KAP-1 Promotes Resection of Broken DNA Ends Not Protected by γ-H2AX and 53BP1 in G₁-Phase Lymphocytes

KAP-1 knockdown limits resection in G₁-phase lymphocytes. (A) Immunoblot (IB) analysis of KAP-1 expression in LigIV^−/−:53BP1^−/− abl pre-B cells expressing either a KAP-1 or control (ctr) shRNA. GAPDH expression is shown as a protein loading control. Molecular mass markers, in kDa, are indicated. (B) Southern blot analysis of pMX-DEL^CJ as described in the legend of Fig. 1A. LigIV^−/−:53BP1^−/−:DEL^CJ abl pre-B cells expressing either ctr, KAP-1, or CtIP shRNA treated with imatinib for the indicated numbers of days were analyzed. Data shown were generated from LigIV^−/−:53BP1^−/−:DEL^CJ-6 abl pre-B cells, and similar results were obtained for LigIV^−/−:53BP1^−/−:DEL^CJ-58 abl pre-B cells. (C) Density plots comparing lane signals from LigIV^−/−:53BP1^−/−:DEL^CJ abl pre-B cells expressing either ctr or KAP-1 shRNA and treated with imatinib for 4 days, as shown in panel B. The signal was normalized for the Eb DNA loading control. Molecular size markers, in kilobases, are shown on the x axis. Peaks representing the unrearranged pMX-DEL^CJ (UR), full-length (2.2-kb) pMX-DEL^CJ coding ends (CE), and resected coding ends are indicated. A.U., arbitrary units. (D) Analysis of chromatin-bound RPA32 in LigIV^−/− and LigIV^−/−:53BP1^−/− abl pre-B cells expressing either ctr, KAP-1, or CtIP shRNA. Cells were treated with imatinib for 2 days and then treated with 5 μg/ml bleocin for 24 h. RPA32 retention in the nucleus was assessed and quantified as the fraction of RPA-positive cells either treated or not treated with bleocin. Data shown were generated from LigIV^−/−:DEL^CJ-7 and LigIV^−/−:53BP1^−/−:DEL^CJ-5 abl pre-B cells. Similar results were obtained for LigIV^−/−:DEL^CJ-10 and LigIV^−/−:53BP1^−/−:DEL^CJ-6 abl pre-B cells.

Expression of human KAP-1 blocks resection of RAG DSBs. (A) Immunoblot analysis of ectopic KAP-1 expression in LigIV^−/−:53BP1^−/− abl pre-B cells expressing either an empty retrovirus (Empty) or a retrovirus encoding Flag–HA–mouse KAP-1 (mKAP-1) or Flag–HA–human KAP-1 (hKAP-1) proteins. Blots were probed with anti-Flag, anti-KAP-1, and anti-GAPDH as a loading control. (B) Southern blot analysis of pMX-DEL^CJ as described in the legend of Fig. 1A. LigIV^−/−:53BP1^−/−:DEL^CJ abl pre-B cells expressing a retrovirus encoding Flag-HA-tagged mKAP-1 or hKAP-1 were treated with imatinib for the indicated numbers of days. (C) Density plots comparing lane signals from LigIV^−/−:53BP1^−/−:DEL^CJ abl pre-B cells in panel B at 4 days of imatinib treatment, as described in the legend of Fig. 2C. Data shown were generated from LigIV^−/−:53BP1^−/−:DEL^CJ-6 abl pre-B cells, and similar results were obtained for LigIV^−/−:53BP1^−/−:DEL^CJ-5 abl pre-B cells.

Effect of KAP-1 mouse-human hybrids on resection of RAG DSBs. (A to D, left) Linear diagrams of mKAP-1, hKAP-1, and human-mouse hybrids, as described in the text. mKAP-1 domains are shown in gray, while hKAP-1 domains are shown in red. All KAP-1 hybrid proteins are Flag tagged at the N terminus. (Right) Density plots comparing lane signals from Southern blot analyses of pMX-DEL^CJ, as described in the legend of Fig. 2C, in LigIV^−/−:53BP1^−/−:DEL^CJ abl pre-B cells expressing a retrovirus encoding the indicated KAP-1 hybrid proteins treated with imatinib for 4 days. Primary data are posted at http://pathology.wustl.edu/labs/sleckman/Tubbs_et_a_MCB_2014_Supplement.pdf. Dotted lines represent KAP-1 hybrids that function similarly to mKAP-1 to promote resection. Solid lines represent KAP-1 hybrids that function to inhibit resection. Data shown were generated from LigIV^−/−:53BP1^−/−:DEL^CJ-6 abl pre-B cells. (E) Probability of disorder, as determined by PrDOS, along the linear amino acid sequences of hKAP-1 and mKAP-1. The gray box indicates the region between amino acids 531 and 548, which is predicted to be disordered.

A single-amino-acid change at position 548 determines KAP-1 functionality during resection of RAG DSBs. (A) Comparison of amino acid discrepancies between mKAP-1 and mKAP-1^h531–548. Human-specific residues are highlighted in red. (B) Immunoblot analysis of LigIV^−/−:53BP1^−/−:DEL^CJ abl pre-B cells expressing either an empty retrovirus (Empty) or a retrovirus encoding the indicated mKAP-1 point mutants. All mutants are Flag tagged at the N terminus. (C) Density plots comparing lane signals from Southern blot analyses of pMX-DEL^CJ, as described in the legend of Fig. 2C, in LigIV^−/−:53BP1^−/−:DEL^CJ-6 abl pre-B cells expressing a retrovirus encoding the indicated mKAP-1 point mutants treated with imatinib for 4 days. Primary data are posted at http://pathology.wustl.edu/labs/sleckman/Tubbs_et_a_MCB_2014_Supplement.pdf. (D) Amino acid sequence surrounding position 548 in mKAP-1, mKAP-1^P548A, hKAP-1, and hKAP-1^A548P. Human-specific residues are highlighted in red. (E) Immunoblot analysis of LigIV^−/−:53BP1^−/−:DEL^CJ abl pre-B cells expressing either a ctr shRNA or KAP-1 shRNA in addition to an empty retrovirus or a retrovirus encoding Flag-HA-tagged mKAP-1, mKAP-1^P548A, hKAP-1, or hKAP-1^A548P. (F) Southern blot analysis of pMX-DEL^CJ, as described in the legend of Fig. 1A. LigIV^−/−:53BP1^−/−:DEL^CJ abl pre-B cells expressing KAP-1 shRNA in addition to a retrovirus encoding mKAP-1, mKAP-1^P548A, hKAP-1, or hKAP-1^A548P were treated with imatinib for the indicated numbers of days. Data shown were generated from LigIV^−/−:53BP1^−/−:DEL^CJ-5 abl pre-B cells, and similar results were obtained for LigIV^−/−:53BP1^−/−:DEL^CJ-6 abl pre-B cells.

KAP-1 is required for opening of hairpin-sealed coding ends in the absence of Artemis. (A) Denaturing Southern blot analysis of pMX-DEL^CJ. EcoRV-digested genomic DNA from Art^−/−:DEL^CJ and LigIV^−/−:DEL^CJ abl pre-B cells treated with imatinib for 2 days were either denatured (D) or left nondenatured (N). Nondenatured (N) and denatured (D) bands distinguishing unrearranged pMX-DEL^CJ (UR), hairpin-sealed coding ends (hCE), and open coding ends (oCE) are labeled. (B) Denaturing Southern blot analysis of pMX-DEL^CJ as described above for panel A. Art^−/−:DEL^CJ, LigIV^−/−:DEL^CJ, and Art^−/−:H2AX^−/−:DEL^CJ abl pre-B cells expressing either an empty retrovirus or a retrovirus encoding Flag-HA-tagged mKAP-1, mKAP-1^P548A, hKAP-1, or hKAP-1^A548P were analyzed after imatinib treatment for the indicated numbers of days. (C) Bar graph showing the percentages of pMX-DEL^CJ coding ends that are either hairpin sealed or open, as quantitated from denaturing Southern blot lanes at 2 days of imatinib treatment in panel B. Data shown were generated from Art^−/−:H2AX^−/−:DEL^CJ-95 abl pre-B cells, and similar results were obtained with Art^−/−:H2AX^−/−:DEL^CJ-124 abl pre-B cells.