MYBL2 and ATM suppress replication stress in pluripotent stem cells

Paloma García,Daniel Blakemore,Agnieszka Gambus,Carl Ward,Eva Petermann,Elena Gonzalez,Miriam Moya,Grant S Stewart,George Murphy,Nuria Vilaplana‐Lopera,Ruba Almaghrabi

doi:10.15252/embr.202051120

Abstract

Replication stress, a major cause of genome instability in cycling cells, is mainly prevented by the ATR‐dependent replication stress response pathway in somatic cells. However, the replication stress response pathway in embryonic stem cells (ESCs) may be different due to alterations in cell cycle phase length. The transcription factor MYBL2, which is implicated in cell cycle regulation, is expressed a hundred to a thousand‐fold more in ESCs compared with somatic cells. Here we show that MYBL2 activates ATM and suppresses replication stress in ESCs. Consequently, loss of MYBL2 or inhibition of ATM or Mre11 in ESCs results in replication fork slowing, increased fork stalling and elevated origin firing. Additionally, we demonstrate that inhibition of CDC7 activity rescues replication stress induced by MYBL2 loss and ATM inhibition, suggesting that uncontrolled new origin firing may underlie the replication stress phenotype resulting from loss/inhibition of MYBL2 and ATM. Overall, our study proposes that in addition to ATR, a MYBL2‐MRN‐ATM replication stress response pathway functions in ESCs to control DNA replication initiation and prevent genome instability.

Highlights

DNA replication is a highly complex process that requires tight regulation to ensure that genome stability is maintained
ATR has been shown to be the principal replication stress-responsive kinase required to maintain fork stability (Paulsen & Cimprich, 2007; Cimprich & Cortez, 2008) and suppress the initiation of replication in the presence of damaged DNA (Costanzo et al, 2003; Syljuasen et al, 2005; Patil et al, 2013; Zeman & Cimprich, 2014). Consistent with this role, our work shows that the inhibition of ATR in both pluripotent stem cells as well as in primary and immortal somatic cells (MEFs) leads to a slow down in replication fork progression
Our data demonstrate a role for ataxia-telangiectasia mutated (ATM), which is normally activated exclusively in response to double-strand breaks (DSB) (Bakkenist & Kastan, 2003; Shiloh, 2003; Lee & Paull, 2004), in regulating the replication stress response in embryonic stem cells (ESCs), in part by facilitating the activation of CHK1

Summary

Introduction

DNA replication is a highly complex process that requires tight regulation to ensure that genome stability is maintained. Obstacles to DNA replication activate the replication stress response pathway, which functions to ensure that the initiation of DNA replication and progression through the cell cycle progression are suppressed, and acts to facilitate the repair and restart of damaged replication forks (Bartek et al, 2004; Aguilera & GomezGonzalez, 2008; Zeman & Cimprich, 2014). ATM is recruited by the MRN complex to DNA ends and is primarily associated with signalling the presence of DNA double-strand breaks (DSB) (Uziel et al, 2003; Lee & Paull, 2005). It is thought that ATM is activated during S-phase only upon MRNdependent recruitment to sites of DSBs, i.e. collapsed replication forks (Bakkenist & Kastan, 2003; Lee & Paull, 2004). It has been hypothesized that ATM could have additional roles in replication control, in non-somatic cell types

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