Figure 6. Irreparable DSBs induced by a 16 hour etoposide treatment lead to an increased interaction of MTA3 with HIC1 and favor its recruitment to the HIC1-response elements in the SIRT1 promoter.

(A) Etoposide-induced non-repairable DSBs lead to an increase of MTA3 interaction with HIC1. HEK293T cells were transfected with the indicated combination of empty FLAG, FLAG-HIC1, and FLAG-MTA3 expression vectors. 32 hours after transfection cells were incubated for 16 hours with 20 μM etoposide (+) or with DMSO (–) as control. After lysis in IPH buffer, cell extracts were co-immunoprecipitated with anti-MTA3 antibodies. The immunoprecipitates as well as 1% of the whole cell extracts were analyzed by SDS/PAGE and transferred to membranes. Relevant pieces of the membranes were cut and analyzed by Western blot with anti-FLAG antibodies to detect MTA3 and HIC1. ΔH2AX and actin levels were used as controls for DSB induction and equal loading, respectively. (B) Etoposide-induced irreparable DSB lead to an increase of MTA3 recruitment on the HiRE in the SIRT1 promoter. Chromatin was prepared from BJ-hTERT fibroblasts mock-treated with DMSO or treated with 80 uM etoposide for 16 hours to induce irreparable DSB and ChIP experiments were performed with antibodies against MTA3 or rabbit IgG. The bound material was eluted and analysed by quantitative PCR using primers flanking the HIC1-responsive elements (HiRE) in the SIRT1 promoter [6], as previously described [46]. GAPDH was used as a nonbinding control. Values that are statistically significantly different are indicated by bars and asterisks as follows: *P < 0.05. NS corresponds to values that are not statistically significantly different.