NFBD1/MDC1, 53BP1 and BRCA1 have both redundant and unique roles in the ATM pathway

Abstract

NFBD1/MDC1, 53BP1 and BRCA1 are DNA damage checkpoint proteins with twin BRCT domains. In order to determine if they have redundant roles in responses to ionizing radiation, we used siRNA and shRNA to deplete NFBD1, 53BP1 and BRCA1 in single, double and triple combinations. These analyses were performed in early passage human foreskin fibroblasts so that checkpoint responses could be assessed in a normal genetic background. We report that NFBD1, 53BP1 and BRCA1 have both unique and redundant functions in radiation-induced phosphorylation and localization events in the ATM-Chk2 pathway. 53BP1, but not NFBD1 and BRCA1, mediates ionizing radiation-induced ATM S1981 autophosphorylation. In contrast, all three mediators collaborate to promote IR-induced Chk2 T68 phosphorylation. NFBD1 and 53BP1, but not BRCA1, work together to mediate pATMS1981, pChk2T68 and NBS1 ionizing radiation induced foci (IRIF). However, the relative importance of NFBD1 and 53BP1 in IRIF formation differ. We also determined the interdependence among mediators in IRIF recruitment. We extend previous findings in cancer cells and mouse cells that NFBD1 is upstream of 53BP1 and BRCA1 to primary human cells. Furthermore, NFBD1 promotes BRCA1 IRIF through both 53BP1-dependent and 53BP1-independent mechanisms.

Figure 1

Effects of knocking down NFBD1, 53BP1 and BRCA1 in single, double, and triple combinations on ATM autophosphorylation. (A) U2-OS cells were transfected with the indicated siRNA oligos. Approximately 48 hours later, cells were irradiated with 5 Gy and lysed in SDS sample buffer one hour later. Western blots were performed using the indicated antibodies. (B) HFFs were infected with the indicated shRNA-expressing retroviruses. Approximately 72 hours later, cells were irradiated with 5 Gy and lysed in SDS sample buffer 15 minutes or one hour later. Western blots were performed using the indicated antibodies. (C) The ratio of BRCA1 to beta-tubulin levels was determined by densitometry. Data from seven independent experiments is represented. (D) Results were confirmed with two different shRNA-expressing retroviruses toward each mRNA of interest. (E) 293T cells were transfected with the indicated plasmids. Transfected 293T cells, along with A-T22IJE-T cells stably transfected with the indicated plasmids were irradiated with 10 Gy. One hour later cells were lysed and anti-FLAG immunoprecipitations were performed. Western blots were performed using the indicated antibodies.

Figure 2

Phospho-ATM S1981 IRIF in HFFs infected with NFBD1, 53BP1 and/or BRCA1 shRNA-expressing retroviruses. (A) HFFs were infected with the indicated shRNA-expressing retroviruses. Approximately 72 hours later, cells were irradiated with 5 Gy. One hour later, cells were fixed and stained with ATM pS1981 antibody. (B) The percentage of cells with at least five phospho-ATM foci. At least fifty cells from each of four independent experiments for the singles and triple and three independent experiments for the doubles were analyzed per condition. (C) Effects of shRNA on protein expression. (D) GM00637 (normal) and GM09607 (AT) cells were irradiated at 5 Gy. One hour later, cells were fixed and stained with ATM pS1981 antibody.

Figure 3

The effects of depleting NFBD1, 53BP1 and BRCA1 in single, double and triple combinations on Chk2 phosphorylation. (A) The same protein extracts analyzed in Figure 1A were immunoblotted for Chk2pT68 and total Chk2. (B) Western blot analysis of the same protein extracts used in Figure 1B, with anti-pT68 Chk2 and anti-Chk2. (C) The ratio of pT68-Chk2 to total Chk2 was determined by densitometry. For the control and single knockdowns, data from seven independent experiments are represented, five with NFBD1a, 53BP1a and BRCA1a and two with NFBD1b, 53BP1b and BRCA1b. For the doubles and triple, data from four independent experiments are represented, three of them with the “a” set of shRNAs and one of them with the “b” set.

Figure 4

Phospho-Chk2 T68 IRIF in HFFs treated with NFBD1, 53BP1 and/or BRCA1 shRNA-expressing retroviruses. (A) HFFs were infected with the indicated shRNA-expressing retroviruses. Approximately 72 hours later, cells were mock-irradiated or irradiated with 5 Gy. One hour later, cells were fixed and analyzed with an anti-pT68 Chk2 antibody. (B) The percentage of cells with at least five phospho-Chk2 foci was determined. At least 40 cells were analyzed in each of three independent experiments for the single and triple knockdowns, and two experiments for the double knockdowns. (C) Analysis of mediator proteins in cells infected with shRNA-viruses. (D) HCT 116 wild type and HCT 116 Chk2^−/− cells were irradiated with 5 Gy. One hour later, cells were fixed and stained for phospho-Chk2.

Figure 5

The effects of mediator knockdowns on NBS1 IRIF. (A) HFFs were infected with retroviruses expressing the indicated shRNAs, irradiated with 15 Gy, and six hours later, stained with anti-NBS1 antibody. The same retrovirus stocks were used in Figure 4. (B) The percentage of cells with at least NBS1 five foci was determined. At least 50 cells were analyzed in each of two independent experiments.

Figure 6

Effects of mediator knockdowns on localization of each other to IRIF. (A and B) HFFs were infected with the indicated shRNA-expressing retroviruses. The same retrovirus stocks were used in Figure 4. Approximately 72 hours later, cells were irradiated with 5 Gy. One hour later, cells were fixed and then stained with the indicated antibodies. (A) NFBD1 IRIF formation in HFFs with 53BP1 and/or BRCA1 knockdowns. (B) 53BP1 IRIF formation in HFFs with NFBD1 and/or BRCA1 knockdowns. (C) BRCA1 IRIF formation in HFFs with NFBD1 and/or 53BP1 knockdowns. HFFs were infected with the indicated shRNA-expressing retroviruses. The same retrovirus preparations were used in Figure 2. Approximately 72 hours later, cells were mock-irradiated or irradiated with 20 Gy. Six hours later, cells were fixed and then stained with anti-BRCA1. (D) The percentage of cells with at least five BRCA1 foci was determined in at least 50 cells from each of two independent experiments per condition.

Figure 7

Summary of results. 53BP1 is crucial for ATM autophosphorylation. ATM phosphorylates H2AX, creating a binding site for NFBD1. NFBD1 stabilizes the interaction with the MRN complex and ATM with the DSB. NFBD1 recruits the RNF8 ubiquitin ligase complex. H2AX is ubiquitinated, causing 53BP1 recruitment and BRCA1 recruitment through Rap80. 53BP1 plays a role in MRN complex and BRCA1 IRIF formation. The MRN complex enhances ATM and Chk2 phosphorylation. NFBD1, 53BP1 and BRCA1 collectively mediate Chk2 phosphorylation.