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. 2017 Jan 1;31(1):46-58.
doi: 10.1101/gad.291807.116. Epub 2017 Jan 23.

PHF11 promotes DSB resection, ATR signaling, and HR

Yi Gong  1 , Naofumi Handa  2   3 , Stephen C Kowalczykowski  2   3 , Titia de Lange  1
Affiliations

PHF11 promotes DSB resection, ATR signaling, and HR

Yi Gong et al. Genes Dev. .

Abstract

Resection of double-strand breaks (DSBs) plays a critical role in their detection and appropriate repair. The 3' ssDNA protrusion formed through resection activates the ATR-dependent DNA damage response (DDR) and is required for DSB repair by homologous recombination (HR). Here we report that PHF11 (plant homeodomain finger 11) encodes a previously unknown DDR factor involved in 5' end resection, ATR signaling, and HR. PHF11 was identified based on its association with deprotected telomeres and localized to sites of DNA damage in S phase. Depletion of PHF11 diminished the ATR signaling response to telomere dysfunction and genome-wide DNA damage, reduced end resection at sites of DNA damage, resulted in compromised HR and misrejoining of S-phase DSBs, and increased the sensitivity to DNA-damaging agents. PHF11 interacted with the ssDNA-binding protein RPA and was found in a complex with several nucleases, including the 5' dsDNA exonuclease EXO1. Biochemical experiments demonstrated that PHF11 stimulates EXO1 by overcoming its inhibition by RPA, suggesting that PHF11 acts (in part) by promoting 5' end resection at RPA-bound sites of DNA damage. These findings reveal a role for PHF11 in DSB resection, DNA damage signaling, and DSB repair.

Keywords: ATR; DSB; EXO1; PHF11; RPA; homologous recombination; resection.

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Figures

Figure 1.
Figure 1.
PHF11 localizes to sites of DNA damage. (A) Myc and γ-H2AX IF on mouse embryonic fibroblasts (MEFs) expressing myc-tagged PHF11 (N-terminal tag) or RPA32 (C-terminal tag) at the indicated times after 3 Gy of IR. (B) Kinetics of PHF11 and RPA32 focus formation compared with γH2AX as in A. (C) IF for myc-PHF11 and 53BP1 foci in asynchronous (Asyn) and G1 cells at 2 h after 3 Gy of IR. G1 cells were collected after mitotic shake-off and plated for 4 h, with IR treatment for the last 2 h. FACS profiles of BrdU-labeled cells (30-min pulse) showed that the G1 population had 0% S-phase cells, whereas the asynchronous population had 30% S-phase cells. (D) PHF11 foci induced by 10 µg/mL Aphi for 6 h, 5 mM HU for 6 h, mitomycin C (MMC) for 6 h after 0.4 µg/mL for 2 h, or CPT for 6 h after 1 µM for 2 h. (E) PHF11 foci at telomeres in the indicated MEFs lacking POT1a/b at 72 h after Cre. (Left) IF for myc-PHF11 (red) and 53BP1 (green). (Right) IF-FISH for myc-PHF11 (red) and telomeric TTAGGG DNA (green). (Blue) DAPI DNA stain; (S) stop allele; (F) floxed allele. (F) Colocalization of RPA32-myc and HA-PHF11 (FH2-PHF11) at Aphi-induced sites of damage and at telomeres lacking POT1a/b. Cells as in E without Cre treatment (control, Aphi) or at 72 h after Cre (ΔPOT1a/b). (G) Effect of ATR on PHF11 localization to telomeres lacking POT1a/b using IF-FISH as in E at 72 h after Cre. (H) Quantification of TIFs (telomere dysfunction-induced foci) (Takai et al. 2003) formed by 53BP1 and PHF11 in ATR-proficient and ATR-deficient MEFs lacking POT1a/b at their telomeres. Averages and SDs are from three independent experiments. (****) P < 0.0001, unpaired Student's t-test.
Figure 2.
Figure 2.
PHF11 promotes ATR signaling. (A) Immunoblotting for the effect of PHF11 deletion on Chk1 phosphorylation and expression of DDR factors. The indicated RPE1 cell lines lacking PHF11 (sg1 and sg2) with or without complementation with FH2-PHF11 were tested alongside the parental cells with and without CPT treatment. (B) Immunoblot for the effect of PHF11 knockout on Chk1-P and Chk2-P in response to HU. Cells were as in A. (C) Immunoblot for the effect of PHF11 knockout on phosphorylation of Chk1 and Chk2 after IR. Cells were as in A. (D) PHF11 shRNA knockdown diminishes the DDR at telomeres lacking POT1a/b. IF for γH2AX, MDC1, and 53BP1 (red) and TTAGGG FISH for telomeric DNA (green) in POT1a/b double-knockout (DKO) MEFs at 72 h after Cre. (Blue) DAPI DNA stain. (Fourth panel) Cells complemented with shRNA-resistant FH2-PHF11. (E) Quantification of the TIF response as in D. Average values and SDs are from three independent experiments. (****) P < 0.0001; (*) P < 0.05, unpaired Student's t-test.
Figure 3.
Figure 3.
PHF11 affects formation of ssDNA at sites of DNA damage. (A) IF for endogenous RPA32 and 53BP1 in the indicated RPE1 cells with and without PHF11 at 4 h after 3 Gy of IR. Cells are as in Figure 2A. (B) IF for endogenous RPA in wild-type and PHF11 knockout cells at 4 h after 3 Gy of IR. Cells were incubated with 10 mM EdU for 4 h after IR to identify S-phase cells with IR-induced RPA foci. Percentages of cells with the shown EdU pattern that contain >10 RPA foci are shown below the images. (C) Quantification of IR-induced RPA32 foci in cells with and without PHF11 (as in A). Average values and SDs are from three independent experiments. (****) P < 0.0001; (***) P < 0.001; (**) P < 0.01, unpaired Student's t-test. (D) PHF11 shRNA knockdown reduces accumulation of RPA at sites of DNA damage. MEFs expressing RPA32-myc examined by IF at 72 h after PHF11 shRNA infection. (Left) RPA foci (Myc IF; red) at telomeres (FISH; green) lacking POT1a/b at 72 h after Cre. (Middle and right) RPA foci (Myc IF; red) colocalizing with 53BP1 (53BP1 IF; green) after HU or IR treatment as indicated in cells not treated with Cre. (E) Examples of the effect of PHF11 depletion on RPA32-myc accumulation at IR-induced DSBs at the indicated time points after 10 Gy of IR (as in C). (F) Effect of PHF11 shRNA on hyperresection at telomeres lacking POT1a/b. Example of an in-gel assay for single-stranded telomeric DNA after PHF11 knockdown at the indicated times after Cre treatment of POT1a/1b double-knockout (DKO) MEFs. (G) Quantification of the telomeric overhang signals assayed as in F with two distinct shRNAs. Single-stranded TTAGGG signals were normalized to the total TTAGGG signal in the same lane. The normalized signal for cells lacking PHF11 shRNA and not treated with Cre was set to 1, and the other values are given relative to this value. Averages are from three independent experiments and SDs. (**) P < 0.01, unpaired Student's t-test. (H) Detection of BrdU in native DNA after treatment with HU in cells infected with PHF11sh9 (two examples shown) and the vector control. Cells were labeled with BrdU for 24 h and then treated with 2 mM HU for 4 h before processing for α-BrdU IF in native DNA. (I) Quantification of the effect of PHF11 depletion on detection of BrdU in native DNA after HU treatment. Assay is as in H. Values represent averages and SDs from three independent experiments. (*) P < 0.05, unpaired Student's t-test.
Figure 4.
Figure 4.
PHF11 affects DSB repair. (A) Effect of PHF11 deletion on HR efficiency measured using the DR-GFP reporter assay. The indicated cells containing the DR-GFP substrate were infected with lentiviral-I-SceI, and GFP+ cells were scored after 48 h. The percentage of GPF+ cells from PHF11 knockout cell lines sg1 and sg2 with and without complementation with FH2-PHF11 were normalized to the value for wild-type cells (set at 100%). Averages are from three independent experiments and SDs. (****) P < 0.0001; (***) P < 0.001, unpaired Student's t-test. (B) Metaphase spreads from MEFs treated with 5 µM olaparib for 18 h after treatment with PHF11 or BRCA1 shRNAs. Examples of misrejoined chromosomes are highlighted with red arrows. (C) Quantification of chromosomal abnormalities in cells as in B. (**) P < 0.01. n = 3. (D) Distributions of the frequency of chromosomal aberration (chromosome and chromatid breaks, fragments, and misrejoined chromosomes) in cells as in B and the vector control. (E) Survival of the indicated cell lines with and without PHF11 after treatment with CPT, IR, and MMC. Cells were treated in triplicate with the indicated concentrations/doses of CPT, MMC, or IR.
Figure 5.
Figure 5.
PHF11 interacts with RPA and resection factors. (A) Schematic of PHF11 and deletion mutants. (B) Coimmunoprecipitation of the indicated endogenous proteins with FH2-PHF11 (wild-type and the indicated mutants) transfected into 293T cells. FH2-eGFP and FH2-TRF1 were used as negative controls. (C) Dimerization of PHF11 through its C terminus. FH2-PHF11 and the indicated deletion mutants were cotransfected with myc-tagged PHF11. The ability of FH2-PHF11 alleles to coimmunoprecipitate the myc-tagged PHF11 was evaluated by immunoblotting after HA immunoprecipitation. FH2-tagged TIN2 served as a negative control. Asterisks indicate degradation products and nonspecific bands. (D) Deletion of the C terminus of PHF11, but not the ePHD finger domain, leads to a defect in chromatin binding. MEFs expressing the indicated PHF11 mutants were fractionated ([WCL] whole-cell lysate; [CP] cytoplasmic proteins; [NP] nuclear proteins; [CB] chromatin bound) and analyzed by immunoblotting for PHF11 and α-tubulin. (E) Coommassie-stained gel of purified recombinant GST-PHF11 and the GST control. (F) Recombinant PHF11 binds recombinant EXO1. GST-PHF11 and the GST control were used to pull down purified recombinant human EXO1, and bound proteins were evaluated by immunoblotting. (G,H) Pull-down of recombinant RPA with GST-PHF11 and the GST control. RPA70 and RPA32 were detected by immunoblotting.
Figure 6.
Figure 6.
PHF11 negates the inhibition of EXO1 by RPA. (A) Schematic of DNA used for resection experiments in B–F. (B) RPA inhibits EXO1-dependent resection, and PHF11 overcomes this inhibition. Resection assays with 0.25 nM EXO1 and the indicated RPA and PHF11 concentrations. (C,D) Quantification of stimulation of EXO1 (two concentrations) by PHF11 in the presence of RPA. The mean and SE are from three independent experiments. (E) Resection assays without EXO1 either with or without RPA, showing that PHF11 is not contaminated with a nuclease. (F) Quantification of experiments done without RPA showing no effect of PHF11 on EXO1-dependent resection of the 3′ tailed DNA substrate. The mean and SE are from three independent experiments. Error bars are smaller than the symbols. (G) No effect of PHF11 on inhibition of EXO1 by RPA on ssDNA.
Figure 7.
Figure 7.
EXO1-independent effects of PHF11. (A) Immunoblot for the effect of PHF11 shRNA (sh2) knockdown in EXO1-proficient and EXO1-deficient MEFs on phosphorylation of Chk1 after HU. (B) Quantification of chromosomal abnormalities from EXO1-proficient and EXO1-deficient MEFs infected with vector or PHF11 sh2 after treatment with 2 µM olaparib for 18 h. Averages are from at least three independent experiments and SDs. (****) P < 0.0001; (*) P < 0.05, unpaired Student's t-test.
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