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

总结 胚胎干细胞（ESC）在培养中无限自我更新，同时保留产生所有细胞的能力。 身体的类型。小鼠ESC通常维持在血清和LIF中，其捕获类似于细胞凋亡的状态。 正常甲基化，植入后上胚层，而在MEK 1/2抑制剂存在下的ESC培养物 而GSK 3，称为"2i"，捕获类似于植入前上胚层的低甲基化的幼稚状态。作为 Wnt激活（通过GSK3抑制剂）和MAPK抑制（通过MEK 1/2抑制剂）重现了 在早期胚胎的环境中，2i诱导的低甲基化提供了一个易处理的和强大的离体系统， 研究植入前胚胎内基因组甲基化模式的重编程。值得注意的是， 甲基化模式不仅受到外部信号的影响，而且受到性染色体的影响， 与男性胚胎干细胞相比甲基化程度较低。雌性特异性低甲基化的过程及其意义 与天真国家的联系仍然不完全清楚。我们最近发现， 通过药理学抑制MEK 1/2或上调X连锁MAPK磷酸酶的MAPK途径 DUSP9分别是2i诱导的和雌性特异性低甲基化的基础。出乎意料的是，我们发现， MAPK途径的抑制也损害基因组稳定性和ESC的发育潜力。 在这里，我们概述了3个互补的目的，剖析MAPK途径影响DNA的机制 多能细胞中的甲基化通过性染色体或外部信号。具体目标1： 缩小MAPK通路中负责低甲基化的上游和下游成分 并测试通过蛋白质组学方法鉴定的候选靶标。我们将进一步探索 幼稚外胚层内基因组低甲基化缺失的后果。在具体目标2中，我们将测试 女性ESC中DUSP9的候选靶点，并将结果与目标1整合以定义相似性和差异性 性别依赖和环境（2i）诱导的低甲基化之间的关系。我们将进一步描述自我- 我们在缺乏Dusp9等位基因的ESC中发现了更新缺陷，并评估了其对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性甲基化差异是否起源于胚胎干细胞分化前。 或植入后胚胎。在具体目标3中，我们将研究我们所认为的 MAPK信号传导和DNA甲基化之间的关系在幼稚的人类胚胎干细胞中是保守的， 可以利用这些信息来培养更稳定的人类细胞。具体而言，我们将评估滴定是否 靶向MAPK信号传导的抑制剂或替代MEK抑制剂的使用增加了DNA甲基化， 降低基因组不稳定性。总的来说，我们的工作将探索MAPK信号传导之间的分子联系， 以及DNA甲基化、基因组稳定性和多能细胞的发育潜力， 剖析基本机制和定义人类干细胞的改善条件。

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.