Figure 2. SIRT6 mediates KDM2A displacement from chromatin to enhance NHEJ. (A) Schematic representation of the GFP-based NHEJ reporter, which represents an RNA Pol II-transcribed gene. DSBs are introduced by transient expression of I-SceI enzyme. The positions of the primer used in this study are indicated by small arrows below: primers for quantification of the preRNA (small black arrows); quantification of DNA DSBs (small red arrows), chromatin IP (blue arrows). (B) Quantification of the cutting efficiency at different time points after transfection with I-SceI vector. DSBs were quantified as a ratio between the product obtained with primers amplifying across the break (red arrows) to a PCR product from a gene away from DSB site (Supplementary Figure 2). The experiment was repeated three times. (C) KDM2A dissociation from broken chromatin is SIRT6-dependent. Time-course chromatin-IP was performed using antibody against endogenous KDM2A in human skin fibroblasts harboring chromosomally-integrated NHEJ GFP reporter with I-SceI sites. Western blot shows depletion of SIRT6. (D) H3K36me2 accumulation post DSB induction is SIRT6-dependent. ChIP analysis of H3K36me2, at different time points post transfection with I-SceI vector using antibody specific for H3K36me2. qPCR was performed using primers positioned 50 bp downstream of TSS. (E) Non-ribosylatable KDM2A R1020W mutant bids to DSBs more efficiently than the wild type protein. Human skin fibroblasts carrying I-SceI reporter cassette were transfected with wild type or mutant (R1020W) KDM2A and I-SceI encoding vectors. At 12 hours post transfection cells were harvested followed by ChIP-qPCR with antibodies against FLAG. Western blot showing the levels of wild type and mutant FLAG-KDM2A proteins in cells after transfection. (F) Cells depleted of KDM2A have enhanced NHEJ repair efficiency. Cells were treated with siRNA to KDM2A four days before transfection with I-SceI. NHEJ efficiency was measured by reactivation of the GFP reporter normalized to transfection efficiency (DsRed) 48 h after I-SceI transfection. Western blot shows KDM2A depletion. (G) Downregulation of KDM2A reduces the number of 53BP1 foci. KDM2A knocked down cells were grown to confluency and fixed for IF experiment using antibody against 53BP1. On the right; quantification of the IF image obtained from 100 nuclei. All experiments were repeated at least three times. (H) Ku70 recruitment to DSB site requires SIRT6, and is counteracted by KDM2A. Time course of ChIP experiment using antibody against Ku70. The experiment was repeated three times. *p < 0.05; **p <0.01.