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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

FA core complex forms foci at sites of DNA damage.(A) U2OS cells were transfected with siControl or siFANCA and treated with mitomycin C (MMC) 60ng/ml for 24h before fixation. Cells were immunostained with the indicated antibodies. (B) Cells were left untreated or treated with 60ng/ml MMC, 2.5 μM cisplatin, 250μM hydroxyurea (HU) or 10μM AZD2281 for 24h, or treated with 10Gy ionizing radiation (IR) 8h before fixation. Then cells were immunostained with anti-FANCA antibody. Representative images are shown. (C) U2OS cells or U2OS expressing 3xFLAG-FANCD2 were treated with MMC 60ng/ml for 24h and immunostained with anti-FANCA, FLAG and γH2AX antibodies. (D) Localized DNA damage induced with a 450nm laser in U2OS cells expressing EGFP-FANCD2. Cells were fixed 30min after irradiation and immunostained with the indicated antibodies. (E) U2OS cells were transfected with the indicated siRNAs, untreated or treated with MMC 60ng/ml for 24h and immunostained with anti-FANCA or anti-FANCG antibodies. Foci/cell were counted using an automated software. Data represent mean values ± SD of three independent experiments. (*) Indicates p < 0.05; (n.s.) indicates no statistical significance (Compared to siControl).
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pgen.1005563.g001: FA core complex forms foci at sites of DNA damage.(A) U2OS cells were transfected with siControl or siFANCA and treated with mitomycin C (MMC) 60ng/ml for 24h before fixation. Cells were immunostained with the indicated antibodies. (B) Cells were left untreated or treated with 60ng/ml MMC, 2.5 μM cisplatin, 250μM hydroxyurea (HU) or 10μM AZD2281 for 24h, or treated with 10Gy ionizing radiation (IR) 8h before fixation. Then cells were immunostained with anti-FANCA antibody. Representative images are shown. (C) U2OS cells or U2OS expressing 3xFLAG-FANCD2 were treated with MMC 60ng/ml for 24h and immunostained with anti-FANCA, FLAG and γH2AX antibodies. (D) Localized DNA damage induced with a 450nm laser in U2OS cells expressing EGFP-FANCD2. Cells were fixed 30min after irradiation and immunostained with the indicated antibodies. (E) U2OS cells were transfected with the indicated siRNAs, untreated or treated with MMC 60ng/ml for 24h and immunostained with anti-FANCA or anti-FANCG antibodies. Foci/cell were counted using an automated software. Data represent mean values ± SD of three independent experiments. (*) Indicates p < 0.05; (n.s.) indicates no statistical significance (Compared to siControl).

Mentions: We carefully optimized immunostaining methods (described in Methods) and successfully detected nuclear foci of FANCA, FANCC, FANCE, FANCF, FANCG and FANCL in U2OS cells treated with MMC (Fig 1A). All of these foci were reduced in FANCA-depleted cells, suggesting that foci formation of the FA core complex proteins is FANCA-dependent and are likely to represent foci formation of the canonical FA core complex. Specificity of the antibodies was also confirmed by western blotting (S1G, S2E, S3C and S3D Figs). The formation of FANCA, FANCG and FANCE foci was induced by treatment with various DNA damaging agents (MMC, cisplatin, hydroxyurea, a PARP inhibitor (AZD2281) and ionizing radiation (IR)), in U2OS cells (Fig 1B and S1A Fig). FANCA and FANCG foci were also observed in HeLa cells and fibroblasts (S1B, S1C and S1E–S1G Fig). FANCA substantially colocalized with FANCD2 and γH2AX, but not TRF1, suggesting that FA core complex proteins localize at sites of DNA damage and not at telomeres (Fig 1C and S1D Fig). Recruitment of FANCA, FANCG, FANCC and FANCF to laser-induced localized DNA damage was also detected (Fig 1D). We were also successful in detecting foci formation of exogenously expressed myc-tagged FANCG (S4 Fig). In this case, detection of the foci was dependent on the level of expression. While high FANCG expression (pMMP-FANCG construct) resulted in pan-nuclear FANCG staining, foci were clearly detected (both with antibodies against FANCG and MYC-tag) when a low expressing (pLentiX1-mycFANCG) was used (S4A and S4B Fig). Both high-expression and low-expression FANCG constructs rescued MMC sensitivity at the same level (S4C Fig).


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)

FA core complex forms foci at sites of DNA damage.(A) U2OS cells were transfected with siControl or siFANCA and treated with mitomycin C (MMC) 60ng/ml for 24h before fixation. Cells were immunostained with the indicated antibodies. (B) Cells were left untreated or treated with 60ng/ml MMC, 2.5 μM cisplatin, 250μM hydroxyurea (HU) or 10μM AZD2281 for 24h, or treated with 10Gy ionizing radiation (IR) 8h before fixation. Then cells were immunostained with anti-FANCA antibody. Representative images are shown. (C) U2OS cells or U2OS expressing 3xFLAG-FANCD2 were treated with MMC 60ng/ml for 24h and immunostained with anti-FANCA, FLAG and γH2AX antibodies. (D) Localized DNA damage induced with a 450nm laser in U2OS cells expressing EGFP-FANCD2. Cells were fixed 30min after irradiation and immunostained with the indicated antibodies. (E) U2OS cells were transfected with the indicated siRNAs, untreated or treated with MMC 60ng/ml for 24h and immunostained with anti-FANCA or anti-FANCG antibodies. Foci/cell were counted using an automated software. Data represent mean values ± SD of three independent experiments. (*) Indicates p < 0.05; (n.s.) indicates no statistical significance (Compared to siControl).
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Related In: Results  -  Collection

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pgen.1005563.g001: FA core complex forms foci at sites of DNA damage.(A) U2OS cells were transfected with siControl or siFANCA and treated with mitomycin C (MMC) 60ng/ml for 24h before fixation. Cells were immunostained with the indicated antibodies. (B) Cells were left untreated or treated with 60ng/ml MMC, 2.5 μM cisplatin, 250μM hydroxyurea (HU) or 10μM AZD2281 for 24h, or treated with 10Gy ionizing radiation (IR) 8h before fixation. Then cells were immunostained with anti-FANCA antibody. Representative images are shown. (C) U2OS cells or U2OS expressing 3xFLAG-FANCD2 were treated with MMC 60ng/ml for 24h and immunostained with anti-FANCA, FLAG and γH2AX antibodies. (D) Localized DNA damage induced with a 450nm laser in U2OS cells expressing EGFP-FANCD2. Cells were fixed 30min after irradiation and immunostained with the indicated antibodies. (E) U2OS cells were transfected with the indicated siRNAs, untreated or treated with MMC 60ng/ml for 24h and immunostained with anti-FANCA or anti-FANCG antibodies. Foci/cell were counted using an automated software. Data represent mean values ± SD of three independent experiments. (*) Indicates p < 0.05; (n.s.) indicates no statistical significance (Compared to siControl).
Mentions: We carefully optimized immunostaining methods (described in Methods) and successfully detected nuclear foci of FANCA, FANCC, FANCE, FANCF, FANCG and FANCL in U2OS cells treated with MMC (Fig 1A). All of these foci were reduced in FANCA-depleted cells, suggesting that foci formation of the FA core complex proteins is FANCA-dependent and are likely to represent foci formation of the canonical FA core complex. Specificity of the antibodies was also confirmed by western blotting (S1G, S2E, S3C and S3D Figs). The formation of FANCA, FANCG and FANCE foci was induced by treatment with various DNA damaging agents (MMC, cisplatin, hydroxyurea, a PARP inhibitor (AZD2281) and ionizing radiation (IR)), in U2OS cells (Fig 1B and S1A Fig). FANCA and FANCG foci were also observed in HeLa cells and fibroblasts (S1B, S1C and S1E–S1G Fig). FANCA substantially colocalized with FANCD2 and γH2AX, but not TRF1, suggesting that FA core complex proteins localize at sites of DNA damage and not at telomeres (Fig 1C and S1D Fig). Recruitment of FANCA, FANCG, FANCC and FANCF to laser-induced localized DNA damage was also detected (Fig 1D). We were also successful in detecting foci formation of exogenously expressed myc-tagged FANCG (S4 Fig). In this case, detection of the foci was dependent on the level of expression. While high FANCG expression (pMMP-FANCG construct) resulted in pan-nuclear FANCG staining, foci were clearly detected (both with antibodies against FANCG and MYC-tag) when a low expressing (pLentiX1-mycFANCG) was used (S4A and S4B Fig). Both high-expression and low-expression FANCG constructs rescued MMC sensitivity at the same level (S4C Fig).

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