The Fanconi anemia pathway has a dual functionin Dickkopf-1 transcriptional repressionCaroline C. Huarda, Cédric S. Tremblaya,1, Audrey Magrona, Georges Lévesquea,b, and Madeleine Carreaua,c,2

aCentre Hospitalier Université Laval Research Center, Québec, QC, Canada G1V 4G2 and Departments of bPsychiatry and Neurosciences and cPediatrics,Université Laval, Québec, QC, Canada G1V 0A6

Edited by David Hockenbery, Fred Hutchinson Cancer Research Center, Seattle, WA, and accepted by the Editorial Board December 26, 2013 (received forreview July 26, 2013)

Fanconi anemia (FA) is an inherited bone marrow failure syndromeassociated with a progressive decline in hematopoietic stem cells,developmental defects, and predisposition to cancer. These variousphenotypic features imply a role of FA proteins in molecular eventsregulating cellular homeostasis. Interestingly, we previously foundthat the Fanconi C protein (FANCC) interacts with the C-terminal-binding protein-1 (CtBP1) involved in transcriptional regulation.Here we report that FANCC with CtBP1 forms a complex withβ-catenin, and that β-catenin activation through glycogen syn-thase kinase 3β inhibition leads to FANCC nuclear accumulationand FA pathway activation, as measured by the Fanconi D2 protein(FANCD2) monoubiquitination. β-catenin and FANCC nuclear entryis defective in FA mutant cells and in cells depleted of the FanconiA protein or FANCD2, suggesting that integrity of the FA pathwayis required for FANCC nuclear activity. We also report that FANCCwith CtBP1 acts as a negative regulator of Dickkopf-1 (DKK1) ex-pression, and that a FA disease-causing mutation in FANCC abro-gates this function. Our findings reveal that a defective FApathway leads to up-regulation of DKK1, a molecule involvedin hematopoietic malignancies.

Fanconi anemia (FA) is an inherited bone marrow failure(BMF) syndrome transmitted through both autosomal and

X-linked modes (1, 2). FA is associated with congenital malfor-mations, genome instability, and a predisposition to cancer. Ge-netic and functional complementation approaches have helpeddefine 16 gene products that cooperate in a molecular pathwaytermed the FA pathway (3, 4). This FA pathway is activated inresponse to cellular stress that causes interruptions in the repli-cation or transcription processes (5). Mutations in any of these FAgenes cause clinical features characteristic of FA, with BMF themost prevalent manifestation.BMF in patients with FA results from a progressive decline in

hematopoietic stem cells (HSCs), suggesting that FA proteins playa role in the maintenance of these cells. The mechanism by whichFA proteins or the FA pathway act in protecting these cells remainsunclear, however. Findings from several groups have shown that theFA proteins act in various cellular functions, including apoptosissuppression, cytokine signaling, and transcriptional regulation(6). Indeed, we recently reported that the Fanconi C protein(FANCC) directly interacts with the C-terminal-binding pro-tein 1 (CtBP1) involved in transcriptional regulation (7). CtBP1and its isoforms are involved in many cellular activities, includingGolgi fission and cellular division, but predominantly in tran-scriptional repression (8). Many known DNA-binding tran-scription factors mediate transcriptional repression in a CtBP1-dependent manner, including Wnt/β-catenin/T-cell factor, bonemorphogenic protein 1/transforming growth factor β, and GATAfactors. Analysis of CtBP1 protein complexes have revealed thepresence of histone-modifying factors as well as DNA-bindingtranscription factors, implying that CtBP1 exerts its transcrip-tional regulation effects through recruitment of cofactors (8, 9).Interaction between FANCC and CtBP1 suggests a role of FA

proteins in transcriptional regulation. In fact, we previouslyshowed that depletion of either the CtBP1 or FA proteinsmodulates Wnt pathway genes, of which the Dickkopf-1 gene(DKK1) is the most up-regulated gene (7). DKK1 belongs to the

Dickkopf family of secreted molecules, which antagonize Wntsignaling by sequestering the Wnt receptors low-density lipopro-tein receptor-related protein 5/6 and Kremen (10). DKK1 wasinitially characterized for its role in developmental morphogenesisof the head, eyes, limbs, and vertebrae. The absence of Dkk1in KO mice is associated with many congenital malformationsreminiscent of features of FA, including anophthalmia and pol-ysyndactyly (11–14), whereas DKK1 up-regulation is often foundin cancers, including multiple myeloma, hepatoblastoma, Wilms’tumor, and breast, head, neck, and oral cancers (15). These typesof cancers are seen in patients with FA (16). In addition, up-regulation of DKK1 has been shown to accelerate cell cycling ofHSCs and to progressively diminish the regenerative function ofHSCs after transplantation (17).The fact that FA is associated with congenital abnormalities,

cancers, and HSC defects (18–22) similar to those associated withderegulation of DKK1 prompted us to investigate the functionalrole of FANCC–CtBP1 interaction in DKK1 transcriptional regu-lation. Here we demonstrate that FANCC forms a complex withCtBP1 and β-catenin. Activation of β-catenin through inhibition ofglycogen synthase kinase 3 beta (GSK3β) induces nuclear accu-mulation of FANCC similar to β-catenin, activation of the FApathway measured by the monoubiquitnation of the Fanconi D2protein (FANCD2), and, subsequently, transcriptional regulationof the DKK1 gene. We also demonstrate that FANCC negativelyregulates DKK1 via CtBP1, and that disease-causing mutations inFANCC abrogates this function. Our findings reveal that FANCCis involved in the transcriptional regulation of DKK1.

Resultsβ-Catenin Activation Induces FANCC Nuclear Accumulation. Based onour earlier observations that FANCC interacts with CtBP1 and thatboth proteins are involved in the regulation of the Wnt/β-catenin

Significance

Fanconi anemia (FA) is a devastating disease associated witha progressive bone marrow failure (BMF) and clonal pro-liferation of primitive hematopoietic cells that leads to leuke-mia. In an effort to understand the molecular basis of BMF andleukemogenesis in FA, we recently uncovered a unique func-tion of proteins associated with FA in transcriptional regulationthat translates into elevated levels of the signaling moleculeDickkopf-1 (DKK1). Overproduction of DKK1 has been shownto alter functions in hematopoiesis and to promote hemato-logic malignancies. Thus, our findings represent a crucial step inthe development of strategies aimed at preventing BMF and/orclonal hematopoiesis in patients with FA.

Author contributions: M.C. designed research; C.C.H., C.S.T., and A.M. performed re-search; C.C.H., G.L., and M.C. analyzed data; and C.C.H., G.L., and M.C. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. D.H. is a guest editor invited by the EditorialBoard.1Present address: Australian Centre for Blood Diseases, Central Clinical School, MonashUniversity, Melbourne, VIC 3004, Australia.2To whom correspondence should be addressed. E-mail: madeleine.carreau@crchul.ulaval.ca.

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target genes, along with a previous study showing immunoprecip-itation (IP) of β-catenin with FANCC (7, 23), we sought to de-termine whether FANCC with CtBP1 forms a complex withβ-catenin. We performed IP experiments in human HEK293T cellsexpressing FANCC with β-catenin and other components of theFA core complex. Cell extracts were subjected to IP using anti-FANCA, anti-FANCC, or anti–β-catenin antibodies. Western blotanalyses of the immunoprecipitates showed that the FA proteinsFANCA and FANCC coimmunoprecipitated with β-cateninand endogenous CtBP1 (Fig. 1A). Western blot analysis ofimmunoprecipitates also showed the presence of FANCE andFANCF, suggesting that β-catenin forms a complex with FAcore complex proteins.Next, to determine whether accumulation of β-catenin favors

this interaction, we performed IP studies using endogenousprotein extracts from cells treated with the GSK3β inhibitorslithium chloride (LiCl) or CT99021, which are known to induceaccumulation and nuclear entry of β-catenin. Our results showthat treatment with GSK3β inhibitors LiCl or CT99021 inducedcomplex formation between FANCA, FANCC, β-catenin, andCtBP1, whereas complex formation between FANCC and CtBP1or FANCA and β-catenin occurred regardless of treatment (Fig.1 B–D). These results suggest that FANCC in association withCtBP1 forms a complex with β-catenin after GSK3β inhibition.Taken together, these results imply that interaction between FAproteins and β-catenin occurs in the nucleus.To detect the cellular localization of FANCC, CtBP1, and

β-catenin, we performed immunofluorescence experiments inHeLa cells. The results showed that FANCC localized withβ-catenin to the cytoplasm, whereas CtBP1 was found mainly inthe nucleus (Fig. 2A). To reconcile the IP data, we performedimmunofluorescence studies in cells treated with the GSK3βinhibitors LiCl or BIO/GSK3 inhibitor IX. As expected, ourresults show that β-catenin accumulates into the nucleus afterGSK3β inhibition. Surprisingly, FANCC also accumulates intothe nucleus after GSK3β inhibition, with a fourfold increase in

nuclear FANCC using LiCl, a competitor for magnesium, as wellas a twofold increase in nuclear FANCC using the ATP pocketinhibitor BIO/GSK3 inhibitor IX (Fig. 2A). In addition, over-expression of β-catenin, which induces its nuclear accumulation,resulted in a similar change in the localization of FANCC (Fig.2A, fourth panel). Consequently FANCC, β-catenin, and CtBP1localize together in the nucleus after GSK3β inhibition.We next performed similar experiments with primary fibroblast

cells. Our results show that treatment of these cells with LiCl in-duced nuclear accumulation of β-catenin and FANCC, confirmingthe results observed in HeLa cells (Fig. 2B). Although nuclearstaining of β-catenin appears to be elevated in untreated PD432cells compared with HeLa cells, GSK3β inhibition induceda threefold increase in β-catenin nuclear accumulation in both celllines. In addition, nuclear and cytoplasmic fractionation studiesshowed that FANCC and β-catenin protein levels increased aftertreatment with CT99021, with a 1.3-fold increase in nuclearFANCC and a 2-fold increase in β-catenin (Fig. 2C). These resultsconfirm our immunofluorescence data and suggest that activationof β-catenin promotes the nuclear accumulation of FANCC.We next sought to determine whether the nuclear entry of

β-catenin requires FANCC. First, we evaluated the nuclear entryof β-catenin in patient-derived FANCC-deficient cells (PD331)harboring the R548X mutation and FANCC-corrected cells(PD331/C). Cells were treated with GSK3β inhibitors LiCl orCT99021, followed by anti–β-catenin staining and visualizationby confocal microscopy. We found that β-catenin failed to prop-erly accumulate and localize to the nucleus in FANCC-deficientcells, whereas FANCC-corrected cells showed strong nuclearstaining of β-catenin after GSK3β inhibition (Fig. 3A). In addi-tion, PD331/C cells showed stronger nuclear FANCC stainingafter GSK3β inhibition compared with untreated cells (Fig. 3A,Right), corresponding to a twofold increase in nuclear FANCC(Fig. 3B). CtBP1 depletion did not affect the nuclear trans-location of β-catenin or FANCC, as demonstrated by strongβ-catenin and FANCC nuclear staining in CtBP1i and controlcells transfected with nontargeting siRNA (Fig. 3C). These resultssuggest that FANCC, but not CtBP1, is required for efficientnuclear translocation or accumulation of β-catenin. Indeed,Western blot analyses showed that β-catenin accumulationrequires FANCC, as demonstrated by the reduced β-cateninlevels in patient-derived FANCC-deficient cells (PD331) aftertreatment with GSK3β inhibitors compared with those inFANCC-corrected cells (Fig. 3D). No significant differences in

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Fig. 1. FANCC forms a complex with CtBP1 and β-catenin. (A) HEK293T cellswere cotransfected with HA-FANCC, FANCA, Myc-FANCE, Myc-FANCF,FANCG, and FANCL coding vectors. WCEs were subjected to IP with anti-bodies against FANCA, FANCC, or β-catenin and immunoblotted with theindicated antibodies. (B–D) WCEs from untreated HEK293T cells (B) or cellstreated with the GSK3β inhibitors LiCl (C) or CT99021 (D) were subjected toIP using anti-FANCA, anti-FANCC, anti-CtBP1, or anti–β-catenin antibodiesand immunoblotted with the indicated antibodies. A longer exposure timeis shown below the blot (overexposed). Negative IP controls were per-formed using rabbit IgG (R). Representative experiments out of three totalexperiments are shown. Numbers indicate molecular weight.

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Fig. 2. β-catenin activation induces FANCC nuclear translocation. (A) HeLa cellstreated with LiCl or BIO or after β-catenin transfection were stained with anti-FANCC, anti–β-catenin, and anti-CtBP1. (B) PD432 fibroblasts treated with LiClwere stainedwith anti-FANCC and anti–β-catenin antibodies and TOPRO-3. Dataare representative of three experiments in which at least 25 cells were analyzedat 100×magnification. Numbers indicate the mean nuclear/cytoplamic intensityratio. (C)Western blot analysis of nuclear (N) and cytoplasmic (C) protein extractsfrom PD432 treated or not treated with CT99021 with the indicated antibodies.Numbers indicate the ratio of the protein detected compared with TBP (nuclear)or α-tubulin (cytoplasmic) and normalized to that of untreated cells.

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β-catenin levels were observed in untreated cells, suggesting thatFANCC plays a role in β-catenin accumulation (Fig. 3E). Takentogether, our results suggest that the FANCC protein plays a rolein accumulation of β-catenin.

FANCC and β-Catenin Nuclear Translocation Is Dependent on aFunctional FA Pathway. Because FANCC is a component of theFA pathway, we sought to determine whether nuclear entry ofFANCC and β-catenin is dependent on a functional FA pathway.To so so, we performed immunofluorescence and cellular frac-tionation studies in FA-deficient cells. First, we performed im-munofluorescence labeling, followed by confocal microscopy, incells depleted of FANCA (FANCAi). In these FANCAi cells, wefound that β-catenin and FANCC remained in the cytoplasmafter GSK3β inhibition, in contrast to control cells transfectedwith nontargeting shRNA (Fig. 4A).We next performed similar experiments in patient-derived

FANCA-mutant cells (PD720) and FANCA-corrected cells(PD720/A). We observed that in FANCA-mutant cells, bothβ-catenin and FANCC are localized mainly to the cytoplasm,whereas correction of the cells with FANCA promoted a four-fold to fivefold increase in β-catenin and FANCC nuclearstaining after activation of β-catenin using GSK3β inhibitors(Fig. 4B).To determine whether the diminished nuclear entry of β-cat-

enin is limited to cells deficient in the FA core complex proteins,including FANCA and FANCC, we used FANCD2-depletedcells (FANCD2i cells), FANCD2 being a downstream compo-nent of the FA pathway. The results indicate that β-cateninand FANCC failed to accumulate in the nucleus after GSK3β

inhibition in FANCD2-depleted cells (Fig. 4C). We then per-formed similar experiments in patient-derived FANCD2-mutantcells (PD20) and FANCD2-corrected cells (PD20/D2). Ourresults show that β-catenin levels were significantly higher inPD20/D2 cells compared with noncorrected PD20 cells (Fig. 4D).Taken together, the foregoing results suggest that nuclear

accumulation of FANCC and β-catenin requires a functional FApathway. Thus, we evaluated FA pathway activity, as measuredby the monoubiquitination of FANCD2 after inhibition ofGSK3β or overexpression of β-catenin. We performed Westernblot analyses with protein extracts from HeLa cells treated withLiCl, BIO/GSK3 inhibitor IX, or CT99021 and with cells trans-fected with β-catenin. Our results show that inhibition of GSK3β,but not overexpression of β-catenin, promoted the accumulationof the monoubiquitinated form of FANCD2 (FANCD2-L) (Fig.4E). Consistent with the role of the FA core complex in mono-ubiquitination of FANCD2, cells mutated in FANCC (PD331)failed to monoubiquitinate FANCD2 after GSK3β inhibition ormitomycin C treatment, whereas monoubiquitination of FANCD2was restored in PD331/C cells.

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Fig. 3. Nuclear entry of β-catenin requires FANCC. (A and B) PD331 andPD331/C cells treated with CT99021 or LiCl were stained with anti-FANCC,anti–β-catenin, and DAPI and observed at 100× magnification. (B) Datashown are mean intensity ratios of nuclear to cytoplasm FANCC represen-tative of two experiments in which at least 25 cells were analyzed. (C) CtBP1icells treated with LiCl were stained with anti-FANCC, anti–β-catenin, andanti-CtBP1 antibodies and were visualized at 100× magnification. (D)Western blot analysis of WCEs from PD331 and PD331/C cells treated or nottreated with CT99021 with the indicated antibodies. A representative ex-periment out of six total experiments is shown. (E) Graph displaying themean relative β-catenin/α-tubulin ratio ± SEM in PD331 and PD331/C cellsafter CT99021 treatment from six independent blots (Lower). *P ≤ 0.05.

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Fig. 4. Nuclear translocation of β-catenin is dependent on a functional FApathway. (A–C) FANCAi cells (A), PD720 and PD720/A fibroblasts (B), andFANCD2i cells (C) were treated with LiCl and stained with anti-β-catenin andanti-FANCC antibodies and visualized at 100× magnification. Data shownare representative of two experiments in which at least 25 cells were ana-lyzed. Numbers indicate the mean nuclear/cytoplamic intensity ratio. (D)Graph displaying the mean relative intensity ratios of nuclear to cytoplasmicβ-catenin in PD20 and PD20/D2 cells after treatment with LiCl and CT99021from two separate experiments in which at least 25 cells were analyzed. (E)Western blot analysis of FANCD2 in WCEs from HeLa, PD331, and PD331/Ccells treated or not treated with LiCl, BIO, CT99021, or mitomycin C ortransfected with β-catenin or empty vector (control). (F) Luciferase assayperformed in COS-1 cells transfected with the TCF/LEF reporter along withβ-catenin and various concentrations of FA core complex expression vectors(FANCA, FANCC, FANCE, FANCF, FANCG, and FANCL). (G and H) Luciferaseassays performed in HeLa cells transfected with the TCF/LEF reporter alongwith β-catenin, TCF4, and increasing amounts of FANCC (G) or FANCD2 (H)expression vectors as indicated. (I) Luciferase assays performed in PD331 andPD331/C cells transfected with the TCF/LEF reporter and exposed to LiCl orCT99021 before analysis. Numbers of experiments performed in duplicateare indicated in each graph. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

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Because the foregoing results suggest that the FA pathwaymay positively impact the transcriptional activity of β-catenin, weevaluated β-catenin activity using the T cell factor (TCF)/lymphoidenhancer binding factor (LEF) reporter assay. We found thatoverexpression of FA core-complex components increasedβ-catenin–mediated transcription of the TCF/LEF reporter ina dose-dependent manner (Fig. 4F). However, overexpression ofFANCC alone did not influence β-catenin–mediated activa-tion of the TCF/LEF reporter (Fig. 4G), whereas overexpression ofFANCD2 increased β-catenin–mediated transcription of the TCF/LEF reporter in a dose-dependent manner (Fig. 4H). These resultsimply that the FA pathway acting through FANCD2 is required forefficient β-catenin activity.To confirm these results, we used FA patient-derived mutant

PD331 cells and found that these cells had significantly reducedβ-catenin activity compared with FANCC-corrected cells(PD331/C), as demonstrated by lower TCF/LEF reporter acti-vation after GSK3β inhibition using LiCl or CT99021 (Fig. 4I).Taken together, our results suggest that FANCC is required forefficient β-catenin nuclear entry and subsequent activity. Con-sequently, FANCC may affect the transcriptional regulation ofβ-catenin target genes.

FA Proteins Act as Transcriptional Repressors of DKK1. Based on ourpreviously reported data showing abnormal expression of Wnt/β-catenin target genes, specifically DKK1, in FA-deficient cells(7), we investigated the role of FANCC in the transcriptionalregulation of DKK1. To do so, we cloned the human DKK1promoter into the pGL3 luciferase reporter vector. First, wetransfected the DKK1 reporter construct in cells expressing in-creasing amounts of FANCC. Our results show that FANCC wasable to significantly repress DKK1 reporter activity in a dose-dependent manner (Fig. 5A), but to a lesser extant than CtBP1,a known repressor (Fig. 5B). These results suggest that FANCCtranscriptional repression activity requires a cofactor. Indeed,cotransfection of FANCC with CtBP1 led to further decrease inDKK1 transcriptional activity (Fig. 5C).We next evaluated the repression capacity of FANCC har-

boring the L554P disease-causing mutation (FANCCL554P;Fig. 5D). Increasing amounts of FANCCL554P lead to a dose-dependent activation of the DKK1 promoter, suggesting thatthe mutated form of FANCC lost its capacity to repress DKK1.Indeed, FANCCL554P had no effect on CtBP1-mediated tran-scriptional repression of the DKK1 promoter (Fig. 5E). Theseresults imply that CtBP1 and FANCC act together as negativeregulators of DKK1 expression, and that disease-causing muta-tions in FANCC negatively affect this function. Indeed, this ideais supported by results obtained in patient-derived FANCC-deficient cells (PD331), whereas stronger DKK1 promoter ac-tivation is found compared with that in FANCC-corrected cells(PD331/C) (Fig. 5F). These results suggest that FANCC withthe corepressor CtBP1 negatively regulates DKK1 expression.To determine whether efficient DKK1 repression requires

a functional FA pathway, we evaluated DKK1 transcriptionalactivity in FA-deficient cells. Our results show that the silencingof FANCD2 led to a fivefold induction of DKK1 reporter ac-tivity similar to that found in FANCC mutant cells and CtBP1-depleted cells (Fig. 6A). Silencing of FANCD2 and CtBP1 led toa further increase in DKK1 reporter activation. These results areconsistent with our previous findings (7) showing a threefold tofourfold increase in DKK1 mRNA and protein expression inFANCD2-depleted cells. Western blot analyses performed inpatient-derived FANCD2-mutant cells (PD20) confirmed theresults obtained in FANCD2i cells showing elevated levels ofDKK1, whereas complementation with the FANCD2 gene(PD20/D2) reduced DKK1 protein levels to normal (Fig. 6B).These results suggest that a functional FANCD2 protein is re-quired for DKK1 repression; however, increasing amounts of theFANCD2 protein did not significantly impact the DKK1 reporter,suggesting an indirect effect (Fig. 6C). Taken together, these

results suggest that the FA pathway acts as a transcriptionalrepressor of DKK1.

DiscussionWe recently reported an interaction between the FANCC pro-tein and the transcriptional corepressor CtBP1 (7). Our previousstudy highlighted the implications of FA proteins along withCtBP1 in the transcriptional regulation of Wnt pathway-responsivegenes including DKK1, a Wnt antagonist. Because DKK1 is a targetof TCF/β-catenin–mediated transcription as well as a Wnt antag-onist acting as a negative feedback loop (10, 24), we investigatedthe functional implication of FA proteins in β-catenin signaling and

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Fig. 5. FANCC and CtBP1 act as transcriptional repressors of DKK1. (A–E)Luciferase assays performed in HeLa cells transfected with the DKK1 pro-moter reporter construct along with equimolar amounts of β-catenin andTCF4 expression vectors and with control empty vectors or various concen-trations of FANCC (A), CtBP1 (B), FANCC with CtBP1 (C), FANCCL554P (D), orFANCCL554P with CtBP1 (E) as indicated. (F) Luciferase assays performed inpatient-derived FANCC-deficient (PD331) and FANCC-corrected (PD331/C)cells transfected with the DKK1 promoter reporter construct and treatedwith CT99021 or LiCl. The number of experiments performed in duplicate isindicated in each graph. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

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Fig. 6. FANCD2-deficient cells have increased DKK1 expression. (A) Lucif-erase assays performed in FANCD2i, CtBP1i, FANCD2i/CtBP1i, or control cells(control) transfected with the DKK1 reporter and assayed for luciferase ac-tivity at 24 h after transfection. (B) Western blot analysis of WCEs from PD20and PD220/D2 cells treated with CT99021 with the indicated antibodies(Upper). Mean relative expression of the DKK1/α-tubulin ratio ± SEM relativeto untreated cells from two independent blots (Lower). **P ≤ 0.01; ***P ≤0.001. (C) Luciferase assays performed in HeLa cells transfected with theDKK1 reporter along with β-catenin, TCF4 expression vectors, and increasingconcentrations of FANCD2, as indicated.

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DKK1 transcriptional regulation. In the present study, we havedemonstrated that FANCC forms a complex with β-catenin andCtBP1. We also have shown that FANCC localizes to the nucleuswith both β-catenin and CtBP1 after activation of β-catenin ei-ther by its overexpression or by inhibition of GSK3β. We alsoprovide evidence that FANCC is required for the efficient nu-clear translocation of β-catenin after GSK3β inhibition. Theseresults imply that efficient nuclear accumulation of β-cateninwould be prevented in cells with a defective FA pathway.Indeed, we observed that in FANCA- and FANCD2-depleted

cells, as well as in patient-derived FA mutant cells, the majorityof β-catenin remains restricted to the cytoplasm after activationof the Wnt pathway. These results imply that the lack of a func-tional FANCC protein or the absence of a functional FA path-way would impede the transcriptional activity of β-catenin. Asexpected, transcriptional activation of the β-catenin/TCF re-porter was reduced in patient-derived FA mutant cells comparedwith their corrected counterparts. These data are consistent withour previous results showing reduced β-catenin/TCF reporteractivation in FA-depleted cells (7).In addition, we found that overexpression of FA core complex

components triggered transcriptional activation of the β-catenin/TCF reporter in dose-dependent manner, whereas overexpressionof only FANCC had no effect on β-catenin/TCF reporter acti-vation. Furthermore, overexpression of FANCD2 promotedtranscription of the β-catenin/TCF reporter similar to that ofFA core complex components, suggesting that the FA corecomplex-mediated activation of the β-catenin/TCF reporter occursthrough the FA pathway downstream component FANCD2. Inaddition, we observed that GSK3β inhibition triggered activationof the FA pathway as measured by formation of the long form ofFANCD2. Recent studies support a role of the FA core complexvia FANCD2 in transcription, both activation and repression(25–27). Taken together, those studies and our results imply thatFA core complex activity may modulate β-catenin function. In-deed, this is supported by a study by Dao et al. (23), whichshowed that β-catenin activity is facilitated by FA core complexubiquitin ligase activity. Our findings support a role of the FApathway in β-catenin nuclear accumulation and subsequent activity.Our findings suggest that a defective FA pathway leading to

diminished nuclear β-catenin and, consequently, diminishedβ-catenin/TCF activity would negatively affect β-catenin targetgenes. Indeed, our previous microarray data showed that FA-deficient cells demonstrate down-regulation of Wnt/β-catenintarget genes involved in signal activation. However, we also ob-served that other Wnt signal modulators, such as DKK1, were up-regulated in these FA-deficient cells (7), suggesting a dualfunction of FA proteins. Our present data provide evidence oftranscriptional regulation of DKK1 by FANCC, as demonstratedby dose-dependent diminished DKK1 promoter activation after in-creasing amounts of FANCC. In addition, overexpression ofFANCC and CtBP1 led to further DKK1 transcriptional repression.Consistent with the role of FANCC in DKK1 repression, the

FANCC protein harboring the disease-causing mutation L554Pfailed to repress transcription of the DKK1 promoter. Moreover,patient-derived FANCC mutant cells (PD331) showed increasedDKK1 transcriptional activation. These effects would result inelevated levels of DKK1. Indeed, we previously showed thatFancC-deficient mice have increased Dkk1 serum levels (7).Taken together, our data suggest that FANCC with CtBP1 actsas a corepressor of DKK1.Our findings support a model (Fig. 7) in which the FA pathway

acts in transcriptional regulation of the DKK1 gene. Here weshow that FANCC accumulates in the nucleus in response toβ-catenin activation. Once in the nucleus, FANCC and itsbinding partner CtBP1 repress transcription of DKK1. Con-comitantly, via FANCD2, the FA core complex enhances thetranscriptional activation of β-catenin/TCF target genes andsubsequently represses DKK1. Consequently, the absence of afunctional FA pathway would prevent FANCC from entering thenucleus and repressing the DKK1 gene, leading to overproduction

of DKK1. In addition, lack of a functional FA core complex wouldlead to reduced activation of β-catenin/TCF target genes andDKK1 repression. Overall, our data provide evidence of a dualmechanism through which the FA pathway acts in transcriptionalregulation of the DKK1 gene.Our results are relevant to the FA disease, because over-

production of Dkk1 from bone marrow niche cells has been asso-ciated with altered HSC function, leading to impaired self-renewalcapacity (17). Impaired self-renewal capacity is a hallmark of FA-deficient HSCs (20–22). In addition, overproduction of Dkk1induces cell cycling of primitive hematopoietic cells that generallyare maintained in a quiescent state (17, 28). FA-deficient primitivehematopoietic cells were shown to have an accelerated cell cycle(29). Taken together, these findings support our model and providea clue to explaining the failure of bone marrow in patients with FA.Further investigation is needed to determine whether DKK1 servesas a potential therapeutic target to prevent exhaustion of primitivebone marrow cells.

Materials and MethodsCell Lines and Culture Conditions. HEK293T (American Type Culture Collectionand Cedarlane Laboratories), HeLa (American Type Culture Collection), andPD432, PD720 (FA-A), PD331 (FA-C), PD20 (FA-D2), and PD20-corrected (PD20/D2) (gifts fromDr.Markus Grompe, OregonHealth Science University, Portland,OR and the Fanconi Anemia Research Fund) fibroblast cell lineswere cultured inDMEM supplemented with 10% FBS and grown at 37 °C in a humidified at-mosphere containing 5% CO2. The knockdown of CtBP1 (CtBP1i) and FA genes(FANCAi and FANCD2i) in HeLa cells was performed using different pLKO.1plasmids carrying shRNAs targeting FANCA (TRCN0000118982), FANCD2(TRCN0000082840), or CtBP1 (TRCN0000013738), as described previously (7).A lentivirus expression system was used to stably complement PD331 withWT FANCC cDNA (PD331/C). Functional correction of PD720 cells was doneby transfection of the FANCA-coding plasmid (PD720/A). Stealth small in-hibitory RNAs (siRNAs) for use against CtBP1 and the negative control werepurchased from Invitrogen. Where indicated, cells were exposed to GSK3βinhibitors LiCl (Sigma-Aldrich), BIO/GSK3 inhibitor IX (Calbiochem), CT99021(Cayman Chemical), or mitomycin C (Sigma-Aldrich). Cells were treated withLiCl at 100 mM for 3 h (short exposure) or 50 mM for 16 h (long exposure).Cells were treated with the BIO/GSK3β inhibitor IX at 5 μM for 16 h. CT99021was used at 10 μM for 3 h (short exposure) or at 1 μM for 16 h (long exposure),and mitomycin C was used at 100 nM for 16 h.

DNA Constructs and Antibodies. All FA gene constructs used in this study havebeen described previously (7, 30). The pCEP4-FANCCL554P plasmid was generously

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Fig. 7. Proposed model of a dual mechanism by which the FA pathwayregulates expression of DKK1. (Left) In the presence of an intact FA pathway(FA-proficient), FANCC and β-catenin efficiently accumulate and localize intothe nucleus after GSK3β inhibition. In the nucleus, FANCC forms a complexwith CtBP1 and β-catenin and represses DKK1. The FA core complex viaFANCD2 enhances expression of other β-catenin/TCF target genes. FANCD2influences the stability or expression of FA core complex proteins and, sub-sequently, DKK1 expression. (Right) In the absence of a functional FApathway (FA-deficient), FANCC and β-catenin do not efficiently accumulateinto the nucleus. As a result, FANCC fails to efficiently repress DKK1. Lack ofFA core complex activity results in reduced expression of β-catenin/TCF tar-gets and increased DKK1 expression.

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provided by Dr. Maureen Hoatlin, Oregon Health & Science University, Port-land, OR. The FANCD2 expression plasmid (pIRESneo-FANCD2) was generouslyprovided by Markus Grompe, Oregon Health & Science University. The pRc/CMV-T7-CtBP1 plasmid was a gift from Dr. G. Chinnadurai, Saint Louis Uni-versity Health Sciences Center, St. Louis, MO. The pCMV6-XL5-β-cateninplasmid was obtained from OriGene Technologies. The TCF4 expressionconstruct was obtained from Upstate Cell Signaling Solutions. The pGL3-basic was obtained from Promega. The following antibodies were used: anti-FANCA (C-20; Santa Cruz Biotechnology or Novus Biologicals), anti-FANCC(31) (8F3, a gift from Dr. M. Hoatlin, Oregon Health & Science University,Abcam, or Novus Biologicals), anti-FANCD2 (Novus Biologicals), anti-CtBP1(Millipore or BD Biosciences), anti–β-catenin (R&D Systems or Santa CruzBiotechnology), anti-DKK1 (R&D Systems), anti-hemagglutinin (HA) (12CA5;Roche Diagnostics), anti-cMyc (9E10; Santa Cruz Biotechnologies), anti–α-tu-bulin (Sigma-Aldrich), anti–TATA-binding protein (TBP; Abcam), anti-goat(Calbiochem), anti-mouse and anti-rabbit (Santa Cruz Biotechnologies), anddonkey anti-rabbit Alexa Fluor 488, anti-mouse Alexa Fluor 555, and anti-goat Alexa Fluor 680 (Invitrogen).

Cellular Fractionation and Immunoprecipitation. Whole-cell extracts (WCEs)from HEK293T or HeLa cells were subjected to IP, immunoblot analysis, or cellfractionation studies as described previously (30). For IP, equal amounts ofprotein were incubated overnight at 4 °C with 2 μg of antibodies, followedby incubation with protein G or A-agarose beads (Calbiochem) or protein-Gmagnetic beads (Invitrogen). Immunoprecipitates were resolved by SDS/PAGE and subjected to Western blot analysis with specific antibodies, asindicated in each figure. For cell fractionation studies, protein extracts weresubjected to cellular separation and preparation using NE-PER nuclear andcytoplasmic extraction reagents (ThermoScientific) according to themanufacturer’s instructions.

Immunofluorescence. For detecting the cellular localization of FANCC, β-catenin,and CtBP1, HeLa, PD432, PD720, PD720/A, PD331, and PD331/C cells were eitherfixed in methanol-acetone (3:7 vol/vol) or in 4% paraformaldehyde and per-meabilized with 0.1% saponin or 0.3% Triton X-100 before standard immu-nofluorescent staining. The slides were mounted with DAPI-Fluoromount-G(Southern Biotech) for fluorescence microscopy. Cell nuclei were labeled withTOPRO-3 (Invitrogen) or DAPI (Sigma-Aldrich) for confocal microscopy. Cellswere visualized with a Nikon E800 fluorescent microscope equipped with a C1

confocal system (Nikon Canada) or were observed under a Zeiss Axio ImagerM2microscope equipped with an AxioCamMRm digital camera and AxioVision4.8 software (Zeiss).

Transcriptional Reporter Assays. The DKK1 promoter spanning the −1037 to+163 region of the gene was amplified by PCR using the forward sequenceprimer 5-CTCCCTAGAAAGGGTATTG-3 and the reverse primer 5-AGATA-GGACCCTTTCAAGG-3, and then cloned into the pGL3-basic luciferase re-porter vector (pGL3-DKK1). For the DKK1 promoter reporter or TCF/LEFreporter assays, cells were transfected with the plasmids pGL3-DKK1 or M50Super 8X TOPFlash (Addgene; plasmid 12456) along with the Renilla lucif-erase control plasmid (Promega). The total amount of plasmid DNA wasequalized between the transfections using empty vectors.

For β-catenin–mediated activation of DKK1 promoter and for the TCF/LEFreporter assays, cells were treated at 16 h after transfection of the luciferasereporter vectors with LiCl (50 mM) or CT99021 (10 μM). Cell extracts wereprepared at 24–48 h after transfection and assayed for luciferase activitywith the Promega Dual Luciferase Reporter Assay System according to themanufacturer’s instructions, using an automated plate reader (Tecan Infinite200). Luciferase activity was normalized to Renilla luciferase and expressedas the mean fold change ± SEM relative to control cells.

Statistical Analyses. Data are expressed as mean ± SEM. Statistical analyseswere performed using GraphPad Prism version 5.0b, and paired and un-paired two-tailed Student t tests were used to compare the means. AP value < 0.05 was considered significant.

ACKNOWLEDGMENTS. We thank Dr. M. Hoatlin for providing the anti-FANCC 8F3 monoclonal antibodies and the pCEP4-HA-FANCCL554P and Dr.M. Grompe for FA cells and the FANCD2 expression plasmid. We thank AnnieGagnon for her work related to siRNA against CtBP1 and Marie-ChantalDelisle for her technical expertise. This work was supported in part by grantsfrom the Canadian Institutes of Health Research in partnership with theCanadian Blood Services Blood Utilisation and Conservation Initiative grant(M.C. and G.L.), the Canadian Leukemia and Lymphoma Society, and scholar-ships from Canadian Institutes of Health Research in partnership with theCanadian Fanconi Anemia Research Fund (C.C.H.) and the Foundation ofStars (C.C.H., C.S.T., and A.M.).

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