Kruppel-associated Box (KRAB)-associated co-repressor (KAP-1) Ser-473 phosphorylation regulates heterochromatin protein 1β (HP1-β) mobilization and DNA repair in heterochromatin

Abstract

The DNA damage response encompasses a complex series of signaling pathways that function to regulate and facilitate the repair of damaged DNA. Recent studies have shown that the repair of transcriptionally inactive chromatin, named heterochromatin, is dependent upon the phosphorylation of the co-repressor, Krüppel-associated box (KRAB) domain-associated protein (KAP-1), by the ataxia telangiectasia-mutated (ATM) kinase. Co-repressors, such as KAP-1, function to regulate the rigid structure of heterochromatin by recruiting histone-modifying enzymes, such HDAC1/2, SETDB1, and nucleosome-remodeling complexes such as CHD3. Here, we have characterized a phosphorylation site in the HP1-binding domain of KAP-1, Ser-473, which is phosphorylated by the cell cycle checkpoint kinase Chk2. Expression of a nonphosphorylatable S473A mutant conferred cellular sensitivity to DNA-damaging agents and led to defective repair of DNA double-strand breaks in heterochromatin. In addition, cells expressing S473A also displayed defective mobilization of the HP1-β chromodomain protein. The DNA repair defect observed in cells expressing S473A was alleviated by depletion of HP1-β, suggesting that phosphorylation of KAP-1 on Ser-473 promotes the mobilization of HP1-β from heterochromatin and subsequent DNA repair. These results suggest a novel mechanism of KAP-1-mediated chromatin restructuring via Chk2-regulated HP1-β exchange from heterochromatin, promoting DNA repair.

FIGURE 1.

KAP-1 is phosphorylated on Ser-473 following DNA damage. A, a multiple sequence alignment of the KAP-1 HP1-binding domain (HP1BD) in different species. B, 293T cells transfected with wild-type FLAG-KAP-1-WT or FLAG-KAP-1-S473A were mock-irradiated or treated with IR (10 Gy) or UVC (50 J/m²) (as indicated) and harvested 1 h after treatment. Cellular protein extracts were prepared and immunoblotted with the indicated antibodies. P-KAP-1, phospho-KAP-1. C, 293T cells were mock-irradiated or treated with IR (10 Gy) and harvested at the indicated time points. Cellular protein extracts were prepared and immunoblotted with the indicated antibodies. D, HeLa cells were mock-irradiated or treated with the indicated dose of IR and harvested 1 h after irradiation. Cellular protein extracts were prepared and immunoblotted with the indicated antibodies. P-ATM, phospho-ATM. E, HeLa cells were synchronized in S-phase using thymidine. Cells were treated with 6 Gy of IR at the indicated times after thymidine release, and extracts were taken after 1 h. Extracts were immunoblotted with the indicated antibodies.

FIGURE 2.

KAP-1 Ser-473 phosphorylation is dependent upon Chk2 and ATM. A, 293T cells transfected with control or Chk2 siRNA were mock-irradiated or treated with IR (10 Gy) and harvested 1 h after treatment. Cellular protein extracts were prepared and immunoblotted with the indicated antibodies. P-KAP-1, phospho-KAP-1. B, HCT15 cells stably expressing HA-Chk2 or HA alone were mock-irradiated or treated with IR (10 Gy) and harvested 1 h after treatment. Cellular protein extracts were prepared and immunoblotted with the indicated antibodies. C, C3ABR (ATM^+/+) or L3 (ATM^−/−) cells were mock-irradiated or treated with IR (10 Gy) and harvested 1 h after treatment. Cellular protein extracts were prepared and immunoblotted with the indicated antibodies.

FIGURE 3.

Cells expressing FLAG-KAP-1 S473A are sensitive to DNA damage. A and B, colony survival in HeLa cells. Cells were transfected with FLAG-CMV, FLAG-KAP-1 WT, or FLAG-KAP-1 S473A and treated with the indicated dose of IR (A) or camptothecin (B). Cells were left to form colonies for 10 days before staining with methylene blue. The data shown represent the average and S.E. of three independent experiments. C, HeLa cells were transfected with FLAG-CMV, FLAG-KAP-1 WT, or FLAG-KAP-1 S473A and treated with IR (6 Gy). Cell extracts were taken at 0.5 and 2 h after IR and immunoblotted with the indicated antibodies. P-KAP-1, phospho-KAP-1; P-ATM, phospho-ATM.

FIGURE 4.

Cells expressing FLAG-KAP-1 S473A display defective foci resolution in heterochromatin following DNA damage. A–D, HeLa cells were transfected with FLAG-CMV, FLAG-KAP-1 WT, or FLAG-KAP-1 S473A and treated with 2 Gy of IR before fixation and staining with γH2AX (A and B) or 53BP1 (C and D) antibodies at the indicated times. Representative images are shown in B and D, and graphical representation of data is shown in A and C. The results shown represent the average and S.E. of three independent experiments. The foci in 50–100 cells were counted in each independent experimental condition. * indicates a two-tailed p value of < 0.01. E and F, γH2AX foci that persist in S473A-expressing cells are associated with heterochromatin. Growth-arrested NIH3T3 cells were transfected with FLAG-KAP-1 WT or S473A. After 24 h, cells were mock-treated or treated with 2 Gy of IR and harvested at the indicated times after IR. Cells were stained with γH2AX (green) and DAPI (blue). Foci were scored as heterochromatin-associated positive or negative. The results shown represent the average and S.E. of three independent experiments. The association between γH2AX foci and heterochromatin in 40–50 cells was quantified in each independent experimental condition. ** indicates a two-tailed p value of < 0.0001.

FIGURE 5.

Phosphorylation of KAP-1 Ser-473 is required for HP1-β mobility following DNA damage. A–C, FRAP analysis of EGFP-HP1-β in heterochromatin, with and without laser-induced DNA damage, in cells stably expressing FLAG-KAP-1 WT (A), FLAG-KAP-1 S473A (B), or FLAG-KAP-1 S473E (C). Data show mean recovery curves (symbols) ± S.E. (n = 12), fitted with a one-phase association equation (solid lines; see “Experimental Procedures” for Equations 1 and 2).

FIGURE 6.

HP1-β knockdown alleviates the requirement for KAP-1 Ser-473 phosphorylation. A–C, HeLa cells were transfected with HP1-β siRNA for 24 h and then transfected with FLAG-CMV, FLAG-KAP-1 WT, or FLAG-KAP-1 S473A. Transfected cells were treated with 2 Gy of IR 48 h after siRNA transfection before fixation and staining with γH2AX (A and C) or 53BP1 (B and C) antibodies at the indicated times. The data shown represent the average and S.E. of three independent experiments. * indicates a two-tailed p value of < 0.04. ** indicates a two-tailed p value of < 0.02. Con si, control siRNA.