The Molecular Mechanism And Function Of Chromatin-associated Protein ELMSAN1 In DNA Damage Response

Objective:Genomic information is not transmitted faithfully from generation to generation.For example,genomic variations can be generated from DNA replication infidelity and unequal chromosome segregation.Natural decay of DNA molecules is also a fundamental source of changing genomic information.In addition,cellular and organismal exposure to exogenous genotoxic agents such as ultraviolet(UV)light,oxidative stress,chemical mutagens,and radiation can lead to a variety of modifications on DNA constituents,resulting in genome alterations.Genomic instability is a characteristic of most cancers.Historical treatment of cancer by radiotherapy and DNA-damaging chemotherapy is based on this principle,yet it is accompanied by significant collateral damage to normal tissue and unwanted side effects.Targeted therapy based on inhibiting the DNA damage response(DDR)in cancers offers the potential for a greater therapeutic window by tailoring treatment to patients with tumors lacking specific DDR functions.Therefore,the study of DNA damage response mechanism is is crucially important to finding new targets of oncotherapy.The MTA family containing conserved EGL-27 and MTA1 homology 2(ELM2)domain and SWI3,ADA2,Nco R,and TFIII-B(SANT)domain have been found to be involved in DNA damage response.However,whether ELMSAN1 can play a part in DNA damage response which similarly contain a conserved ELM2-SANT domain remains undiscovered.Here,we reveal the specific mechanism of ELMSAN1 in DNA damage response and provide a new potential target for oncotherapy.Methods:First,we explore whether ELMSAN1 could be recruited to the DNA double-strand break site by UVA-laser(365nm)and immunofluorescence staining.Next,we used DRGFP and EJ5-GFP reporter systems to investigate whether ELMSAN1 is involved in HR and NHEJ.Then,we identified the interaction of ELMSAN1 with TDIF1,and HDAC1/2 by immunoaffinity purification and silver staining combined with mass spectrometry analysis,and verified the mass spectrometry results by co-immunoprecipitation.Moreover,we used UVA-laser to induce DNA damage,and examined the effect of knocking down TDIF1 and HDAC1/2 on the recruitment of ELMSAN1.In addition,we tested the effect of knocking down ELMSAN1 and TDIF1 on the recruitment of Ku70 by laser micro-irradiation,and explained the how ELMSAN1 contributes to NHEJ.Finally,the effects of ELMSAN1 on DNA repair and cell survival under IR were examined by comet assay and MTS.Results:1.ELMSAN1 is recruited to damage sites in a TDIF1-dependent manner.2.ELSMAN1 is physically associated with TDIF1 and HDAC1/2.3.ELMSAN1 depletion compromised NHEJ and resulted in a persistent accumulation of Ku70 at sites of DNA damage.4.ELMSAN1 is required for cells to cope with IR-induced DNA damage.5.ELSMAN1 is implicated in ovarian cancer carcinogenesis progression and patient survival.Conclusion:In this study,we identified a chromatin protein containing conserved ELM2-SANT domain,ELMSAN1,could be recruited to DSB sites in TDIF1 dependent manner.ELMSAN1 and TDIF1 could promote NHEJ,at least in part by restraining unproductive Ku sliding and instead maintaining Ku at DSB termini to mediate repair.Finally,we found ELMSAN1 is required for cells to cope with IR-induced DNA damage.In conclusion,we found that ELMSAN1 plays a crucial role in DNA damage response and NHEJ repair,thus maintaining genomic stability,suggesting that ELMSAN1 may be a potential drug target for oncotherapy.