c-Myc suppression of DNA double-strand break repair

Abstract

c-Myc is a transcriptional factor that functions as a central regulator of cell growth, proliferation, and apoptosis. Overexpression of c-Myc also enhances DNA double-strand breaks (DSBs), genetic instability, and tumorigenesis. However, the mechanism(s) involved remains elusive. Here, we discovered that γ-ray ionizing radiation-induced DSBs promote c-Myc to form foci and to co-localize with γ-H2AX. Conditional expression of c-Myc in HO15.19 c-Myc null cells using the Tet-Off/Tet-On inducible system results in down-regulation of Ku DNA binding and suppressed activities of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and DNA end-joining, leading to inhibition of DSB repair and enhanced chromosomal and chromatid breaks. Expression of c-Myc reduces both signal and coding joins with decreased fidelity during V(D)J recombination. Mechanistically, c-Myc directly interacts with Ku70 protein through its Myc box II (MBII) domain. Removal of the MBII domain from c-Myc abrogates its inhibitory effects on Ku DNA binding, DNA-PKcs, and DNA end-joining activities, which results in loss of c-Myc's ability to block DSB repair and V(D)J recombination. Interestingly, c-Myc directly disrupts the Ku/DNA-PKcs complex in vitro and in vivo. Thus, c-Myc suppression of DSB repair and V(D)J recombination may occur through inhibition of the nonhomologous end-joining pathway, which provides insight into the mechanism of c-Myc in the development of tumors through promotion of genomic instability.

Figure 1

IR induces formation of c-Myc foci and DSBs. (A) H1299 cells were treated with increasing doses of IR (0–10 Gy). After radiation, c-Myc and γ-H2AX were analyzed immediately by immunofluorescent staining. (B) H1299 cells were treated with IR (5 Gy). Cells were harvested at various time points as indicated. c-Myc and γ-H2AX were analyzed by immunofluorescent staining.

Figure 2

Conditional expression of c-Myc downregulates Ku DNA binding activity and suppresses DSB repair leading to increased genetic instability. (A) HO15.19 cells bearing inducible Tet-Off/Tet-On c-Myc gene were established as described in Materials and Methods section. c-Myc was turned on in three independent stable clones by the addition of DOX (1 µg/ml) for 24 hours. Expression levels of c-Myc, Ku70, and Ku86 were analyzed by Western blot. (B) DNA binding activity of Ku70 or Ku86 in c-Myc-Off or c-Myc-On cells was measured using a Ku70/86 DNA Repair Kit. Error bars represent ± SD. (C and D) The c-Myc-Off and c-Myc-On HO15.19 cells were exposed to 5 Gy of IR. Cells were harvested at various time points. DSBs were determined by PFGE or analysis of γ-H2AX by immunostaining. The number of γ-H2AX foci per cell was determined on a cell-to-cell basis by the quantitative analysis of at least 30 randomly chosen cells as described [58]. The percentage of γ-H2AX foci-positive cells was determined by analyzing 100 randomly chosen cells as described [34]. Error bars represent ± SD. (E and F) c-Myc was turned on by addition of DOX for 4 weeks. Then, percentage of abnormal metaphases in the c-Myc-Off or c-Myc-On cells was quantified by T-FISH analysis. At least 30 metaphases per culture were analyzed. Error bars represent ± SD. Frequency of cytogenetic abnormality per metaphase in c-Myc-Off or c-Myc-On cells. Each experiment was repeated three times and error bars represent ± SD.

Figure 3

c-Myc interacts with Ku70. (A) H1299 cells were treated with increasing doses of IR (0–15 Gy). co-IP was carried out using c-Myc antibody. Ku70 and c-Myc were analyzed by Western blot. (B) H1299 cells were treated with IR (5 Gy). co-IP was performed with anti-Myc antibody or normal IgG in the presence or absence of 150 KU of DNase I. Ku70 and c-Myc were analyzed by Western blot. (C) Purified recombinant c-Myc was incubated with purified Ku70 in lysis buffer at 4°C for 2 hours. A co-IP was performed using a c-Myc antibody or normal IgG control. Ku70 and c-Myc were analyzed by Western blot.

Figure 4

Ku70 directly interacts with c-Myc through its MBII domain, which is essential for c-Myc suppression of DSB repair. (A) WT or each of the c-Myc deletion mutants was transfected into HO15.19 cells. Expression levels of c-Myc were analyzed by Western Blot. Additionally, a co-IP was performed using Ku70 antibody. The Ku-associated c-Myc was analyzed by Western blot. White arrow(s) points c-Myc or c-Myc deletion mutant protein band(s). (B) DNA binding activity of Ku70 or Ku86 in HO15.19 cells expressing WT or each of the c-Myc deletion mutants was measured using a Ku70/86 DNA Repair Kit. Error bars represent ± SD. (C–E) HO15.19 cells expressing WT or each of the c-Myc deletion mutants were exposed to 5 Gy of IR. After 24 hours, DSBs were measured by PFGE or analysis of γ-H2AX by immunostaining with quantitative evaluation as described in the legend of Figure 2.

Figure 5

c-Myc directly disrupts the Ku/DNA-PKcs complex. (A and B) The Ku70/DNA-PKcs or Ku86/DNA-PKcs complex was coimmunoprecipitated from HO15.19 cells and incubated with purified c-Myc (10–20 ng/ml) in EBC lysis buffer at 4°C for 2 hours. After centrifugation, the resulting supernatant and immunocomplex beads were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Ku, c-Myc, and DNA-PKcs that bound to Ku or unbound DNA-PKcs present in the supernatant were then analyzed by Western blot. (C) HO15.19 cells expressing WT or each of the c-Myc deletion mutants were disrupted in EBC lysis buffer. co-IP was performed using an agarose-conjugated Ku70 or Ku86 antibody, respectively. The Ku-associated DNA-PKcs was then analyzed by Western blot. Normal rabbit IgG was used as a control.

Figure 6

Depletion of endogenous c-Myc by RNAi enhances Ku DNA binding, DNA-PKcs, and DNA end-joining activities in association with accelerated DSB repair. (A) H460 cells expressing high levels of endogenous c-Myc were transfected with c-Myc siRNA or control siRNA. Expression of c-Myc was analyzed by Western blot. (B–D) Ku DNA binding, DNA-PKcs, and DNA end-joining activities were measured in H460 cells expressing c-Myc siRNA and control siRNA. (E–H) H460 cells expressing c-Myc siRNA or control siRNA were exposed to 5 Gy of IR. After 24 hours, DSBs were measured by PFGE or analysis of γ-H2AX by immunostaining with quantitative evaluation as described in the legend of Figure 2.