Priority Research Paper Volume 12, Issue 12 pp 11165—11184

SIRT6 mono-ADP ribosylates KDM2A to locally increase H3K36me2 at DNA damage sites to inhibit transcription and promote repair

Figure 3. SIRT6 is required for inhibition of transcription upon induction of DNA DSB. (A) SIRT6 is required for RNA Pol II displacement upon DSB. Time course ChIP analysis of initiating (S5 phosphorylated) RNA Pol II levels on the TSS region of the GFP in NHEJ reporter upon DSB induction. (B) Time course ChIP analysis of initiating RNA Pol II levels on the TSS region of INTS4 gene, which does not undergo a DSB (negative control to A). (C) qRT-PCR analysis of GFP premature RNA expression at different time points after transfection with I-SceI vector. (D) qRT-PCR analysis of INTS4 premature RNA expression (control for C). (E) SIRT6 recruitment to DSB peaks within 12 h post DSB induction. Chromatin IP of endogenous SIRT6 in skin fibroblasts. Chromatin samples were analyzed by qPCR using primers flanking I-SceI cut sites with antibodies against SIRT6 or IgG. The positions of primers in NHEJ reporter and INTS4 gene are shown in Figures 2A and Supplementary Figure 2. The experiments were repeated three times. *p < 0.05.