An ERK5-KLF2 signalling module regulates early embryonic gene expression and telomere rejuvenation in stem cells.

Helen A Brown,Saria Mansoor,Nora Dieguez-Martinez,Marios P Stavridis,Houjiang Zhou,Greg M Findlay,Robert Gourlay,Diana Rios-Szwed,Liam Mcmulkin,Matthias Trost,Julien Peltier,Charles A.C Williams,Rosalia Fernandez-Alonso,Jose M Lizcano,Rachel Toth

doi:10.1042/bcj20210646

Abstract

The ERK5 MAP kinase signalling pathway drives transcription of naïve pluripotency genes in mouse Embryonic Stem Cells (mESCs). However, how ERK5 impacts on other aspects of mESC biology has not been investigated. Here, we employ quantitative proteomic profiling to identify proteins whose expression is regulated by the ERK5 pathway in mESCs. This reveals a function for ERK5 signalling in regulating dynamically expressed early embryonic 2-cell stage (2C) genes including the mESC rejuvenation factor ZSCAN4. ERK5 signalling and ZSCAN4 induction in mESCs increases telomere length, a key rejuvenative process required for prolonged culture. Mechanistically, ERK5 promotes ZSCAN4 and 2C gene expression via transcription of the KLF2 pluripotency transcription factor. Surprisingly, ERK5 also directly phosphorylates KLF2 to drive ubiquitin-dependent degradation, encoding negative feedback regulation of 2C gene expression. In summary, our data identify a regulatory module whereby ERK5 kinase and transcriptional activities bi-directionally control KLF2 levels to pattern 2C gene transcription and a key mESC rejuvenation process.

Highlights

Embryonic Stem Cells (ESCs) can self-renew or differentiate along any lineage in the adult body, a property known as pluripotency [1]
We developed a quantitative proteomics workflow employing complementary strategies to activate and inhibit ERK5 signalling in mouse ESCs (mESCs) (Figure 1B)
Previous work identifies Klf2 as an ERK5 target gene [6,16,17,18,19], where Klf2 transcription is driven by the ERK5 transcriptional activation domain and ERK5-mediated phosphorylation of MEF2 transcription factors at the Klf2 promoter [16]

Summary

Introduction

Embryonic Stem Cells (ESCs) can self-renew or differentiate along any lineage in the adult body, a property known as pluripotency [1]. ERK5 signalling promotes the naïve state by driving the expression of pluripotency genes [6], the wider molecular targets and biological functions of the ERK5 pathway in mESCs have not been identified. These mechanisms together are hypothesised to control ERK5-dependent gene expression and proteome dynamics. We use state-of-the-art quantitative proteomics to identify the ERK5-responsive proteome in mESCs. Within a specific cohort of ERK5-dependent proteins is ZSCAN4, a key member of a network of early embryonic 2-cell stage specific (2C) genes [8] that promotes the attainment of naïve pluripotency [9,10] and stem cell ‘rejuvenation’ in vitro [11]. Our data provide molecular insight into ERK5 kinase and transcriptional functions in mESCs, which directionally modulate KLF2 levels to pattern early embryonic 2C gene transcription and a key mESC rejuvenation process

Results

Discussion

Experimental procedures