The Mechanism Of P16^Ink4a Regulated Telomeric DNA Replication In MTR^-/-Wrn^-/- Werner Syndrome Mouse Cells

Telomeres is a cap-like nucleoprotein complex located at the ends of linear chromosomes in eukaryotic cells,consisting of short tandem repeats and Shelterin complexes bound on them to protect chromosome integrity and participate in regulating the cell cycle.Telomeric DNA is enriched in guanine that can form the G4body（G-quadruplex）structure by Hoogsteen hydrogen bonding,and the 3’-single stranded overhang can fold back into double stranded DNA to form T-loop and D-loop,these telomeric secondary structures can inhibit abnormal DDR（DNA Damage Response）signaling pathway on telomeres,which is important for the structure and function of telomeres.However,if these secondary structures of telomeres fail to dynamically unfold and refold as the replication fork progresses during replication,the replication stress will be accumulated and the stalled replication forks leading to DDR.By recruiting damage repair related enzymes to the stalled replication forks,the telomeric DNA is cleaved,which impairs the genomic stability and leads to chromosomal aberrations that cause senescence and tumors.The unfolding of secondary telomeric DNA structures requires the cooperation of multiple proteins,and the Rec Q helicase WRN plays an important role in this process.WRN helicase mutation can lead to Werner Syndrome（WS）.In 2004,Chang’s and Du’s lab established the WS mouse model separately by knocking out Wrn and m Terc genes（RNA template in telomerase）,which provides an ideal system to studying abnormal telomere dysfunction and its regulatory mechanisms.p16^Ink4a（hereinafter referred to as p16）encodes a Cyclin-Dependent Kinase（CDK）inhibitor protein,which induces cell senescence and apoptosis and inhibits the occurrence of tumors mainly by participating in the regulation of cell cycle.In the previous study,we found that the average life span of p16-TKO（triple knock out of m TR,Wrn and p16 genes）mice was significantly prolonged and aging-related phenotypes such as osteoporosis were significantly alleviated compared with WS mice（double knock out of m TR and Wrn,hereinafter referred to as DKO）mice.Moreover,the proliferation capacity of p16-TKO MEFs（Mouse Embryo Fibroblasts）increased and the loss of p16 was observed to reverse the rapid loss of telomeres in DKO cells.Therefore,this study investigated the molecular mechanism of the regulation of telomeric DNA replication by p16 gene in the WS genetic background.Include:（1）p16 depletion promoted the maintenance of telomere length and function.The FISH experiment demonstrated that loss of p16 not only promoted the maintence of telomere length in MEFs of DKO mice,but also significantly reduced the level of abnormal telomere signals.（2）The absence of p16 did not activate the ALT pathway in DKO cells.Since telomerase gene m TR was knocked out in DKO cells,the loss of p16 may activate ALT pathway,which is an telomerase-independent telomere maintaining pathways.We proved that loss of p16 effectively reduced the level of T-SCE in DKO cells,but had no effect on the levels of C-Circle and APBs,which are the classic ALT phenotypes.（3）The loss of p16 reduced the level of telomere damage in DKO cells.WB and IF experiments showed that the expression level ofγ-H2AX and the micronucleus（a marker of genomic instability）were significantly reduced in p16-TKO cells,while TIF experiments demonstrated that loss of p16 reduced the co-localization ofγ-H2AX at telomeres in DKO cells.（4）The loss of p16 alleviated the replication stress caused by G4 structures on telomeres.It has been reported that loss of Wrn lead to the abnormal unfolding of the G4 bodies during replication,hindering the advancement of the replication forks and causing the accumulation of replication stress.Using a G4-body stabilizer,PDS（Pyridostatin）,we found that PDS significantly inhibited the proliferation of DKO MEF,while p16-TKO cells were significantly less sensitive to PDS.Similarly,when p16 was knocked down in U2OS cells,the sensitivity of cells to PDS was also reduced.BLM,another important protein in the Rec Q helicase family,also plays a major role in the telomere G4 unfolding during replication.WB results showed that the protein expression of BLM was elevated in p16-TKO cells.In conclusion,this study shows that in the genetic background of m TR^-/-Wrn^-/-,loss of p16 can ensure the progress of replication forks at telomeres by up-regulating BLM expression to promote the unwinding of G4 structures during telomere DNA replication,which maintain the length and function of telomere and delaying the aging progress in WS mice.This study preliminarily explored the molecular mechanism of p16 regulating telomere maintenance in WS aging mice,providing new ideas and targets for clinical research on aging-related disease and tumors.