Double Strand Breaks Can Initiate Gene Silencing and SIRT1-Dependent Onset of DNA Methylation in an Exogenous Promoter CpG Island
Figure 3
Effects of knockdown of SIRT1 by siRNA.
(A) Cells were transiently transfected for three consecutive days with non-target siRNA (NT) or SIRT1 siRNA (SIRT1), treated with tetracycline to induce HA-I-SceI as indicated, and crosslinked and sonicated for ChIP analyses. A western blot was performed for anti-SIRT1, anti-acetyl lysine 382 of p53, anti-HA, and anti-β-actin using a portion of the sonicated sample. (B) Changes in ChIP results at the SCE site with SIRT1 knockdown. ChIP was performed using material from (A) and antibodies against SIRT1 and H4K16ac. Representative gels (top panels) are shown for PCR using primers in the SCE promoter region. The average change in recruitment to the promoter over input as measured by ChIP was quantitated (bottom panels) by gel densitometry for three PCRs with error bars indicating the standard error. (C) Changes in ChIP results at the TK site with SIRT1 knockdown. Using ChIP samples from (B) PCR was performed using primers in the body of the gene labeled TK with representative gels (top panels) and quantitation of results as in (B) (bottom panels). (D) Effects of knockdown of SIRT1 on phospho-H2AX recruitment kinetics. ChIP was performed using antibodies against phospho-H2AX. Representative gels (top panels) are shown for PCR using primers in the promoter region. Graphs for quantitation (bottom panel) are shown using the SCE primers, and error bars indicate the standard error for three PCRs. (E) Effects of knockdown of SIRT1 on the kinetics of break repair. Input DNA was used from the ChIP samples from (B). PCR was performed using primers on either side of the cut site with the 3′ primer being unique to the exogenous E-cad promoter. PCR using genomic GAPDH primers is used as a loading control. The average change in PCR across the break site over GAPDH was quantitated by gel densitometry for four PCRs with error bars indicating standard error (bottom panel).