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 (Xia et al., 2019). 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 indicated that TCP was a non-reversible inhibitor of LSD1 and LSD2, 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. (Binda et al., 2010; Fang et al., 2010 )

(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, which is interesting. I think we may have used different parameters in the confocal laser shooting process(Ancelin et al., 2016). 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

References

Xia W, Xu J, Yu G, Yao G, Xu K, Ma X, Zhang N, Liu B, Li T, Lin Z, Chen X, Li L, Wang Q, Shi D, Shi S, Zhang Y, Song W, Jin H, Hu L, Bu Z, Wang Y, Na J, Xie W, Sun YP. Resetting histone modifications during human parental-to-zygotic transition. Science. 2019 Jul 26;365(6451):353-360. doi: 10.1126/science.aaw5118. Epub 2019 Jul 4. PMID: 31273069.

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.

Fang R, Barbera AJ, Xu Y, Rutenberg M, Leonor T, Bi Q, Lan F, Mei P, Yuan GC, Lian C, Peng J, Cheng D, Sui G, Kaiser UB, Shi Y, Shi YG. Human LSD2/KDM1b/AOF1 regulates gene transcription by modulating intragenic H3K4me2 methylation. Mol Cell. 2010 Jul 30;39(2):222-33. doi: 10.1016/j.molcel.2010.07.008. PMID: 20670891; PMCID: PMC3518444.

Ancelin K, Syx L, Borensztein M, Ranisavljevic N, Vassilev I, Briseño-Roa L, Liu T, Metzger E, Servant N, Barillot E, Chen CJ, Schüle R, Heard E. Maternal LSD1/KDM1A is an essential regulator of chromatin and transcription landscapes during zygotic genome activation. Elife. 2016 Feb 2;5:e08851. doi: 10.7554/eLife.08851. PMID: 26836306; PMCID: PMC4829419.

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.

Figures and data

Mapping H3K4me2 in mouse (C57BL6/N) gametes and pre-implantation embryos.

Comparison of the H3K4me2 between sperm and oocyte.

Resetting H3K4me2 during mouse pre-implantation development.

Pervasive H3K4me2 in CpG-rich regulatory regions in mouse embryos.

Chromatin states at specific stages and H3K4me2 mode chart during mouse maternal-to-zygotic transition.

Validation of H3K4me2 CUT&RUN data in Human K562 cell line, oocytes and early embryos.

H3K4me2 in mouse gametes.

H3K4me2 in late 2-cell embryos.

H3K4me2 in mouse 4/8-cell embryos.

Chromatin states at specific stages and distal CcREs.