Single-cell analysis of Non-CG methylation dynamics and gene expression in human oocyte maturation

Abstract

Oocyte maturation is a coordinated process that is tightly linked to reproductive potential. A better understanding of gene regulation during human oocyte maturation will not only answer an important question in biology, but also facilitate the development of in vitro maturation technology as a fertility treatment. We generated single-cell transcriptome and use previously published single-cell methylome data from human oocytes at different maturation stages to investigate how genes are regulated during oocyte maturation, focusing on the potential regulatory role of non-CG methylation.DNMT3B, a gene encoding a key non-CG methylation enzyme, is one of the 1000 genes upregulated in mature oocytes, which may be at least partially responsible for the increased non-CG methylation as oocytes mature. Non-CG differentially methylated regions (DMRs) between mature and immature oocytes have multiple binding motifs for transcription factors, some of which bind with DNMT3B and may be important regulators of oocyte maturation through non-CG methylation. Over 98% of non-CG DMRs locate in transposable elements, and these DMRs are correlated with expression changes of the nearby genes. Taken together, this data indicates that global non-CG hypermethylation during oocyte maturation may play an active role in gene expression regulation, potentially through the interaction with transcription factors. KEY WORDSDNA methylation, oocyte maturation, gene expression 1 3 proximity of LINE1 elements to the AR gene. When we looked in the UCSC genome browser, there are cluster of non-CG hypermethylated LINE1 elements upstream of the AR gene ( Figure 4). In our gene expression data AR is expressed in GV and expression drops until it is undetectable in MII oocytes. AR expression is important in early follicle maturation and in knockout mouse models, females have many phenotypes associated with reduced fertility 42 . AR is also important for germinal vesicle breakdown, the process by which the GV oocyte resumes meiosis to become the mature MII oocyte 42 . TMEFF2 is also a gene potentially regulated by proximal hypermethylated LINE1 elements ( Figure 4). TMEFF2 is upregulated in early oocyte development in the primordial/primary follicle stage 43 and in our data we observed downregulation of TMEFF2 from GV to MII. Therefore non-CG hypermethylation in the MII stage oocyte could be a means to suppress LINE1 elements that help regulate the expression of early oocyte maturation genes. DISCUSSIONWhile many studies have looked at the genetic and epigenetic contributions of the oocyte to embryonic development, less focus has been on regulatory factors during oocyte maturation. Here we used RNA-seq and WGBS data generated in single-cell oocytes at three different timepoints of oocyte maturation. We use oocytes from patients undergoing ART and therefore one major limitation of our study is the potential heterogeneity of the patients samples. In our three timepoints we observed less RNA transcripts and general downregulation of gene expression as the oocyt...