A rewiring of DNA replication mediated by MRE11 exonuclease underlies primed-to-naive cell de-differentiation

Mouse embryonic stem cells (mESCs) in the primed pluripotency state, that resembles the post-implantation epiblast, can be de-differentiated in culture to anaivestate that resembles the pre-implantation inner cell mass.We report that primed-to-naive mESC transition entails a significant slowdown of DNA replication forks and the compensatory activation of dormant origins. Using iPOND (“isolation of proteins on nascent DNA”) coupled to mass spectrometry, we identify key changes in replisome composition that are responsible for these effects. Naive mESC forks are enriched in MRE11 nuclease and other DNA repair proteins. MRE11 is recruited to newly synthesized DNA in response to transcription-replication conflicts, and its inhibition or genetic downregulation in naive mESCs is sufficient to restore the fork speed observed in primed cells. Notably, MRE11 is required for the efficient primed-to-naive mESC transition, demonstrating a direct link between DNA replication dynamics and the mESC de-differentiation process Overall design: RNA-Seq of untreated primed mESCs and after a 6-day 2i treatment, alone or in combination with two different concentrations of a MRE11 inhibitor.

处于始发态多能性状态的小鼠胚胎干细胞（mESCs），其表型类似植入后上胚层，可在体外培养中被去分化为类似植入前内细胞团的原始态细胞。本研究发现，始发态向原始态mESCs的转化过程伴随DNA复制叉的显著减慢，以及休眠复制起点的代偿性激活。我们采用结合质谱分析的新生DNA结合蛋白分离技术（isolation of proteins on nascent DNA, iPOND），鉴定出介导上述效应的复制体组成关键改变。原始态mESCs的复制叉富集MRE11核酸酶及其他DNA修复蛋白。MRE11可响应转录-复制冲突被招募至新合成的DNA区域，在原始态mESCs中抑制MRE11或通过遗传学手段下调其表达，即可恢复始发态细胞中的复制叉速度。值得注意的是，MRE11对于始发态向原始态mESCs的高效转化是必需的，这证实了DNA复制动力学与mESCs去分化过程之间存在直接关联。

总体实验设计：未处理的始发态mESCs，以及经6天2i培养基单独处理、或联合两种不同浓度MRE11抑制剂处理的细胞的RNA测序。