The Molecular Mechanism Of FBL Regulating DNA Damage Repair

DNA repair is an important self-defence mechanism.It can help rebuild DNA structure when cells suffer from serious injury with repair proteins and other related factors.DNA damage repair related genes play a key role in DNA damage response system,and their functional changes play an important role in the occurrence,development and treatment of cancer.Fibrillarin(FBL)is a highly conserved nucleolar methyltransferase responsible for methylation of both ribosomal RNA and proteins.Under normal physiological conditions,FBL is located in the nucleolus.FBL is a nucleocytoplasmic protein that consists of two unique functional domains including an N-terminal glycine-and arginine-rich(GAR)domain and a C-terminal methyltrans-ferase-like domain.FBL as methyltransferase,the role of FBL in ribosome biogenesis,breast cancer proliferation and aging has been well established to date.However,the role of FBL in DNA damage repair and maintaining genomic stability is still unclear.Here,we describe a distinct role of FBL in DNA damage response.We find that FBL is highly expressed in a wide variety of cancers and high FBL expression levels are associated with poor prognosis and worse survival outcome in cancer patients.FBL knockdown significantly sensitizes tumor cells to Mitomycin C(MMC)or Cisplatin in vitro and in vivo.FBL-deficient cells display accumulation of unrepaired DSBs and defective HR.In order to study the molecular mechanism of FBL in cells,this project constructed the full length of FBL into the SFB(S-Flag-SBP tag)vector,and constructed a stable cell line overexpressing FBL.Affinity chromatography combined with mass spectrometry technology to screen FBL interacting proteins.Co-IP and GST pull down assay show that FBL interacts directly with Y-box binding protein-1(YBX1)protein.Follow up research findings that both MMC and Cisplatin treatment induce YBX1 nuclear accumulation,thereby increased the association between FBL and YBX1 in nucleus.Importantly,we identify BRCA1 as one of the FBL-regulated targets through RNA sequencing and show that depletion of FBL affected the localization of YBX1 into nucleus and reduced the binding of YBX1 to the promoter of BRCA1 gene,thereby diminishing the expression of BRCA1.Furthermore,depletion of FBL significantly enhances the sensitivity of xenografts to DNA damaging drug in murine.Taken together,this study examined the molecular mechanisms and biological functions of FBL in DNA damage repair,and the following conclusions are obtained.1.FBL expression is up-regulated in a variety of human tumor tissues;2.The deletion of FBL inhibits the proliferation of tumor cells and is sensitive to DNA damage drugs;3.FBL participates in HR mediated DNA damage repair in an enzyme independent manner;4.FBL interacts with YBX1;5.FBL affects the nuclear localization of YBX1 to regulate DNA damage;6.FBL affects the binding of YBX1 to BRCA1 promoter and regulates the transcriptional activation of BRCA1;7.FBL deletion enhances the sensitivity of tumors to DNA damage drugs.In conclusion,this study discovered that FBL contributed to DNA damage repair by transcriptionally regulating BRCA1 expression via YBX1,suggesting that targeting this pathway could be a potential strategy to enhance the efficacy of DNA damaging agents for cancer therapy.