A novel ubiquitin ligase is deficient in Fanconi anemia

© 2003 Nature Publishing Group http://www.nature.com/naturegenetics

LETTERS

A novel ubiquitin ligase is deficient in Fanconi anemia Amom Ruhikanta Meetei1, Johan P de Winter2, Annette L Medhurst2, Michael Wallisch3, Quinten Waisfisz2, Henri J van de Vrugt2, Anneke B Oostra2, Zhijiang Yan1, Chen Ling1, Colin E Bishop4, Maureen E Hoatlin3, Hans Joenje2 & Weidong Wang1 Fanconi anemia is a recessively inherited disease characterized by congenital defects, bone marrow failure and cancer susceptibility1,2. Cells from individuals with Fanconi anemia are highly sensitive to DNA-crosslinking drugs, such as mitomycin C (MMC). Fanconi anemia proteins function in a DNA damage response pathway involving breast cancer susceptibility gene products, BRCA1 and BRCA2 (refs. 1,2). A key step in this pathway is monoubiquitination of FANCD2, resulting in the redistribution of FANCD2 to nuclear foci containing BRCA1 (ref. 3). The underlying mechanism is unclear because the five Fanconi anemia proteins known to be required for this ubiquitination have no recognizable ubiquitin ligase motifs. Here we report a new component of a Fanconi anemia protein complex, called PHF9, which possesses E3 ubiquitin ligase activity in vitro and is essential for FANCD2 monoubiquitination in vivo. Because PHF9 is defective in a cell line derived from an individual with Fanconi anemia, we conclude that PHF9 (also called FANCL) represents a novel Fanconi anemia complementation group (FA-L). Our data suggest that PHF9 has a crucial role in the Fanconi anemia pathway as the likely catalytic subunit required for monoubiquitination of FANCD2. We recently immunoisolated a complex called BRAFT, which contains all five core complex Fanconi anemia proteins (FANCA, FANCC, FANCE, FANCF and FANCG) together with BLM (the helicase involved in Bloom syndrome), topoisomerase IIIα (Topo IIIα), replication protein A (RPA) and several polypeptides of unknown function (called Fanconi anemia–associated polypeptides (FAAPs) or BLM-associated polypeptides (BLAPs); ref. 4; Fig. 1a). In the presence of 0.7 M salt, BLM, Topo IIIα, RPA and BLAP75 were completely dissociated from the Fanconi anemia proteins. The remaining subcomplex, which comprised four FAAPs (of 250, 100, 90 and 43 kDa) and the five known Fanconi anemia proteins, is probably the Fanconi anemia protein core complex that has previously been suggested to exist but never isolated to this level of purity5–7. We identified FAAP43 by mass spectrometry as PHF9 (PHD finger protein 9; see Fig. 3). Several pieces of evidence suggest that PHF9 is a

stable component of the Fanconi anemia core complex. First, PHF9 was coimmunoprecipitated from nuclear extract by two antibodies to FANCA (Fig. 1b). Second, four of five known Fanconi anemia core complex proteins (all but FANCE) were coimmunoprecipitated by an antibody to PHF9 (Fig. 1b). Third, all known core complex proteins, including FANCE, were detected after immunoprecipitation with an antibody to FLAG in 293 cells stably expressing FLAG-tagged PHF9 (Fig. 1c). Fourth, PHF9 cofractionated with FANCA by Superose 6 gel-filtration analysis of HeLa cell extract (Fig. 1d). The levels of PHF9 in nuclear extracts of lymphoblasts from individuals with Fanconi anemia complementation groups A, E and B (FA-A, FA-E and FA-B, respectively) were significantly lower than that in wild-type cells (Fig. 2a). The levels of PHF9 in the nuclear extracts of FA-E and FA-A cells recovered to wild-type levels after these cells were complemented by ectopic expression of the corresponding genes (Fig. 2b,c). We detected PHF9 in both the nuclear and cytoplasmic extracts of wild-type cells (Fig. 2c). In lysates from two FA-A cell lines, however, the level of PHF9 was markedly lower than wild-type in the nuclear extract but was normal in the cytoplasm, suggesting that the nuclear accumulation of PHF9 depends on FANCA. This feature of PHF9 is reminiscent of Fanconi anemia core complex components FANCA and FANCC, whose nuclear localization depends on other core complex proteins6,8–10. This provides additional evidence that PHF9 is a component of the Fanconi anemia complex. PHF9 encodes three potential WD40 repeats and a PHD-type zinc-finger motif (Fig. 3a). WD40 repeats are known to mediate protein-protein interactions11, and several PHD fingers are reported to possess E3 ubiquitin ligase activity12–14. These PHD finger ligases all have a conserved tryptophan between cysteines 6 and 7 (ref. 15), which might distinguish ligases from non-ligases. Notably, the PHD finger of PHF9 contains this conserved tryptophan (Fig. 3a), suggesting that PHF9 could be a ubiquitin ligase. Indeed, two GST fusion proteins encompassing the PHD finger of PHF9 had ubiquitin ligase activity (Fig. 3b,c), whereas mutant proteins with alanine substituted for the first or the second conserved cysteine residues had no activity, indicating that the PHD finger is essential for the ubiquitin ligase activity of PHF9.

1Laboratory

of Genetics, National Institute on Aging, National Institutes of Health, 333 Cassell Drive, TRIAD Center Room 3000, Baltimore, Maryland 21224, USA. of Clinical Genetics and Human Genetics, Free University Medical Center, Van der Boechorststraat 7, NL-1081 BT Amsterdam, The Netherlands. 3Division of Molecular Medicine, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97239, USA. 4Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Texas 77030, USA. Correspondence should be addressed to W.W. ([email protected]). 2Department

Published online 14 September 2003; doi:10.1038/ng1241

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Figure 1 PHF9 is a component of the Fanconi anemia core complex. (a) Silver-stained SDS gel showing the BRAFT complex components immunoisolated (IP) from HeLa cell nuclear extracts by an antibody (7489) against FANCA (lane 1). The Fanconi anemia core complex (lane 2) was obtained by immunoprecipitation under high-stringency-washing conditions (0.75 M salt), which removed BLM, Topo IIIα, BLAP75 and RPA70 (marked by arrows). All the polypeptides listed in the figure, including PHF9, were identified by mass spectrometry. (b) Immunoblotting shows that PHF9 and Fanconi anemia proteins coimmunoprecipitated from nuclear extracts. Two different FANCA antibodies (7489 and 5G9) and one PHF9 antibody were used for immunoprecipitation (IP). Preimmune sera for both FANCA and PHF9 were used in control immunoprecipitations (mock). Nuclear extract (NE) is also shown. The absence of FANCE in PHF9 immunoprecipitate could be explained by its displacement by PHF9 antibody. (c) Immunoblotting shows that five Fanconi anemia proteins were immunoprecipitated by an antibody to FLAG from Ebna-293 cells stably expressing FLAG-PHF9 (lanes 1 and 2). As a control, none of the Fanconi anemia proteins were immunoprecipitated from the parental cell line (lanes 3 and 4). (d) Immunoblotting analysis shows cofractionation of PHF9 and FANCA by Superose 6 gel filtration. One peak corresponds to the 1.5-MDa BRAFT complex, and a second peak corresponds to a complex of approximately 600 kDa. Fractions corresponding to known molecular weight markers are indicated at the bottom.

When we knocked down PHF9 expression in HeLa cells by siRNA, we observed significantly lower levels of both basal and MMC-induced monoubiquitinated FANCD2 (Fig. 4a), as well as fewer FANCD2 nuclear foci in response to MMC treatment (Fig. 4b). This indicates that PHF9 is essential for both FANCD2 monoubiquitination and redistribution into subnuclear foci. In the

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presence of MMC, the percentage of cells containing chromosomal aberrations was significantly higher among PHF9-knock-down cells than among control cells (Table 1). These chromosomal aberrations are hallmarks of Fanconi anemia cells, suggesting that, like other Fanconi anemia proteins, PHF9 is essential for maintaining chromosomal stability.

HS C9 3 EU FA 41 0 EU FA FA 41 NC 0+ E-H A

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Figure 2 Nuclear localization of PHF9 is defective in cells from several Fanconi anemia complementation groups. (a) Immunoblotting shows the level of PHF9 in nuclear extracts prepared from lymphoblasts of the eight known Fanconi anemia complementation groups and from wild-type cells (WT). The levels of FANCA and FANCG are shown for comparison. The crossreactive bands above and below the PHF9 band can be used as loading controls. (b) Immunoblotting shows that complementation with FANCE cDNA restored the level of PHF9 in nuclear extract of FA-E lymphoblasts (“WT”). A crossreactive band (marked with asterisk) can be used as a loading control. HA, hemagglutinin; WT, wild-type. (c) Immunoblotting shows that nuclear localization of PHF9 was defective in FA-A cells and that this defect was corrected after complementation with FANCA cDNA (“WT”). A crossreactive band (marked with asterisk) can be used as a loading control. FANCG is unstable in FA-A cells (see panel a), and FANCA does not localize in the nucleus in FA-B cells, consistent with previous findings5,6. NE, nuclear extract; WT, wild-type.

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a

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Figure 3 PHF9/Pog contains a PHD finger–type E3 ubiquitin ligase motif and has autoubiquitin ligase activity in vitro. (a) Alignment of PHF9 sequences from different species. PHF9 is a human homolog of the mouse Pog gene product (80% identity throughout entire protein). D. melanogaster (dro) and A. gambiae (ano) have homologs of FANCD2 and PHF9 but not of the five Fanconi anemia core complex proteins (FANCA, FANCC, FANCE, FANCF and FANCG), suggesting that aspects of the FANCD2 monoubiquitination mechanism are not conserved in invertebrates. The three predicted WD40 repeats and the PHD finger are marked. The conserved cysteines and histidine in the PHD finger are indicated below the figure. The arrow indicates a conserved tryptophan residue present in all PHD finger–type E3 ligases but absent in PHD finger proteins that are non-ligases15. The PHD fingers of E3 ligases have recently been proposed to be variations of ring domains30. (b) Results from autoubiquitination assay show that two recombinant GST-PHF9 fusion proteins, GST-PHF9B (amino acids 275–375) and GST-PHF9C (amino acids 200–375), which contain the PHD finger, have autoubiquitination activity. An autoradiograph measuring radioactivity from 32P-labeled GST-ubiquitin (Ub; top) and the same SDS gel stained with Coomassie blue (bottom) are shown. GST alone and GST fused to an N-terminal region of PHF9 (GSTPHF9A; amino acids 60–170) were used as negative controls. A GST-fusion protein with known E3 ligase activity, GST-Mekk1, was used as a positive control. The presence or absence of 32P-labeled GST-ubiquitin, E1 and E2 (Ubc4) are indicated at the top. (c) A Coomassie blue–stained SDS gel showing that two different point mutations in the PHD finger of PHF9, C307A and C310A, inactivate its autoubiquitination activity. The different GST fusion proteins are shown at the top.

The mouse homolog of PHF9, named Pog (for proliferation of germ cells), was recently identified as the gene underlying the phenotype of gcd (germ cell–deficient) mice16. The gcd mice, like mice carrying null Pog alleles owing to targeted disruption, are less fertile and have defective proliferation of germ cells, characteristics that are also found in Fanconi anemia knockout mice17–22. Moreover, bone marrow cells isolated from Pog-knockout mice were hypersensitive to MMC (data not shown). These data in mice are consistent with PHF9/Pog being associated with Fanconi anemia. We screened for mutations in PHF9 in lymphoblastoid cell lines from individuals with FA-B and FA-I whose underlying genetic defect is unknown and whose cell lines lack FANCD2 monoubiquitination but found no mutations (data not shown). However, we detected little or no PHF9 protein in a cell line, EUFA868, from an individual with Fanconi anemia of unassigned complementation group (Fig. 5a).


PHF9 cDNA from this cell line lacked exon 11, thus removing the conserved PHD finger and part of the third WD40 repeat (Fig. 5b). The genomic DNA from this individual showed a homo- or hemizygous insertion of 177 bp into a pyrimidine-rich sequence at the splice junction between intron 10 and exon 11 (Fig. 5c). As pyrimidine-rich sequences are known to serve as signals for intron-exon junctions, this insertion probably disturbs splicing at this particular junction, resulting in the observed deletion. The phenotype of cells from the EUFA868 cell line resembles that of other Fanconi anemia cells, including the absence of monoubiquitinated FANCD2 (Fig. 5d) and hypersensitivity to MMC (Fig. 5e). These Fanconi anemia defects were corrected by ectopic expression of PHF9. We conclude that PHF9 is the gene inactivated in EUFA868 cells, and that this individual represents a novel Fanconi anemia complementation group (FA-L).

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Figure 4 PHF9-knockdown cells have less monoubiquitinated FANCD2 and fewer FANCD2 FANCD2 foci nuclear foci. (a) Immunoblotting shows that 100.0 0 foci suppression of PHF9 expression in HeLa cells 90.0 1–5 foci by siRNA reduces the levels of 80.0 6–10 foci – + – + – + monoubiquitinated FANCD2 in cells that are MMC 70.0 10+ foci FANCD2-L either untreated or treated with MMC. 60.0 FANCD2-S Immunoblotting of BLM is shown as a loading 50.0 control. As a control, an oligo with a scrambled 40.0 sequence did not knock down PHF9 expression 30.0 PHF9 or reduce the level of monoubiquitinated 20.0 FANCD2. The residual level of 10.0 BLM monoubiquitinated FANCD2 in the cells 0.0 HeLa HeLa + HeLa + HeLa + HeLa + HeLa + transfected with PHF9 oligo could be due to a MMC scrambled scrambled PHF9 PHF9 small percentage of cells that were not siRNA RNA oligo RNA oligo siRNA transfected by the siRNA oligo. (b) Histogram + MMC oligo oligo + of immunofluorescence analysis of FANCD2 MMC nuclear foci in HeLa cells in which PHF9 expression was knocked down by siRNA. Cells that are untransfected or transfected with a control oligo were used as controls. The presence or absence of MMC is indicated. Nuclear foci larger than 0.6 µm2 were electronically counted. Nuclei were tabulated in groups containing 0, 1–5, 6–10 and 10+ foci per nucleus.

PHF9 carrying a point mutation in its PHD finger (C307A) that inactivates its ubiquitin ligase activity (Fig. 3c) did not complement the Fanconi anemia–associated defects of EUFA868 cells (Fig. 5d,e), consistent with the notion that the ubiquitin ligase activity of PHF9 is required for FANCD2 monoubiquitination. But the amount of FANCA that coimmunoprecipitated with the mutant protein was lower than that observed in wild-type cells (Fig. 5f and Supplementary Fig. 1 online), suggesting that this mutation could also affect the interaction between PHF9 and other components of the Fanconi anemia complex. Although BRCA1 has been proposed as a possible ubiquitin ligase for FANCD2 (ref. 3), two recent studies using siRNA-knockdown and gene-knockout methods have indicated that BRCA1 is not essential for FANCD2 monoubiquitination23,24. In agreement with the latter results, we reproducibly observed both basal and MMC-induced FANCD2 monoubiquitination in a BRCA1-mutant cell line but detected no FANCD2 monoubiquitination in the PHF9-defective cell line (Fig. 5g). Because correction of the BRCA1-mutant cell line by BRCA1 transfection leads to more monoubiquitinated FANCD2, BRCA1 might affect the stability of monoubiquitinated FANCD2 or the efficiency of FANCD2 monoubiquitination. This is consistent Table 1 Spontaneous and MMC-induced chromosomal abnormalities after siRNA-induced knockdown of PHF9 Pretreatment None Mock-transfected Control oligo-transfected PHF9 oligo-transfected

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aCells in metaphase were examined for chromatid-type interchange aberrations and scored as aberrant if they featured at least one triradial, quadriradial or other type of interchange figure. bSignificantly different from control oligo-transfected MMC-treated cells (two-sample χ2 test, P = 0.001).

HeLa cells were exposed to MMC (120 nM, 24 h) after a pretreatment siRNA protocol designed to decrease expression of PHF9.

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with a recent siRNA study showing that depletion of BRCA1 results in less monoubiquitination of FANCD2 (ref. 24). The absence of FANCD2 foci in irradiated BRCA1-mutant cells3,23 clearly suggests that BRCA1 is involved in the Fanconi anemia pathway. Taken together, however, these data imply that BRCA1 may modulate the ubiquitination process but that PHF9 is probably the primary ubiquitin ligase for FANCD2. Future studies will be required to elucidate the precise roles of PHF9 and BRCA1. In summary, we have identified PHF9, which encodes a novel E3 ubiquitin ligase, as a new gene associated with Fanconi anemia (FAL). This is the first such gene identified by protein association and the first gene associated with Fanconi anemia that encodes a product with a catalytic activity. The finding that PHF9 and five other components of the newly isolated Fanconi anemia protein core complex are all required for FANCD2 monoubiquitination suggests that the Fanconi anemia core complex is directly involved in the ubiquitination of FANCD2, with PHF9 probably serving as the crucial catalytic subunit and other components functioning as regulatory subunits. METHODS Subjects and cell culture. We obtained Fanconi anemia cell lines from the European Fanconi Anemia Research Consortium cell repository. These cell lines were established according to European Consortium guidelines, which require informed consent from the individuals or their legal representatives. We diagnosed individuals with Fanconi anemia on the basis of clinical symptoms in combination with a standard chromosomal breakage test showing hypersensitivity to crosslinking agents before establishing lymphoblastoid cell lines. We maintained the cell lines in RPMI 1640 culture medium supplemented with 1 mM glutamine, 1 mM sodium pyruvate and 10% fetal calf serum. We transfected HSC72 cells with a pIRESneo construct (Clontech) encoding FANCA with a C-terminal His tag and selected stable cell lines with 400 µg neomycin per ml culture medium. We transfected EUFA410 and EUFA868 cells with pCEP4 constructs (Invitrogen) encoding FANCE with a C-terminal hemagglutinin tag or PHF9 with an N-terminal FLAG tag, respectively. We selected stable cell lines in the presence of hygromycin (100 µg ml–1). We carried out the MMC-induced growth inhibition test as described25. We grew EBNA-293T cells in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum in the presence of penicillin and streptomycin. We generated stable transfectants from EBNA-293T cells by selection in hygromycin (400 µg ml–1; Invitrogen), which we initiated 48 h after transfection with pCEP4-FLAG-PHF9. We obtained HeLa S3 cells from the National Cell Culture Center.



LETTERS Cell extraction and fractionation. We prepared cytoplasmic and nuclear extracts in Figure 2c as described26. In other experiments, we isolated nuclei from lymphoblastoid cells with hypotonic buffer and Nonidet P-40 as described6. After extensive washing with hypotonic buffer, we isolated nuclear proteins by high salt extraction (20 mM HEPES-KOH (pH7.9), 420 mM NaCl, 25% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol and protease inhibitors). We separated equal amounts of cytosolic and nuclear proteins on 8–16% Tris-glycine gels (Invitrogen) and immunoblotted them with different antibodies. For FANCD2 western blotting, we separated samples by electrophoresis on 3–8% NuPAGE Tris-acetate gels (Invitrogen). For gel filtration analysis, we applied the HeLa S3 nuclear extract (10 mg) directly to a Superose 6 column (HR16/50; Amersham) equilibrated with column running buffer containing 20 mM HEPES (pH 7.9), 200 mM NaCl, 1 mM dithiothreitol, 0.1mM phenylmethylsulfonyl fluroide and 10% glycerol.

a

d

We collected fractions of 1.5 ml and analyzed them (20 µl each) by SDS–PAGE (8–16% gradient Tris-glycine gel; Invitrogen) and immunoblotting. Antibodies. Antibodies against FANCA, FANCF, FANCG, FANCC and BLM have been described4,6,27. FANCE antibody was provided by K. J. Patel9 (Department of Investigative Medicine, Addenbrookes Hospital, University of Cambridge, Cambridge, UK). The rabbit PHF9 polyclonal antibody was raised against a chimeric protein containing a C-terminal region of PHF9 (amino acids 200–375) fused to maltose binding protein and affinity-purified. The FANCD2 antibody was made similarly against an N-terminal region of FANCD2 (amino acids 155–330) fused to maltose binding protein and affinity-purified. Immunoprecipitation. We immunoisolated BRAFT complex from HeLa S3 nuclear extract as described4. To immunoprecipitate Fanconi anemia core

e

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Figure 5 PHF9 is deficient in an individual with Fanconi anemia. (a) Immunoblotting shows that little or no PHF9 protein was detected in the whole-cell extract from a cell line (EUFA868) established from an individual with Fanconi anemia. (b) Sequence showing the mutated region of PHF9 cDNA from cell line EUFA868. Exon 11 is completely deleted, resulting in a transcript in which exon 10 is directly linked to exon 12. This creates a change in reading frame at codon 275 and might result in a truncated protein lacking the C-terminal PHD finger. (c) Genomic sequence of wild-type PHF9 (top) and PHF9 from EUFA868 cells (bottom) near the mutated region. The pyrimidine-rich sequence, which is probably a signal for the splicing junction between intron 10 and exon 11, is underlined. This sequence was disrupted by an insertion of an 177-bp DNA fragment. Database searches indicate that this 177-bp DNA is derived from intron 11, which is about 2,000 bp downstream. (d) Immunoblotting shows that EUFA868 cells lack monoubiquitinated FANCD2, which is corrected by ectopic expression of wild-type PHF9 (WT) but not by PHF9 with a point mutation in the PHD finger (C307A; mu) that inactivated its ubiquitin ligase activity. (e) Analysis of MMC-induced growth inhibition shows that ectopic expression of wild-type PHF9 protein (WT), but not the C307A mutant (mu), complements the MMC hypersensitivity of EUFA868 cells. The result shown is a representative of four independent assays, three including wild-type PHF9 protein and two including mutant PHF9. (f) Immunoblotting shows that the amount of FANCA coimmunoprecipitating with FLAG-PHF9 mutant protein (C307A; mu) is lower than the amount that coimmunoprecipitates with the wild-type (WT). (g) Immunoblotting shows FANCD2 monoubiquitination in response to MMC treatment in the BRCA1-mutant cell line (HCC1937), the same cell line complemented by stable expression of BRCA1, and the PHF9-deficient cell line (EUFA868). Cells were exposed to MMC for 0, 24 and 36 h as indicated. The level of PHF9 is also shown. A 100-kDa polypeptide crossreacting with PHF9 antibody is shown as a loading control.


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LETTERS complex, we diluted 1 ml (8 mg ml–1) of nuclear extract five times with IP buffer (20 mM HEPES, pH 7.9, 500 mM NaCl, 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride and 10% glycerol) and incubated it at least 12 h with 5 µg of affinity-purified rabbit polyclonal antibody to FANCA (7489) crosslinked to protein A beads (Amersham Pharmacia). We washed the immunoprecipitate four times with IP buffer containing 700 mM NaCl. We eluted the complex from beads with 100 mM glycine-HCl buffer (pH 2.5) and subjected it to SDS–PAGE and immunoblotting analysis. For mass spectrometric analysis, we visualized proteins by staining with Coomassie blue, excised them from the gel and analyzed them by liquid chromatography and mass spectrometry. The mass spectrometric data are not shown but are available on request. We carried out immunoprecipitations from total cell lysates in lysis buffer (50 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 1.0% Triton-X100 and 200 mM NaCl) in the presence of a protease inhibitor cocktail (Complete; Roche Molecular Biochemicals). We immunoprecipitated FLAG-tagged proteins with anti-FLAG M2 agarose affinity gel (Sigma) for 12 h at 4 °C from 3 mg of total cell lysate. Immunoprecipitated material was washed three times for 15 min each in lysis buffer and eluted with FLAG peptide (150 µg ml–1) in phosphate-buffered saline. In vitro autoubiquitination assay. We carried out in vitro autoubiquitination assays as described14. Briefly, the reaction mixture contained E1 (100–300 nM, Calbiochem), E2 (recombinant His-Ubc4, 1–5 µM), 32P-GST-Ub (6–10 µM), GST-PHF9 fusion proteins (1–2 µg), ATP (1–2 mM), Tris-HCl (50 mM, pH 7.5), MgCl2 (2.5 mM) and dithiothreitol (0.5 mM). After a 90-min incubation at room temperature, we stopped reactions by adding 4× SDS-gel loading buffer. We resolved the samples on 8–16% Tris-glycine SDS-gel (Invitrogen) and analyzed them either by autoradiography or by staining with Coomassie. siRNA experiment. We generated a 19-nucleotide double-stranded siRNA oligo against PHF9 using nucleotides GACAAGAGCTGTATGCACT (Dharmacon Research). We transfected HeLa cells with the PHF9 siRNA oligo or a scrambled control oligo using Oligofectamine following manufacturer’s instructions (Invitrogen). After 48 h, we seeded the cells either on chamber slides (for microscopic analysis) or in tissue culture flasks and incubated them for another 24 h with or without MMC (40 ng ml–1). We then collected the cells by trypsinization and processed them for immunoblotting or metaphase spreads. We evaluated metaphases for chromatid-type aberrations essentially as described28. Sequencing and mutation analysis. We generated four overlapping PHF9 cDNA fragments of 250–350 bp from total RNA by RT–PCR. We identified the mutation causing skipping of exon 11 in individual EUFA868 by PCR amplification of exon 11 from genomic DNA using a primer pair 40–60 bp away from the exonintron boundary. All primer pairs contained T7 and SP6 promoter sequences. We then sequenced fragments with universal CY5.5-labeled T7 and SP6 primers using a Thermo Sequenase primer cycle sequencing kit (Amersham). We analyzed products on a Visible Genetics automatic DNA sequencer. Immunofluorescence. We carried out immunofluorescence analysis as described29. We captured images with the Applied Precision “Image Restoration System” with a 40× oil lens, a Nikon TE200 inverted fluorescent microscope with standard filter sets, halogen illumination with API light homogenizer, a CH350L Camera (500 kHz, 12-bit, 2Mp, KAF 1400 GL, 1317 × 1035, liquid cooled) and DeltaVision software. Deconvolution using the iterative constrained algorithm of Sedat and Agard and additional image processing was done on an SGI Octane workstation. Quantification of foci was computerized using ImageJ (US National Institutes of Health). We counted at least 80 cells in 6–10 different fields for each experiment. GenBank accession numbers. Human PHF9, XP_002688.3; mouse ortholog, NP_080199; Drosophila melanogaster ortholog, AAF54486.1; Anopheles gambiae ortholog, EAA04802. Note: Supplementary information is available on the Nature Genetics website. ACKNOWLEDGMENTS We thank K. J. Patel for FANCE antibody and communicating unpublished data, M. Grompe for FANCD2 cDNA, Z. Lu and T. Hunter for MEKK1, Y. Qiong for ubiquitin vectors, J. Chen for BRCA1 cells, Y. Yang for ubiquitin reagents, N.

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Sherman for mass spectrometry identification, J. Qin and L. Li for advice, D. Schlessinger for critical reading of the manuscript and the National Cell Culture Center for providing cells. W.W. has received funding from the Ellison Medical Foundation and Rett Syndrome Research Foundation. This work was also supported by the Dutch Cancer Society (A.L.M. and H.J.V.), the Netherlands Organization for Health Research and Development (J.P.W. and Q.W.) and the US National Institutes of Health (W.W. and M.E.H.). COMPETING INTERESTS STATEMENT The authors declare that they have no competing financial interests. Received 1 May; accepted 25 August 2003 Published online at http://www.nature.com/naturegenetics/ 1. Joenje, H. & Patel, K.J. The emerging genetic and molecular basis of Fanconi anaemia. Nat. Rev. Genet. 2, 446–459 (2001). 2. D’Andrea, A.D. & Grompe, M. The Fanconi anaemia/BRCA pathway. Nat. Rev. Cancer 3, 23–34 (2003). 3. Garcia-Higuera, I. et al. Interaction of the Fanconi anemia proteins and BRCA1 in a common pathway. Mol. Cell 7, 249–262 (2001). 4. Meetei, A.R. et al. A multiprotein nuclear complex connects Fanconi anemia and bloom syndrome. Mol. Cell. Biol. 23, 3417–3426 (2003). 5. Garcia-Higuera, I., Kuang, Y., Naf, D., Wasik, J. & D’Andrea, A.D. Fanconi anemia proteins FANCA, FANCC, and FANCG/XRCC9 interact in a functional nuclear complex. Mol. Cell. Biol. 19, 4866–4873 (1999). 6. de Winter, J.P. et al. The Fanconi anemia protein FANCF forms a nuclear complex with FANCA, FANCC and FANCG. Hum. Mol. Genet. 9, 2665–2674 (2000). 7. Medhurst, A.L., Huber, P.A., Waisfisz, Q., de Winter, J.P. & Mathew, C.G. Direct interactions of the five known Fanconi anaemia proteins suggest a common functional pathway. Hum. Mol. Genet. 10, 423–429 (2001). 8. Yamashita, T. et al. The fanconi anemia pathway requires FAA phosphorylation and FAA/FAC nuclear accumulation. Proc. Natl. Acad. Sci. USA 95, 13085–13090 (1998). 9. Pace, P. et al. FANCE: the link between Fanconi anaemia complex assembly and activity. EMBO J. 21, 3414–3423 (2002). 10. Taniguchi, T. & D’Andrea, A.D. The Fanconi anemia protein, FANCE, promotes the nuclear accumulation of FANCC. Blood 100, 2457–2462 (2002). 11. Smith, T.F., Gaitatzes, C., Saxena, K. & Neer, E.J. The WD repeat: a common architecture for diverse functions. Trends Biochem. Sci. 24, 181–185 (1999). 12. Coscoy, L., Sanchez, D.J. & Ganem, D. A novel class of herpes virus-encoded membrane-bound E3 ubiquitin ligases regulates endocytosis of proteins involved in immune recognition. J. Cell. Biol. 155, 1265–1273 (2001). 13. Hewitt, E.W. et al. Ubiquitylation of MHC class I by the K3 viral protein signals internalization and TSG101-dependent degradation. EMBO J. 21, 2418–2429 (2002). 14. Lu, Z., Xu, S., Joazeiro, C., Cobb, M.H. & Hunter, T. The PHD domain of MEKK1 acts as an E3 ubiquitin ligase and mediates ubiquitination and degradation of ERK1/2. Mol. Cell 9, 945–956 (2002). 15. Coscoy, L. & Ganem, D. PHD domains and E3 ubiquitin ligases: viruses make the connection. Trends Cell. Biol. 13, 7–12 (2003). 16. Agoulnik, A.I. et al. A novel gene, Pog, is necessary for primordial germ cell proliferation in the mouse and underlies the germ cell deficient mutation, gcd. Hum. Mol. Genet. 11, 3047–3053 (2002). 17. Chen, M. et al. Inactivation of Fac in mice produces inducible chromosomal instability and reduced fertility reminiscent of Fanconi anaemia. Nat. Genet. 12, 448–451 (1996). 18. Whitney, M.A. et al. Germ cell defects and hematopoietic hypersensitivity to gammainterferon in mice with a targeted disruption of the Fanconi anemia C gene. Blood 88, 49–58 (1996). 19. Cheng, N.C. et al. Mice with a targeted disruption of the Fanconi anemia homolog Fanca. Hum. Mol. Genet. 9, 1805–1811 (2000). 20. Nadler, J.J. & Braun, R.E. Fanconi anemia complementation group C is required for proliferation of murine primordial germ cells. Genesis 27, 117–123 (2000). 21. Yang, Y. et al. Targeted disruption of the murine Fanconi anemia gene, Fancg/Xrcc9. Blood 98, 3435–3440 (2001). 22. Koomen, M. et al. Reduced fertility and hypersensitivity to mitomycin C characterize Fancg/Xrcc9 null mice. Hum. Mol. Genet. 11, 273–281 (2002). 23. Vandenberg, C.J. et al. BRCA1-independent ubiquitination of FANCD2. Mol. Cell 12, 247–254 (2003). 24. Brunn, D. et al. siRNA depletion of BRCA1, but not BRCA2, causes increased genome instability in Fanconi anemia cells. DNA Repair advance online publication, 16 July 2003 (doi:10.1016/S1568-7864(03)00112-5). 25. Joenje, H. et al. Classification of Fanconi anemia patients by complementation analysis: evidence for a fifth genetic subtype. Blood 86, 2156–2160 (1995). 26. Dignam, J.D., Martin, P.L., Shastry, B.S. & Roeder, R.G. Eukaryotic gene transcription with purified components. Methods Enzymol. 101, 582–598 (1983). 27. Waisfisz, Q. et al. A physical complex of the Fanconi anemia proteins FANCG/XRCC9 and FANCA. Proc. Natl. Acad. Sci. USA 96, 10320–10325 (1999). 28. Joenje, H., Arwert, F., Eriksson, A.W., de Koning, H. & Oostra, A.B. Oxygen-dependence of chromosomal aberrations in Fanconi’s anaemia. Nature 290, 142–143 (1981). 29. Hoatlin, M.E. et al. The Fanconi anemia group C gene product is located in both the nucleus and cytoplasm of human cells. Blood 91, 1418–1425 (1998). 30. Aravind L., I., L.M., Koonin, E.V. Scores of rings but no PHDs in ubiquitin signaling. Cell Cycle 2, 123–126 (2003).
