Limits...
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.

Show MeSH

Related in: MedlinePlus

Increased resection of 53BP1−/− cells is CtIP dependent. (A) Analysis of spontaneous genomic instability in metaphases from B cells isolated from mice of the indicated genotypes. Genomic instability measured in five independent experiments is normalized to levels seen in CtIP-deficient B cells (n > 200 metaphases/genotype; P = 0.4179: two-tailed paired Student’s t test). (B) Constitutive levels of p53 phosphorylation in the CtIPΦ/– and CtIPΦ/– 53BP1−/− double mutant B cells. (C) Western blot analysis showing levels of CtIP expression in MEFs infected with CtIP shRNA (shCtIP). (D) Fold increase of mean BrdU fluorescence of irradiated (30 Gy) versus unirradiated MEFs of the indicated genotypes that were either infected or not with ShCtIP. The mean and SD of three independent experiments is shown (significance tests were analyzed with one-tailed paired Student’s t test; *, P < 0.05). (E) Percentage of cells with >5 RAD51 foci after 10 Gy irradiation relative to WT (16.5% of WT cells were positive for RAD51 foci). For each experiment, >600 cells were counted. (F) Analysis of genomic instability in metaphases from B cells treated with PARPi (1 µM; 16 h; n > 200 metaphases/genotype were counted; the mean of 4 independent experiments is reported relative to BRCA1-deficient cells; significance tests were analyzed with two-tailed paired Student’s t test; *, P < 0.05; n.s., not significant). (G) Analysis of genomic instability in B cells after PARPi treatment (1 µM for 16 h). The graph shows the average number of aberrant chromosome structures per cell measured in three independent experiments relative to genome instability in BRCA1-deficient cells (n = 150 metaphases/genotype; 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.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4042650&req=5

fig2: Increased resection of 53BP1−/− cells is CtIP dependent. (A) Analysis of spontaneous genomic instability in metaphases from B cells isolated from mice of the indicated genotypes. Genomic instability measured in five independent experiments is normalized to levels seen in CtIP-deficient B cells (n > 200 metaphases/genotype; P = 0.4179: two-tailed paired Student’s t test). (B) Constitutive levels of p53 phosphorylation in the CtIPΦ/– and CtIPΦ/– 53BP1−/− double mutant B cells. (C) Western blot analysis showing levels of CtIP expression in MEFs infected with CtIP shRNA (shCtIP). (D) Fold increase of mean BrdU fluorescence of irradiated (30 Gy) versus unirradiated MEFs of the indicated genotypes that were either infected or not with ShCtIP. The mean and SD of three independent experiments is shown (significance tests were analyzed with one-tailed paired Student’s t test; *, P < 0.05). (E) Percentage of cells with >5 RAD51 foci after 10 Gy irradiation relative to WT (16.5% of WT cells were positive for RAD51 foci). For each experiment, >600 cells were counted. (F) Analysis of genomic instability in metaphases from B cells treated with PARPi (1 µM; 16 h; n > 200 metaphases/genotype were counted; the mean of 4 independent experiments is reported relative to BRCA1-deficient cells; significance tests were analyzed with two-tailed paired Student’s t test; *, P < 0.05; n.s., not significant). (G) Analysis of genomic instability in B cells after PARPi treatment (1 µM for 16 h). The graph shows the average number of aberrant chromosome structures per cell measured in three independent experiments relative to genome instability in BRCA1-deficient cells (n = 150 metaphases/genotype; 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: Loss of 53BP1 normalizes the HR defects in BRCA1-deficient cells, at least in part by increasing DSB resection (Bunting et al., 2010). To determine what impact 53BP1 deletion might have on CtIP−/−-associated genomic instability, we generated CtIP/53BP1 double knockout B cells by crossing CD19cre/+CtIPco/− with 53BP1−/− mice (Fig. 2 A). We found that CtIP/53BP1 double-mutant B cells harbored spontaneous chromosomal aberrations and constitutive DNA damage signaling similar to the levels observed in CtIP single-mutant cells (Fig. 2, A and B). Thus, in contrast to its ability to rescue BRCA1-associated phenotypes, loss of 53BP1 does not restore genomic integrity in the absence of CtIP.


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)

Increased resection of 53BP1−/− cells is CtIP dependent. (A) Analysis of spontaneous genomic instability in metaphases from B cells isolated from mice of the indicated genotypes. Genomic instability measured in five independent experiments is normalized to levels seen in CtIP-deficient B cells (n > 200 metaphases/genotype; P = 0.4179: two-tailed paired Student’s t test). (B) Constitutive levels of p53 phosphorylation in the CtIPΦ/– and CtIPΦ/– 53BP1−/− double mutant B cells. (C) Western blot analysis showing levels of CtIP expression in MEFs infected with CtIP shRNA (shCtIP). (D) Fold increase of mean BrdU fluorescence of irradiated (30 Gy) versus unirradiated MEFs of the indicated genotypes that were either infected or not with ShCtIP. The mean and SD of three independent experiments is shown (significance tests were analyzed with one-tailed paired Student’s t test; *, P < 0.05). (E) Percentage of cells with >5 RAD51 foci after 10 Gy irradiation relative to WT (16.5% of WT cells were positive for RAD51 foci). For each experiment, >600 cells were counted. (F) Analysis of genomic instability in metaphases from B cells treated with PARPi (1 µM; 16 h; n > 200 metaphases/genotype were counted; the mean of 4 independent experiments is reported relative to BRCA1-deficient cells; significance tests were analyzed with two-tailed paired Student’s t test; *, P < 0.05; n.s., not significant). (G) Analysis of genomic instability in B cells after PARPi treatment (1 µM for 16 h). The graph shows the average number of aberrant chromosome structures per cell measured in three independent experiments relative to genome instability in BRCA1-deficient cells (n = 150 metaphases/genotype; 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.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4042650&req=5

fig2: Increased resection of 53BP1−/− cells is CtIP dependent. (A) Analysis of spontaneous genomic instability in metaphases from B cells isolated from mice of the indicated genotypes. Genomic instability measured in five independent experiments is normalized to levels seen in CtIP-deficient B cells (n > 200 metaphases/genotype; P = 0.4179: two-tailed paired Student’s t test). (B) Constitutive levels of p53 phosphorylation in the CtIPΦ/– and CtIPΦ/– 53BP1−/− double mutant B cells. (C) Western blot analysis showing levels of CtIP expression in MEFs infected with CtIP shRNA (shCtIP). (D) Fold increase of mean BrdU fluorescence of irradiated (30 Gy) versus unirradiated MEFs of the indicated genotypes that were either infected or not with ShCtIP. The mean and SD of three independent experiments is shown (significance tests were analyzed with one-tailed paired Student’s t test; *, P < 0.05). (E) Percentage of cells with >5 RAD51 foci after 10 Gy irradiation relative to WT (16.5% of WT cells were positive for RAD51 foci). For each experiment, >600 cells were counted. (F) Analysis of genomic instability in metaphases from B cells treated with PARPi (1 µM; 16 h; n > 200 metaphases/genotype were counted; the mean of 4 independent experiments is reported relative to BRCA1-deficient cells; significance tests were analyzed with two-tailed paired Student’s t test; *, P < 0.05; n.s., not significant). (G) Analysis of genomic instability in B cells after PARPi treatment (1 µM for 16 h). The graph shows the average number of aberrant chromosome structures per cell measured in three independent experiments relative to genome instability in BRCA1-deficient cells (n = 150 metaphases/genotype; 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: Loss of 53BP1 normalizes the HR defects in BRCA1-deficient cells, at least in part by increasing DSB resection (Bunting et al., 2010). To determine what impact 53BP1 deletion might have on CtIP−/−-associated genomic instability, we generated CtIP/53BP1 double knockout B cells by crossing CD19cre/+CtIPco/− with 53BP1−/− mice (Fig. 2 A). We found that CtIP/53BP1 double-mutant B cells harbored spontaneous chromosomal aberrations and constitutive DNA damage signaling similar to the levels observed in CtIP single-mutant cells (Fig. 2, A and B). Thus, in contrast to its ability to rescue BRCA1-associated phenotypes, loss of 53BP1 does not restore genomic integrity in the absence of CtIP.

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