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FANCI Regulates Recruitment of the FA Core Complex at Sites of DNA Damage Independently of FANCD2.

Castella M, Jacquemont C, Thompson EL, Yeo JE, Cheung RS, Huang JW, Sobeck A, Hendrickson EA, Taniguchi T - PLoS Genet. (2015)

Bottom Line: Here we show that the FA core complex proteins are recruited to sites of DNA damage and form nuclear foci in S and G2 phases of the cell cycle.ATR kinase activity, an intact FA core complex and FANCM-FAAP24 were crucial for this recruitment.Additionally, BRCA1 was required for efficient FA core complex foci formation.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Divisions of Human Biology and Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America.

ABSTRACT
The Fanconi anemia (FA)-BRCA pathway mediates repair of DNA interstrand crosslinks. The FA core complex, a multi-subunit ubiquitin ligase, participates in the detection of DNA lesions and monoubiquitinates two downstream FA proteins, FANCD2 and FANCI (or the ID complex). However, the regulation of the FA core complex itself is poorly understood. Here we show that the FA core complex proteins are recruited to sites of DNA damage and form nuclear foci in S and G2 phases of the cell cycle. ATR kinase activity, an intact FA core complex and FANCM-FAAP24 were crucial for this recruitment. Surprisingly, FANCI, but not its partner FANCD2, was needed for efficient FA core complex foci formation. Monoubiquitination or ATR-dependent phosphorylation of FANCI were not required for the FA core complex recruitment, but FANCI deubiquitination by USP1 was. Additionally, BRCA1 was required for efficient FA core complex foci formation. These findings indicate that FANCI functions upstream of FA core complex recruitment independently of FANCD2, and alter the current view of the FA-BRCA pathway.

No MeSH data available.


Related in: MedlinePlus

USP1 is required for FA core complex foci formation.(A) U2OS cells were transfected with the indicated siRNAs and treated with 10Gy IR. The cells were fixed and stained 8 hours later. Cells with > 5 foci were counted and the percentage of positive cells is shown (n = 3, mean ± SD). (*) Indicates p<0.05; (Compared to siControl for each foci, respectively). The lower panel shows a western blot corresponding to samples shown in upper panel. (B) Cells were pre-treated with ML323 at the indicated dose 2 hours before irradiation (10 Gy IR). Cells were fixed and stained 8 hours later. Graph shows cells containing >5 foci (n = 3, mean ± SD). (*) Indicates p<0.05; (Compared to Untreated sample for each foci, respectively). (C) Cells were transfected with the indicated combinations of siRNA and plasmid 48 hours before treatment (IR 10 Gy, fixed 8 hours later). The percentage of cells containing > 5 FANCG foci (n = 3, mean ± SD) is shown. (*) Indicates p<0.05. (n.s.) indicates no statistical significance. In samples transfected with USP1 wild-type or USP1 C90S, only USP1-expressing cells (Myc-positive) were included in the analysis. (D) Representative images corresponding to the experiment detailed in panel C.
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pgen.1005563.g007: USP1 is required for FA core complex foci formation.(A) U2OS cells were transfected with the indicated siRNAs and treated with 10Gy IR. The cells were fixed and stained 8 hours later. Cells with > 5 foci were counted and the percentage of positive cells is shown (n = 3, mean ± SD). (*) Indicates p<0.05; (Compared to siControl for each foci, respectively). The lower panel shows a western blot corresponding to samples shown in upper panel. (B) Cells were pre-treated with ML323 at the indicated dose 2 hours before irradiation (10 Gy IR). Cells were fixed and stained 8 hours later. Graph shows cells containing >5 foci (n = 3, mean ± SD). (*) Indicates p<0.05; (Compared to Untreated sample for each foci, respectively). (C) Cells were transfected with the indicated combinations of siRNA and plasmid 48 hours before treatment (IR 10 Gy, fixed 8 hours later). The percentage of cells containing > 5 FANCG foci (n = 3, mean ± SD) is shown. (*) Indicates p<0.05. (n.s.) indicates no statistical significance. In samples transfected with USP1 wild-type or USP1 C90S, only USP1-expressing cells (Myc-positive) were included in the analysis. (D) Representative images corresponding to the experiment detailed in panel C.

Mentions: FANCA,FANCG, FANCC and FANCL foci formation were impaired in USP1-depleted U2OS cells and in cells treated with a USP1 specific inhibitor, ML323 [37] (Fig 7A and 7B and S12A Fig). Increasing concentrations of ML323 resulted in decreased FANCA and FANCD2 foci, without affecting BRCA1 or γH2AX foci (Fig 7B). ML323 treatment resulted in increased FANCD2 and FANCI ubiquitination, confirming USP1 inhibition (S12B Fig). To confirm the siRNA and inhibitor data, USP1-depleted cells were complemented using an siRNA-resistant USP1 cDNA. Due to the high instability of overexpressed wild-type USP1 protein, a form of USP1 that is unable to cleave itself, GG670/671AA, (described in [38]) was used. The catalytic function of GG670/671AA mutant of USP1 is comparable to wild-type USP1 [38]. The FA core complex and FANCD2 foci defect was rescued when wild-type USP1, but not the catalytic inactive form (USP1 C90S), was expressed (Fig 7C and 7D and S12C Fig). In both cases, an overall reduction in the number of cells with foci was observed, likely due to cellular toxicity of USP1 overexpression. These data indicate that catalytic activity of USP1 is required to promote efficient recruitment of FA core complex and FANCD2 to sites of DNA damage. They also indicate that impaired FA core complex recruitment at sites of DNA damage does not necessarily translate into deficient FANCD2-FANCI ubiquitination.


FANCI Regulates Recruitment of the FA Core Complex at Sites of DNA Damage Independently of FANCD2.

Castella M, Jacquemont C, Thompson EL, Yeo JE, Cheung RS, Huang JW, Sobeck A, Hendrickson EA, Taniguchi T - PLoS Genet. (2015)

USP1 is required for FA core complex foci formation.(A) U2OS cells were transfected with the indicated siRNAs and treated with 10Gy IR. The cells were fixed and stained 8 hours later. Cells with > 5 foci were counted and the percentage of positive cells is shown (n = 3, mean ± SD). (*) Indicates p<0.05; (Compared to siControl for each foci, respectively). The lower panel shows a western blot corresponding to samples shown in upper panel. (B) Cells were pre-treated with ML323 at the indicated dose 2 hours before irradiation (10 Gy IR). Cells were fixed and stained 8 hours later. Graph shows cells containing >5 foci (n = 3, mean ± SD). (*) Indicates p<0.05; (Compared to Untreated sample for each foci, respectively). (C) Cells were transfected with the indicated combinations of siRNA and plasmid 48 hours before treatment (IR 10 Gy, fixed 8 hours later). The percentage of cells containing > 5 FANCG foci (n = 3, mean ± SD) is shown. (*) Indicates p<0.05. (n.s.) indicates no statistical significance. In samples transfected with USP1 wild-type or USP1 C90S, only USP1-expressing cells (Myc-positive) were included in the analysis. (D) Representative images corresponding to the experiment detailed in panel C.
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pgen.1005563.g007: USP1 is required for FA core complex foci formation.(A) U2OS cells were transfected with the indicated siRNAs and treated with 10Gy IR. The cells were fixed and stained 8 hours later. Cells with > 5 foci were counted and the percentage of positive cells is shown (n = 3, mean ± SD). (*) Indicates p<0.05; (Compared to siControl for each foci, respectively). The lower panel shows a western blot corresponding to samples shown in upper panel. (B) Cells were pre-treated with ML323 at the indicated dose 2 hours before irradiation (10 Gy IR). Cells were fixed and stained 8 hours later. Graph shows cells containing >5 foci (n = 3, mean ± SD). (*) Indicates p<0.05; (Compared to Untreated sample for each foci, respectively). (C) Cells were transfected with the indicated combinations of siRNA and plasmid 48 hours before treatment (IR 10 Gy, fixed 8 hours later). The percentage of cells containing > 5 FANCG foci (n = 3, mean ± SD) is shown. (*) Indicates p<0.05. (n.s.) indicates no statistical significance. In samples transfected with USP1 wild-type or USP1 C90S, only USP1-expressing cells (Myc-positive) were included in the analysis. (D) Representative images corresponding to the experiment detailed in panel C.
Mentions: FANCA,FANCG, FANCC and FANCL foci formation were impaired in USP1-depleted U2OS cells and in cells treated with a USP1 specific inhibitor, ML323 [37] (Fig 7A and 7B and S12A Fig). Increasing concentrations of ML323 resulted in decreased FANCA and FANCD2 foci, without affecting BRCA1 or γH2AX foci (Fig 7B). ML323 treatment resulted in increased FANCD2 and FANCI ubiquitination, confirming USP1 inhibition (S12B Fig). To confirm the siRNA and inhibitor data, USP1-depleted cells were complemented using an siRNA-resistant USP1 cDNA. Due to the high instability of overexpressed wild-type USP1 protein, a form of USP1 that is unable to cleave itself, GG670/671AA, (described in [38]) was used. The catalytic function of GG670/671AA mutant of USP1 is comparable to wild-type USP1 [38]. The FA core complex and FANCD2 foci defect was rescued when wild-type USP1, but not the catalytic inactive form (USP1 C90S), was expressed (Fig 7C and 7D and S12C Fig). In both cases, an overall reduction in the number of cells with foci was observed, likely due to cellular toxicity of USP1 overexpression. These data indicate that catalytic activity of USP1 is required to promote efficient recruitment of FA core complex and FANCD2 to sites of DNA damage. They also indicate that impaired FA core complex recruitment at sites of DNA damage does not necessarily translate into deficient FANCD2-FANCI ubiquitination.

Bottom Line: Here we show that the FA core complex proteins are recruited to sites of DNA damage and form nuclear foci in S and G2 phases of the cell cycle.ATR kinase activity, an intact FA core complex and FANCM-FAAP24 were crucial for this recruitment.Additionally, BRCA1 was required for efficient FA core complex foci formation.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Divisions of Human Biology and Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America.

ABSTRACT
The Fanconi anemia (FA)-BRCA pathway mediates repair of DNA interstrand crosslinks. The FA core complex, a multi-subunit ubiquitin ligase, participates in the detection of DNA lesions and monoubiquitinates two downstream FA proteins, FANCD2 and FANCI (or the ID complex). However, the regulation of the FA core complex itself is poorly understood. Here we show that the FA core complex proteins are recruited to sites of DNA damage and form nuclear foci in S and G2 phases of the cell cycle. ATR kinase activity, an intact FA core complex and FANCM-FAAP24 were crucial for this recruitment. Surprisingly, FANCI, but not its partner FANCD2, was needed for efficient FA core complex foci formation. Monoubiquitination or ATR-dependent phosphorylation of FANCI were not required for the FA core complex recruitment, but FANCI deubiquitination by USP1 was. Additionally, BRCA1 was required for efficient FA core complex foci formation. These findings indicate that FANCI functions upstream of FA core complex recruitment independently of FANCD2, and alter the current view of the FA-BRCA pathway.

No MeSH data available.


Related in: MedlinePlus