Author response:
eLife Assessment
This study provides valuable mechanistic insight into the mutually exclusive distributions of the histone variant H2A.Z and DNA methylation by testing two hypotheses: (i) that DNA methylation destabilizes H2A.Z nucleosomes, thereby preventing H2A.Z retention, and (ii) that DNA methylation suppresses H2A.Z deposition by ATP-dependent chromatin remodeling complexes. Through a series of well-designed and carefully executed experiments, findings are presented in support of both hypotheses. However, the evidence in support of either hypothesis is incomplete, so that the proposed mechanisms underlying the enrichment of H2A.Z on unmethylated DNA remain somewhat speculative.
We would like to thank the editor and reviewers for their critical assessments of our manuscript. While we do acknowledge the limitations of our work, we believe that our results provide important mechanistic insights into the long-standing question of how H2A.Z is preferentially enriched in hypomethylated genomic DNA regions. First, our structural and biochemical data suggest that DNA methylation increases the openness and physical accessibility of H2A.Z, albeit the effect is relatively subtle and is sequence-dependent. Second, using Xenopus egg extracts and synthetic DNA templates, we provide the first clear and direct evidence that DNA methylation-sensitive H2A.Z deposition is due to the H2A.Z chaperone SRCAP-C, corroborated by our discovery that SRCAP-C binding to DNA is suppressed by DNA methylation. Although the molecular details by which DNA methylation inhibits binding of SRCAP-C is an important area of future study, in our current manuscript, we do provide evidence that directly links the presence of SRCAP-C to the establishment of the DNA methylation/H2A.Z antagonism in a physiological system. Thanks to criticisms by the reviewers, we realized that we did not clearly state in our Abstract that the impact of DNA methylation on intrinsic H2A.Z nucleosome stability is relatively subtle, although we did explain these observations and limitations in the main text. In our revised manuscript, we are willing to edit the text to better clarify the criticisms raised by the reviewers.
Public Reviews:
Reviewer #1 (Public review):
Summary:
The authors considered the mechanism underlying previous observations that H2A.Z is preferentially excluded from methylated DNA regions. They considered two non-mutually exclusive mechanisms. First, they tested the hypothesis that nucleosomes containing both methylated DNA and H2A.Z might be intrinsically unstable due to their structural features. Second, they explored the possibility that DNA methylation might impede SRCAP-C from efficiently depositing H2A.Z onto these DNA methylated regions.
Their structural analyses revealed subtle differences between H2A.Z-containing nucleosomes assembled on methylated versus unmethylated DNA. To test the second hypothesis, the authors allowed H2A.Z assembly on sperm chromatin in Xenopus egg extracts and mapped both H2A.Z localization and DNA methylation in this transcriptionally inactive system. They compared these data with corresponding maps from a transcriptionally active Xenopus fibroblast cell line. This comparison confirmed the preferential deposition or enrichment of H2A.Z on unmethylated DNA regions, an effect that was much more pronounced in the fibroblast genome than in sperm chromatin. Furthermore, nucleosome assembly on methylated versus unmethylated DNA, along with SRCAP-C depletion from Xenopus egg extracts, provided a means to test whether SRCAP-C contributes to the preferential loading of H2A.Z onto unmethylated DNA.
Strengths:
The strength and originality of this work lie in its focused attempt to dissect the unexplained observation that H2A.Z is excluded from methylated genomic regions.
Weaknesses:
The study has two weaknesses. First, although the authors identify specific structural effects of DNA methylation on H2A.Z-containing nucleosomes, they do not provide evidence demonstrating that these structural differences lead to altered histone dynamics or nucleosome instability. Second, building on the elegant work of Berta and colleagues (cited in the manuscript), the authors implicate SRCAP-C in the selective deposition of H2A.Z at unmethylated regions. Yet the role of SRCAP-C appears only partial, and the study does not address how the structural or molecular consequences of DNA methylation prevent efficient H2A.Z deposition. Finally, additional plausible mechanisms beyond the two scenarios the authors considered are not investigated or discussed in the manuscript.
Although we acknowledge the limitations of our study and are willing to expand our discussion to more thoroughly discuss these points, we believe our manuscript provides several important mechanistic insights which this reviewer may not have fully appreciated.
Our first conclusion that H2A.Z nucleosomes on methylated DNA are more open and accessible compared to their unmethylated counterparts is supported by both our cryo-EM study and the restriction enzyme accessibility assay. Although the physical effect of DNA methylation is relatively subtle and is likely sequence dependent, as we clearly noted within the manuscript, the difference does exist and is valuable information for the chromatin field at large to consider.
The second major conclusion of our manuscript is that SRCAP-C exhibits preferential binding to unmethylated DNA over methylated DNA, and that SRCAP-C represents the major mechanism that can explain the biased deposition of H2A.Z to unmethylated DNA in Xenopus egg extracts. Furthermore, our experiments using Xenopus egg extract clearly demonstrated that H2A.Z is deposited by both DNA-methylation sensitive and insensitive mechanisms. Depletion of SRCAP-C almost completely eliminated the levels of DNA-methylation-sensitive H2A.Z deposition and reduced the total level of H2A.Z on chromatin to less than half of that seen in non-depleted extract. This result demonstrated that DNA methylation-sensitive H2A.Z loading is primarily regulated by SRCAP-C, at least in our experimental context where transcription, replication, and other epigenetic modifications are not involved. It is likely that additional mechanisms do further contribute, implicated by our sequencing experiments, particularly at regions with active transcription, and we have noted these possibilities and the rationale for their existence in the Discussion.
Our study also suggests that a SRCAP-independent, DNA methylation-insensitive mechanism of H2A.Z loading exists, which we suspect to be mediated by Tip60-C. In line with this possibility, our data suggest that Tip60-C binds DNA in a DNA methylation-insensitive manner in Xenopus egg extract. Since antibodies to deplete Tip60-C from Xenopus egg extract are currently unavailable, we were unable to directly test that hypothesis and decided not to include Tip60-C into our final model as we lacked experimental evidence for its role. However, whether or not Tip60-C is the complex responsible for the DNA methylation-insensitive pathway does not influence our final conclusion that SRCAP-C plays a major role in DNA methylation-sensitive H2A.Z loading. We are planning to edit our manuscript to more comprehensively discuss these points.
Please note that while Berta et al reported that DNA methylation increases at H2A.Z loci in tumors defective in SRCAP-C, they selected those regions based off where H2A.Z is typically enriched within normal tissues (Berta et al., 2021). They did not show data indicating whether H2A.Z is still retained specifically at those analyzed loci upon mutation of SRCAP-C subunits. Thus, although we greatly admire their work and are pleased that many of our findings align with theirs, their paper did not directly address whether SRCAP-C itself differentiates between DNA methylation status nor the impact that has on H2A.Z and DNA methylation colocalization. In contrast, our Xenopus egg extract system, where de novo methylation is undetectable (Nishiyama et al., 2013; Wassing et al., 2024) offers a unique opportunity to examine the direct impact of DNA methylation on H2A.Z deposition using controlled synthetic DNA substrates. Corroborated with our demonstration that DNA binding of SRCAP-C is suppressed by DNA methylation, we believe that our manuscript provides a specific mechanism that can explain the preferential deposition of H2A.Z at hypomethylated genomic regions.
Reviewer #2 (Public review):
This manuscript aims to elucidate the mechanistic basis for the long-standing observation that DNA methylation and the histone variant H2A.Z occupy mutually exclusive genomic regions. The authors test two hypotheses: (i) that DNA methylation intrinsically destabilizes H2A.Z nucleosomes, thereby preventing H2A.Z retention, and (ii) that DNA methylation suppresses H2A.Z deposition by ATP-dependent chromatin-remodelling complexes. However, neither hypothesis is rigorously addressed. There are experimental caveats, issues with data interpretation, and conclusions that are not supported by the data. Substantial revision and additional experiments, including controls, would be required before mechanistic conclusions can be drawn. Major concerns are as follows:
We appreciate the critical assessment of our manuscript by this reviewer. Although we acknowledge the limitations of our study and will revise the manuscript to better describe them, we would like to respectfully argue against the statement that our "conclusions […] are not supported by the data".
(1) The cryo-EM structure of methylated H2A.Z nucleosomes is insufficiently resolved to address the central mechanistic question: where the methylated CpGs are located relative to DNA-histone contact points and how these modifications influence H2A.Z nucleosome structure. The structure provides no mechanistic insights into methylation-induced destabilization.
The fact that the DNA resolution in the methylated structure was not high enough to resolve the positions of methylated CpGs despite a high overall resolution of 2.78 Å implies that 1) the Sat2R-P DNA was not as stably registered as the 601L sequence, requiring us to create two alternative Sat2R-P atomic models to account for the variable positioning in our samples, and 2) that the presence of DNA methylation increases that positional variability. We understand that one may prefer to see highly resolved density around each methylation mark, but we do believe that our inability to accomplish that is actually a feature rather than a weakness and has important biological implications. The decrease in local DNA resolution on the methylated Sat2R-P structure compared to its unmethylated counterpart is meaningful and suggests to us that DNA methylation weakens overall DNA wrapping and positioning on the nucleosome, supported by the increased flexibility seen at the linker DNA ends as well as an increase in the population of highly shifted nucleosomes amongst the methylated particles. Additionally, one major view in the DNA methylation/nucleosome stability field is that the presence of DNA methylation can make DNA stiffer and harder to bend, causing opening and destabilization of nucleosomes (Ngo et al., 2016). The increased opening of linker DNA ends and accessibility of methylated H2A.Z nucleosomes in our hands also aligns with such an idea, again suggesting decreased histone-DNA contact stability on methylated DNA substrates. We plan to revise the writing in our manuscript to better reflect these ideas.
The experimental system also lacks physiological relevance. The template DNA sequence is artificial, despite the existence of well-characterised native genomic sequences for which DNA methylation is known to inhibit H2A.Z incorporation. Alternatively, there are a number of studies examining the effect of DNA methylation on nucleosome structure, stability, DNA unwrapping, and positioning. Choosing one of these DNA sequences would have at least allowed a direct comparison with a canonical nucleosome. Indeed, a major omission is the absence of a cryo-EM structure of a canonical nucleosome assembled on the same DNA template - this is essential to assess whether the observed effects are H2A.Z-specific.
The reviewer raises a fair question about whether canonical H2A would experience the same DNA methylation-dependent structural effects. We had considered solving the H2A structures, however, ultimately decided against it for a few reasons. First, there already exists crystal structures of canonical H2A nucleosomes using a DNA sequence highly similar to our Sat2R-P with and without the presence of DNA methylation (PDB: 5CPI and 5CPJ). The authors of this study did not see any physical differences present in their structures (Osakabe et al., 2015). Additionally, we had included canonical H2A conditions within our restriction enzyme accessibility assay and did not see a significant impact of DNA methylation on those samples (Fig 3). Because of the previous report and our own negative data, we expected that only limited additional insights would be obtained from the canonical H2A structures and decided not to pursue that analysis.
One of the primary reasons we chose the Sat2R-P sequence was, as noted above, that there already was a published study examining how DNA methylation affects nucleosome structure using a variant of this sequence which we could compare to our results, as the reviewer has suggested. We did have to modify the sequence, namely by making it palindromic, in order to increase the final achievable resolution. We viewed the Sat2R-P sequence as an attractive candidate because it is physiologically relevant; the initial sequence was taken directly from human satellite II. Several modifications were made for technical reasons, including making the sequence palindromic as described above and also ensuring that each CpG is recognizable by a methylation-sensitive restriction enzyme so that we could be certain about the degree of methylation on our substrates. These practical concerns outweighed the necessity of maintaining a strict physiological sequence to us. However, we still believe the final Sat2R-P more closely mimics physiological sequences than Widom 601. Additionally, human satellite II is a highly abundant sequence in the human genome that is known to undergo large methylation changes on the onset of many disorders, like cancer, as well as during aging. Thus, there are interesting biological questions surrounding how the methylation state of this particular sequence affects chromatin structure. Furthermore, it has been reported that satellite II is devoid of H2A.Z (Capurso et al., 2012). Beyond those reasons, the satellite II sequence is generally interesting to our lab because we have been studying genes involved in ICF syndrome, where hypomethylation of satellite II sequences forms one of the hallmarks of this disorder (Funabiki et al., 2023; Jenness et al., 2018; Wassing et al., 2024). We understand that sequence context plays a large role in nucleosome wrapping and stability. This is why we strived to test multiple sequences in each of our assays. We do agree that it would be interesting to use DNA sequences where H2A.Z binding has already been described to be affected in a DNA methylation-dependent manner, forming an exciting future study to pursue.
Furthermore, the DNA template is methylated at numerous random CpG sites. The authors' argument that only the global methylation level is relevant is inconsistent with the literature, which clearly demonstrates that methylation effects on canonical nucleosomes are position-dependent. Not all CpG sites contribute equally to nucleosome stability or unwrapping, and this critical factor is not considered.
We did not argue that only the global methylation level is relevant. We also would appreciate it if the reviewer could provide specific references that "clearly demonstrates that methylation effects on canonical nucleosomes are position-dependent". We are aware of a series of studies conducted by Chongli Yuan's group, including one testing the effect of placing methylated CpGs at different positions along the Widom 601 sequence. In that study (Jimenez-Useche et al., 2013), they did find that positioning of mCpGs has differential impacts on the salt resistance of the nucleosomes, with 5 tandem mCpG copies at the dyad causing the most dramatic nucleosome opening whereas having mCpGs only at the DNA major grooves, but not elsewhere, increased nucleosome stability. However, they did also find that methylation of the original Widom 601 sequence also caused destabilization, albeit to a lesser degree, and another study by the same group (Jimenez-Useche et al., 2014) also found that CpG methylation decreased nucleosome-forming ability for all tested variants of the Widom 601 sequence, regardless of CpG density or positioning.
Other studies monitored how distribution of methylated CpGs correlates with nucleosome positioning (Collings et al., 2013; Davey et al., 1997; Davey et al., 2004). However, these studies assessed the sequence-dependent effects specifically on nucleosome assembly during in vitro salt dialysis, which is a different physical process than the one our manuscript focuses on, especially when considering the fact that H2A.Z is deposited onto preassembled H2A-nucleosome. Our cryo-EM analysis examines the structural changes induced by DNA methylation on already formed nucleosomes rather than the process of formation. Thus, probing accessibility changes using a restriction enzyme was the more appropriate biochemical assay to verify our structures.
We do very much agree that DNA context can influence nucleosome stability under different conditions. A study of molecular dynamics simulations concluded that the "combination of overall DNA geometrical and shape properties upon methylation" makes nucleosomes resistant to unwrapping (Li et al., 2022), while another modeling study suggests that DNA methylation impacts nucleosome stability in a manner dependent on DNA sequence, where "[s]trong binding is weakened and weak binding is strengthened" (Minary and Levitt, 2014). While G/C-dinucleotides are preferentially placed at major groove-inward positions in the nucleosomes in vivo (Chodavarapu et al., 2010; Segal et al., 2006) and G/C-rich segments are excluded from major groove-outward positions in Widom 601-like nucleosomes (Chua et al., 2012), methylated CpG dinucleotides are preferably, if not exclusively, located at major groove-outward positions in vivo. Mechanisms behind this biased mCpG positioning on the nucleosome remain speculative, likely caused by a combination of multiple factors, but the fact that we did not observe clear structural impacts using the Widom 601L sequence, where mCpGs are located at the major groove-outward and -inward positions ((Chua et al., 2012) and our structure), deserves a space for discussion. On the other hand, positioning of mCpG on satellite II-derived sequences that we used in this study was based on a physiological sequence, and thus it may not be appropriate to say that those CpGs are placed at multiple "random" positions. Although we decided not to discuss the position of 5mC on our Sat2R nucleosome structure due to ambiguous base assignments, neither of our two atomic models is consistent with an idea that DNA methylation repositions the CpG to the outward major grooves. As the potential contribution of how DNA methylation affects the nucleosome structure via modulating DNA stiffness has been extensively studied (Choy et al., 2010; Li et al., 2022; Ngo et al., 2016; Perez et al., 2012), we believe that it is appropriate to consider overall DNA properties along the whole DNA sequence, though we are willing to discuss potential positional effects in the revised manuscript.
Perhaps one of the most important points that we did not emphasize enough in our original manuscript was that in contrast to the subtle intrinsic effect of DNA methylation that was DNA sequence dependent, we observed SRCAP-dependent preferential H2A.Z deposition to unmethylated DNA over methylated DNA in both 601 and satellite II DNAs. In the revised manuscript, we will make the value of comparative studies on 601 and satellite II in two distinct mechanisms.
Finally, and most importantly, the reported increase in accessibility of the methylated H2A.Z nucleosome is negligible compared with the much larger intrinsic DNA accessibility of the unmethylated H2A.Z nucleosome. These data do not support the authors' hypothesis and contradict the manuscript's conclusions. Claims that methylated H2A.Z nucleosomes are "more open and accessible" must therefore be removed, and the title is misleading, given that no meaningful impact of DNA methylation on H2A.Z nucleosome stability is demonstrated.
We respectfully disagree with this reviewer's criticism. We investigated the potential impact of DNA methylation on nucleosome stability to the best of our abilities through complementary assays and reported our observations. The effect of DNA methylation is smaller than the difference between H2A.Z and H2A, but we were able to see an effect. It is also not uncommon for small differences to have functional impacts in biological systems. We agree that further testing is required to determine whether this subtle effect is functionally important, and it remains the subject of future research due to the many technical challenges associated with addressing said question. We would like to note that 18 years have passed since Daniel Zilberman first reported the antagonistic relationship between H2AZ and DNA methylation (Zilberman et al., 2008) but very few studies have since directly tested specific mechanistic hypotheses. We believe that our study lays the groundwork for exciting future investigation that better elucidates the pathways that contribute to this antagonism and will have meaningful impacts on the field in general. However, thanks to the reviewer's criticism, we realized that we did not clearly state in the Abstract the relatively subtle effect of DNA methylation on the intrinsic H2A.Z nucleosome stability. Therefore, we will accordingly revise the Abstract to make this point clearer.
(2) The cryo-EM structures of methylated and unmethylated 601L H2A.Z nucleosomes show no detectable differences. As presented, this negative result adds little value. If anything, it reinforces the point that the positional context of CpG methylation is critical, which the manuscript does not consider.
We believe the inclusion and factual reporting of negative data is important for the scientific community as one of the major issues currently in biology research is biased omission of negative data. We considered eLife as a venue to publish this work for this reason. We understand that the reviewer believes our 601L structures may detract from the overall message of our manuscript. We believe this data rather emphasizes the importance of DNA sequence context, something that the reviewer also rightfully notes. It is standard practice in the nucleosome field to use the Widom 601 sequence, along with its variants. Our experience has shown that use of an artificially strong positioning sequence may mask weaker physical effects that could play a physiological role. Thus, we were careful to validate all further assays with multiple DNA sequences and believed it important to report these sequence-dependent effects on nucleosome structure.
(3) Very little H3 signal coincides with H2A.Z at TSSs in sperm pronuclei, yet this is neither explained nor discussed (Supplementary Figure 10D). The authors need to clarify this.
Our H3 signal, which represents the global nucleosome population, is more broadly distributed across the genome than H2A.Z, which is known to localize at specific genomic sites. Since both histone types were sequenced to similar read depths, H3 peaks are generally shallower than H2A.Z and peak heights cannot be directly compared (i.e. they should be represented in separate appropriate data ranges).
(4) In my view, the most conceptually important finding is that H2A.Z-associated reads in sperm pronuclei show ~43% CpG methylation. This directly contradicts the model of strict mutual exclusivity and suggests that the antagonism is context-dependent. Similarly, the finding that the depletion of SRCAP reduces H2A.Z deposition only on unmethylated templates is also very intriguing. Collectively, these result warrants further investigation (see below).
(5) Given that H2A.Z is located at diverse genomic elements (e.g., enhancers, repressed gene bodies, promoters), the manuscript requires a more rigorous genomic annotation comparing H2A.Z occupancy in sperm pronuclei versus XTC-2 cells. The authors should stratify H2A.Z-DNA methylation relationships across promoters, 5′UTRs, exons, gene bodies, enhancers, etc., as described in Supplementary Figure 10A.
(below is response to (4) and (5) together)
We agree that the substantial presence of co-localized H2A.Z and DNA methylation specifically in the sperm pronuclei samples and the changes in pattern between nuclear types are highly interesting and require further investigation. However, we faced technical challenges in our sequencing experiments that made us refrain from conducting a more detailed analysis for fear of over-interpreting potential artifacts. These challenges mainly stemmed from the difficulties in collecting enough material from Xenopus egg extracts and Tn5’s innate bias towards accessible regions of the genome. Because of this, open regions of the genome tend to be overrepresented in our data (as noted in our Discussion), making it challenging to rigorously compare methylation profiles and H2A.Z/H3 associated genomic elements.
While the degree of separation seems to be dependent on nuclei type, we still believe the antagonism exists in both the sperm pronuclei and XTC-2 samples when comparing H2A.Z methylation profiles to the corresponding H3 condition. Our study also demonstrates that H2A.Z is preferentially deposited to hypomethylated DNA in a manner dependent of SRCAP-C (the loss of SRCAP only reduces H2A.Z on unmethylated substrates) but an additional methylation-insensitive H2A.Z deposition mechanism also exists. We realized that this interesting point was not clearly highlighted in Abstract, so we will revise it accordingly.
(6) Although H2A.Z accumulates less efficiently on exogenous methylated substrates in egg extract, substantial deposition still occurs (~50%). This observation directly challenges the strong antagonistic model described in the manuscript, yet the authors do not acknowledge or discuss it. Moreover, differences between unmethylated and methylated 601 DNA raise further questions about the biological relevance of the cryo-EM 601 structures.
As depicted in Figure 6 and described in the Discussion, we clearly indicated that both methylation-sensitive and methylation-insensitive pathways exist to deposit H2A.Z within the genome. We also directly stated in our Discussion that a substantial proportion of H2A.Z colocalizes with DNA methylation both in our study as well as in previous reports, which is of major interest for future study. Additionally, we further discussed how the absence of transcription in Xenopus eggs is a likely reason for the more limited effect of DNA methylation restricting H2A.Z deposition in our egg extract system.
As noted in our response to (2), the lack of a clear impact on our 601L structures implies that this is due to the extraordinarily strong artificial nucleosome positioning capacity of the 601 sequence and its variants. Since 601 is heavily used in chromatin biology, including within DNA methylation research, such negative data are still useful to include and publish.
(7) The SRCAP depletion is insufficiently validated i.e., the antibody-mediated depletion of SRCAP lacks quantitative verification. A minimum of three biological replicates with quantification is required to substantiate the claims.
We are willing to address this concern. However, please note that our data showed that methylation-dependent H2A.Z deposition is almost completely erased upon SRCAP depletion, indicating functionally effective depletion. The specificity of the custom antibody against Xenopus SRCAP was verified by mass spectrometry. Additionally, we have obtained the same effect using another commercially available SRCAP antibody, though we did not include this preliminary result in our original manuscript. Due to its relatively low abundance and high molecular weight, SRCAP western blot signals are weak, making it challenging to quantify the degree of depletion. We also believe that the value of quantification in this context, with the points noted above, is rather limited. In the past, our lab has published papers on depleting the H3T3 kinase Haspin from Xenopus egg extracts (Ghenoiu et al., 2013; Kelly et al., 2010) but were never able to detect Haspin via western blot. This protein was only detected by mass spectrometry specifically on nucleosome array beads with H3K9me3 (Jenness et al., 2018). However, depletion of Haspin was readily monitored by erasure of H3T3ph, the enzymatic product of Haspin. In these experiments, it was impossible, and not critical, to quantitatively monitor the depletion of Haspin protein in order to investigate its molecular functions. Similarly, in this current study, the important fact is that depletion of SRCAP suppressed methylation-sensitive H2A.Z deposition and quantifying the degree of SRCAP depletion would not have a major impact on this conclusion.
(8) It appears that the role of p400-Tip60 has been completely overlooked. This complex is the second major H2A.Z deposition complex. Because p400 exhibits DNA methylation-insensitive binding (Supplementary Figure 14), it may account for the deposition of H2A.Z onto methylated DNA. This possibility is highly significant and must be addressed by repeating the key experiments in Figure 5 following p400-Tip60 depletion.
We are aware that the Tip60 complex is a very likely candidate for mediating DNA methylation-insensitive H2A.Z deposition, which is why we tested whether DNA binding of p400 is methylation sensitive. Therefore, the reviewer's statement that we "completely overlooked" Tip60-C’s role does not fairly report on our efforts. We wished to test the potential contribution of Tip60-C, but, unfortunately, the antibodies we currently have available to us were not successful in depleting the complex from egg extract. Since we had no direct experimental evidence indicating the role Tip60-C plays, we decided to take a conservative approach to our model and leave the methylation-insensitive pathway as mediated by something still unidentified. While further investigating Tip60-C’s contribution to this pathway is of definite value, we do not believe that it impacts our major conclusion that SRCAP-C is the main mediator responsible for H2A.Z deposition on unmethylated DNA and thus remains a subject for future study.
(9) The manuscript repeatedly states that H2A.Z nucleosomes are intrinsically unstable; however, this is an oversimplification. Although some DNA unwrapping is observed, multiple studies show that H3/H4 tetramer-H2A.Z/H2B interactions are more stable (important recent studies include the following: DOI: 10.1038/s41594-021-00589-3; 10.1038/s41467-021-22688-x; and reviewed in 10.1038/s41576-024-00759-1).
We understand that the H2A.Z stability field is highly controversial. We have introduced the many conflicting reports that have been published in the field but can further expand on the controversies if desired. We also understand that the term “nucleosome stability” is broad and encompasses many physical aspects. As noted in a prior response, we will better specify our use of the term within the manuscript. In our assays, we are most focused on the DNA wrapping stability of the nucleosome and have consistently seen in our hands that H2A.Z nucleosomes are much more open and accessible compared to canonical H2A on satellite II-derived sequences, regardless of methylation status. However, we do understand that many groups have observed the opposite findings while others have obtained results similar to us. We reported on our findings of the general H2A.Z stability with the hopes to help clarify some of the field’s controversies.
In summary, the current manuscript does not present a convincing mechanistic explanation for the antagonism between DNA methylation and H2A.Z. The observation that H2A.Z can substantially coexist with DNA methylation in sperm pronuclei, perhaps, should be the conceptual focus.
We appreciate this reviewer’s advice. However, please note that the first author who led this project has already successfully defended their PhD thesis primarily based on this project, making it impractical and unrealistic to completely change the focus of this manuscript to include an entirely new avenue of research. We believe that our data provide important insights into the mechanisms by which H2A.Z is excluded from methylated DNA, particularly via the DNA methylation-sensitive binding of SRCAP-C, which has never been described before. We agree that many questions are still left unanswered, including the exact molecular mechanism behind how DNA methylation prevents SRCAP-C binding. We have preliminary data that suggest none of the known DNA-binding modules of SRCAP-C, including ZNHIT1, by themselves can explain this sensitivity. This implies that domain dissection in the context of the holo-SRCAP complex is required to fully address this question. We believe this represents a very exciting future avenue of study; however, it does not negate our finding that SRCAP-C itself is important for maintaining the DNA methylation/H2A.Z antagonism. Therefore, we respectfully disagree with this reviewer's summary statement, which misleadingly undermines the impact of our work.
Reviewer #3 (Public review):
Summary:
Histone variant H2A.Z is evolutionarily conserved among various species. The selective incorporation and removal of histone variants on the genome play crucial roles in regulating nuclear events, including transcription. Shih et al. aimed to address antagonistic mechanisms between histone variant H2A.Z deposition and DNA methylation. To this end, the authors reconstituted H2A.Z nucleosomes in vitro using methylated or unmethylated human satellite II DNA sequence and examined how DNA methylation affects H2A.Z nucleosome structure and dynamics. The cryo-EM analysis revealed that DNA methylation induces a more open conformation in H2A.Z nucleosomes. Consistent with this, their biochemical assays showed that DNA methylation subtly increases restriction enzyme accessibility in H2A.Z nucleosomes compared with canonical H2A nucleosomes. The authors identified genome-wide profiles of H2A.Z and DNA methylation using genomic assays and found their unique distribution between Xenopus sperm pronuclei and fibroblast cells. Using Xenopus egg extract systems, the authors showed SRCAP complex, the chromatin remodelers for H2A.Z deposition, preferentially deposit H2A.Z on unmethylated DNA.
Strengths:
The study is solid, and most conclusions are well-supported. The experiments are rigorously performed, and interpretations are clear. The study presents a high-resolution cryo-EM structure of human H2A.Z nucleosome with methylated DNA. The discovery that the SRCAP complex senses DNA methylation is novel and provides important mechanistic insight into the antagonism between H2A.Z and DNA methylation.
We are grateful that this reviewer recognizes the importance of our study.
Weaknesses:
The study is already strong, and most conclusions are well supported. However, it can be further strengthened in several ways.
(1) It is difficult to interpret how DNA methylation alters the orientation of the H4 tail and leads to the additional density on the acidic patch. The data do not convincingly support whether DNA methylation enhances interactions with H2A.Z mono-nucleosomes, nor whether this effect is specific to methylated H2A.Z nucleosomes.
The altered H4 tail orientation and extra density seen on the acidic patch were incidental findings that we thought could be interesting for the field to be aware of but decided not to follow up on as there were other structural differences that were more directly related to our central question. We do believe that the above two differences are linked to each other because we used a highly purified and homogenous sample for cryo-EM analysis and the H4 tail/acidic patch interaction is a well characterized contact that mediates inter-nucleosome interactions. Additionally, other groups have reported that the presence of DNA methylation causes condensation of both chromatin and bare DNA (cited within our manuscript), though the mechanics behind this phenomenon remain to be elucidated. We believed that our structure data may also align with those findings. However, the reviewer is fair in pointing out that we do not provide further experimental evidence in verifying the existence of these increased interactions. We can revise our writing to clarify that these points are currently hypotheses rather than validated results.
(2) It remains unclear whether DNA methylation alters global H2A.Z nucleosome stability or primarily affects local DNA end flexibility. Moreover, while the authors showed locus-specific accessibility by HinfI digestion, an unbiased assay such as MNase digestion would strengthen the conclusions.
We would like to thank the reviewer for bringing up these issues. Although our current data cannot explicitly clarify these possibilities, we favor an idea that DNA methylation specifically alters histone to DNA contacts and that this effect is felt globally across the entire nucleosome rather than only at specific locations. The intrinsic flexibility of linker DNA ends means that that region tends to exhibit the greatest differences under different physical influences, hence the focus on characterizing that area; flexibility of a thread on a spool is most pronounced at the ends. However, we also found that the DNA backbone of H2A.Z on methylated DNA had a lower local resolution compared to its unmethylated counterpart, despite that structure having a higher global resolution, which suggested to us that DNA positioning along the nucleosome is overall weaker under the presence of DNA methylation. This is corroborated by the increased population of open/shifted structures in our classification analysis. The reviewer raises a fair point about the use of a specific restriction enzyme versus MNase. We agree that our accessibility assay is highly influenced by the position of the restriction site and have previously seen that moving the cut site too close to the linker DNA end will abolish any DNA methylation-dependent differences. We did initially attempt an MNase digestion-based assay, but the data were not as reproducible as with the use of a specific restriction enzyme. We do not know the reason behind this irreproducibility though we believe that the processivity of MNase could make it difficult to capture subtle effects like those induced by DNA methylation on already highly accessible H2A.Z nucleosomes. Overall, while we believe that DNA methylation does exert a physical effect, its subtlety may explain the many contradictory studies present within the DNA methylation and nucleosome stability field.
References
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