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 ...