PRC2 and EHMT1 regulate H3K27me2 and H3K27me3 establishment across the zygote genome

Abstract

The formation of zygote is the beginning of mammalian life, and dynamic epigenetic modifications are essential for mammalian normal development. H3K27 di-methylation (H3K27me2) and H3K27 tri-methylation (H3K27me3) are marks of facultative heterochromatin which maintains transcriptional repression established during early development in many eukaryotes. However, the mechanism underlying establishment and regulation of epigenetic asymmetry in the zygote remains obscure. Here we show that maternal EZH2 is required for the establishment of H3K27me3 in mouse zygotes. However, combined immunostaining with ULI-NChIP-seq (ultra-low-input micrococcal nuclease-based native ChIP-seq) shows that EZH1 could partially safeguard the role of EZH2 in the formation of H3K27me2. Meanwhile, we identify that EHMT1 is involved in the establishment of H3K27me2, and that H3K27me2 might be an essential prerequisite for the following de novo H3K27me3 modification on the male pronucleus. In this work, we clarify the establishment and regulatory mechanisms of H3K27me2 and H3K27me3 in mouse zygotes.

Introduction

Life begins with the union of a sperm and an oocyte. Understanding how epigenetic modifications are regulated has been a major challenge in developmental biology, and, so far, the regulatory mechanisms of histone modification changes in zygotes is far from clear. H3K27me3 is a hallmark of facultative heterochromatin in numerous organisms, which is catalyzed by PRC2 (polycomb repressive complex 2) comprised of EED (embryonic ectoderm development), EZH1/2 (enhancer of zeste 1/2), and SUZ12 (suppressor of zeste 12). It is interesting that the phenotypes of Ezh2 and Eed maternal knockout are inconsistent. The former displays a severe growth retardation¹, while the latter shows significant reduced litter size and a significant overgrowth^2,3. Unlike EZH2, which is indispensable, EZH1 is not necessary for the development of mouse⁴. Ezh1 depletion neither reduced global H3K27me2/3 levels nor impacted viability and fertility in mice^4,5. However, it is also reported that PRC2-EZH1 directly and robustly represses transcription from chromatinized templates and compacts chromatin in the absence of the methyltransferase cofactor SAM⁵. Interestingly, PRC2 displays post-translational modifications of automethylation in its own subunits to modulate its histone methyltransferase activity⁶, which then is demonstrated through its EZH1/2-mediated automethylation activity⁷.

H3K27me3 is a mark of facultative heterochromatin and gene silencing. Maternal H3K27me3 can function as an imprinting mark. Specifically, Inoue et al.⁸ proved that H3K27me3 modification on differential histones in the parent genome was a new regulatory mechanism for gene imprinting independent of DNA methylation. Erhardt et al.¹ reported that X-inactivation (XCI) was stably propagated thereafter in zygotes lacking maternal EZH2. In a follow-up study, Inoue et al.³ recently demonstrated that loss of maternal H3K27me3 induced maternal ** was performed by Samtools with MAPQ more than 30 and self-coded Perl scripts. The duplication reads were removed by Picard to obtain the non-redundant reads. SNP split was utilized to split the maternal and paternal reads alignment with the allele-specific C57BL6 and PWK mm10 genome. SICER was utilized to call peaks (windows 200, Gap 600, q value 1e−5) with SICER.sh module and the differential peaks were found with SICER-df.sh module³⁰. The peaks filtered by length more than 1000 bp and fold change more than 5 were annotated by Chip Seeker for gene category analysis and Cluster profiler for gene function annotation such as KEGG and GO analysis. The depth and coverage of ChIP-sequencing data were calculated by Bed tools with the 5 kb windows and self-coded python scripts, respectively, which were visualized by ggpubr and ggplot packages in R with the Wilcoxon rank test.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.