Transcriptome and DNA methylation profiling during the NSN to SN transition in mouse oocytes

During oocyte maturation, the transition from a non-surrounded nucleolus (NSN) to a surrounded nucleolus (SN) stage coincides with dramatic chromatin reconfiguration and transcriptional silencing and is crucial for developmental competence. To study the transcriptome and DNA methylation dynamics during the NSN to SN transition we used single cell (sc)M&T-seq to generate scRNA-seq and sc bisulphite-seq data from GV oocytes, classified as NSN or SN by Hoechst staining of their nuclei. Transcriptome analysis showed a lower number of detected transcripts in SN oocytes as well as downregulation of 576 genes, which were enriched for processes related to mRNA processing. We used the RNA-seq data to generate a classifier, which can infer chromatin stage in scRNA-seq data sets, based on their transcription profile. The classifier was successfully tested in multiple published datasets of mouse models with a known skew in NSN:SN ratios. DNA methylation analysis showed increased methylation in SN compared to NSN oocytes, which was most pronounced in regions with intermediate levels of DNA methylation. Overlap with ChIP-seq data for the histone modifications H3K36me3, H3K4me3 and H3K27me3 showed that regions gaining methylation in SN oocytes are enriched for overlapping H3K36me3 and H3K27me3, which is unusual as these marks do not typically coincide. We believe that these late-methylating SN regions are in regions with high chromatin plasticity and that the overlap of H3K36me3 and H3K27me3 may indicate a transient switch or heterogeneity on a single-cell level. RNA-seq was performed for oocytes belonging to either the NSN or SN stage in development to identify transcriptomic differences在卵母细胞成熟过程中，非环绕核仁（non-surrounded nucleolus, NSN）向环绕核仁（surrounded nucleolus, SN）阶段的转变伴随剧烈的染色质重塑与转录沉默，且对卵母细胞的发育潜能至关重要。为研究NSN向SN转变过程中的转录组与DNA甲基化动态变化，本研究采用单细胞（single cell, sc）M&T-seq技术，从经细胞核Hoechst染色分类为NSN型或SN型的生发泡卵母细胞（germinal vesicle oocyte, GV oocyte）中获取了单细胞RNA测序（single cell RNA sequencing, scRNA-seq）与单细胞亚硫酸氢盐测序（single cell bisulphite sequencing, sc bisulphite-seq）数据。转录组分析结果显示，SN型卵母细胞中检测到的转录本数量更少，且有576个基因呈现下调表达，这些下调基因显著富集于mRNA加工相关的生物学过程。本研究利用RNA测序数据构建了一款分类器，可基于转录谱推断单细胞RNA测序数据集对应的染色质阶段，该分类器在多个已发表的、NSN:SN比例存在已知偏倚的小鼠模型数据集上均成功通过测试。DNA甲基化分析显示，相较于NSN型卵母细胞，SN型卵母细胞的甲基化水平更高，这一差异在DNA甲基化水平处于中等的区域最为显著。通过与组蛋白修饰H3K36me3、H3K4me3及H3K27me3的染色质免疫共沉淀测序（chromatin immunoprecipitation sequencing, ChIP-seq）数据进行交集分析，本研究发现SN型卵母细胞中发生甲基化的区域显著富集有H3K36me3与H3K27me3的共同修饰区域，这一现象较为罕见，因为这两种组蛋白修饰通常不会同时出现。我们推测，这些晚期甲基化的SN区域位于染色质可塑性较高的区域，而H3K36me3与H3K27me3的共同修饰可能提示单细胞水平上存在瞬时的表观遗传转换或细胞异质性。本研究还对发育进程中处于NSN或SN阶段的卵母细胞开展了RNA测序，以鉴定二者的转录组差异。