Resetting of H3K4me2 during mammalian parental-to-zygote transition

  1. Chong Wang
  2. Yang Li
  3. Yaqian Wang
  4. Yong Shi
  5. Xiangrui Meng
  6. Wenbo Li
  7. Jia Guo
  8. Kaiyue Hu
  9. Hao Chen
  10. Jiawei Xu

  • Curated by eLife

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    eLife Assessment

    This important study reports dynamic reprogramming of global H3K4me2 during the oocyte-to-embryo transition in mice. The data presented support the main conclusion and are generally convincing. The work will be of interest to researchers working on epigenetic reprogramming during embryonic development.

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Abstract

Upon sperm and oocyte fertilization, the embryo undergoes drastic histone modification reprogramming during pre-implantation development. In this study, we investigated the erasure and re-establishment of H3K4me2 in mouse GV, MII, and embryos using an improved approach called Cleavage Under Targets and Release Using Nuclease (CUT&RUN) with high-throughput sequencing. We found that H3K4me2 extensively occurs as a non-canonical pattern in mouse GV oocytes and early embryos, and parental H3K4me2 was erased by maternal LSD2 during forming the pronucleus in the zygote. The H3K4me2 was erased from GV to MII oocyte and was re-established in the late two-cell stage, removing this epigenetic burden is crucial for ZGA. We then revealed the H3K4me2 locates widespread in the CpG-rich and hypomethylated regulatory regions in the four-cell stage, and the eight-cell stage, but dramatic changes occurred in the inner cell mass (ICM) in mouse embryos. Then, these CpG-rich H3K4me2 regulatory regions became either activated or repressed. To summarize, in this study, we elucidated the pattern of H3K4me2 transition from parent to zygote and H3K4me2 profile during early embryo development. Our findings might provide deeper insights into epigenetic reprogramming during early development and in vitro fertilization in mammalian.

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  1. eLife

    eLife Assessment

    This important study reports dynamic reprogramming of global H3K4me2 during the oocyte-to-embryo transition in mice. The data presented support the main conclusion and are generally convincing. The work will be of interest to researchers working on epigenetic reprogramming during embryonic development.

  2. eLife

    Reviewer #2 (Public review):

    Chong Wang et al. investigated the role of H3K4me2 during the reprogramming processes in mouse preimplantation embryos. The authors show that H3K4me2 is erased from GV to MII oocytes and re-established in the late 2-cell stage by performing Cut & Run H3K4me2 and immunofluorescence staining. Erasure and re-establishment of H3K4me2 have not been studied well, and profiling of H3K4me2 in germ cells and preimplantation embryos is valuable to understanding the reprogramming process and epigenetic inheritance.

    (1) "The authors' assertion that H3K27me3 did not change from GV to MII stage (Author response) is noted; however, this data was not provided. To validate the technical success of the CUT&RUN protocol in MII oocytes-a stage characterized by chromatin condensation and low DNA input-it is essential that the authors provide their internal H3K27me3 data as a positive control.

    Without showing that a stable mark (like H3K27me3) can be successfully mapped in their MII samples, the 'disappearance' of H3K4me2 peaks cannot be distinguished from a technical failure of the assay at this developmental stage. Furthermore, I remain skeptical of the inclusion of the first polar body as a DNA quantity, as polar body chromatin is often undergoing degradation and may not reflect the epigenetic state of the oocyte itself.

    (2) I remain concerned by the inconsistencies regarding KDM1A (LSD1) expression. The authors claim in the text and Figure 4A that KDM1A is 'rarely expressed' during mouse embryonic development. However, their own Extended Data Figure 3A shows high expression of KDM1A in oocytes, 4-cell, and 8-cell stages. Both figures reportedly use published RNA-seq data. The authors must explain how the same gene in the same developmental stages can appear 'rarely expressed' in one figure and 'expressed' in another.

    (3) Page 6 (Line 161-165) The H3K4me2 demethylases KDM1B (LSD2) were also highly expressed in growing oocytes but showed decreased expression in MII oocytes (Figure S3A, see also revised heatmap). We have re-analyzed the expression data and corrected the heatmap normalization; the revised Figure S3A now accurately shows that Kdm1b is highly expressed in growing oocytes, with lower levels in 8- week and MII oocytes, consistent with its role in maternal imprint establishment.

    The expression data in growing oocytes are missing in Fig S3A.

    (4) The authors' proposal to use transcriptome data to confirm TCP specificity is insufficient (Author reply). Since TCP is a small-molecule inhibitor of protein activity, its primary effect is not the reduction of mRNA transcripts, but the inhibition of the enzymes themselves.

    To support their claim that the observed H3K4me2 resetting and developmental arrest are specifically due to KDM1B inhibition, I recommend:

    a. Perform Western Blot for KDM1A/B

    b. Use a Selective Inhibitor: Use a more selective KDM1A inhibitor (e.g. GSK-LSD1)

    c. Address the Discrepancy: Provide a clear biological explanation for why chemical inhibition leads to 4-cell arrest while a maternal KO of KDM1B survives to E10."

    (5) "The authors' response that IF and CUT&RUN cannot be compared because of 'different analysis models' is not scientifically sound in this context.

    Quantitative Contradiction: A 1,000-fold increase in global IF signal must be reflected in the genomic landscape. If only 251 genes show a gain in H3K4me2 in the CUT&RUN data, this represents a negligible fraction of the genome, directly contradicting the 'dramatic increase' claimed in the IF data.

    Lack of Spike-in Normalization: Did the authors use a spike-in for their CUT&RUN? Without global normalization, CUT&RUN only reports relative changes. If H3K4me2 increased everywhere, a non-normalized CUT&RUN library would fail to show it, making the data misleading. Currently, the two datasets provide two different versions of biological reality. A third validation (e.g., Spike-in Normalized CUT&RUN) would be recommended to determine which dataset is accurate."

  3. eLife

    Reviewer #3 (Public review):

    Summary:

    The study "Resetting of H3K4me2 during mammalian parental-to-zygote transition" provides valuable insights into the dynamic changes in H3K4me2 during early embryonic development.

    Strengths:

    The findings provide valuable insights into the temporal and spatial dynamics of H3K4me2 and its potential role in zygotic genome activation (ZGA).

    Weaknesses:

    Key areas for improvement include enhancing the innovation and novelty of the study, providing robust functional validation, establishing a clear model for H3K4me2's role, and addressing technical and presentation issues. While the findings are significant, the current manuscript falls short in several critical areas. Addressing these major and minor issues will significantly strengthen the study's contribution to the field of epigenetic reprogramming and embryonic development.

    Comment on revised version:

    It would be better for the author to directly provide some experimental or analytical data rather than discussing and defending.

  4. eLife

    Author response:

    The following is the authors’ response to the original reviews.

    Public Reviews:

    Reviewer #1 (Public Review):

    (1) The study emphasizes H3K4me2, which often serves as a precursor to H3K4me3, a well-studied modification during early development. Analyzing the new H3K4me2 dataset alongside published H3K4me3 data is crucial for comprehensively understanding epigenetic reprogramming post-fertilization and the interplay between histone modifications. However, the current analysis is preliminary and lacks depth.

    We fully agree with this valuable suggestion. Our research group has previously systematically profiled H3K4me3 dynamics in human and mouse early embryos, and the relevant results have been published in Science (2019). The core objective of the current study is to explore the erasure, re-establishment and biological functions of H3K4me2 during mammalian parental-to-zygote transition. To enrich our analysis, we have now integrated our H3K4me2 data with publicly available H3K4me3 datasets for joint analysis. The results clearly demonstrate that H3K4me2 is not merely a precursor of H3K4me3. These two histone marks present distinct genome-wide distribution patterns and perform independent regulatory roles in embryonic epigenetic reprogramming. We have added the joint analysis results and relevant discussions in the revised manuscript to elaborate the crosstalk between H3K4me2 and H3K4me3.

    Manuscript Revisions

    All supplementary analyses and discussions are located in the Results and Discussion sections, Results section (Page 6, Lines 156–165) (Page 7, Lines 197–201) (Page 10, Lines 274–278) and Discussion section (Page 13- 14, Lines 374–382) (Page 15, Lines 424–429.

    (2) Tranylcypromine (TCP) is known as an irreversible inhibitor of monoamine oxidase and LSD1. While the authors suggest TCP inhibits the expression of LSD2, this assertion is questionable. Given TCP's potential non-specific effects in cells, conclusions related to the experiments using TCP should be made with caution.

    We highly appreciate this important reminder about the off-target effects of TCP. We have supplemented two classic literatures (J Am Chem Soc, 2010; Mol Cell, 2010) which have proven that TCP acts as an irreversible inhibitor targeting both LSD1 (KDM1A) and LSD2 (KDM1B). According to our transcriptome and protein detection data, the endogenous expression level of LSD1 is extremely low in mouse early embryos. Therefore, LSD2 is the primary functional target of TCP in our embryonic experimental system. All conclusions derived from TCP treatment experiments are described prudently in the full text to avoid over-interpretation.

    Manuscript Revisions

    Relevant supplements are made in the Results section (Page 7, Lines 197–201).

    (3) Some batches of H3K4me2 antibody are known to cross-react with H3K4me3. Has the H3K4me2 antibody used in CUT&RUN been tested for such cross-reactivity? Heatmaps in the figures indeed show similar distribution for H3K4me2 and H3K4me3, further raising concerns about antibody specificity.

    Thank you for raising this critical question regarding antibody specificity, which is essential for the reliability of CUT&RUN experiments. The H3K4me2 antibody used in this study was purchased from Millipore (Cat. No. 07030). Based on the manufacturer’s product specification and our internal verification, this antibody has very low cross-reactivity with H3K4me3.The similar distribution shown in heatmaps is not caused by antibody contamination. Instead, it reflects the inherent spatial correlation between H3K4me2 and H3K4me3 on chromatin in early embryos.

    (4) Certain statements lack supporting references or figures (examples on page 9 can be found on line 245, line 254, and line 258).

    We apologize for the inadequate citation in the original manuscript. We have comprehensively checked the full text and added standard peer-reviewed references to all statements without literature support. Specifically, we have supplemented corresponding references for the content on Page 9, Line 259 and Line 266 as suggested. We also completed a full-text inspection to fix similar problems in other positions.

    Manuscript Revisions

    References are supplemented on Page 9, Line 259 and Line 266.

    (5) Extensive language editing is recommended to clarify ambiguous sentences. Additionally, caution should be taken to avoid overstatement - most analyses in this study only suggest correlation rather than causality.

    We fully accept this suggestion. We have thoroughly revised all ambiguous, redundant and grammatically problematic sentences throughout the manuscript to improve readability and academic rigour. Furthermore, we have carefully modified all overstated expressions. For all experimental results and bioinformatics analyses, we only use words such as correlate with, suggest, indicate to describe correlative relationships. All inappropriate causal inferences have been completely removed to ensure objective presentation of our data.

    Manuscript Revisions

    Full manuscript is polished and revised.

    Reviewer #2 (Public Review):

    (1) The authors claim that the Cut & Run worked for MII oocytes, zygotes, and the 2-cell embryos. However, it is unclear if H3K4me2 is erased during the stage or if the Cut & Run did not work for these samples. To support the hypothesis of the erasure of H3K4me2, the authors conducted immunofluorescence staining, and H3k4me2 was undetected in the MII oocyte, PN5, and 2-cell stage. However, the published papers showed strong staining of H3K4me2 at the zygote stage and 2-cell stage ((Ancelin et al., 2016; Shao et al., 2014)). The authors need to cite these papers and discuss the contradictory findings.

    The authors used 165 MII oocytes and 190 GV oocytes for the Cut & Run. The amount of DNA in MII oocytes is halved because of the emission of the first polar body. Would it be a reason that H3K4me2 has fewer H3K4me2 peaks in MII oocytes?

    Thank you for putting forward these thoughtful questions. Firstly, we have cited two published literatures (Ancelin et al., 2016; Shao et al., 2014) in the revised manuscript and discussed the inconsistent immunofluorescence results. The main reason for the discrepancy lies in different confocal microscope parameters including laser power, gain and exposure time adopted by different laboratories. In our study, we used unified imaging parameters to continuously observe samples from GV oocytes to blastocysts, so weak H3K4me2 signals at zygote and two-cell stages could not be detected. When we adjust parameters specifically for these stages, weak fluorescence signals can be observed. We have elaborated this point in the Discussion section.

    Secondly, we clarify that the reduction of H3K4me2 peaks in MII oocytes is not caused by decreased DNA content. Although MII oocytes extrude the first polar body during maturation, we collected the polar body together with oocytes in all CUT&RUN experiments, so the total DNA content of MII samples is not reduced. Combined with previous studies on human oocytes, we confirm that the loss of H3K4me2 peaks from GV to MII stage is a real physiological epigenetic change accompanying oocyte meiotic maturation and chromatin remodeling.

    (2) The authors claim that Kdm1a is rarely expressed during mouse embryonic development (Figure 4A). However, the published paper showed that KDM1a is present in the zygote and 2-cell stage using immunostaining and western blotting ((Ancelin et al., 2016)). Additionally, this paper showed that depletion of maternal KDM1A protein results in developmental arrest at the two-cell stage, and therefore, KDM1a is functionally important in early development. The authors should have cited the paper and described the role of KDM1a in early embryos.

    We apologize for the ambiguous expression in the original manuscript. What we described is a relative expression level: in mouse early embryos, the expression of KDM1A is lower than KDM1B, rather than the absolute absence of KDM1A.

    (3) The authors used the published RNA data set and interpreted that KDM1B (LSD2) was highly expressed at the MII stage (Figure S3A). However, the heat map shows that KDM1B expression is high in growing oocytes but not at 8w_oocytes and MII oocytes. The authors need to interpret the data accurately.

    We sincerely apologize for the data misinterpretation caused by improper data normalization in the original heatmap. We have completely re-normalized the RNA-seq data and redrawn Supplementary Figure S3A.

    The updated heatmap clearly shows that KDM1B is highly expressed in growing oocytes, while its expression decreases in 8-week oocytes and MII oocytes. Combined with Figure 4A, we have rewritten the description of KDM1B expression trends across different oocyte stages, and all textual descriptions are now consistent with the corrected data.

    Manuscript Revisions

    Supplementary Figure S3A is remade; data interpretation is revised in the Results section. Supplementary Figure S3A (remade); Results section (Page 42).

    (4) All embryos in the TCP group were arrested at the four-cell stage. Embryos generated from KDM1b KO females can survive until E10.5 (Ciccone et al., 2009); therefore, TCP-treated embryos show a more severe phenotype than oocyte-derived KDM1b deleted embryos. Depletion of maternal KDM1A protein results in developmental arrest at the two-cell stage ((Ancelin et al., 2016)). The authors need to examine whether TCP treatment affects KDM1a expression. Western blotting would be recommended to quantify the expression of KDM1A and KDM1B in the TCP-treated embryos.

    We dig the transcriptome data to confirm the specificity of TCP to KDM1b. In addition, the intervention of TCP on the whole fertilized egg in this study increased the H3K4me2 content, and the embryo development retarding effect was more significant than that obtained by crossing with normal paternal lines after knocking down KDM1B from the mother.

    (5) H3K4me2 is increased dramatically in the TCP-treated embryos in Figure 4 (the intensity is 1,000 times more than the control). However, the Cut & Run H3K4me2 shows that the H3K4me2 signal is increased in 251 genes and decreased in 194 genes in the TCP-treated embryos. The authors need to explain why the gain of H3K4me2 is less evident in the Cut & Run data set than in the immunofluorescence result.

    Thank you for this valuable question. The inconsistent data performance between immunofluorescence (IF) and CUT&RUN is determined by the essential differences between the two technical principles.

    Immunofluorescence is a global semi-quantitative method, which reflects the total content of H3K4me2 in the whole nucleus. The 1000-fold increase refers to the overall fluorescence intensity of the nucleus. In contrast, CUT&RUN combined with high-throughput sequencing is a locus-specific quantitative method, which detects H3K4me2 enrichment changes at individual gene loci. Different analytical models and threshold settings also lead to differences in final data presentation.

  5. Version published to 10.7554/elife.99417.2 on eLife
  6. Version published to 10.7554/elife.99417 on eLife
  7. Version published to 10.1101/2022.05.05.490401v4 on bioRxiv
  8. Version published to 10.1101/2022.05.05.490401v3 on bioRxiv
  9. Version published to 10.7554/elife.99417.1 on eLife
  10. eLife

    eLife assessment

    This study presents an important finding of dynamic reprogramming of global H3K4me2 during mouse oocyte-to-embryo transition. While the H3K4me2 epigenome data is convincing, the interpretation and the potential mechanistic claims of the authors are incomplete in the current shape with the primary concerns regarding the contribution of Kdm1b or Kdm1a, as well as the specificity of the inhibitor and the antibody. The work will be of interest to researchers interested in epigenetic reprogramming.

  11. eLife

    Reviewer #1 (Public Review):

    By mapping H3K4me2 in mouse oocytes and pre-implantation embryos, the authors aim to elucidate how this histone modification is erased and re-established during the parental-to-zygotic transition, as well as how the reprogramming of H3K4me2 regulates gene expression and facilitates zygotic genome activation.

    Employing an improved CUT&RUN approach, the authors successfully generated H3K4me2 profiling data from a limited number of embryos. While the profiling experiments are very well executed, several weaknesses, particularly in data analysis, are apparent:

    (1) The study emphasizes H3K4me2, which often serves as a precursor to H3K4me3, a well-studied modification during early development. Analyzing the new H3K4me2 dataset alongside published H3K4me3 data is crucial for comprehensively understanding epigenetic reprogramming post-fertilization and the interplay between histone modifications. However, the current analysis is preliminary and lacks depth.

    (2) Tranylcypromine (TCP) is known as an irreversible inhibitor of monoamine oxidase and LSD1. While the authors suggest TCP inhibits the expression of LSD2, this assertion is questionable. Given TCP's potential non-specific effects in cells, conclusions related to the experiments using TCP should be made with caution.

    (3) Some batches of H3K4me2 antibody are known to cross-react with H3K4me3. Has the H3K4me2 antibody used in CUT&RUN been tested for such cross-reactivity? Heatmaps in the figures indeed show similar distribution for H3K4me2 and H3K4me3, further raising concerns about antibody specificity.

    (4) Certain statements lack supporting references or figures (examples on page 9 can be found on line 245, line 254, and line 258).

    (5) Extensive language editing is recommended to clarify ambiguous sentences. Additionally, caution should be taken to avoid overstatement - most analyses in this study only suggest correlation rather than causality.

  12. eLife

    Reviewer #2 (Public Review):

    Chong Wang et al. investigated the role of H3K4me2 during the reprogramming processes in mouse preimplantation embryos. The authors show that H3K4me2 is erased from GV to MII oocytes and re-established in the late 2-cell stage by performing Cut & Run H3K4me2 and immunofluorescence staining. Erasure and re-establishment of H3K4me2 have not been studied well, and profiling of H3K4me2 in germ cells and preimplantation embryos is valuable to understanding the reprogramming process and epigenetic inheritance.

    (1) The authors claim that the Cut & Run worked for MII oocytes, zygotes, and the 2-cell embryos. However, it is unclear if H3K4me2 is erased during the stage or if the Cut & Run did not work for these samples. To support the hypothesis of the erasure of H3K4me2, the authors conducted immunofluorescence staining, and H3k4me2 was undetected in the MII oocyte, PN5, and 2-cell stage. However, the published papers showed strong staining of H3K4me2 at the zygote stage and 2-cell stage ((Ancelin et al., 2016; Shao et al., 2014)). The authors need to cite these papers and discuss the contradictory findings.

    The authors used 165 MII oocytes and 190 GV oocytes for the Cut & Run. The amount of DNA in MII oocytes is halved because of the emission of the first polar body. Would it be a reason that H3K4me2 has fewer H3K4me2 peaks in MII oocytes than GV oocytes?

    In Figure 3C, 98% (13,183/13,428) of H3K4me2 marked genes in GV oocytes overlap with those in the 4-cell stage. Furthermore, 92% (14,049/15,112) of H3K4me2 marked genes in sperm overlap with those in the 4-cell stage. Therefore, most regions maintain germ line-derived H3K4me2 in the 4-cell stage. The authors need to clarify which regions of germ line-derived H3K4me2 are maintained or erased in preimplantation embryos. Additionally, it would be interesting to investigate which regions show the parental allele-specific H3K4me2 in preimplantation embryos since the authors used hybrid preimplantation embryos (B6 x DBA).

    (2) The authors claim that Kdm1a is rarely expressed during mouse embryonic development (Figure 4A). However, the published paper showed that KDM1a is present in the zygote and 2-cell stage using immunostaining and western blotting ((Ancelin et al., 2016)). Additionally, this paper showed that depletion of maternal KDM1A protein results in developmental arrest at the two-cell stage, and therefore, KDM1a is functionally important in early development. The authors should have cited the paper and described the role of KDM1a in early embryos.

    (3) The authors used the published RNA data set and interpreted that KDM1B (LSD2) was highly expressed at the MII stage (Figure S3A). However, the heat map shows that KDM1B expression is high in growing oocytes but not at 8w_oocytes and MII oocytes. The authors need to interpret the data accurately.

    (4) All embryos in the TCP group were arrested at the four-cell stage. Embryos generated from KDM1b KO females can survive until E10.5 (Ciccone et al., 2009); therefore, TCP-treated embryos show a more severe phenotype than oocyte-derived KDM1b deleted embryos. Depletion of maternal KDM1A protein results in developmental arrest at the two-cell stage ((Ancelin et al., 2016)). The authors need to examine whether TCP treatment affects KDM1a expression. Western blotting would be recommended to quantify the expression of KDM1A and KDM1B in the TCP-treated embryos.

    (5) H3K4me2 is increased dramatically in the TCP-treated embryos in Figure 4 (the intensity is 1,000 times more than the control). However, the Cut & Run H3K4me2 shows that the H3K4me2 signal is increased in 251 genes and decreased in 194 genes in the TCP-treated embryos (Fold changes > 2, P < 0.01). The authors need to explain why the gain of H3K4me2 is less evident in the Cut & Run data set than in the immunofluorescence result.

    References

    Ancelin, K., ne Syx, L., Borensztein, M., mie Ranisavljevic, N., Vassilev, I., Briseñ o-Roa, L., Liu, T., Metzger, E., Servant, N., Barillot, E., Chen, C.-J., Schü le, R., & Heard, E. (2016). Maternal LSD1/KDM1A is an essential regulator of chromatin and transcription landscapes during zygotic genome activation. https://doi.org/10.7554/eLife.08851.001

    Ciccone, D. N., Su, H., Hevi, S., Gay, F., Lei, H., Bajko, J., Xu, G., Li, E., & Chen, T. (2009). KDM1B is a histone H3K4 demethylase required to establish maternal genomic imprints. Nature, 461(7262), 415-418. https://doi.org/10.1038/nature08315

    Shao, G. B., Chen, J. C., Zhang, L. P., Huang, P., Lu, H. Y., Jin, J., Gong, A. H., & Sang, J. R. (2014). Dynamic patterns of histone H3 lysine 4 methyltransferases and demethylases during mouse preimplantation development. In Vitro Cellular and Developmental Biology - Animal, 50(7), 603-613. https://doi.org/10.1007/s11626-014-9741-6

  13. eLife

    Reviewer #3 (Public Review):

    Summary:

    This study explores the dynamic reprogramming of histone modification H3K4me2 during the early stages of mammalian embryogenesis. Utilizing the advanced CUT&RUN technique coupled with high-throughput sequencing, the authors investigate the erasure and re-establishment of H3K4me2 in mouse germinal vesicle (GV) oocytes, metaphase II (MII) oocytes, and early embryos.

    Strengths:

    The findings provide valuable insights into the temporal and spatial dynamics of H3K4me2 and its potential role in zygotic genome activation (ZGA).

    Weaknesses:

    The study primarily remains descriptive at this point. It would be advantageous to conduct further comprehensive functional validation and mechanistic exploration.
    Key areas for improvement include enhancing the innovation and novelty of the study, providing robust functional validation, establishing a clear model for H3K4me2's role, and addressing technical and presentation issues. The text would benefit from the introduction of a novel conceptual framework or model that provides a clear explanation of the functional consequences and molecular mechanisms underlying H3K4me2 reprogramming in the transition from parental to early embryonic development.

    While the findings are significant, the current manuscript falls short in several critical areas. Addressing major and minor issues will significantly strengthen the study's contribution to the field of epigenetic reprogramming and embryonic development.

  14. eLife

    Author response:

    Public Reviews:

    Reviewer #1 (Public Review):

    By mapping H3K4me2 in mouse oocytes and pre-implantation embryos, the authors aim to elucidate how this histone modification is erased and re-established during the parental-to-zygotic transition, as well as how the reprogramming of H3K4me2 regulates gene expression and facilitates zygotic genome activation.

    Employing an improved CUT&RUN approach, the authors successfully generated H3K4me2 profiling data from a limited number of embryos. While the profiling experiments are very well executed, several weaknesses, particularly in data analysis, are apparent:

    (1) The study emphasizes H3K4me2, which often serves as a precursor to H3K4me3, a well-studied modification during early development. Analyzing the new H3K4me2 dataset alongside published H3K4me3 data is crucial for comprehensively understanding epigenetic reprogramming post-fertilization and the interplay between histone modifications. However, the current analysis is preliminary and lacks depth.

    Thank you very much for your valuable suggestions. The data of histone H3K4me3 in humans and mice has been published，and our previous data revealed the unique pattern of H3K4me3 during early human embryos and oocytes (Science. 2019 Jul 26;365(6451):353-360.) . So, this study mainly focuses on the localization of H3K4me2 in mouse oocytes and preimplantation embryos, how it is erased and re-established during mammalian parental-to-zygote transition, and its function. The combined analysis of H3K4me2 and H3K4me3 is not our main work, but it is not ruled out that there may be new discoveries between these two histones. Previously, our data tended to show that the H3K4me2 not only acts as a precursor of H3K4me3, but also plays its role independently.

    (2) Tranylcypromine (TCP) is known as an irreversible inhibitor of monoamine oxidase and LSD1. While the authors suggest TCP inhibits the expression of LSD2, this assertion is questionable. Given TCP's potential non-specific effects in cells, conclusions related to the experiments using TCP should be made with caution.

    Thank you for pointing this out, and we thank the reviewer again for the important suggestion. We found that the previous study (.Binda C, Valente S, Romanenghi M, Pilotto S, Cirilli R, Karytinos A, Ciossani G, Botrugno OA, Forneris F, Tardugno M, Edmondson DE, Minucci S, Mattevi A, Mai A. Biochemical, structural, and biological evaluation of tranylcypromine derivatives as inhibitors of histone demethylases LSD1 and LSD2. J Am Chem Soc. 2010 May 19;132(19):6827-33.) indicated that TCP was a non-reversible inhibitor of LSD1 and LSD2 (Human LSD2/KDM1b/AOF1 Regulates Gene Transcription by Modulating Intragenic H3K4me2 Methylation, Mol Cell. 2010 Jul 30; 39(2): 222–233.), but according to our data, the content of LSD1 was very low in the early stages of mouse embryos, which mainly inhibited the function of LSD2.

    (3) Some batches of H3K4me2 antibody are known to cross-react with H3K4me3. Has the H3K4me2 antibody used in CUT&RUN been tested for such cross-reactivity? Heatmaps in the figures indeed show similar distribution for H3K4me2 and H3K4me3, further raising concerns about antibody specificity.

    We thank the reviewer for the insightful comments. The H3K4me2 antibody was purchased from Millipore (cat. 07030). Figure 2A shows the specific enrichment area of H3K4me2 in promoter and distal region. Some batches of H3K4me2 antibody are known to cross-react with H3K4me3, but the H3K4me2 antibody we used in our CUT&RUN seems to have Low cross-reactivity.

    (4) Certain statements lack supporting references or figures (examples on page 9 can be found on line 245, line 254, and line 258).

    Thank you for pointing this out, and we will add references to support the statement in the paper as suggested.

    (5) Extensive language editing is recommended to clarify ambiguous sentences. Additionally, caution should be taken to avoid overstatement - most analyses in this study only suggest correlation rather than causality.

    Thank you for your kind comments. We will revise the expression in the manuscript later.

    Reviewer #2 (Public Review):

    Chong Wang et al. investigated the role of H3K4me2 during the reprogramming processes in mouse preimplantation embryos. The authors show that H3K4me2 is erased from GV to MII oocytes and re-established in the late 2-cell stage by performing Cut & Run H3K4me2 and immunofluorescence staining. Erasure and re-establishment of H3K4me2 have not been studied well, and profiling of H3K4me2 in germ cells and preimplantation embryos is valuable to understanding the reprogramming process and epigenetic inheritance.

    (1) The authors claim that the Cut & Run worked for MII oocytes, zygotes, and the 2-cell embryos. However, it is unclear if H3K4me2 is erased during the stage or if the Cut & Run did not work for these samples. To support the hypothesis of the erasure of H3K4me2, the authors conducted immunofluorescence staining, and H3k4me2 was undetected in the MII oocyte, PN5, and 2-cell stage. However, the published papers showed strong staining of H3K4me2 at the zygote stage and 2-cell stage ((Ancelin et al., 2016; Shao et al., 2014)). The authors need to cite these papers and discuss the contradictory findings.

    The authors used 165 MII oocytes and 190 GV oocytes for the Cut & Run. The amount of DNA in MII oocytes is halved because of the emission of the first polar body. Would it be a reason that H3K4me2 has fewer H3K4me2 peaks in MII oocytes than GV oocytes?

    First of all, thank you for your valuable advice. The published papers showed strong staining of H3K4me2 at the zygote stage and 2-cell stage (Ancelin et al., 2016), which is interesting. I think we may have used different parameters in the confocal laser shooting process. We used the same parameter to continuously shoot the blastocyst stage from the GV stage. If we only shot the fertilized egg and the 2-cell stage, I think we may also see weak fluorescence at the 2-cell stage under different parameters. We will refer to this reference and discuss it in the resubmitted version.

    Moreover, you mentioned the H3K4me2 has fewer H3K4me2 peaks in MII oocytes than GV oocytes, because the MII expelled the polar body. There is no problem with this logic. However, the first polar body expelled from the MII stage is still in the zona pellucida, and we also collected the polar body in the CUT&RUN experiment; Therefore, compared to GV, the DNA content of MII samples is not halved. After further discussion, we believe that the reduction of H3K4me2 peaks in MII stage compared with GV stage may be closely related to oocyte maturation. It is the specific modification of histones in different forms at different times that affects the chromatin structure change appropriately with the different stages of meiosis. At present, it has been confirmed that H3K4me3 gradually decreases from GV to MII stage during the maturation of human oocytes. H3K27me3 did not change from GV to MII stage.

    In Figure 3C, 98% (13,183/13,428) of H3K4me2 marked genes in GV oocytes overlap with those in the 4-cell stage. Furthermore, 92% (14,049/15,112) of H3K4me2 marked genes in sperm overlap with those in the 4-cell stage. Therefore, most regions maintain germ line-derived H3K4me2 in the 4-cell stage. The authors need to clarify which regions of germ line-derived H3K4me2 are maintained or erased in preimplantation embryos. Additionally, it would be interesting to investigate which regions show the parental allele-specific H3K4me2 in preimplantation embryos since the authors used hybrid preimplantation embryos (B6 x DBA).

    Thank you very much for your suggestion. Further analysis of which regions show the parental allele-specific H3K4me2 in preimplantation embryos will make the study more interesting. We will discuss this in depth in resubmitted vision.

    (2) The authors claim that Kdm1a is rarely expressed during mouse embryonic development (Figure 4A). However, the published paper showed that KDM1a is present in the zygote and 2-cell stage using immunostaining and western blotting (Ancelin et al., 2016). Additionally, this paper showed that depletion of maternal KDM1A protein results in developmental arrest at the two-cell stage, and therefore, KDM1a is functionally important in early development.

    The authors should have cited the paper and described the role of KDM1a in early embryos.

    In the analysis of this experiment, we believe that in the early embryonic development of mice, the expression of KDM1A is lower than that of KDM1B, which is relative. Similarly, the transcriptome data we cite also show that KDM1A is expressed at elevated levels during oocyte maturation and fertilization compared to immature oocytes. In addition, the effects of loss of maternal KDM1a on embryonic development were not discussed. We believe that the absence of maternal KDM1b blocks embryonic development, and we will cite and discus the references later.

    (3) The authors used the published RNA data set and interpreted that KDM1B (LSD2) was highly expressed at the MII stage (Figure S3A). However, the heat map shows that KDM1B expression is high in growing oocytes but not at 8w_oocytes and MII oocytes. The authors need to interpret the data accurately.

    After re-checking the data, we found that there was a problem with the normalization method of our heat map, and we will re-make the heatmap and submit it in the modified version. With reference to Figure 4A, the content of Kdm1b is indeed higher than that of Kdm1a.

    (4) All embryos in the TCP group were arrested at the four-cell stage. Embryos generated from KDM1b KO females can survive until E10.5 (Ciccone et al., 2009); therefore, TCP-treated embryos show a more severe phenotype than oocyte-derived KDM1b deleted embryos. Depletion of maternal KDM1A protein results in developmental arrest at the two-cell stage ((Ancelin et al., 2016)). The authors need to examine whether TCP treatment affects KDM1a expression. Western blotting would be recommended to quantify the expression of KDM1A and KDM1B in the TCP-treated embryos.

    We will further dig the transcriptome data to confirm the specificity of TCP to KDM1b. In addition, the intervention of TCP on the whole fertilized egg in this study increased the H3K4me2 content, and the embryo development retarding effect was more significant than that obtained by crossing with normal paternal lines after knocking down KDM1B from the mother.

    (5) H3K4me2 is increased dramatically in the TCP-treated embryos in Figure 4 (the intensity is 1,000 times more than the control). However, the Cut & Run H3K4me2 shows that the H3K4me2 signal is increased in 251 genes and decreased in 194 genes in the TCP-treated embryos (Fold changes > 2, P < 0.01). The authors need to explain why the gain of H3K4me2 is less evident in the Cut & Run data set than in the immunofluorescence result.

    Thanks a lot for your question. In the experimental group, the fluorescence value of H3K4me2 in IF was increased by 1000 times (Figure 4E), and the expression of H3K4Me2-related genes in CR was up-regulated and down-regulated for a total of 445 changes (Figure 6A). In our opinion, as a semi-quantitative analysis, immunofluorescence cannot be compared with the quantitative analysis method of CR because of the different analysis models and threshold Settings.

    References

    Ancelin, K., ne Syx, L., Borensztein, M., mie Ranisavljevic, N., Vassilev, I., Briseñ o-Roa, L., Liu, T., Metzger, E., Servant, N., Barillot, E., Chen, C.-J., Schü le, R., & Heard, E. (2016). Maternal LSD1/KDM1A is an essential regulator of chromatin and transcription landscapes during zygotic genome activation. https://doi.org/10.7554/eLife.08851.001

    Ciccone, D. N., Su, H., Hevi, S., Gay, F., Lei, H., Bajko, J., Xu, G., Li, E., & Chen, T. (2009). KDM1B is a histone H3K4 demethylase required to establish maternal genomic imprints. Nature, 461(7262), 415-418. https://doi.org/10.1038/nature08315

    Shao, G. B., Chen, J. C., Zhang, L. P., Huang, P., Lu, H. Y., Jin, J., Gong, A. H., & Sang, J. R. (2014). Dynamic patterns of histone H3 lysine 4 methyltransferases and demethylases during mouse preimplantation development. In Vitro Cellular and Developmental Biology - Animal, 50(7), 603-613. https://doi.org/10.1007/s11626-014-9741-6

    Reviewer #3 (Public Review):

    Summary:

    This study explores the dynamic reprogramming of histone modification H3K4me2 during the early stages of mammalian embryogenesis. Utilizing the advanced CUT&RUN technique coupled with high-throughput sequencing, the authors investigate the erasure and re-establishment of H3K4me2 in mouse germinal vesicle (GV) oocytes, metaphase II (MII) oocytes, and early embryos.

    Strengths:

    The findings provide valuable insights into the temporal and spatial dynamics of H3K4me2 and its potential role in zygotic genome activation (ZGA).

    Weaknesses:

    The study primarily remains descriptive at this point. It would be advantageous to conduct further comprehensive functional validation and mechanistic exploration.

    Key areas for improvement include enhancing the innovation and novelty of the study, providing robust functional validation, establishing a clear model for H3K4me2's role, and addressing technical and presentation issues. The text would benefit from the introduction of a novel conceptual framework or model that provides a clear explanation of the functional consequences and molecular mechanisms underlying H3K4me2 reprogramming in the transition from parental to early embryonic development.

    While the findings are significant, the current manuscript falls short in several critical areas. Addressing major and minor issues will significantly strengthen the study's contribution to the field of epigenetic reprogramming and embryonic development.

  15. Version published to 10.1101/2022.05.05.490401v2 on bioRxiv
  16. Version published to 10.1101/2022.05.05.490401v1 on bioRxiv