A wave of minor de novo DNA methylation initiates in mouse 8-cell embryos and co-regulates imprinted X- chromosome inactivation

Reviewer #1 (Public Review):

Summary:

In this study, Yue et al. re-processed publicly available DNA methylation data (published in 2012 and 2017 from the Meissner lab) from pre- and post-implantation mouse embryos. Against the global wave of genome-wide reduction of DNA methylation occurring during pre-implantation development, they detected a slight increase (~1% on average) of DNA methylation at gene promoter regions during the transition from 8-cell to blastocyst stage. They claim that many such promoters are located in the X chromosome. Subsequently, they knocked down Dnmt3b (presumably because of its upregulation during the transition from the 8-cell to blastocyst stage) and detected the aberrant patterning of H3K27me3 in the mutant female embryos. Based on this observation, they claim that imprinted X-chromosome inactivation is impaired in the Dnmt3b-Kd pre-implantation embryos. Finally, they propose a model where such an increase of DNA methylation together with H3K27me3 regulates imprinted X-chromosome inactivation in the pre-implantation embryos. While their observation is of potential interest, the current version of the work fails to provide enough evidence to support their conclusions. Below are suggestions and comments on the manuscript.

Major issues:

1. Sex of the embryos of the genome-wide bisulfite-sequencing data
The authors re-analyzed publicly available genome-wide DNA methylation data from the Meissner lab published in 2012 and 2017. The former used reduced representation bisulfite sequencing (RRBS) and the latter used whole-genome bisulfite sequencing (WGBS). Based mainly on the RRBS data, Yue et al. detected de novo DNA methylated promoters during the transition from 8-cell to blastocyst against the global wave of genome-wide DNA demethylation. They claim that such promoter regions are enriched at the "inactive" X chromosome. However, it would be difficult to discuss DNA methylation at inactive X-chromosomes as the RRBS data were derived from a mixture of male and female embryos. It would also be notable that the increase of DNA methylation at these promoter regions is ~1% on average. Such a slight increase in DNA methylation during pre-implantation development could also be due to the developmental variations between the embryos or between the sexes of embryos.

2. Imprinted X-chromosome inactivation and evaluation of H3K27me3 (related to Figures 2C, D; 3F; Figure2-supplement 2 F, G; Figure3-supplement 3G)
Based on the slight change in the H3K27me3 signals in the Dnmt3b-Kd blastocysts, the authors claim that imprinted X-chromosome inactivation is impaired in the mutant embryo. It would be not easy to reach this conclusion from such a rough analysis of H3K27me3 presented in Figure 2C, D. Rigorous quantification/evaluation of the H3K27me3 signals in the Dnmt3b-Kd embryos should be considered. Additional evidence for the impairment of H3K27me3 in the mutant embryos should also be provided (expression of a subset of X-linked genes by RNA-FISH or RT-PCR etc.). Though technically challenging, high-resolution genome-wide approach such as ChIP-seq of H3K27me3 in the Dnmt3b-kd female embryos (with traceable SNPs between maternal and paternal X chromosome to distinguish inactive and active X-chromosome) could more precisely evaluate regions that lose H3K27me3 in the X-chromosome (de novo DNA methylated promoters from 8-cell to blastocyst, for example).

3. Analysis of the developmental potential of Dnmt3b-kd embryos
While the authors claim that Dnmt3b-mediated de novo DNA methylation plays an important role in imprinted X-chromosome inactivation, it remains unclear whether the analysis presented in Figure 4 is derived from "female" embryos. This analysis seemed confusing as the authors claim that de novo DNA methylation in the promoter regions during the transition from 8-cell to blastocyst regulates imprinted X-chromosome inactivation, but this should not happen in the male embryos. Was the impairment of embryonic proliferation and differentiation observed in both male and female embryos? Or is this specific to the female embryos? We think that the sex of the embryos would be critical for the analysis presented in Figure 4.

Reviewer #2 (Public Review):

Summary:

Here, Yue et al. set out to determine if the low DNMT3B expression that is observed prior to de novo DNA methylation (before the blastocyst stage) has a function. Re-analyzing existing DNA methylation data from Smith et al. (2012) they find a small DNA methylation gain over a subset of promoters and gene bodies, occurring between the 8-cell and blastocyst stages, and refer to this as "minor de novo DNA methylation". They attempt to assess the relevance/functionality of this minor DNA methylation gain, and report reduced H3K27me3 in Dnmt3b knockdown (KD) trophoblast cells that normally undergo imprinted X-chromosome inactivation (iXCI) before the blastocyst stage. In addition, they assess the proliferation, differentiation, metabolic function, implantation rate, and live birth rate of Dnmt3b KD blastocysts.

Strengths:

Working with early embryos is technically demanding, making the well-designed experiments from this manuscript useful to the epigenetics community. Particularly, the DNMT3B expression and 5-mC staining at different embryonic stages.

Weaknesses:

- Throughout the manuscript, please represent DNA methylation changes as delta DNA methylation instead of fold change.

- Detailed methods on the re-analysis of the DNA methylation data from Smith et al. 2012 are missing from the materials and methods section. Was a minimum coverage threshold used?

- Detailed methods on the establishment and validation of Dnmt3b KO blastocysts and 5-aza-dC treated blastocysts are missing (related to Figure 2).

- Detailed methods on the re-analysis of the ChIPseq data from Liu et al. 2016 are missing from the materials and methods section.

- Some of the data represented in bar graphs does not look convincing/significant. Maybe this data can be better represented differently, such as in box plots or violin plots, which would better represent the data.

- The relevance and rationale for experiments using 5-aza-dC treatment is unclear.