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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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Increased spontaneous genomic instability and DNA damage signaling in CtIP-deficient B cells. (A) Expression of CtIP, KAP1 phosphorylation, and p53 phosphorylation as detected by Western blotting in CtIP-deficient and WT B cells. (B) Representative cell cycle analysis of CtIP-deficient (blue line) and WT (red line) B cells. After 2 d in culture, cells were fixed, stained with PI, and analyzed by flow cytometry. (C) Cellular division measured by CFSE dye dilution detected by FACS. One out of three experiments is shown. (D) Frequency of YFP+ cells relative to the percentage at day 0 (the day in which B cells were isolated). (E) Analysis of spontaneous genomic instability in metaphases from B cells isolated from mice of indicated genotypes. Graph shows the mean number of aberrant chromosome structures per cell measured in 3 independent experiments (n = 255 metaphases/genotype; P = 0.0131; two-tailed paired Student’s t test). (F) Percentage of CtIP-deficient B cells with normal chromosomes or with spontaneous chromosomal abnormalities is quantified. Data are derived from 10 independent experiments (n > 600 metaphases; P = 0.0013: two-tailed paired Student’s t test). (G) Percentage of cells with >5 RAD51 foci after 10 Gy irradiation relative to WT. For each experiment more than 600 cells were counted. The mean and SD of 3 independent experiments is shown (P = 0.0258; one-tailed paired Student’s t test). (H) Analysis of genomic instability in metaphase spreads before and after PARPi treatment (1 µM for 16 h) in B cells (n = 50 metaphases). Percentage of radials, chromatid, and chromosome breaks is quantified. (I) Mean percentage of radial structures among total aberrations counted in 4 different experiments (n > 200 metaphases) in BRCA1 or CtIP-deficient B cells upon PARPi treatment (1 µM for 16 h; P = 0.0103; two-tailed paired Student’s t test). For each independent experiment, one mouse per genotype was used.
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fig1: Increased spontaneous genomic instability and DNA damage signaling in CtIP-deficient B cells. (A) Expression of CtIP, KAP1 phosphorylation, and p53 phosphorylation as detected by Western blotting in CtIP-deficient and WT B cells. (B) Representative cell cycle analysis of CtIP-deficient (blue line) and WT (red line) B cells. After 2 d in culture, cells were fixed, stained with PI, and analyzed by flow cytometry. (C) Cellular division measured by CFSE dye dilution detected by FACS. One out of three experiments is shown. (D) Frequency of YFP+ cells relative to the percentage at day 0 (the day in which B cells were isolated). (E) Analysis of spontaneous genomic instability in metaphases from B cells isolated from mice of indicated genotypes. Graph shows the mean number of aberrant chromosome structures per cell measured in 3 independent experiments (n = 255 metaphases/genotype; P = 0.0131; two-tailed paired Student’s t test). (F) Percentage of CtIP-deficient B cells with normal chromosomes or with spontaneous chromosomal abnormalities is quantified. Data are derived from 10 independent experiments (n > 600 metaphases; P = 0.0013: two-tailed paired Student’s t test). (G) Percentage of cells with >5 RAD51 foci after 10 Gy irradiation relative to WT. For each experiment more than 600 cells were counted. The mean and SD of 3 independent experiments is shown (P = 0.0258; one-tailed paired Student’s t test). (H) Analysis of genomic instability in metaphase spreads before and after PARPi treatment (1 µM for 16 h) in B cells (n = 50 metaphases). Percentage of radials, chromatid, and chromosome breaks is quantified. (I) Mean percentage of radial structures among total aberrations counted in 4 different experiments (n > 200 metaphases) in BRCA1 or CtIP-deficient B cells upon PARPi treatment (1 µM for 16 h; P = 0.0103; two-tailed paired Student’s t test). For each independent experiment, one mouse per genotype was used.

Mentions: The analysis of CtIP functions in vivo has been hampered by the lethality of CtIP knockout mice, with mutant embryos dying at embryonic day E3.5 (Chen et al., 2005). To circumvent this, we generated a B cell–specific deletion of CtIP by intercrossing CD19cre mice (Rickert et al., 1997) with mice carrying conditional and alleles (CtIPco/−; Chen et al., 2008; Bothmer et al., 2013). The resulting CtIP- (CtIPΦ/−) B cells expressed little or no CtIP protein as determined by Western blot analysis (Fig. 1 A). Upon ex vivo stimulation with LPS and IL-4 cells, splenic B cells enter the cell cycle and undergo class switch recombination (CSR). Despite the fact that CtIP- mice invariably experience early embryonic death (Chen et al., 2005), CtIPΦ/− B cells were able to proliferate, exhibited a normal cell cycle distribution (Fig. 1 B), and underwent a similar number of cellular divisions as WT cells, as determined by CFSE dye dilution (Fig. 1 C). Moreover, CtIP-deficient B cells have similar level of class switching compared with WT (Bothmer et al., 2013).


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 spontaneous genomic instability and DNA damage signaling in CtIP-deficient B cells. (A) Expression of CtIP, KAP1 phosphorylation, and p53 phosphorylation as detected by Western blotting in CtIP-deficient and WT B cells. (B) Representative cell cycle analysis of CtIP-deficient (blue line) and WT (red line) B cells. After 2 d in culture, cells were fixed, stained with PI, and analyzed by flow cytometry. (C) Cellular division measured by CFSE dye dilution detected by FACS. One out of three experiments is shown. (D) Frequency of YFP+ cells relative to the percentage at day 0 (the day in which B cells were isolated). (E) Analysis of spontaneous genomic instability in metaphases from B cells isolated from mice of indicated genotypes. Graph shows the mean number of aberrant chromosome structures per cell measured in 3 independent experiments (n = 255 metaphases/genotype; P = 0.0131; two-tailed paired Student’s t test). (F) Percentage of CtIP-deficient B cells with normal chromosomes or with spontaneous chromosomal abnormalities is quantified. Data are derived from 10 independent experiments (n > 600 metaphases; P = 0.0013: two-tailed paired Student’s t test). (G) Percentage of cells with >5 RAD51 foci after 10 Gy irradiation relative to WT. For each experiment more than 600 cells were counted. The mean and SD of 3 independent experiments is shown (P = 0.0258; one-tailed paired Student’s t test). (H) Analysis of genomic instability in metaphase spreads before and after PARPi treatment (1 µM for 16 h) in B cells (n = 50 metaphases). Percentage of radials, chromatid, and chromosome breaks is quantified. (I) Mean percentage of radial structures among total aberrations counted in 4 different experiments (n > 200 metaphases) in BRCA1 or CtIP-deficient B cells upon PARPi treatment (1 µM for 16 h; P = 0.0103; two-tailed paired Student’s t test). 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

fig1: Increased spontaneous genomic instability and DNA damage signaling in CtIP-deficient B cells. (A) Expression of CtIP, KAP1 phosphorylation, and p53 phosphorylation as detected by Western blotting in CtIP-deficient and WT B cells. (B) Representative cell cycle analysis of CtIP-deficient (blue line) and WT (red line) B cells. After 2 d in culture, cells were fixed, stained with PI, and analyzed by flow cytometry. (C) Cellular division measured by CFSE dye dilution detected by FACS. One out of three experiments is shown. (D) Frequency of YFP+ cells relative to the percentage at day 0 (the day in which B cells were isolated). (E) Analysis of spontaneous genomic instability in metaphases from B cells isolated from mice of indicated genotypes. Graph shows the mean number of aberrant chromosome structures per cell measured in 3 independent experiments (n = 255 metaphases/genotype; P = 0.0131; two-tailed paired Student’s t test). (F) Percentage of CtIP-deficient B cells with normal chromosomes or with spontaneous chromosomal abnormalities is quantified. Data are derived from 10 independent experiments (n > 600 metaphases; P = 0.0013: two-tailed paired Student’s t test). (G) Percentage of cells with >5 RAD51 foci after 10 Gy irradiation relative to WT. For each experiment more than 600 cells were counted. The mean and SD of 3 independent experiments is shown (P = 0.0258; one-tailed paired Student’s t test). (H) Analysis of genomic instability in metaphase spreads before and after PARPi treatment (1 µM for 16 h) in B cells (n = 50 metaphases). Percentage of radials, chromatid, and chromosome breaks is quantified. (I) Mean percentage of radial structures among total aberrations counted in 4 different experiments (n > 200 metaphases) in BRCA1 or CtIP-deficient B cells upon PARPi treatment (1 µM for 16 h; P = 0.0103; two-tailed paired Student’s t test). For each independent experiment, one mouse per genotype was used.
Mentions: The analysis of CtIP functions in vivo has been hampered by the lethality of CtIP knockout mice, with mutant embryos dying at embryonic day E3.5 (Chen et al., 2005). To circumvent this, we generated a B cell–specific deletion of CtIP by intercrossing CD19cre mice (Rickert et al., 1997) with mice carrying conditional and alleles (CtIPco/−; Chen et al., 2008; Bothmer et al., 2013). The resulting CtIP- (CtIPΦ/−) B cells expressed little or no CtIP protein as determined by Western blot analysis (Fig. 1 A). Upon ex vivo stimulation with LPS and IL-4 cells, splenic B cells enter the cell cycle and undergo class switch recombination (CSR). Despite the fact that CtIP- mice invariably experience early embryonic death (Chen et al., 2005), CtIPΦ/− B cells were able to proliferate, exhibited a normal cell cycle distribution (Fig. 1 B), and underwent a similar number of cellular divisions as WT cells, as determined by CFSE dye dilution (Fig. 1 C). Moreover, CtIP-deficient B cells have similar level of class switching compared with WT (Bothmer et al., 2013).

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