Genome-wide DNA methylation maps of mouse embryonic fibroblasts and reprogramming cells modulated by cell cycle and Dnmt1 expression. Mus musculus

Dramatic change in DNA methylation patterns and levels both globally and/or locus-specifically is associated with the process of cell linage specification and somatic reprogramming. We found the expression of DNA methyltransferase 1 (Dnmt1) is tightly regulated by the features of cell cycle, which we referred as cell cycle-directed Dnmt1 expression adjustment. To further investigate how DNA methylation is affected by this mechanism, we applied reduced representation bisulphate sequencing (RRBS) to map genome-wide DNA methylation on mouse embryonic fibroblasts (MEFs). To characterize how this mechanism affects somatic reprogramming, samples of MEFs induced by Yamanaka factors (Sox2, Klf4, Oct4, cMyc) was included. The cell cycle was accelerated by shRNA targeted to p53, and the expression of Dnmt1 was manipulated genetically. We find 5mC level remains constant in regardless of cell proliferation rate, a result from the adjusted expression of Dnmt1. However, repressed expression of Dnmt1 in fast-proliferating cells results in global DNA demethylation. The patterns of DNA methylation and demethylation affected by this mechanism is sensitive to CpG densities. Generally, the results demonstrate cell cycle-directed Dnmt1 expression adjustment as an mechanism for the insurance of the stability of genomic 5mC inheritance. Overall design: Reduced representation bisulphate sequencing in cell cycle modulated MEFs and SKOM induced reprogramming cells.

DNA甲基化模式与水平在全基因组范围或位点特异性层面的显著改变，与细胞谱系特化及体细胞重编程过程密切相关。我们发现，DNA甲基转移酶1（DNA methyltransferase 1, Dnmt1）的表达严格受细胞周期特征调控，我们将这一过程称为细胞周期定向的Dnmt1表达调控。为进一步探究该机制如何影响DNA甲基化过程，我们采用简化代表性亚硫酸氢盐测序（reduced representation bisulphate sequencing, RRBS）对小鼠胚胎成纤维细胞（mouse embryonic fibroblasts, MEFs）的全基因组DNA甲基化水平进行图谱绘制。为表征该机制对体细胞重编程的影响，我们纳入了经山中伸弥因子（Yamanaka factors, Sox2、Klf4、Oct4、cMyc）诱导的MEFs样本。我们通过靶向p53的短发夹RNA（shRNA）加速细胞周期进程，并通过遗传学手段操控Dnmt1的表达。我们发现，无论细胞增殖速率如何，5-甲基胞嘧啶（5mC）水平均保持恒定，这一现象源于Dnmt1表达的适应性调控。然而，在快速增殖的细胞中抑制Dnmt1表达会引发全基因组DNA去甲基化。该机制所影响的DNA甲基化与去甲基化模式对CpG密度敏感。综上，本研究结果表明，细胞周期定向的Dnmt1表达调控是维持基因组5mC遗传稳定性的保障机制。整体实验设计：对经细胞周期调控的MEFs及SKOM诱导的重编程细胞开展简化代表性亚硫酸氢盐测序。