Dissecting Erk and Gsk3 transcription regulation activity during early differentiation of mouse embryonic stem cells

Abstract

Embryonic stem (ES) cells are cells able to give rise to any somatic cell types. A lot is known about how ES cell pluripotency is maintained, but only a little is known about how differentiation is promoted. In particular, cell fate decisions are regulated by interactions within transcriptional and signalling networks. Indeed, recently it has been discovered that pluripotency of mouse ES cells can be maintained using inhibitors of the Extracellular-signal Regulated Kinase (Erk) and Glycogen Synthase Kinase 3 (Gsk3) pathways (called "2i condition"). Erk and Gsk3 pathways have been previously associated with differentiation and developmental processes and with the regulation of gene expression. These observations suggest that Erk and Gsk3 pathways play a key role in the decision-making process of ES cells.The aim of this study is to understand how activation of the Erk and Gsk3 pathways (upon 2i removal) contributes to cell fate decisions. A genome-wide RNAi screen was recently performed in our laboratory and identified factors that facilitate pluripotency maintenance or that are involved in promoting the exit of ES cells from pluripotency after activation of the Erk and Gsk3 pathways. This PhD project aims to understand the mechanisms by which some of the identified transcription regulators contribute to cell fate decisions, with particular focus on how MAPK and Gsk3 pathway activation might give rise to the transcription and epigenetic profiles which are required for ES cells to exit from the pluripotent state. In this study the behaviour of Erk and Gsk3 during early differentiation was analysed and several transcription regulators that potentially act downstream of these pathways were identified, among which the transcription factors Jun and Elk1. Both Jun and Elk1 are well-known Erk targets. Jun expression is upregulated within days upon 2i removal and the activatory phosphorylation of Elk1 is induced within minutes upon 2i withdrawal. The study highlighted a role played by Jun in mouse ES cells undergoing differentiation in regulating the expression of adhesion-associated genes. Furthermore, the analysis of Elk1 binding sites (using Chromatin Immunoprecipitation followed by next generation sequencing) revealed a redundant role of Elk1 and Elk4 in inducing the expression of a novel long non-coding RNA, Ite1 (Inducible Transcript in ESCs), which is upregulated after days upon 2i removal in ES cells that are exiting the pluripotent state.

Date of Award	1 Aug 2015
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Andrew Sharrocks (Supervisor) & Paul Shore (Supervisor)