The role of Alu methylation and physiologic-replication independent-endogenous DNA double strand breaks (phy-RIND-EDSBs) on genomic instability prevention

Abstract

Here, we aimed to study the epigenetic marks involving genomic instability, a common event in aging. We focused on two epigenetic marks. First is methylation of Alu element, commonly studied IRS which was proposed to associate with aging characteristics. Second is endogenous DNA double strand breaks (EDSBs), which one of the genomic instability mechanisms in non-dividing cells, change in homeostasis during aging. First, a negative correlation between the methylation of Alu in the blood-derived DNA and endogenous DNA damages was found in healthy people. Then we used siRNA-Alu to direct Alu element in human immortalized cell lines and human diploid fibroblasts. Increase in Alu methylation level was observed in cells with siRNA-Alu transfection. The de novo methylation induced by siRNA-Alu was sequence specific and sustainable. Moreover, the Increasing level of Alu methylation induced by siRNA-Alu depended on siRNA binding sites having insertion or deletion and percent identity value. Interestingly, Alu methylation altered celluar phenotypes. Hypermethylated Alu cells proliferated faster than control cells, lower endogenous DNA damages and more resist to DNA damage agents. Therefore, we proposed that siRNA-Alu promoting Alu methylation has high potential to prevent genomic instability. In the same way, in regular physiologic condition, a eukaryote relieves tension of the genome by maintaining a different kind of EDSBs, called physiologic replication independent EDSBs (phy-RIND-EDSBs). In this study, genome-wide EDSB PCR and high throughput sequencing were used to quantify sequence and classify RIND-EDSBs to explore a mechanism of how genomic instability occurs and induces senescence in non-dividing cells. We discovered a reduction of phy-RIND-EDSBs, DSB repair defect and production and retention of pathologic RIND-EDSBs (path-RIND-EDSBs) in aging cells. Path-RIND-EDSBs can occur spontaneously, and they induce the global repair of EDSBs, including phy-RIND-EDSBs. Alternately, lowered numbers of phy-RIND-EDSBs increase a generation of path-RIND-EDSBs. Accompany with DSB repair defects, path-RIND-EDSBs are retained and reduce viability in chronologically aging cells. These evidences indicated that phy-RIND-EDSBs play an epigenetic role in preventing genomic instability due to the generation and retention of path-RIND-EDSBs. As a result, taken together, Alu methylation and phy-RIND-EDSB may be used as a target to reduce genomic instability to prevent celluar aging.