© 2011 Nature America, Inc. All rights reserved. be addressed to K.J.P. ([email protected]). Biology Center, Kyoto University, Kyoto, Japan. 1 ( of chromosomal aberrations, mean quantification right, ; structured radial indicates arrowhead (left, aberrations chromosomal promotes ( assay. viability cell MTS by tested when formaldehyde ( exposure. formaldehyde exogenous after stability Figure 1 that damage DNA of source physiological the be cannot molecules 15 of one agents DNAthat tain cross-link chemotherapeutic any of con cer by caused damage DNA repairs evolutionarily eventually that an pathway in served inactivation together operate products germline FA Most through arises FA cancer to predisposition significant a have and attrition cell stem with FAIndividuals afflicted are prone to abnormal development and survival. cell vertebrate in repair cross-link DNA and catabolism formaldehyde of action combined the for role fundamental a define results pathway. DNA-repair (FA) These anemia Fanconi the and ADH5 between cells avian in interaction lethal synthetic a report we Here DNA. damage can that aldehyde reactive simple, toxic, a formaldehyde, generate to predicted is TakataMinoru Rosado V Ivan DNA-repair pathway anemia in cells deficient for the Fanconi Formaldehyde catabolism is essential NATURE STRUCTURAL & MOLECULAR BIOLOGY MOLECULAR & NATURE STRUCTURAL Received 29 June; accepted 16 September; published online 13 November 2011; ( treatment and release. ( c b a a Medical Research Council Laboratory of Molecular Biology, Cambridge, UK. e ) Induction of ) ) Formaldehyde exposure in human ) DT40 knockouts 0.01 Survival (%) 100 0.1 10 1 03 , encoding the main formaldehyde-detoxifying , enzyme, formaldehyde-detoxifying main the encoding ,

Formaldehyde The FA DNA-repair pathway ensures genome genome ensures pathway FAThe DNA-repair FANCB Wild type 06 FANCB 09 1 G – 2 -H2AX after 2-h formaldehyde pulse pulse formaldehyde 2-h after -H2AX , Frédéric Langevin & Ketan J Patel J Ketan & knockout cells are sensitive to exogenous exogenous to sensitive are cells knockout 0 XRCC2 (  120 M) d ) 150 FANCL , KU70 Wild type FANC b DT40 knockout cells are hypersensitive to formaldehyde; formaldehyde; to hypersensitive are cells knockout DT40 1 and and o B , 3 s.e.m., s.e.m., – FANCB 1 formaldehyde , Gerry P , CrossanGerry REV1 3 – University University of Cambridge, Department of Medicine, Addenbrooke’s Hospital, Cambridge, UK. should Correspondence n − are not hypersensitive to formaldehyde; formaldehyde; to hypersensitive not are cells cells = 2). = 2).

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100 50 metaphases 0.1 10 10 20 30 40 1 0 2 2– 03 Laboratory Laboratory of DNA Damage Signaling, Department of Late Effect Studies, Radiation Wild type 4 1 - - –– . . Chromosome gaps Chromatid breaks Chromosome breaks Chromatid gaps Radial d Formaldehyde 06 o ++ lines, revealing a fundamental role for the FA pathway in protecting protecting FA in the pathway for role fundamental a revealing lines, endogenous formaldehyde accumulation in DNA repair–deficient cell detoxification and DNA repair. Here we examine the consequences of cells that of action enzymatic the combined through threat this against protect hypothesized we DNA, attack can and abundant is both the (HCHO), aldehydesimplestreactive formaldehyde to contrast in cells, within generated pathway DNA-repair FA-associated the by repair necessitating damage DNA endogenous eukaryotes. complex all in conserved are FAgenes the why reason the be cannot linkers cross- chemotherapeutic FAthe Furthermore, precipitates phenotype. FANCB of the an inactivation carrying counterpart its isogenic Methods Supplementary formaldehyde genotoxicity in a human FA gene–deficient cell line (see FA complex the core of independently HR by repaired be to shown been also has damage aldehyde this to sensitivity selective translesion synthesis (TLS) or (HR) show gene downstreampathwayFA the in deficient lines cell repair–deficient DNA chicken specifically, More genotoxic. be can cells against this reactive aldehyde. i : 1 Recently we showed that acetaldehyde is a potential source of of source potential a is acetaldehyde that showed we Recently Previous work has demonstrated that exogenous formaldehyde formaldehyde exogenous that demonstrated has work Previous FANCB 0 09 . 1 0 3 XPA -deficient cells are very sensitive to formaldehyde ( formaldehyde to sensitive very are cells -deficient 8 – 01

/ n ( µ s -deficient cells are mildly sensitive to formaldehyde. M) m 20 FANCL b . 2 1 150 c 7 BRIE TAP-FANCL FANCL FANCL Wild type 02 + HCHO 3 -complemented cell line is resistant to formaldehyde. formaldehyde. to resistant is line cell -complemented 1 1 2 9 . We chose to extend these findings by testing testing by findings these extend We to . chose . However, acetaldehyde is not abundantly abundantly not is acetaldehyde However, . –/ –/ Wild type – –

). We used the human B cell line NALM-6 and + F 1 34 0 Recovery . Given that endogenous formaldehyde formaldehyde endogenous that Given . e Survival (%) COMMUNICATIONS 0.01 100 0.1 10 5 1 03 6 1 1 . Formaldehyde-induced DNA Formaldehyde-induced . + HCHO 02 Formaldehyde 06 1 09 FANC 34 Recovery 0 B – (  M) 120 FANCB 5 6 150 FANCD2  Histone H3  Hours FANCL REV XRCC2 XPA KU7 Wild type Fig. 1 Fig. -H2AX -tubulin gene 1 – 0 –/ –/ – – –/ –/–/ a 1 – 3 ), ), 1 – . ,

© 2011 Nature America, Inc. All rights reserved. genous formaldehyde under these conditions ( growth was not compromised, nor did it show increased 4a sensitivity Fig. to exo ( disruption gene through strain DT40 knockout genous formaldehyde can pool be genotoxic, we generated an by (encoded dehydrogenase5 of action the through formaldehyde generated endogenously ( orthologs AlkB by bases DNA methylated of dealkylation and ases demethyl Jumonji the by demethylation histone of byproduct a interstrand cross-links. include not may damage DNA formaldehyde-induced that suggests XRCC3 synthesis ( enhanced formaldehyde sensitivity in cells lines mutant for translesion ( repair excision (specifically, mutant nucleotide for the in gene deficient those of exception the with major pathways of DNA repair were largely resistant to formaldehyde, ( nents of the FA to cells pathway formaldehyde so far sensitized tested way using mutant chicken DT40 cell lines. Mutations in all the compo FA the of path DNA-repair components downstream and upstream of (DSBs). breaks phosphorylation Ser139 ( of H2AX histone induction enhanced showed also ( formation structure the radial and breakage some chromo with chromatid-type of correlated accumulation sensitivity this and lacking both G exact test. ns, nonsignificant.) ( independent experiments. ( values obtained from seven plates from two at 50 cells per well into 96-well plates; mean assay of FACS GFP bars define s.e.m. ( values of two independent experiments. Error GFP B treatment for FANCD2 (top), GFP (middle) and -negative ( ( DT40 cells by addition of 4-OH tamoxifen. conditional deletion of lethal in DT40 cells. ( Figure 2 BRIE 2 line, which lost FANCD2 and GFP expression upon treatment with with treatment upon expression GFP and FANCD2 lost which line, ADH5 deficient and sion of the treatment to leads Cre inducible exci 4-OH-tamoxifen recombinase, condFANCD2 GFP DT40 and FANCL provedalleles).This unsuccessful for(0/138 by strain this the in either of disruption pathway FA the inactivate to attempted we Next b -H2AX in FACS GFP Fig. Fig. 1 Fig. 1 Fig. Supplementary Fig. 3 Fig. Supplementary -tubulin (bottom). (

) ) Western blot of FACS GFP-positive (+) or We comprehensively tested formaldehyde sensitivity in both both in sensitivity formaldehyde tested comprehensively We Formaldehyde is generated endogenously within the nucleus as as nucleus the within endogenously generated is Formaldehyde + ADH5 expression cassette flanked by flanked cassette expression and GFP and gene in this strain to create the FANCD2 e d , , ), ), suggesting that the combined inactivation of the FA pathway

). In contrast to earlier results by others b and and RAD51C FANCD2 F ). The ). − REV1 ) ) sorted cells after 4-OH tamoxifen FANCD2-IRES-GFP could be synthetically lethal. We therefore constructed a constructed We therefore lethal. synthetically be could FANCD2 COMMUNICATIONS Supplementary Fig. 1 Fig. Supplementary − GFP ) ( sorted cells representing mean Fig. Fig. 2 − ADH5 and and and and + / , n G − and GFP and negative ( negative RAD52 c HA) ( -H2AX) = 2). ( 2). = strain that carries a conditional − and and ) ) Growth curve of FACS a and GFP and ) ) Strategy outlining the ADH5 FANCD2 REV3 a ). Owing to the presence of a 4-OH-tamoxifen– − / ADH5 ) − P d FANCC 10 strain was readily obtained; this mutant’s this obtained; readily was strain and and < 0.0001, Fisher’s ) ) Clonogenic survival are synthetically − , ) or homologous recombination ( ) recombination or homologous sorted cells plated 14 + FANCD2 in in e sorted cells i. 1 Fig. , . ) ) Induction of 1 RAD54 5 cassette, rendering the cells cells the rendering cassette, ADH5 ADH5 . The cell prevents accumulation of of accumulation prevents cell The . XPA (one allele) or ). Cell lines deficient in the other other the in deficient lines Cell ). c lox -null -null ), a marker of double strand strand double of marker a ), ), ), which showed mild sensitivity − ) ) ( P sites (hitherto referred to as to referred (hitherto P sites 1 GFP 6 Supplementary Fig. Supplementary 2 condFANCD2 ADH5 condFANCD2 . To test whether the endo the To whether . test

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ADH IRES TetO -ME suppressed this accumulation. this suppressed -ME ) components of the FA pathway. Our observation that FA- + / , − (encoding the enzyme that catabolizes acetaldehyde), acetaldehyde), catabolizes that enzyme the (encoding GFP ADH5 FANCD2 ADH FANCD2 G 5 –/

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− − GFP +/– ++ +/– + cells were obtained were obtained cells cells ( cells ANCD2 − M ADH5 / ADH5 ) or downstream ) or downstream − knockout strain strain knockout ADH5 − − M / cells ( cells − cells. We puri cells. cells but only but only cells B Fig. 2 Fig. TAP + B –/ –/ -ME, giving giving -ME, TetO expression, – – -ME to the the to -ME : Fig. Fig. 3 : FANCL  GFP FANCD2-S FANCD2-L FANCD2 ( FANCD2 Histone H3  -tubulin -H2AX Fig. 3 Fig. Fig. 3 Fig. - Fig. 3 Fig. FANCL ø e ADH5 ADH5 ). TAP- e ), ), in d − b c / ). ). ). ). ). ). − -

© 2011 Nature America, Inc. All rights reserved. DT40 clonogenic assays and assisted in the generation of generation in the and assays assisted clonogenic DT40 I.V.R. performed the majority of the experiments presented. F.L. contributed to I.V.R. and K.J.P. the designed study and the experiments, and wrote the paper. Anaemia Research Fund and Children’s Trust, Leukaemia respectively. invaluable help with flow cytometry. I.V.R. and F.L. are funded by the Fanconi insight and to M. Daly and F. Zhang (MRC Laboratory of Molecular forBiology) J. Sutherland and K. Lang (MRC Laboratory of Molecular forBiology) chemical Japan) for kindly providing NALM-6 human cell lines. We are grateful to We thank N. Adachi and H. Koyama (Kihara Institute for Biological Research, B the availableon informationSupplementaryis Note: FA. in cells stem blood and germ of attrition progressive the explain therefore might genotoxicity formaldehyde tions reliant might on be particularly FA pathway repair. Endogenous with a cell lineages certain propensity for histone extensive modifica byproduct of histone demethylation, our results lead us to predict that cell pools stem of emergence the and multicellularity with FA coevolved genes FA genes is only found in higher eukaryotes; perhaps the expansion of potently gen more and/or abundant more either be to appears dehyde whose absence is not synthetically lethal bars, s.e.m. ( ADH5 of two independent experiments. Error bars, s.e.m. ( with TAP-FANCL. ( or DT40 cells by addition of doxycycline. ( Figure 3 NATURE STRUCTURAL & MOLECULAR BIOLOGY MOLECULAR & NATURE STRUCTURAL AUTH A c a i CK o

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–1 –1 ADH ADH 1 2 3 4 0 1 2 3 4 0 0246 0 18. 17. 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. reprints/index.html. http://www.nature.com/at online available is information permissions and Reprints Published online at http://www.nature.com/nsmb/. The authors declare no competing interests.financial line. FANCD2cell the inducible provided and cell lines. G.P.C. helped with analysis of chromosome breaks. M.T. generated C 5 5 O +/ –/

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Days inDO 246 MP Zhang, X.Y.Zhang, G. Mosedale, B. F.J.Iborra, Sedgwick, & E. T.Seeberg, R.F.& P.O.,Johansen, Falnes, Lindahl, R.P., Hausinger, T.F., Henshaw, S.C., Trewick, H. Koyama, & N. Y.,Adachi, Nomura, T.Noda, J.R. Ridpath, Y.Shi, & N. Mosammaparast, K.J. Patel, & M.J. Arends, I.V., G.P.,Rosado, F.,Crossan, Langevin, K.J. Patel, & A.F. Alpi, W., Niedzwiedz, I.V., Rosado, H. Joenje, J.P.Winter,& de H. Joenje, & K.J. Patel, K.J. Patel, & H. Joenje, Y.Kim, C. Stoepker, G.P.Crossan, D.A. Williams, & L.U. Muller, Nature (2011). 53–58 (2009). 4360–4370

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