Fanconi Anemia (FA) Binding Protein FAAP20 Stabilizes FA Complementation Group a (FANCA) and Participates in Interstrand Cross-Link Repair

Fanconi anemia (FA) binding protein FAAP20 stabilizes FA complementation group A (FANCA) and participates in interstrand cross-link repair

Justin Wai Chung Leunga, Yucai Wanga, Ka Wing Fonga, Michael Shing Yan Huenb, Lei Lia, and Junjie Chena,1

aDepartment of Experimental Radiation Oncology, University of Texas MD Anderson Cancer Center, Houston, TX 77030; and bGenome Stability Research Laboratory, Department of Anatomy, University of Hong Kong, Hong Kong Special Administrative Region, China

Edited* by Stephen J. Elledge, Harvard Medical School, Boston, MA, and approved February 6, 2012 (received for review November 13, 2011)

The Fanconi anemia (FA) pathway participates in interstrand cross- proliferating cell nuclear antigen (PCNA) monoubiquitination link (ICL) repair and the maintenance of genomic stability. The FA (26). It remains to be resolved how PCNA monoubiquitination is core complex consists of eight FA proteins and two Fanconi anemia- linked with the activation of the FA pathway. FANCA ∼ associated proteins (FAAP24 and FAAP100). The FA core complex Among all of the FA genes, mutations in ( 60%), FANCC (∼14%), and FANCG (∼10%) account for over 80% of has ubiquitin ligase activity responsible for monoubiquitination of fi the FANCI-FANCD2 (ID) complex, which in turn initiates a cascade of the mutations identi ed in patients (27). However, FANCA, biochemical events that allow processing and removal of cross- FANCC, and FANCG are orphan proteins that do not share extensive sequence homology with other proteins. Thus, it is still linked DNA and thereby promotes cell survival following DNA fi unknown how these proteins function in the FA pathway. We damage. Here, we report the identi cation of a unique component reason that the identification of new FA-associated proteins may of the FA core complex, namely, FAAP20, which contains a RAD18- fi help us understand how these orphan proteins participate in like ubiquitin-binding zinc- nger domain. Our data suggest that DNA repair. In this study, we report the identification of C1orf86 FAAP20 promotes the functional integrity of the FA core complex isoform2 as a previously undescribed FANCA-interacting protein via its direct interaction with the FA gene product, FANCA. Indeed, (Fanconi anemia-associated protein 20 kDa, hereafter referred as somatic knockout cells devoid of FAAP20 displayed the hallmarks FAAP20). Genetic inactivation of FAAP20 revealed many fea- of FA cells, including hypersensitivity to DNA cross-linking agents, tures of FA cells, highlighting that FAAP20 is a key component of chromosome aberrations, and reduced FANCD2 monoubiquitina- the FA core complex and participates in ICL repair. tion. Taking these data together, our study indicates that FAAP20 is an important player involved in the FA pathway. Results FAAP20 Is a Unique Component of the FA Core Complex. We per- mitomycin C | DNA repair | foci formed tandem affinity purification (TAP) using FANCA as bait to identify FANCA-associated proteins. After excluding general con- taminants, such as heat-shock proteins and ribosomal proteins, we anconi anemia (FA) is a rare recessive genetic disorder char- fi A Facterized by bone marrow failure, congenital developmental identi ed FAAP20 as a potential FANCA-binding partner (Fig. 1 ). defects, and cancer predisposition (1–4). Cellular features of FA FAAP20 (LOC1999990 isoform 2) encodes a 20-kDa protein with unknown function (gene name: C1orf86 isoform 2; accession number: include chromosomal instability and hypersensitivity to cross- fi linking agents (5). Fifteen FA complementation genes have been NP_872339.2). To con rm its association with FANCA, we per- fi formed a reverse purification using FAAP20 as bait and showed that identi ed so far. These genes form several complexes to orches- fi A trate interstrand cross-linking (ICL) repair. The FA core complex FANCA and FANCG copuri ed with FAAP20 (Fig. 1 ), indicating is composed of eight of the FA gene products (FANCA, FANCB, that FAAP20 is a potential component of the FA core complex. FANCC, FANCE, FANCF, FANCG, FANCL, and FANCM), in addition to FAAP24 and FAAP100 (6, 7), and acts as an E3 ligase FAAP20 Binds Directly to FANCA. To verify that FAAP20 interacts to ubiquitinate FANCI/FANCD2 (I/D2) complex (8–11). The with FANCA, we coexpressed triple-tagged (SFB-tag: S-protein tag, monoubiquitinated FANCI/FANCD2 complex interacts with FLAG epitope, tag and streptavidin-binding peptide tag) FANCA Fanconi anemia-associated nuclease 1 (FAN1), which has exo- with Myc-tagged FAAP20 or FANCG. As expected, we observed nuclease and endonuclease activity that may unhook the ICL, a robust binding of SFB-tagged FANCA with Myc-tagged FANCG. In addition, we found a strong interaction between FANCA and facilitate translesion synthesis, and promote downstream homol- B ogous recombination (HR) repair (12–15). FAAP20 (Fig. 1 ), suggesting that they exist in the same complex. Besides the FA core complex and FANCI/FANCD2, there are Moreover, we showed that endogenous FANCA coimmunopreci- pitated with endogenous FAAP20 and this interaction occurs in- several other FA proteins that likely act downstream of FANCI/ C FANCD2 and participate in HR repair. These proteins include dependently of mitomycin C (MMC) treatment (Fig. 1 ). We generated a series of internal deletion mutants of FANCA BRCA2 (FANCD1) and PALB2 (FANCN), both of which are D essential for HR repair (16–18). Another downstream FA pro- (FANCA-D1 to FANCA-D5) (Fig. 1 ) and observed that two of fi them, FANCA-D3 and FANCA-D4, failed to interact with tein, BACH1 (FANCJ), is also a bona de double-strand break E repair factor (19). BRCA2/FANCD1, PALB2/FANCN, and FAAP20 (Fig. 1 ), indicating that FAPP20 binds to the middle BACH1/FANCJ are all recruited to ICL sites (20), indicating that region of FANCA. In addition, we performed a pull-down assay they are directly involved in ICL repair. More recently, mutations using bacterial expressed GST-fused FAAP20 and maltose- in two other genes, RAD51C/FANCO and SLX4/FANCP, were CELL BIOLOGY identified in patients with FA phenotypes (21–23), suggesting that there may be additional FA genes responsible for this disease. Author contributions: J.W.C.L., M.S.Y.H., L.L., and J.C. designed research; J.W.C.L., Y.W., Not all FA proteins function in a linear pathway involved in ICL M.S.Y.H., and K.W.F. performed research; J.W.C.L., Y.W., K.W.F., M.S.Y.H., L.L., and J.C. repair. Many of the downstream FA proteins are involved in HR analyzed data; and J.W.C.L., Y.W., K.W.F., M.S.Y.H., L.L., and J.C. wrote the paper. repair and associated with breast cancer susceptibility (16–19, 24, The authors declare no conflict of interest. 25). These proteins all have functions besides ICL repair. More *This Direct Submission article had a prearranged editor. recently, another DNA repair protein, RAD18, which is best known 1To whom correspondence should be addressed. E-mail: [email protected]. for its role in UV lesion bypass, has also been shown to participate This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. in the activation of the FA pathway via its ability to promote 1073/pnas.1118720109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1118720109 PNAS | March 20, 2012 | vol. 109 | no. 12 | 4491–4496 Downloaded by guest on October 2, 2021 AB SFB-FANCA Myc-FANCG Myc-FAAP20 WB: Flag (FANCA) FANCG

WB: Myc

FAAP20 Fig. 1. FAAP20 is a FANCA-binding protein. (A) 293T cells 2% Input Pull down: stably expressed SFB-FANCA or SFB-FAAP20, respectively, Streptividine were subjected to TAP and mass spectrometry analysis. Red beads indicates the bait protein and blue indicates the known or putative-associated proteins. Number of peptides recovered from mass spectrometry analysis is also presented. (B) 293T C AAP20 cells were transfected with constructs encoding SFB-FANCA FANCA 2% Input IP:IgG IP:F along with constructs encoding Myc-FANCG or Myc-FAAP20. D FANCA MMC FANCA 11-673) WB: FANCA Coprecipitation and immunoblotting were carried out as FANCA indicated. (C) Lysates prepared from control or MMC-treated FANCA WB: FAAP20 293T cells were incubated with protein A agarose beads FANCA conjugated with rabbit IgG or anti-FAAP20 antibodies.

02P Western blotting was performed using indicated antibodies. Pulldown

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1D- 2D-ACNA 3 4D 5D-ACNA

1D-ACNAF 2 3D-AC 4D-

5D of FANCA used in this study. (E) 293T cells were transfected

% T TSG

D-ACNAF

D-AC

-ACNAF

- SG

ACN AC

ACNAF

A with constructs encoding SFB-FAAP20 along with constructs 2

CNAF

NAF

NAF NA WB: MBP (FANCG) encoding Myc-tagged wild-type or deletion mutants of

F F WB: MBP FAAP20. Precipitation and immunoblotting were conducted WB:Flag (FANCA 674-1208) (FAAP20) as indicated. (F) Pull-down assays were performed using bacterially expressed and puriﬁed GST-FAAP20 and MBP- WB:Myc Coomassie (FANCA) Staining fused FANCA (residues 674–1032) or FANCG. Immunoblot- ting were conducted using anti-MBP antibody.

binding protein (MBP)-fused FANCA (aa674-1208) or MBP-fused FANCA (Fig. 2D), suggesting that this N-terminal motif within FANCG. GST-FAAP20 pulled down MBP-fused FANCA (resi- FAAP20 is necessary for FAAP20-FANCA interaction. dues 674–1208), but not MBP-fused FANCG (Fig. 1F), suggesting that FAAP20 binds directly to FANCA, but not to FANCG. FAAP20 Contains an Evolutionarily Conserved RAD18-like Ubiquitin- We then used a series of FAAP20 internal-deletion mutants to Binding Zinc-Finger Domain. We noticed that FAAP20 contains an map a FANCA-binding region on FAAP20 (Fig. 2A). Although evolutionarily conserved ubiquitin-binding zinc-finger (UBZ) 4-type wild-type FAAP20 coprecipitated with FANCA, two N-terminal domain that belongs to the RAD18 family of zinc-finger (ZNF) deletion mutants of FAAP20 (FAAP20-D1 and FAAP20-D2) domains (Fig. 3A). This domain is also present in other DNA failed to do so (Fig. 2B). To further narrow down the residues on damage-repair proteins, such as FAN1, RAD18, and SLX4/ FAAP20 that are responsible for FAAP20-FANCA interaction, FANCP, which are involved in the FA pathway (Fig. 3B). Prompted we aligned the human FAAP20 sequence with those of FAAP20 by the ability of these zinc-finger domains in recognizing ubiquitin, from other species and noted three highly conserved motifs at we examined whether FAAP20 would also bind to ubiquitin the N terminus of FAAP20 (residues 40–45, residues 76–81, and through its UBZ domain. Indeed, GST-FAAP20 was able to pull residues 83–87). Thus, we constructed three alanine substitution down endogenous ubiquitin chains, whereas GST alone or the GST- mutants, 6A1 (40WAELLR/AAAAAA45), 6A2 (76EVFTVG/ FAAP20 C147/150A mutant failed to do so (Fig. 3C). FAAP20 AAAAAA81), and 6A3 (83 KTFSWT/AAAAAA87), to disrupt interacted with both K48- and K63-linked ubiquitin chains without each of these conserved motifs (Fig. 2C), respectively, and ex- any notable preference (Fig. 3D). Given that monoubiquitinated amined their ability to interact with FANCA. The 6A1 mutant FANCD2 is important for the function of the FA pathway (9), we completely abolished the interaction between FAAP20 and tested but did not observe any appreciable binding of FAAP20 with

A UBZ BD SFB-FANCA SFB-FANCA

6A1 WB: C Myc WT FAAP20 FAAP20-D1 FAAP20-D2 FAAP20-D3 FAAP20-D4

Homo Sapien 35 60 Flag WT FAAP20 FAAP20-6A1 FAAP20-6A2 FAAP20-6A3 WB: Rattus norvegicus 35 64 (FANCA) Flag (FANCA)

Mus musculus 25 53 2% Myc

Input 2%

Canis familiaris 46 71 (FAAP20) Input Myc (FAAP20) 6A2 6A3 Flag (FANCA) Flag (FANCA)

61 90 own: bead 65 96 bead Myc Myc (FAAP20)

(FAAP20) down: Pull

54 86 d Pull

Streptavidine Streptavidine 72 104

Fig. 2. A conserved N-terminal region of FAAP20 is required for its interaction with FANCA. (A) Illustration of deletion mutants of FAAP20 used in this study. (B and D) 293T cells were transfected with plasmids encoding SFB-FANCA along with those encoding Myc-tagged wild-type or mutants of FAAP20. Pre- cipitation was conducted using streptavidin beads and immunoblotting was performed using anti-Flag or anti-Myc antibodies as indicated. (C) Alignment of the N terminus of FAAP20 from different species. The conserved amino acids are shaded in black. Three mutants of FAAP20 with six alanine substitutions were generated (6A1, 6A2, and 6A3).

4492 | www.pnas.org/cgi/doi/10.1073/pnas.1118720109 Leung et al. Downloaded by guest on October 2, 2021 Fig. 3. FAAP20 contains a RAD18-like A Homo Sapien 140 173 C Pull down UBZ domain at its C terminus. (A) Mus Musculus 136 169 Gallus Gallus 149 182 Alignment of the RAD18-like UBZ do- Tetraodon Nigroviridis 108 142 main of FAAP20 from different species. ** Identical residues are shaded in black. C1orf86 isoform 2 2% Input GST C147/150A GST-FAAP20 GST-FAAP20 (B) Alignment of FAAP20 UBZ domain B RAD18 with other RAD18-like UBZ domains. FAN1 SLX4 The conserved Cys and His residues that deﬁnethetwodyadsofthe WRNIP1 ubiquitin-binding ZNF domain are C1orf124 ZBTB1 WB:Ub shaded in red. The asterisks denote the conserved Cys147 and Cys150 residues mutated in the the FAAP20 C147/150A D K48 K63 mutant. (C) FAAP20 binds to ubiquitin via its RAD18-like UBZ domain. GST Chromatin fr. Chromatin fr. E -MMC +MMC pull-down experiments were carried Coomassie

Staining ZBU- out using 293T lysates and GST, GST- Z 1% Input GST-FAAP20 GST

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M F ubiquitin chains and GST-fusion pro- C WB:GST Coomassie teins, as indicated. (E) Chromatin (Ub-GST) Staining lysates were prepared from control or Coomassie MMC-treated cells. In vitro pull-down WB:GST Staining experiments were performed using indicated GST-fusion proteins and Im- munoblotting was conducted using anti-FANCD2 antibody. (F) FAAP20 binds to monoubiquitin. In vitro pull-down assays were performed using ub-GST and immobilized MBP-fusion proteins, as indicated.

monoubiquitinated FANCD2 in vitro, whereas the FAN1-UBZ The FA core complex acts as an ubiquitin ligase that mono- (residues 1–100) domain was able to do so (Fig. 3E). We also ex- ubiquitinates the FANCI/FANCD2 complex during S phase and amined whether or not FAAP20 would bind to monoubiquitin. In also upon exposure to cross-linking agents. Although only FANCL this regard, we performed an in vitro pull-down assay using bacte- has intrinsic E3 ligase activity, depletion of any component of the FA rial-expressed carboxyl terminal-tagged ubiquitin (ub-GST). We core complex compromises FANCI/FAND2 monoubiquitination observed that MBP-FAAP20 and MBP-FAN1-UBZ interacted (1, 8, 28). Thus, monoubiquitination of FANCD2 has been used as with ub-GST, but MBP or MBP-FAAP20-C147/150A did not (Fig. a surrogate marker for the integrity or the activation of the FA 3F). Thus, although FAAP20 contains a ubiquitin-binding domain, pathway. Because FAAP20 is a FANCA-associated protein, we the physiological partners of this UBZ domain remain unknown. reasoned that FAAP20-deficient cells might display defect in FANCD2 ubiquitination. Western blotting analysis revealed the fi FAAP20 Is Required for ICL Repair. To further substantiate the role presence of both unmodi ed and monoubiquitinated forms of of FAAP20 in the FA pathway, we used the eChIP assay with a FANCD2 in parental HCT116 cells, and an increase of mono- fi ubiquitinated FANCI and FANCD2 after MMC treatment (Fig. de ned ICL (20) to test whether FAAP20 localize to ICL sites C (Fig. 4A). Indeed, we observed a fourfold enrichment of FAAP20 4 ). In contrast, ubiquitination of FANCI and FANCD2 was A largely abrogated in FAAP20- and FANCL-deficient cells (Fig. at cross-linked substrates (Fig. 4 ), suggesting that FAAP20 is C recruited to ICLs in vivo. As a positive control, FANCD2 was also 4 ), suggesting that like FANCL, FAAP20 is also a component of enriched at ICL substrates. the FA core complex and contributes to the activation of the FA pathway following MMC treatment. Interestingly, we observed a To study the function of FAAP20 in the FA pathway and ICL fi repair, we transiently knocked down FAAP20 in U2OS using siRNA reduction in FANCA protein level in FAAP20-de cient cells (Fig. 4C), indicating that as a FANCA-binding protein, FAAP20 and examined FANCD2 monoubiquitnation, as well as FANCD2 A may stabilize FANCA in the cell. In addition to reduced foci formation following MMC treatment (Fig. S1 ). U2OS cells with FANCD2 monoubiquitnation, FAAP20-deficient cells also dis- FAAP20 down-regulation showed a reduction of FANCD2 mono- played defective FANCD2 foci formation (Fig. 4 D and E). ubiquitination and FANCD2 foci formation (Fig. S1). However, Moreover, increased G2/M accumulation and genomic instability, monoubiquitnation of FANCD2 was not absent in FAAP20 knock- including radial chromosome formation and chromosome breaks, down cells. This finding could be because of incomplete knockdown fi are also the hallmarks of FA cells (29). As a matter of fact, we of FAAP20 by siRNA. Alternatively, this nding may suggest that observed all of these phenotypes in FAAP20-deficient cells (Fig. 4 FAAP20 is not as important as other FA core components. G and H). These data further validated that FAAP20 is a com- To test whether FAAP20 is essential for the activation of the FA ponent of the FA pathway. pathway, we decided to generate a FAAP20-deficient cell line in fi HCT116 colon carcinomas and a control FANCL-de cient HCT116- Binding to FANCA Is Required for FAAP20 Function in the FA Pathway. derivative cell line. After targeting both alleles with virus vectors We reconstituted FAAP20-deficient cells with wild-type FAAP20 containing homology arms of exon 2 and 3 of FAAP20 and exon 2 and observed that the FANCA level, MMC hypersensivity, and CELL BIOLOGY and 3 of FANCL, we screened for clones with the correctly targeted MMC-induced FANCD2 monoubiquitination and foci formation alleles by PCR analysis (Fig. S2)andconfirmed the absence of were all restored (Fig. 5). These data strongly suggest that the FAAP20 or FANCL in these cell lines by Western blotting (Fig. 4C). defects observed in FAAP20-deficient cells are a result of the loss Because the FA pathway is important for ICL repair, de- of FAAP20 in these cells. pletion of any of the FA proteins leads to hypersensitivity to To understand whether the FANCA-binding activity and the DNA cross-linking agents. As expected, we observed that both UBZ domain of FAAP20 are important for its function in ICL FAAP20- and FANCL-deficient cells exhibited enhanced sensi- repair, we also reconstituted the FAAP20-deficient cells with tivity to MMC. It is worth noting that FAAP20-deficient cells FAAP20-6A1 and FAAP20-C147/150A mutants, respectively. As showed lower sensitivity than FANCL-deficient cells (Fig. 4B). expected, FAAP20-6A1 could not restore FANCA level and thus

Leung et al. PNAS | March 20, 2012 | vol. 109 | no. 12 | 4493 Downloaded by guest on October 2, 2021 CTL C A Substrate B EBV OriP eCHIP PCR primer HCT116 FAAP20 FANCL XL Wildtype -/- -/-

% %

Substrate lavivr ICL MMC - + - + - + WB FAAP20 FANCD2 FANCD2 (L)

u FANCD2

lleC FANCD2 (S) FANCI (L) FANCI FANCI (S) FANCA (% of input) (% of input) FANCL Enrichment at ICL Enrichment at ICL Enrichment at ICL Enrichment at ICL FAAP20 CTL XL CTL XL MMC (nM) α-Tubulin

-MMC +MMC D DAPI FANCD2 DAPI FANCD2 E

% icof 5> 5> icof %

HCT116 H -MMC +MMC

htiw G1-61.9% G1-45.5% S-11.4% S-17.5%

G2/M-26.7% G2/M-37%

C FAAP20-/- HCT116

G1-56.1% G1-39.5% HCT116 FAAP20 FANCL S-10.9% S-11.5% -/- -/- G2/M-33% G2/M-49% FANCL-/- G FAAP20-/- HCT116 FAAP20-/- F G1-54.7% G1-27.9% S-9.1% S-8.7% G2/M-36.2% G2/M-63.4% FANCL-/- per 50 cells number of aberration HCT116 HCT116 FANCL-/- FANCL-/- FAAP20-/- FAAP20-/- -MMC +MMC

Fig. 4. FAAP20 deficiency sensitizes cells to ICL damage and leads to genomic instability. (A) Schematic representation of the plasmid substrates used in the eChIP assay. The presence of psoralen-ICL and PCR primer locations are indicated. Relative enrichment of FAAP20 at ICLs was calculated by normalizing com- parative concentration from real-time PCR of each sample against that of its input. Error bars represent SD from three independent experiments. CTL, control; XL, cross-linked. (B) Clonogenic survival assay of HCT116 cells, FANCL-deficient cells, and FAAP20-deficient cells following MMC treatment. (C) Whole-cell extracts were prepared from HCT116 cells, FAAP20-deficient cells, or FANCL-deficient cells mock-treated or treated with MMC for 24 h. Western blotting was conducted using indicated antibodies. (D) HCT116 cells, FAAP20-deficient cells, and FANCL-deficient cells were mock-treated or treated with 1 μM MMC for 24 h. Immu- nostaining was performed using anti-FANCD2 antibody and cells were counterstained with DAPI, as indicated. (Magnification: 100×.) (E) Quantification results were the average of two independent experiments and were presented as mean ± SEM. More than 300 cells were counted in each experiment. (F) HCT116 cells or FAAP20-deficient cells were exposed to a low dose of MMC and then treated with colcemid. A representative micrograph shows radial chromosome formation and chromosome breaks marked by arrows that were observed in FAAP20-deficient cells. (Magnification: 100×.) (G) Quantification of chromosome aberration were the average of two independent experiments using wild-type, FAAP20−/− cells, and FANCL−/− cells. The data were presented as mean ± SEM. (H) HCT116 cells or FAAP20-deficient cells were mock-treated or treated with 50 nM MMC. Cell-cycle distributions were analyzed by FACS and presented as percentages of cells in G1, S, or G2/M phases.

this mutant of FAAP20 could not rescue MMC sensitivity or it is known that the loss of any component in the FA core complex MMC-induced FANCD2 monoubiquitination and foci formation would lead to similar defects in FANCD2 ubiquitination and MMC in FAAP20-deficient cells (Fig. 5). On the other hand, the C147/ sensitivity, it is not yet clear how these subcomplexes function to- 150A mutant of FAAP20 rescued all of the above defects ob- gether. An early study indicates that FANCA and FANCG stabilize served in FAAP20-deficient cells (Fig. 5). Taken together, these each other and promote the nuclear localization of the FA core data indicate that FANCA-binding, but not its UBZ domain, is complex (34). This finding is similar to the situation in the present critical for FAAP20 function in ICL repair. study. FANCA and FANCG consistently copurified with FAAP20 (Fig. 1A), suggesting that these three components likely form Discussion a stable subcomplex. The destabilization of FANCA in FAAP20- In this study, we identified FAAP20 as a unique component of deficient cells could, in part, contribute to the defects observed in the FA core complex. FAAP20 binds directly to FANCA and thesecells.ItisnoteworthytopointoutthatunlikeotherFAcells, stabilizes FANCA in the cell. The FANCA binding is essential residual FANCD2 monoubiquitination was detectable in FAAP20- for its function in the FA pathway, which is at least one mech- deficient cells. This phenotype is similar to that observed in Δ Δ anism of how it functions in the FA core complex. FANCM 2/ 2 mouse embyrionic fibroblasts (35). The significance The FA core complex comprises several subcomplexes, including of this observation is currently unclear. FANCL-FANCB-FAAP100 (4), FANCC-FANCE-FANCF (30), FAAP20 contains a RAD18-like ZNF domain that binds to FANCA-FANCG (31, 32), and FANCM-FAAP24 (33). Although ubiquitin. At least three RAD18 UBZ domain-containing proteins,

4494 | www.pnas.org/cgi/doi/10.1073/pnas.1118720109 Leung et al. Downloaded by guest on October 2, 2021 FAAP20-/- AB

6A1 Wildtype +FAAP20 +FAAP20 +FAAP20 HCT116 FAAP20-/- C147/150A WB MMC - + - + - + - + - + FANCD2 (L) FANCD2 FANCD2 (S)

FANCA

Cell survival % α-Tubulin FAAP20-/- SFB-FAAP20

FAAP20 Endogenous MMC (nM) FAAP20

C -MMC +MMC D DAPI FLAG FANCD2 DAPI FLAG FANCD2 Wildtype +FAAP20 6A1 FAAP20-/- +FAAP20

Cell with >5 foci %

+wildtype +6A1 +C147/150A FAAP20-/- +FAAP20 C147/150A

Fig. 5. FANCA-binding is required for FAAP20 function in the FA pathway. (A) Clonogenic survival assay of HCT116 cells, FAAP20-deficient cells, and FAAP20- deficient cells reconstituted with wild-type FAAP20, the 6A1, or the C147/150A mutant of FAAP20. (B) Whole-cell extracts were prepared from HCT116 cells, FAAP20- deficient cells, and FAAP20-deficient cells complemented with indicated constructs with or without MMC treatment. Western blotting was conducted using indicated antibodies. (C)FAAP20-deficient cells reconstituted with indicated SFB-tagged wild-type or mutant FAAP20 were mock-treated or treated with MMC for 24 h. Immunostaining was performed using anti-Flag and anti-FANCD2 antibodies. Cells were counterstained with DAPI as indicated. (Magnification: 100×.) (D)Quan- tification results were the average of two independent experiments and were presented as mean ± SEM. More than 100 cells were counted in each experiment.

RAD18, FAN1, and SLX4/FANCP, are known to play critical roles expression in mammalian cells, and GST or MBP tag for expression in bacteria, in the FA pathway (12–15, 21, 22, 26, 36, 37). In particular, the respectively. UBZ domain of FAN1 binds directly to monoubiquitinated FANCD2 and thus recruits FAN1 to ICL sites to carry out its TAP of Protein Complexes. TAP was performed as previously described (38). function in ICL repair (12–15). However, in the case of FAAP20, Briefly, 293T cells were transfected with constructs encoding SFB-tagged we showed that the UBZ domain of FAAP20 is not critical for FANCA or FAAP20 and selected with media containing puromycin (2 μg/mL). FANCD2 monoubiquitination and MMC sensitivity. Further study Cell lines stably expressing these tagged proteins were confirmed by West- fl is required to elucidate the cellular function of this highly con- ern blotting and immuno uorescence staining. For TAP, cells were lysed in served UBZ domain of FAAP20. NETN buffer (20 mM Tris-HCl, pH 8, 100 mM NaCl, 1 mM EDTA, 0.5% Non- idet P-40, 1 mM MgCl2)for20minin48C. The crude lysates were cleared by In conclusion, our data suggest that FAAP20 interacts with × 8 FANCA and participates in the regulation of the FA pathway. It centrifugation at 18,407 g (Eppendorf 5424, Hamburg, Germany) at 4 C is likely that via stabilizing FANCA, FAAP20 modulates the for 30 min and rocked with streptavidin-conjugated beads (Amersham) for 2hat48C. The immunocomplexes were washed with NETN three times and ubiquitin ligase activity of the FA core complex, which in turn eluted with 2 mg/mL biotin. The eluent was then incubated with S-protein regulates the FANCI/FANCD2 monoubiquitination following Agarose beads (Novagen) for 2 h at 4 8C. The beads were then washed three DNA damage. Up to now, no patient was found having mutation times. The protein mixtures were eluted and analyzed by the Taplin Mass with several genes encoding FA pathway-related proteins (in- Spectrometry Facility at Harvard Medical School (Boston, MA). cluding FAN1, FAAP100, and FAAP24). Because the cellular phenotypes of FAAP20-deficient cells are rather mild compared fi Antibodies. The primary antibodies used in this study were as follows: poly- with FANCL-de cient cells, it is possible that patients with clonal anti-C1orf86 isoform 2 (FAAP20) antibody (Sigma-Aldrich; HPA038829); FAAP20 mutation would display mild FA phenotypes and thus anti-myc antibody (Santa Cruz Biotechnology; sc-40); anti-FLAG antibody be difficult to diagnose. Knocking out the FAAP20 gene in mice (Sigma-Aldrich; F1804); polyclonal anti-FANCA and anti-FANCI antibodies may provide some clues to the function of this protein in (Bethyl Laboratories; A301-980A and A301-254A); monoclonal anti-FANCD2 CELL BIOLOGY mammals and may help the diagnosis of these patients. antibody (Santa Cruz Biotechnology; sc-20022); polyclonal anti-FANCD2 antibody (Novus Biologicals; NB100-182); polyclonal anti-MBP antibody (Milli- Materials and Methods pore; 05–912); monoclonal anti-Ub antibody (Millipore; 04–263); monoclonal Plasmids. FAAP20, FANCG, FANCA, and FAN1 cDNAs were purchased from anti-GST (Santa Cruz; SC-138); polyclonal anti-FANCL antibodies were a gen- Open Biosystems. The cDNAs were cloned into the pDONR201 vector using erous gift from Weidong Wang (National institute on Aging, National Insti- Gateway cloning technology (Invitrogen). All of the deletions and point mu- tutes of Health, Baltimore, MD). tations were generated by site-directed mutagenesis using standard protocols. The corresponding entry vectors were transferred into a Gateway-compatible Cell Cultures and Transfection. Human embryonic kidney 293T cells and hu- destination vector harboring an N-terminal triple-tag (S-protein, Flag, and a man colorectal cancer HCT116 cells were cultured in RPMI 1640 and DMEM, streptavidin-binding peptide), HA-Flag epitope tag, or a myc epitope tag for respectively, supplemented with (vol/vol) FBS, 100 units/mL penicillin, and

Leung et al. PNAS | March 20, 2012 | vol. 109 | no. 12 | 4495 Downloaded by guest on October 2, 2021 100 μg/mL streptomycin, and maintained in 5% CO2 at 37 8C. Plasmid and primers derived from the neomycin-resistant gene and the upstream region siRNA transfection was performed using Lipofectamine 2000 and oligo- of the left homologous arm or the downstream region of the right homol- fectamineb (Invitrogen), respectively, according to the manufacturer’s ogous arm. After the first allele was targeted, the neomycin-resistant gene instructions. The coding strand for control siRNA was UCCAGUGAAUCCUU- was excised using viruses expressing Cre-recombinase. Second targeting was GAGGUUU and that for FAAP20 siRNA was UCCGAAAGCACAGAA- performed using the same approach. GACGUUU. All siRNA were purchased from Dharmacon. Clonogenic Assay. Cells were seeded at a density of 700 cells onto 6-cm dishes Immunoprecipitation, GST Pull-Down, and Western Blotting Analysis. Cells were in triplicate. Twenty-four hours after seeding, the cells were treated with the lysed in NETN buffer containing protease inhibitors. For immunoprecipitation of indicated concentrations of MMC for 24 h. Cells were then washed free of endogenous protein complexes, cell extracts were incubated with protein-A drugs and incubated in fresh medium for another 10–14 d. The cells were beads and antibody against FAAP20 for 2 h at 4 8C. For precipitation of SFB- then fixed and stained with 0.5% crystal violet in 20% ethanol. Colonies tagged proteins or pull-down experiments, cell extracts were incubated with containing more than 50 cells were counted. either streptavidin beads or GST-fusion proteins immobilized on glutathione 8 beads for 2 h at 4 C. For in vitro binding assay, ub-GST were eluted with glu- MMC-Induced Radial Chromosome Analysis. Cells were plated in 10-cm dishes tathione and then incubated with beads coated with bacterial expressed MBP, and treated with 0.063 μM MMC for 48 h. After treatment, cells were ex- MBP-FAAP20, MBP-FAAP20 C147/150A, or MBP FAN1-1-100. The beads were posed to colcemid for 8 h, swollen using 0.075 M KCl, and fixed with 3:1 × washed with NETN buffer and proteins were eluted by boiling in 1 Laemmli methanol:acetic acid. Slides were stained with Giemsa and 50 metaphase buffer. Samples were resolved by SDS/PAGE, transferred to polyvinylidene spreads were scored for radials in two independent experiments. difluoride membrane, and immunoblotted with antibodies as indicated. eChIP Assay. Control substrates or substrates containing a defined cross-link fl Immuno uorescence Staining. Cells cultured on coverslips were washed in PBS, were introduced into HEK293T cells stably expressing SFB-tagged FAAP20. fi xed in 3% paraformaldehyde for 15 min and then permeabilized in 0.5% ChIP was carried out as previously described (20). triton solution for 5 min at room temperature. Samples were incubated with primary antibodies for 30 min, washed, and incubated with secondary anti- Cell Cycle Analysis. Cells were exposed to 50 nM MMC and were allowed to bodies for 30 min. Samples were then counterstained with DAPI and mounted grow for 24 h. Cells were trypsinized and fixed in 70% ethanol overnight. Cells on the glass slides with an antifade solution and visualized using a Nikon were then washed in PBS, nuclei were stained with propidium iodide (4 μg/mL), Eclipse 90i fluorescence microscope. treated with RNase (2 μg/mL) at room temperature for 30 min, and analyzed in a flowcytometer using FACS Flow Jo software. Somatic Knockout of FAAP20 and FANCL. For the generation of somatic knockout cells, adeno-associated virus-based strategy was used as previously ACKNOWLEDGMENTS. We thank our colleagues in the J.C. laboratory for described (39). The targeting adeno-associated viruses were packaged in 293T μ insightful discussions and technical assistance. This work was supported in cells by transfecting 3 g of the targeting vector, pHelper, and pRC plasmids. part by the Cancer Prevention Research Institute of Texas, Multi-Investigator Viruses were harvested at 72 h after transfection. Human colon cancer cell Award, Grant RP110465-P2 (to J.C.). J.C. is a recipient of Era of Hope Scholar line HCT116 was infected for 48 h and selected with geneticin for 20 d. The Award W81XWH-05-1-0470 from the Department of Defense and a member geneticin-resistant clones were then screened using genomic PCR with of the MD Anderson Cancer Center (CA016672).

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