The Molecular Mechanism Of Histone Demethylase PHF8 In ATR Activation

Objective:Replication stress is a major source of endogenous DNA damage,causing both single-stranded and double-stranded DNA lesions when unrepaired.Any condition leading to high levels of DNA damage will result in replication stress,which is a source of genome instability and a feature of pre-cancerous and cancerous cells.To deal with DNA damage loads associated with excessive replication,cancerous cells have evolved and acquired particular machineries or programs in response and repair of DNA damage,which is essential for the survival of these cells and renders cells resistant to DNA damage-based therapy.A negative aspect of replication stress is its prominent role in tumorigenesis,while a positive aspect is that it provides a potential target for cancer therapy.ATR(ataxia telangiectasia mutated and Rad3-related)is an apical kinase in sensing and resolving replication stress,but how optimal activation of ATR is achieved remains unclear.Post-tranlational modifications of histone or non-histone proteins including phosphorylation,acetylation and ubiquitination have been implicated in resovling replication stress.However,whether methylation towards histone or non-histone proteins also plays a role in this process remains obscure.Here,we planned to investigate whether protein methylation is involved in ATR activation and aims to reveal the relevant mechanisms associated with replication stress response.Also,we will examine the aberrations of these mechanisms in malignancies and reveal their contributions to genome instability as well as chemoor radio-therapeutic resistance of tumors.Initially,we have screened all members of histone methyltransferases and histone demethylases and identified several candidates in ATR activation.Specifically,we uncovered that histone demethylase PHF8 physically interacts with TopBP1(topoisomerase II binding protein 1),an essential allosteric activator of ATR,and activates ATR in TopBP1-and demethylase activity-dependent manner.We also showed that PHF8 is required for breast cancer cells to counteract replication stress.We believe that understanding the molecular basis of ATR activation and replication stress response is crucial to the understanding of genome stability and will benefit to the therapeutic intervention of tumors.Results:A.PHF8 is required for ATR activation.B.PHF8 is physically associated with TopBP1.C.PHF8 interacts with the BRCT 7+8 of TopBP1 through its C-terminal 22 amino acids.D.The region of PHF8 interacting with TopBP1 and its demethylase activity can promote the activation of ATR signal.E.PHF8 affects TopBP1 recruitment through its interaction region with TopBP1 and its demethylase activity.F.High expression of PHF8 and TopBP1 in breast cancer tissues may lead to poor prognosis of breast cancer patients.G.PHF8 deficiency sensitizes breast cancer cells to PARP inhibitors.Conclusion:In conclusion,we found that histone demethylase PHF8 is a regulator in ATR activation.Specifcally,we found that PHF8 is physically associated with TopBP1,an essential regulator required for activating ATR.Molecular interface mapping revealed that BRCT 7+8 domain of TopBP1 and 22 amino acids of PHF8 C terminal are responsible for their interaction.Interesingly,we demonstrated that both the 22 aa of PHF8 and its demethylase activity are indispensable for the recruitment of TopBP1 thus in promoting ATR activation upon replication stress.Bioinformatic analysis with TCGA datasets revealed that high expression of both PHF8 and TopBP1 indicated poor survival of breast cancer patients.Indeed,we found that PHF8 deficiency render breast cancer cells more volnerable to genotoxic stress agents.This study has given histone demethylase PHF8 the ability to maintain the stability of replication fork by promoting the activation of ATR signal in the process of replication fork arrest and collapse,which not only gives us a new understanding of the regulation of the activation of ATR signaling pathway,but also provides possible targets and therapeutic directions for PHF8 in the treatment of cancer.