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

> **NIH NIH R01** · MASSACHUSETTS GENERAL HOSPITAL · 2021 · $511,569

## Abstract

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 ...

## Key facts

- **NIH application ID:** 10094448
- **Project number:** 1R01HD103612-01
- **Recipient organization:** MASSACHUSETTS GENERAL HOSPITAL
- **Principal Investigator:** Konrad Hochedlinger
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $511,569
- **Award type:** 1
- **Project period:** 2021-04-01 → 2026-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10094448

## Citation

> US National Institutes of Health, RePORTER application 10094448, Dissecting mechanistic links between MAPK signaling, genomic hypomethylation and naive pluripotency (1R01HD103612-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10094448. Licensed CC0.

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