C
onnor Higgins
Master's Thesis Defense
Advisors: Dr. Chris Phiel,
Dr. Jeff Knight and Dr. Carlos Infante
Date: October 9th, 2026
Time: 9:00am – 11:00am
Location: North Classroom 1207
RNA-seq Analysis of ESCs Comparing the Effects of Mettl3 Inhibition to Dual Inhibition of MEK/Gsk-3
Mouse embryonic stem cells (mESCs) are a common cellular model for studying embryonic development. However, there remains much left to be uncovered about how these cells maintain their pluripotent state. LIF (leukemia inhibitory factor) is sufficient to keep mESCs pluripotent. Several years ago, a chemically-defined media containing small molecule inhibitors of mitogen-activated protein kinase kinase (MEK; PD098059) and glycogen synthase kinase-3 (GSK-3; CHIR99021) was shown to replace LIF in the maintenance of ESC pluripotency. Despite this, the mechanisms underlying their action with respect to pluripotency are not fully understood. mRNA methylation (or m6A), a reversible post-transcriptional modification, has recently gained attention in its role in embryonic stem cell pluripotency. The RNA methyltransferase complex, composed of the proteins METTL3, METTL14, and WTAP, promotes methylation, while the fat mass and obesity-associated protein (FTO) acts in opposition, demethylating mRNA. Reducing m6A promotes pluripotency, while increased m6A correlates with differentiation. METTL3, METTL14, WTAP, and FTO have all been shown to be regulated by phosphorylation. Interestingly, MEK and GSK-3, the targets of the 2i-cocktail that promotes pluripotency, are also the kinases that regulate METTL3 and FTO, controlling the modulation of m6A . The aim of this study is to test the hypothesis that the inhibition of the methyltransferase complex using the METTL3 inhibitor STM2457 will result in similar downstream effects on the transcriptome and m6A epitranscriptome of mESCs as the 2i-cocktail. Additionally, these experiments suggest multiple genetic markers of the pluripotent state are partially or fully phenocopied by METTL3 inhibition at either or both levels. Markers showing non-phenocopying patterns suggest that future experiments modulating FTO could help resolve the remainder of the 2i-cocktail’s mechanism of action.