ZMYM2 inhibits NANOG-mediated reprogramming.

Moyra Lawrence,Patrick Lombard,Thorold W Theunissen,David J Adams,José C R Silva

doi:10.12688/wellcomeopenres.15250.1

Abstract

Background: NANOG is a homeodomain-containing transcription factor which forms one of the hubs in the pluripotency network and plays a key role in the reprogramming of somatic cells and epiblast stem cells to naïve pluripotency. Studies have found that NANOG has many interacting partners and some of these were shown to play a role in its ability to mediate reprogramming. In this study, we set out to analyse the effect of NANOG interactors on the reprogramming process. Methods: Epiblast stem cells and somatic cells were reprogrammed to naïve pluripotency using MEK/ERK inhibitor PD0325901, GSK3β inhibitor CHIR99021 and Leukaemia Inhibitory Factor (together termed 2i Plus LIF). Zmym2 was knocked out using the CRISPR/Cas9 system or overexpressed using the PiggyBac system. Reprogramming was quantified after ZMYM2 deletion or overexpression, in diverse reprogramming systems. In addition, embryonic stem cell self renewal was quantified in differentiation assays after ZMYM2 removal or overexpression. Results: In this work, we identified ZMYM2/ZFP198, which physically associates with NANOG as a key negative regulator of NANOG-mediated reprogramming of both epiblast stem cells and somatic cells. In addition, ZMYM2 impairs the self renewal of embryonic stem cells and its overexpression promotes differentiation. Conclusions: We propose that ZMYM2 curtails NANOG's actions during the reprogramming of both somatic cells and epiblast stem cells and impedes embryonic stem cell self renewal, promoting differentiation.

Highlights

Reprogramming is the process whereby a somatic cell is reverted back to a pluripotent state
ZFP281 was selected as a control, as knocking it down had been previously demonstrated to increase Nanog-mediated reprogramming efficiency[24]
In order to address whether these factors impact Nanog-induced reprogramming, Nanog-overexpressing Epiblast stem cells (EpiSCs), which reprogram at low efficiency[10,20], were transiently transfected with siRNA against the target genes of interest (Figure 1A)

Summary

Introduction

Reprogramming is the process whereby a somatic cell is reverted back to a pluripotent state. Reprogramming can be carried out by overexpressing only four factors in somatic cells: Oct[4], Klf[4], Sox[2] and cMyc[1] Together, these factors reset the transcriptional and epigenetic state of the cell to those of a pluripotent cell. NANOG is a homeodomain-containing transcription factor which forms one of the hubs in the pluripotency network and plays a key role in the reprogramming of somatic cells and epiblast stem cells to naïve pluripotency. Embryonic stem cell self renewal was quantified in differentiation assays after ZMYM2 removal or overexpression. ZMYM2 impairs the self renewal of embryonic stem cells and its overexpression promotes differentiation. Conclusions: We propose that ZMYM2 curtails NANOG’s actions during the reprogramming of both somatic cells and epiblast stem cells and impedes embryonic stem cell self renewal, promoting differentiation

Objectives

Methods

Results

Conclusion