Dissecting mechanistic links between MAPK signaling, genomic hypomethylation and naive pluripotency

摘要 胚胎干细胞(ESCs)在培养过程中可无限期自我更新，同时保持产生ALL细胞的能力 身体的类型。小鼠胚胎干细胞通常维持在血清和LIF中，它们捕获的状态类似于 正常甲基化，植入后的上胚层，而胚胎干细胞在MEK1/2抑制剂存在的情况下培养 而GSK3，被称为“2i”，捕捉到了一种低甲基化的天真状态，类似于植入前的外胚细胞。AS WNT激活(通过GSK3抑制剂)和MAPK抑制(通过MEK1/2抑制剂)重述信号 在早期胚胎的环境中，2i诱导的低甲基化提供了一个易于处理和强大的体外系统 研究植入前胚胎内基因组甲基化模式的重新编程。值得注意的是， 甲基化模式不仅受外部信号的影响，还受性染色体的影响，雌性ESCs 与男性ESCs相比，是低甲基化的。女性特异性低甲基化的过程及其机制 与这个天真的国家之间的联系仍然没有完全被理解。我们最近发现，对 药物抑制MEK1/2或上调X-连锁MAPK磷酸酶的MAPK通路 DUSP9分别是2I诱导的和雌性特异的低甲基化的基础。没想到，我们发现 抑制MAPK通路也损害了胚胎干细胞的基因组稳定性和发育潜力。 在这里，我们概述了3个互补的目标，以剖析MAPK途径影响DNA的机制 通过性染色体或外部信号在多能细胞中的甲基化。在具体的目标1中，我们将 缩小MAPK通路的上游和下游负责低甲基化的成分 并测试蛋白质组学方法确定的候选靶点。我们将进一步探索分子 幼稚上胚层内基因组低甲基化缺失的后果。在具体的AIM 2中，我们将测试 DUSP9在女性ESCs中的候选靶点并将结果与目标1相结合以确定异同 在性别依赖和环境(2I)诱导的低甲基化之间。我们将进一步描述自我的特征 我们在同时缺乏Dusp9等位基因的ESCs中发现的更新缺陷及其对DNA的依赖性 甲基化。最后，我们将确定胚胎干细胞中性别特异的甲基化差异是否起源于前 或者植入后的胚胎。在具体的目标3中，我们将调查机械连接是否 在幼稚的人胚胎干细胞中观察到MAPK信号和DNA甲基化之间的保守以及这是否 信息可以被利用来培养更稳定的人类细胞。具体来说，我们将评估滴定是否 靶向MAPK信号转导的抑制剂或使用替代的MEK抑制剂会增加DNA甲基化和 减少基因组的不稳定性。总的来说，我们的工作将探索MAPK信号之间的分子联系 以及DNA甲基化、基因组稳定性和多能细胞的发展潜力，目标是 剖析人类干细胞的基本机制并确定改进的条件。

SUMMARY Embryonic stem cells (ESCs) self-renew indefinitely in culture while retaining the capacity to produce all cell types of the body. Mouse ESCs are typically maintained in serum and LIF, which capture a state resembling the normally methylated, post-implantation epiblast, whereas culture of ESC in the presence of inhibitors of MEK1/2 and GSK3, termed “2i”, captures a hypomethylated, naïve state that resembles the pre-implantation epiblast. As Wnt activation (via GSK3 inhibitor) and MAPK suppression (via MEK1/2 inhibitor) recapitulates the signaling environment of early embryos, 2i-induced hypomethylation offers a tractable and powerful ex vivo system to study the reprogramming of genomic methylation patterns within the pre-implantation embryo. Notably, methylation patterns are not only influenced by external signals but also by sex chromosomes, with female ESCs being hypomethylated compared to male ESCs. The process of female-specific hypomethylation and its connection to the naïve state remain incompletely understood. We recently discovered that suppression of the MAPK pathway through pharmacological inhibition of MEK1/2 or upregulation of the X-linked MAPK phosphatase DUSP9 underlies 2i-induced and female-specific hypomethylation, respectively. Unexpectedly, we found that suppression of the MAPK pathway also compromises genomic stability and the developmental potential of ESCs. Here, we outline 3 complementary aims to dissect the mechanisms by which the MAPK pathway influences DNA methylation in pluripotent cells through either sex chromosomes or external signals. In SPECIFIC AIM 1, we will narrow down the upstream and downstream components of the MAPK pathway responsible for hypomethylation and test candidate targets identified by proteomics approaches. We will further explore the molecular consequences of loss of genomic hypomethylation within the naïve epiblast. In SPECIFIC AIM 2, we will test candidate targets of DUSP9 in female ESCs and integrate results with Aim 1 to define similarities and differences between sex-dependent and environment (2i)-induced hypomethylation. We will further characterize the self- renewal defect we uncovered in ESCs lacking both Dusp9 alleles and assess its dependence on DNA methylation. Lastly, we will determine whether sex-specific methylation differences in ESCs originate from pre- or post-implantation embryos. In SPECIFIC AIM 3, we will investigate whether the mechanistic connection we observed between MAPK signaling and DNA methylation is conserved in naïve human ESCs and whether this information can be exploited to grow more stable human cells. Specifically, we will assess whether the titration of inhibitors that target MAPK signaling or the use of alternative MEK inhibitors increases DNA methylation and decreases genomic instability. Collectively, our work will explore molecular links between MAPK signaling and DNA methylation, genomic stability and developmental potential in pluripotent cells with the goal to dissect basic mechanisms and define improved conditions for human stem cells.