balance of DNMT3B localization from genic regions to heterochromatin regions. Surprisingly, complete abrogation the PWWP domain does not result in DNMT3B redistribution, suggesting the W263A and D266A are gain of function mutations. Finally, the authors demonstrate that the uncharacterized N terminal of DNMT3B is required for heterochromatin targeting, but is dispensable in a W263A mutant context.

    Overall, this is an excellently conducted and well written study. The authors employ orthogonal technologies to conclude DNMT3B's role in the methylation of pericentromeric and constitutive heterochromatin DNA. While evidence already suggested DNMT3B associates with pericentromeric DNA in mouse via HP1a and Suv39h proteins (Lehnertz et al., 2003 Current Biology), Taglini and colleagues widened the scope of this interaction to generalize the association between DNMT3B and constitutive heterochromatin genome-wide. Furthermore, the integration of disease-relevant mutations deconvoluted a standing mystery in clinically observed DNMT3B mutation in ICF-1 syndrome.

    Major comments

    I wonder whether DNMT3A can compensate for the lack of DNMT3B in the 'parental' DNMT3B KO cell line. Both at gene bodies (which seems to be the case) and over heterochromatin. The authors could do a Southern on Satellite II DNA to check methylation levels in the DNMT3A/B double KO to confirm.

    During my reading on the Results section it was unclear the extent of overlap between pericentromeric regions and H3K9me3 domains. If possible, the authors could include pericentromeric DNA annotations (if these exist) or the location of Satellite II DNA (again, assuming reliable annotations exist).

    Does the ΔPWWP mutation change the stability of DNMT3B?

    Do the H3K9me3 domains correspond to LADs? Could mine data from (van Schaik et al., 2020 EMBO Reports).

    Minor comments

    Rescue with DNMT3A instead of DNMT3B showed little gain of DNAme at H3K9me3 domains, revealing a difference in targeting of the DNMTs. However, if the cells normally express DNMT3A, this is an overexpression experiment, which could be discussed.

    Could the authors speculate about the many isoforms of DNMT3B and how they may encode proteins with variable N terminal domains or missing PWWP? Such isoforms were discussed in a 2016 paper using the HCT116 cell line (Duymich et al., 2016 Nature Communications).

    P5. "They also significantly overlapped heterochromatic regions resistant to nuclease digestion identified using Protect-seq in the same cells (Jaccard=0.623, p=2.11x10-30, Fisher's test) (Spracklin and Pradhan, 2020)" has no associated figure, though I'm not sure one is required. Maybe a supplemental table?

    What is the overlap of K9me3 domains in DNMT3B WT & KO cells?

    The title of Fig 2c (right) could be improved as it's not JUST W263A cells, but also 3BWT cells.

    I think Figure 7 model is a tad ambiguous about K9me3/K27me3 marked regions and omits interesting biology, namely, that H3K27me3 is redistributed over K9me3-marked regions in DNMT3B KO cells. Further, different "PWWP mutations" have different effects on DNMT3B targeting.

    Boxplot Fig 1c,e: Would be nice to know the absolute levels of DNAme instead of just the delta (maybe as a supplement).

    Would also be nice to display the p-value between "other domains" and "H3K27me3 only domains" in Fig 1c.

    Are the DNMT3B W263A and 266A mutations found in human diseases?

    Are major satellite II / pericentromeric DNA regions transcribed in the various cell lines?

    Similarly, how come H3K27me3 colocalizes with H3K9me3 in the absence of DNAme? See (Saksouk et al., 2014 Molecular Cell). Could the authors speculate?

    It would be interesting to test whether any of the mutations generated and tested here modify the cooperative binding of DNMT3B with other proteins frequently mutated in human ICF, such as ZBTB24 (ICF-2), CDCA7 (ICF-3) or HELLS (ICF-4).

    Competing interests

    The author declares that they have no competing interests.

  9. Version published to 10.1101/2022.12.19.521050v1 on bioRxiv