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CtIP-mediated resection is essential for viability and can operate independently of BRCA1.

Polato F, Callen E, Wong N, Faryabi R, Bunting S, Chen HT, Kozak M, Kruhlak MJ, Reczek CR, Lee WH, Ludwig T, Baer R, Feigenbaum L, Jackson S, Nussenzweig A - J. Exp. Med. (2014)

Bottom Line: First, using mouse models expressing S327A or T847A mutant CtIP as a sole species, and B cells deficient in CtIP, we show that loss of the CtIP-BRCA1 interaction does not detectably affect resection, maintenance of genomic stability or viability, whereas T847 is essential for these functions.Second, although loss of 53BP1 rescues the embryonic lethality and HR defects in BRCA1-deficient mice, it does not restore viability or genome integrity in CtIP(-/-) mice.Finally, the sensitivity of BRCA1-deficient cells to poly ADP ribose polymerase (PARP) inhibition is partially rescued by the phospho-mimicking mutant CtIP (CtIP-T847E).

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Genome Integrity, Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.

ABSTRACT
Homologous recombination (HR) is initiated by DNA end resection, a process in which stretches of single-strand DNA (ssDNA) are generated and used for homology search. Factors implicated in resection include nucleases MRE11, EXO1, and DNA2, which process DNA ends into 3' ssDNA overhangs; helicases such as BLM, which unwind DNA; and other proteins such as BRCA1 and CtIP whose functions remain unclear. CDK-mediated phosphorylation of CtIP on T847 is required to promote resection, whereas CDK-dependent phosphorylation of CtIP-S327 is required for interaction with BRCA1. Here, we provide evidence that CtIP functions independently of BRCA1 in promoting DSB end resection. First, using mouse models expressing S327A or T847A mutant CtIP as a sole species, and B cells deficient in CtIP, we show that loss of the CtIP-BRCA1 interaction does not detectably affect resection, maintenance of genomic stability or viability, whereas T847 is essential for these functions. Second, although loss of 53BP1 rescues the embryonic lethality and HR defects in BRCA1-deficient mice, it does not restore viability or genome integrity in CtIP(-/-) mice. Third, the increased resection afforded by loss of 53BP1 and the rescue of BRCA1-deficiency depend on CtIP but not EXO1. Finally, the sensitivity of BRCA1-deficient cells to poly ADP ribose polymerase (PARP) inhibition is partially rescued by the phospho-mimicking mutant CtIP (CtIP-T847E). Thus, in contrast to BRCA1, CtIP has indispensable roles in promoting resection and embryonic development.

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CDK-dependent phosphorylation of CtIPT847 is essential for CtIP function. (A) WT and BRCA1Δ11/Δ11 MEFs were irradiated with a 364-nm laser line derived from a LSM510 microscope. After 10-min recovery, cells were processed for immunofluorescence analysis of CtIP (red) and γH2AX (green). Bar, 15 µm. (B) Western blot showing expression of transgenic CtIPWT and CtIPS327A protein and phosphorylated KAP1 and p53 in B cells. (C) Percentage of cells with >5 RAD51 foci after 10 Gy irradiation. Three independent experiments are reported. For each experiment, >600 cells were counted (significance tests were analyzed with two-tailed paired Student’s t test; *, P < 0.05; n.s., not significant). (D) Analysis of total genomic instability in metaphases from B cells isolated from mice of indicated genotypes after treatment for 16 h with 1 µM PARPi. The mean number of chromosome aberrations per cell measured in 3 independent experiments is shown (n = 250 metaphases; significance tests were analyzed with two-tailed paired Student’s t test; *, P < 0.05; n.s., not significant). (E) Levels of transgenic CtIPWT, CtIPT847E, and CtIPT847A protein expression and KAP1 and p53 phosphorylation in mutant B cells detected by Western blotting. (F) Analysis of total genomic instability in metaphases from B cells with or without PARPi treatment. Mean of three independent experiments is shown (significance tests were analyzed with two-tailed paired Student’s t test; *, P < 0.05; n.s., not significant). (G) Percentage of B cells with >5 RAD51 foci after 10 Gy IR. Three independent experiments are reported. >600 cells were counted for each genotype (P = 0.0251; two-tailed paired t test). (H) Western blot showing the level of CtIP expression in infected cells using anti-FLAG antibody (top). WT MEFs expressing FLAG-CtIPWT, FLAG-CtIPT847A, or FLAG-CtIPT847E were treated with Hoechst 33342 and irradiated with the 364-nm laser line and after 20-min recovery FLAG (red) and 53BP1 (green) were detected by immunofluorescence (bottom). Bar, 15 µm. (I) Western blot showing the levels of CtIP protein in B cells from mice of the indicated genotype (top). Genomic instability in B cells isolated from mice of the indicated genotypes, relative to total instability measured in BRCA1-deficient B cells. Cells were treated with 1 µM PARPi for 16 h (bottom). Three independent experiments are reported (significance tests were analyzed with two-tailed paired Student’s t test; *, P < 0.05; n.s., not significant). For each independent experiment, one mouse per genotype was used.
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fig3: CDK-dependent phosphorylation of CtIPT847 is essential for CtIP function. (A) WT and BRCA1Δ11/Δ11 MEFs were irradiated with a 364-nm laser line derived from a LSM510 microscope. After 10-min recovery, cells were processed for immunofluorescence analysis of CtIP (red) and γH2AX (green). Bar, 15 µm. (B) Western blot showing expression of transgenic CtIPWT and CtIPS327A protein and phosphorylated KAP1 and p53 in B cells. (C) Percentage of cells with >5 RAD51 foci after 10 Gy irradiation. Three independent experiments are reported. For each experiment, >600 cells were counted (significance tests were analyzed with two-tailed paired Student’s t test; *, P < 0.05; n.s., not significant). (D) Analysis of total genomic instability in metaphases from B cells isolated from mice of indicated genotypes after treatment for 16 h with 1 µM PARPi. The mean number of chromosome aberrations per cell measured in 3 independent experiments is shown (n = 250 metaphases; significance tests were analyzed with two-tailed paired Student’s t test; *, P < 0.05; n.s., not significant). (E) Levels of transgenic CtIPWT, CtIPT847E, and CtIPT847A protein expression and KAP1 and p53 phosphorylation in mutant B cells detected by Western blotting. (F) Analysis of total genomic instability in metaphases from B cells with or without PARPi treatment. Mean of three independent experiments is shown (significance tests were analyzed with two-tailed paired Student’s t test; *, P < 0.05; n.s., not significant). (G) Percentage of B cells with >5 RAD51 foci after 10 Gy IR. Three independent experiments are reported. >600 cells were counted for each genotype (P = 0.0251; two-tailed paired t test). (H) Western blot showing the level of CtIP expression in infected cells using anti-FLAG antibody (top). WT MEFs expressing FLAG-CtIPWT, FLAG-CtIPT847A, or FLAG-CtIPT847E were treated with Hoechst 33342 and irradiated with the 364-nm laser line and after 20-min recovery FLAG (red) and 53BP1 (green) were detected by immunofluorescence (bottom). Bar, 15 µm. (I) Western blot showing the levels of CtIP protein in B cells from mice of the indicated genotype (top). Genomic instability in B cells isolated from mice of the indicated genotypes, relative to total instability measured in BRCA1-deficient B cells. Cells were treated with 1 µM PARPi for 16 h (bottom). Three independent experiments are reported (significance tests were analyzed with two-tailed paired Student’s t test; *, P < 0.05; n.s., not significant). For each independent experiment, one mouse per genotype was used.

Mentions: The aforementioned findings suggested that CtIP could act independently of BRCA1 to promote resection. Consistent with this, we found that BRCA1 was not required for recruitment of CtIP to DNA damage sites (Fig. 3 A). To explore the physiological role of the CtIP–BRCA1 interaction mediated by CDK-dependent phosphorylation of CtIP at S327 (Yu and Chen, 2004), we generated BAC transgenic mouse models. By introducing the human gene coding for CtIP (hereafter, referred to as CtIPWT) as a transgenic copy carried by a bacterial artificial chromosome (BAC RP11-104H10), we were able to rescue the lethality caused by homozygous deletion of mouse CtIP (Table 2). CtIPWT was expressed at a similar level compared with endogenous CtIP (Fig. 3 B). Moreover, B cells in CtIPWTCtIP−/− mice were equivalent to CtIP+/+ with respect to RAD51 foci formation and genome stability (Fig. 3, C and D).


CtIP-mediated resection is essential for viability and can operate independently of BRCA1.

Polato F, Callen E, Wong N, Faryabi R, Bunting S, Chen HT, Kozak M, Kruhlak MJ, Reczek CR, Lee WH, Ludwig T, Baer R, Feigenbaum L, Jackson S, Nussenzweig A - J. Exp. Med. (2014)

CDK-dependent phosphorylation of CtIPT847 is essential for CtIP function. (A) WT and BRCA1Δ11/Δ11 MEFs were irradiated with a 364-nm laser line derived from a LSM510 microscope. After 10-min recovery, cells were processed for immunofluorescence analysis of CtIP (red) and γH2AX (green). Bar, 15 µm. (B) Western blot showing expression of transgenic CtIPWT and CtIPS327A protein and phosphorylated KAP1 and p53 in B cells. (C) Percentage of cells with >5 RAD51 foci after 10 Gy irradiation. Three independent experiments are reported. For each experiment, >600 cells were counted (significance tests were analyzed with two-tailed paired Student’s t test; *, P < 0.05; n.s., not significant). (D) Analysis of total genomic instability in metaphases from B cells isolated from mice of indicated genotypes after treatment for 16 h with 1 µM PARPi. The mean number of chromosome aberrations per cell measured in 3 independent experiments is shown (n = 250 metaphases; significance tests were analyzed with two-tailed paired Student’s t test; *, P < 0.05; n.s., not significant). (E) Levels of transgenic CtIPWT, CtIPT847E, and CtIPT847A protein expression and KAP1 and p53 phosphorylation in mutant B cells detected by Western blotting. (F) Analysis of total genomic instability in metaphases from B cells with or without PARPi treatment. Mean of three independent experiments is shown (significance tests were analyzed with two-tailed paired Student’s t test; *, P < 0.05; n.s., not significant). (G) Percentage of B cells with >5 RAD51 foci after 10 Gy IR. Three independent experiments are reported. >600 cells were counted for each genotype (P = 0.0251; two-tailed paired t test). (H) Western blot showing the level of CtIP expression in infected cells using anti-FLAG antibody (top). WT MEFs expressing FLAG-CtIPWT, FLAG-CtIPT847A, or FLAG-CtIPT847E were treated with Hoechst 33342 and irradiated with the 364-nm laser line and after 20-min recovery FLAG (red) and 53BP1 (green) were detected by immunofluorescence (bottom). Bar, 15 µm. (I) Western blot showing the levels of CtIP protein in B cells from mice of the indicated genotype (top). Genomic instability in B cells isolated from mice of the indicated genotypes, relative to total instability measured in BRCA1-deficient B cells. Cells were treated with 1 µM PARPi for 16 h (bottom). Three independent experiments are reported (significance tests were analyzed with two-tailed paired Student’s t test; *, P < 0.05; n.s., not significant). For each independent experiment, one mouse per genotype was used.
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fig3: CDK-dependent phosphorylation of CtIPT847 is essential for CtIP function. (A) WT and BRCA1Δ11/Δ11 MEFs were irradiated with a 364-nm laser line derived from a LSM510 microscope. After 10-min recovery, cells were processed for immunofluorescence analysis of CtIP (red) and γH2AX (green). Bar, 15 µm. (B) Western blot showing expression of transgenic CtIPWT and CtIPS327A protein and phosphorylated KAP1 and p53 in B cells. (C) Percentage of cells with >5 RAD51 foci after 10 Gy irradiation. Three independent experiments are reported. For each experiment, >600 cells were counted (significance tests were analyzed with two-tailed paired Student’s t test; *, P < 0.05; n.s., not significant). (D) Analysis of total genomic instability in metaphases from B cells isolated from mice of indicated genotypes after treatment for 16 h with 1 µM PARPi. The mean number of chromosome aberrations per cell measured in 3 independent experiments is shown (n = 250 metaphases; significance tests were analyzed with two-tailed paired Student’s t test; *, P < 0.05; n.s., not significant). (E) Levels of transgenic CtIPWT, CtIPT847E, and CtIPT847A protein expression and KAP1 and p53 phosphorylation in mutant B cells detected by Western blotting. (F) Analysis of total genomic instability in metaphases from B cells with or without PARPi treatment. Mean of three independent experiments is shown (significance tests were analyzed with two-tailed paired Student’s t test; *, P < 0.05; n.s., not significant). (G) Percentage of B cells with >5 RAD51 foci after 10 Gy IR. Three independent experiments are reported. >600 cells were counted for each genotype (P = 0.0251; two-tailed paired t test). (H) Western blot showing the level of CtIP expression in infected cells using anti-FLAG antibody (top). WT MEFs expressing FLAG-CtIPWT, FLAG-CtIPT847A, or FLAG-CtIPT847E were treated with Hoechst 33342 and irradiated with the 364-nm laser line and after 20-min recovery FLAG (red) and 53BP1 (green) were detected by immunofluorescence (bottom). Bar, 15 µm. (I) Western blot showing the levels of CtIP protein in B cells from mice of the indicated genotype (top). Genomic instability in B cells isolated from mice of the indicated genotypes, relative to total instability measured in BRCA1-deficient B cells. Cells were treated with 1 µM PARPi for 16 h (bottom). Three independent experiments are reported (significance tests were analyzed with two-tailed paired Student’s t test; *, P < 0.05; n.s., not significant). For each independent experiment, one mouse per genotype was used.
Mentions: The aforementioned findings suggested that CtIP could act independently of BRCA1 to promote resection. Consistent with this, we found that BRCA1 was not required for recruitment of CtIP to DNA damage sites (Fig. 3 A). To explore the physiological role of the CtIP–BRCA1 interaction mediated by CDK-dependent phosphorylation of CtIP at S327 (Yu and Chen, 2004), we generated BAC transgenic mouse models. By introducing the human gene coding for CtIP (hereafter, referred to as CtIPWT) as a transgenic copy carried by a bacterial artificial chromosome (BAC RP11-104H10), we were able to rescue the lethality caused by homozygous deletion of mouse CtIP (Table 2). CtIPWT was expressed at a similar level compared with endogenous CtIP (Fig. 3 B). Moreover, B cells in CtIPWTCtIP−/− mice were equivalent to CtIP+/+ with respect to RAD51 foci formation and genome stability (Fig. 3, C and D).

Bottom Line: First, using mouse models expressing S327A or T847A mutant CtIP as a sole species, and B cells deficient in CtIP, we show that loss of the CtIP-BRCA1 interaction does not detectably affect resection, maintenance of genomic stability or viability, whereas T847 is essential for these functions.Second, although loss of 53BP1 rescues the embryonic lethality and HR defects in BRCA1-deficient mice, it does not restore viability or genome integrity in CtIP(-/-) mice.Finally, the sensitivity of BRCA1-deficient cells to poly ADP ribose polymerase (PARP) inhibition is partially rescued by the phospho-mimicking mutant CtIP (CtIP-T847E).

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Genome Integrity, Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.

ABSTRACT
Homologous recombination (HR) is initiated by DNA end resection, a process in which stretches of single-strand DNA (ssDNA) are generated and used for homology search. Factors implicated in resection include nucleases MRE11, EXO1, and DNA2, which process DNA ends into 3' ssDNA overhangs; helicases such as BLM, which unwind DNA; and other proteins such as BRCA1 and CtIP whose functions remain unclear. CDK-mediated phosphorylation of CtIP on T847 is required to promote resection, whereas CDK-dependent phosphorylation of CtIP-S327 is required for interaction with BRCA1. Here, we provide evidence that CtIP functions independently of BRCA1 in promoting DSB end resection. First, using mouse models expressing S327A or T847A mutant CtIP as a sole species, and B cells deficient in CtIP, we show that loss of the CtIP-BRCA1 interaction does not detectably affect resection, maintenance of genomic stability or viability, whereas T847 is essential for these functions. Second, although loss of 53BP1 rescues the embryonic lethality and HR defects in BRCA1-deficient mice, it does not restore viability or genome integrity in CtIP(-/-) mice. Third, the increased resection afforded by loss of 53BP1 and the rescue of BRCA1-deficiency depend on CtIP but not EXO1. Finally, the sensitivity of BRCA1-deficient cells to poly ADP ribose polymerase (PARP) inhibition is partially rescued by the phospho-mimicking mutant CtIP (CtIP-T847E). Thus, in contrast to BRCA1, CtIP has indispensable roles in promoting resection and embryonic development.

Show MeSH
Related in: MedlinePlus