supporting online material for -...

www.sciencemag.org/cgi/content/full/1137509/DC1

Supporting Online Material for

An X Chromosome Gene, WTX, Is Commonly Inactivated

in Wilms Tumor

Miguel N. Rivera, Woo Jae Kim, Julie Wells, David R. Driscoll, Brian W. Brannigan, Moonjoo Han, James C. Kim, Andrew P. Feinberg, William L. Gerald, Sara O. Vargas,

Lynda Chin, A. John Iafrate, Daphne W. Bell, Daniel A. Haber*

*To whom correspondence should be addressed. E-mail: [email protected]

Published 4 January 2007 on Science Express

DOI: 10.1126/science.1137509

This PDF file includes:

Materials and Methods Figs. S1 to S4 Tables S1 to S4 References

WTX is inactivated in Wilms tumor

Supporting Online Material Materials and Methods Tumor and tissue samples Frozen Wilms tumor and matched normal tissue samples were obtained from Children’s Hospital Boston, Brigham and Women’s Hospital, Memorial Sloan Kettering Cancer Center and Johns Hopkins Medical Center. All samples were collected with institutional review board approval. Mouse tissues were from C57BL/6 or FVB mice (Charles River Laboratories, Wilmington, MA). DNA was prepared using the Puregene DNA Purification Kit (Gentra, Minneapolis, MN) and RNA was prepared using Trizol (Invitrogen, Carlsbad, CA) or the RNeasy Mini Kit (Qiagen, Valencia, CA) following the manufacturer’s instructions. Array CGH DNA was prepared from tumor material that was at least 90% pure histologically. Long oligonucleotide array CGH was performed as previously described (1). FISH DNA FISH BACs RP11-1105P13 (WTX, chromosome Xq11.1; linear position 63,282,689 NCBI Build 36.1) and RP11-54P15 (5 mb telomeric to WTX, chromosome Xq13.1; linear position 68,481,365) were obtained from BAC/PAC Resources (Children's Hospital, Oakland, CA). BAC DNA was isolated using the Qiagen Plasmid Maxi Kit (Qiagen, Valencia, CA), sequenced to confirm the identity of the clone and labelled using a nick translation kit (Vysis, Downers Grove, IL) with Spectrum Orange-11-dUTP (WTX) or Spectrum Green-11-dUTP (telomeric probe). As a control for X chromosome copy number a centromere X probe (DXZ1) was labelled with biotin and detected with streptavidin-CY5. Three-color FISH was performed on paraffin sections using the manufacturer’s recommended hybridization conditions, with denaturation at 80°C for 5 minutes and overnight hybridization at 37°C. Cells were counterstained with DAPI. Images were acquired using an Olympus BX61 fluorescent microscope equipped with a CCD camera, and analysis was performed with Genus software (Applied Imaging, San Jose, CA). At least 30 nuclei were scored in each assay. 35 non-tumor tissue samples were used as controls for deletions when matched DNA was not available (Cybrdi Inc., Frederick, Maryland). RNA/DNA FISH A fosmid clone overlapping the Xist locus (WI2-935P22/ G248P8779H11; Xq13.2; linear position 72,955,542) was obtained from BAC/PAC Resources (Children's Hospital, Oakland, CA). The fosmid DNA was labelled by nick translation with Spectrum Green-11-dUTP, and used in a triple-hybridization with probes for WTX (spectrum orange) and centromere X (biotin) following standard protocols (2). Frozen tumor sections or touch preparations of frozen tumor tissue were prepared on microscope slides, air-dried for 5 minutes and fixed for 5 minutes in freshly prepared 4% paraformaldehyde at 4 degrees. Nuclei were permeabilized in 2x SSC/ 0.5% triton X-100 for 10 minutes. Following dehydration in ethanol (70% 2 minutes, 90% 2 minutes, 100% 2 minutes), and drying at

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60 degrees for 20 minutes, slides were hybridized with Xist, WTX, and centromere X probes (denaturation at 75 °C for 3 minutes, hybridization at 37°C overnight). Following routine FISH washing (50% formamide/2xSSC twice for 5 minutes at 37°C, and 2X SSC for 5 minutes twice at 37°C), the centromere X probe was detected with streptavidin CY5 and slides were counterstained with DAPI. A minimum of 30 nuclei were scored in each assay. Only cells in which all three signals were clearly visible were counted. The Xist DNA probe effectively labelled Xist RNA on only one X chromosome in multiple normal female tissue preparations. Sequencing The complete WTX sequence was assembled from RACE-PCR and RT-PCR products derived from human and mouse neonatal kidneys. RT-PCR products from multiple sources were used to confirm the results. The assembled human WTX sequence differs from the annotated database sequence (Genbank AK097146) at the 3’ end (Fig. S1). Genomic DNA The WTX gene was amplified from genomic DNA by PCR with primers listed in Table S3. Primers for WT1 are listed in Table S4. Exon 3 of β-catenin was amplified and sequenced using 5’ GCTGATTTGATGGAGTTGGACATGG 3’ as a forward primer and 5’ CTCTTACCAGCTACTTGTTCTTGAG 3’ as a reverse primer. PCR products were purified and subjected to nucleotide sequencing using BigDye v1.1 (Applied Biosystems, Foster City CA). Tumor and reference sequences were aligned using sequence Navigator and Factura softwares (Applied Biosystems) and compared to identify homozygous and heterozygous nucleotide positions. All nucleotide variants were confirmed in at least two independent experiments. cDNA cDNA was prepared from 1 µg of total RNA using Superscript III and random hexamer primers according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). WTX was amplified with primers spanning one intron to prevent the amplification of genomic DNA and sequenced with appropriate internal primers. All sequences were confirmed in at least two independent reactions. The primers used were: Human WTX cDNA forward 5’ ACAATAACCGGGCTAGGAACCTGAC 3’

reverse 5’ ATCATCATCTGGCAAGGCCATCT 3’ internal 5’ AAATGGCCTGTAAAGATCCAGAA 3’

Quantitative PCR (QPCR) Quantitative PCR was performed using Power SYBR Green on an Applied Biosystems 7500 Real time PCR System (Applied Biosystems) using either genomic DNA or cDNA as a substrate. cDNA was synthesized from 1 µg of DNAse treated total RNA using Superscript III and random hexamer primers (Invitrogen). Power SYBR Green Master Mix (Invitrogen) was used in all reactions according to the manufacturer’s protocols. The results were analyzed using Sequence Detection Software version 1.3 (Applied Biosystems) and threshold levels were set during the exponential phase of the PCR reaction. The comparative threshold cycle (Ct) method was used to calculate the amount

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of each target gene relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Applied Biosystems User Bulletin #2: http://docs.appliedbiosystems.com/pebiodocs/04303859.pdfAll primers had approximately equal amplification efficiency when tested with serial dilutions of genomic DNA or cDNA. All reactions were subjected to melt curve analysis and selected products were further tested on agarose gels. The primers used were: Human WTX 5’ (genomic) forward 5’ CATATGCCTGCCAATCAGGAC 3’,

reverse 5’ CAGTCACCTCCAGCACTGACTC 3’ Human WTX 3’ (genomic) forward 5’ AGCTGTACCACACTGTCCGTTC 3’

reverse 5’ AGCTGTCTACCAGGTCCCACAC 3’ Human GAPDH (genomic) forward 5’ ACACCCACTCCTCCACCTTT 3’

reverse 5’ CTGAGCCAGCCACCAGAG 3’ Mouse WTX (cDNA) forward 5’ GCGCAGCAGACAATAAACAG 3’

reverse 5’ TCTTGGGCATCGCTAGAAGT 3’ Mouse WT1 (cDNA) forward 5’ GTGGTGCCCAGTACAGAATACA 3’

reverse 5’ TCTCTTATTGCAGCCTGGGTAT 3’ Mouse GAPDH (cDNA) forward 5’ TGGTGAAGCAGGCATCTGAG 3’

reverse 5’ TGCTGTTGAAGTCGCAGGAG 3’ Northern Blotting Northern blotting was performed following standard protocols. Human fetal kidney RNA used for Northern blotting was purchased from Stratagene (La Jolla, CA). All other RNA samples were prepared as described above. Twenty micrograms of RNA was loaded per lane of 1.2% agarose formaldehyde gels and transferred to Hybond-N+ membranes (Amersham, Piscataway, New Jersey). Blots were subsequently probed with radioactive probes using ExpressHyb (Clontech, Mountain View, CA) and following the manufacturer’s protocol. Radioactive probes were prepared by random primer labeling of cloned fragments of cDNA with radioactive dCTP (Perkin Elmer, Wellesley, MA) using the NEBlot Kit (New England Biolabs, Ipswich, MA) and following the manufacturer’s instructions. The primers used to generate the Northern blot probes were as follows: Human WTX probe forward 5’ CCAGGGCGGCTCAGCGGACAATAAC 3’, reverse 5’ ACCTTGCTCTTCCGGTGACGGCGG 3’ Mouse WTX probe forward 5’ AGAGGCTCCAGCTCAAATCGGAGCA 3’, reverse 5’ GTCTTGCCCTTCCGGTGACGGCGG 3’ Multiple sequence alignment Multiple sequence alignment of human WTX with selected predicted orthologs was performed with Multalin 5.4.1 (3). BLAT (4) was used to identify predicted open reading frames with the highest homology to human WTX in the genomic DNA of various species. The genomic sequences used were: Mus musculus (February 2006 Build 36), Canis familiaris (May 2005 whole genome shotgun assembly v2.0), Gallus gallus (May 2006) and Danio rerio (March 2006 Zv6 assembly).

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http://docs.appliedbiosystems.com/pebiodocs/04303859.pdf


RNA in situ hybridization ISH was performed using standard protocols modified as described previously (5). Frozen tissue sections were fixed in 4% paraformaldehyde, digested with proteinase K (4 μg/ml), treated with acetic anhydride and dehydrated with ethanol. Sections were hybridized overnight with sense and antisense digoxigenin labeled probes prepared with the DIG RNA Labeling Kit (Roche, Basel, Switzerland). Bound probes were detected using an alkaline phosphatase conjugated anti-digoxigenin antibody (Roche) and BM purple (Roche). The primers used to amplify probes were: Human WTX probe forward 5’ CCAGGGCGGCTCAGCGGACAATAAC 3’, reverse 5’ CAGAACCTTTGCTCCGTCCCCCTCC 3’ Human WT1 probe forward 5’ AGCTCCAGCTCAGTGAAATGGACAGAAGGG 3’, reverse 5’ GTGATGGCGGACTAATTCATCTGACCGGGC 3’ Colony formation assays The full WTX open reading frame was cloned by PCR into the pcDNA4TO expression vector (Invitrogen) with a 3’ terminal FLAG tag. HEK-293 and U2OS cells were cultured in DMEM 10% FBS and plated in 12 well plates. Lipofectamine 2000 (Invitrogen) was used for cotransfection of empty vector or WTX expression vector and the selection plasmid pBABE puro. HEK-293 cells were transfected with 1 μg of expression vector and 0.1 μg of selection plasmid. 2 μg of expression vector and 0.5 μg of selection plasmid were used for U2OS cells. 50,000 HEK-293 cells and 20,000 U2OS cells were plated in 10-cm dishes after transfection. Following 2 weeks of selection in puromycin, colonies were counted automatically using Quantity One software (Biorad, Hercules, CA). All assays were performed in triplicate and repeated twice. Apoptosis assay HEK-293 cells were grown on coverslips in 24 well plates and transfected with 0.8 μg of empty vector or WTX expression vector using Lipofectamine 2000 (Invitrogen) following the manufacturer’s protocol. 48 hours after transfection the cells were fixed with 4% formaldehyde, permeabilized with Triton X-100 and incubated with a anti-cleaved caspase-3 antibody (Cell Signalling Technologies, Danvers, MA) followed by a goat anti-rabbit Alexa Fluor 488 conjugated secondary antibody (Invitrogen) at 1:500 and 1:2000 concentrations respectively. Experiments were performed in duplicate and repeated twice.

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A B

(7.5 kb) (7.5 kb)

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l kid

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Human WTX Mouse WTX

Human WTX

METQKDEAAQAKGAAASGSTREQTAEKGAKNKAAEATEGPTSEPSSSGPGRLKKTAMKLF 60

GGKKGICTLPSFFGGGRSKGSGKGSSKKGLSKSKTHDGLSEAAHGPEDVVSEGTGFSLPL

PELPCQFPSSQSAHGALETGSRCKTSVAGATEKAVAEKFPSMPKPKKGLKGFFSSIRRHR 180

KSKVTGAEQSEPGAKGPERVRARPHEHVSSAPQVPCFEETFQAPRKENANPQDAPGPKVS

PTPEPSPPATEKMACKDPEKPMEACASAHVQPKPAPEASSLEEPHSPETGEKVVAGEVNP 300

PNGPVGDPLSLLFGDVTSLKSFDSLTGCGDIIAEQDMDSMTDSMASGGQRANRDGTKRSS

CLVTYQGGGEEMALPDDDDEEEEEEEEVELEEEEEEVKEEEEDDDLEYLWETAQMYPRPN 420

MNLGYHPTTSPGHHGYMLLDPVRSYPGLAPGELLTPQSDQQESAPNSDEGYYDSTTPGFE

DDSGEALGLVRRDCLPRDSYSGDALYEFYEPDDSLENSPPGDDCLYDLHGRSSEMFDPFL 540

NFEPFLSSRPPGAMETEEERLVTIQKQLLYWELRREQLEAQEARAREAHAREAHAREAYT

REAYGREAYAREAHTWEAHGREARTREAQAREVRCRETQVRETQARQEKPVLEYQMRPLG 660

PSVMGLAAGVSGTSQISHRGITSAFPTTASSEPDWRDFRPLEKRYEGTCSKKDQSTCLMQ

LFQSDAMFEPDMQEANFGGSPRRAYPTYSPPEDPEEEEVEKEGNATVSFSQALVEFTSNG 780

NLFSSMSCSSDSDSSFTQNLPELPPMVTFDIADVERDGEGKCEENPEFHNDEDLAASLEA

FELGYYHKHAFNNYHSRFYQGLPWGVSSLPRYLGLPGLHPRPPPAAMALNRRSRSLDTAE 900

TLEMELSNSHLVQGYLESDELQAQQEDSDEEDEEEEEGEWSRDSPLSLYTEPPGAYDWPA

WAPCPLPVGPGPAWISPNQLDRPSSQSPYRQATCCIPPMTMSISLSVPESRAPGESGPQL 1020

ARPSHLHLPMGPCYNLQPQASQSMRARPRDVLLPVDEPSCSSSSGGFSPSPLPQAKPVGI

THGIPQLPRVRPEHPQPQPTHYGPSSLDLSKERAEQGASLATSYSSTAMNGNLAK

Figure S1. WTX is detected as a single band approximately 7.5 kb in size in Northern blots of human fetal kidneys (pooled 18 week and 22 week kidneys) and cells lines (A). A band of the same size is detected in mouse kidneys (B). (C) Amino acid sequence of human WTX. The nuclear localization signal (blue) and coiled-coil domains (red) are shown. The acidic and proline-rich domains are underlined.

1 53 H.sap. MET QKDEAAQAKG AAASGSTREQ TAEKGAKNKA AEATEGPTTSE PSSSGPGRLKC.fam. MET GKDEASQAKG ASASMDTQDQ GAEKGAKNKA AEITEGPVSE PPSSGPGRLR M.mus. MES QQDEAVQTTKG ASTSSDAQDQ GAEKGAKNKT TEATEGPTSET PPLSGPGRLKG.gal. METGCTEEAA RTQSQSAACR QCKGGEQQPE ENVERLSQRS DASATAAEQQ PPQAPSGKLKD.rer. MEIATR CEVGAMRGPS SDSVSHDIPQ PQSPPSVKIR 54 113 H.sap. KTAMKLFGGK KGICTLPSFF GGGRSKGSGK GSSKKGLSKS KTHDGLSEAA HGPEDVVSEG C.fam. KTAMKLFGGK KGICTLPSFF GGGRSKGSGK SISRKGLSKS KTHDGLTTEAA HGPKDIFSEG M.mus. KTAMKLFGGK KGICTLPSFF GGGRSKGSGK VSSKKSLNKS KTHDGLSEAS QGPEDVVIEEG.gal. KTAFKLFGGK RSICTLPSFF SS-RNKGQGK GASKKGLSKS KTHDGISGTA YDEGSGVQLED.rer. KTAFKFFGGR KSICVLPSFF GG-RGRSQRK GSSKTTGVTKS QTTYDGVSRAC WDDLGRSSSE 114 165 H.sap. TGFSLT PLPEL PCQFPSSQSA HGALETGSRC KTSVA----- ---GATEKAT V AEKFPSMPKPC.fam. TSFALT PLPES SCQLPSSQSA HGVLETDSRC KTSVA----- ---GATEKAT E AEKALSLSKPM.mus. TDLSTT PLSKS SAQFPSSQSA NGALEIGSKH KTSGT----- ---EAIEKAG VEKVPSVHKPG.gal. SPSDGSRDSH PCLLPSSQSV HVAIDTSVKF DFGRQDGSPP GSIEGYT EKKP NGDKSSFPRPD.rer. VASGDFEFCS EPQKSQEDH- ---------- ---------- ---------- -GKSQSLPRQ 166 218 H.sap. KKGLKGFFSS IRRHRKSKVT GAEQSEPG-- -----AKGPE RVRARPHEHV SSAPQVPCFEC.fam. KKGLKGFFSS IRRHRKSKVS GAEKSELR-- -----AKGPE GARARPHEYV SSVILHHT-EM.mus. KKSLKSFFSS IRRHRKGKTS GADQSVPG-- -----AKELE GARTTRSHEHV SSISL-PSSEG.gal. KKGLKGLLNS IRRHRKSKVA ECEKTTELS-- -----EWPGD SEETSKAQGT KAETPGTVEED.rer. RRGLRGLFSS IRRHRKNKNV EVEKREALEM SSSFHAKTVP GALPSVSDRG DYHGDSQGEE 219 288 H.sap. ETFQAPRKEN ANPQDAPGPK VSPTPEPSPP ATT EKMACKDP EKPMEACASA HVQPKPAPEAC.fam. DILQAPRKEN AKPQDAPGSK LSLVPEPFPA VIEKTTACKDP EKSVEACASA LMQPKPTLEAT

M.mus. EIFRDTRKEN AKPQDAPGPK MSPAQVHFSP TTEKAACKNP EKLTRTCASE FMQPKPVLEG G.gal. GVPGSVPLAA ACPGSSEDNC LVRTVADT FGE AAEPDWLQAD KGSCEGDVVA VPGGKDDLDAD.rer. LVPDVPNQTT GSECELPLAA TECTIDVTLV PEKRRSRVEM DT KRRRAEEEG IGEDEKTGRQ 289 330 H.sap. SSLEEPHSPE TGT EKVVAGEV N--------P PNGPVGDPLS LLFGDVTSLK SFDSLTGCGDC.fam. SGPEEPHSPE TGT EKVVSEEV N--------P PNGPVGDQLS LLFGDVTSLK SFDSLTGCGDM.mus. GSLEEPHTSE TEGT KVVAGEV N--------P PNGPVGDQLS LLFGDVTSLK SFDSLTGCGDG.gal. KSEVDAVVYT ESNYSHLPVA LHPDLADNDP PSLHSGDQLS LLFGDVTSLK SFDSLTGCGDD.rer. EGLMTYHQPL SAESELDRLA EQNVDVPDGE PPVASCSSEN LVFGDVSSLK SFDSLTGCGD 331 379 H.sap. IIAEQDMDSM TDSMASGGQR ANRDGTKRSS CLVTYQGGGE EMALPDDDD- ---------- C.fam. IIAEQDMDSM TDSMASGGQR ANRDGTKRSS CLVTYQGGGE EMALPDDE-- ---------- M.mus. IIAEQDMDSM TDSMASGGQR ANRDGTKRSS CLVTYQGGGE EMALPDDDDN DDEEEEEEEEG.gal. IIAEPDILSI AESTISVERS --RDTAKRSP CLVTYQGGGE EMAMSEEEYL QQIWDSTAEED.rer. IIADQDDVSV AESSVSAD-R GSRNAGKRSS CFVTYQGGGE EMATTPDEIDA DYLQSLWESE Figure S2. Multiple sequence alignment of human WTX and selected orthologs. Amino acids are color coded according to the degree of identity: 60-80% blue, 80%-100% red. Numbers refer to the human sequence.

380 429 H.sap. ---------- EEEEEEEEVE LEEEEEEVKE EEEDDDLEYL WETAQMYPRP NMNLGYHPTT C.fam. ---------- EEEEEEDEVE LEEEEEDVKE EE-DYDLEYL WASAQMYPKS NLNLSYHPIT M.mus. EEEEEEEEEE EEEEEEEEEE LLEDEEEVKD GEENDDLEYL WASAQIYPRF NMNLGYHTAI G.gal. GRSYEAPLPP VVSSPELQAV SSKLETRGLH EGEVHPYASG AVDGV----- ---------- D.rer. TSNEVCYIPS DRGSDSPS-- ---------- ---------- ---------- ---LTPDQQL 430 489 H.sap. SPGHHGYMLL DPVRSYPGLA PGELLTPQSD QQESAPNSDE GYYDSTTPGF EDDSGEALGLC.fam. SPGHHGYMLL DSVRSYPGLA PGELLTPQSD QQESAPNSDE GYYDSTTPGF EDDSGEALGLM.mus. SPSHQGYMLL DPVQSYPNLG LGELLTPQSD QQESAPNSDE GYYDSTTPGF EDDSGEALGLG.gal. ---------- ---------- --ELLTPQSD QQESAPNSDE GYYDSTTPGP EDETGDGLCE D.rer. SSIRATSSSS PMGITETALT PADLLSPQSD RQESVPNSDE GYYDSTTPGM EEESRERPHQ 490 549 H.sap. VRRDCLPRDS YSGDALYEFY EPDDSLENSP PGDDCLYDLH GRSSEMFDPF LNFEPFLSSRC.fam. IRRDCLPRDS YSGDALYEFY EPDDSLETTSP PVDDCLYDLH GHSSEIFDPF LNFELFSSSRM.mus. AHRDCLPRDS YSGDALYEFY EPDDSLEHSP PGDDCLYDLR GRNSEMLDPF LNLEPF-SSRG.gal. IKKDRLPRDS YSGDALYEFY EPDDTTLMSPS HGEQSLFESK VSHPEIFSYF LDFCLPPEKS D.rer. ER---LPRDS YSGDALYELF EPDDRLLSPS LPPKDAHSFV GAPLQADKSP TNPLYSLAST 550 602 H.sap. PP-------G AMETEEERLV TIQKQLLYWE LRREQLEAQE ARAREAHARE AHAREAYTRE C.fam. PP-------G AMETEEERLV TIQKQLLCWE LRREQLEARE AQ--EACAQE IHTREAYTQE M.mus. PP-------G AMETEEERLV TIQKQLLYWE LRREQREAQE ACAREAHA-- ---------- G.gal. LIQMMDQKRG VMETEEERLA AIQKELLFWE LQREPGLKRL DVPSK----- ---------- D.rer. AIET-----G AMETEEERLS KIQHALLCCE LQNLRSPSKN QLLFH----- ---------- 603 662 H.sap. AYGREAYARE AHTWEAHGRE ARTREAQARE VRCRETQVRE TQARQEKPVL EYQMRPLGPSC.fam. THARETHVRE AHVREAQARE AYAREAQVRE AHAQEAQVRE VQAWQEKPII EYHMRPLGPSM.mus. ---REAYARD THTRESYGRN VRARETTQALE AHSQEGRVQE TKVRQEKPAL EYQMRPLGPSG.gal. ---------- ---------- ---------- ---------E KCPREKQHVE CNTRAANLIG D.rer. ---------- ---------- ---------- ---------- ---------- ---------- 663 721 H.sap. -VMGLAAGVS GTTSQISHRGI TSAFPTTAT T SS EPDWRDFRPL EKRYEGTCSKT KDQSTCLMQLC.fam. SVMGLVAGAS GASQTTSHRAT TSAFPATTASS EPDWRDFRPL EKRFEGICSK KDQSTCLMQLM.mus. -VMGLVAGTTS GGSQTSHRT GT TSAFPATST SS EPDWRDFRPL EKRFEGTCSKT KDQSTCLMQLG.gal. KSQSCLGSEQ IASQALNRSV NGGILVARAE NPEWSDFPGR LCPENCYNSQ KAQGSCLIQLD.rer. ---------- ---------- ---------- ----SDCFYD DSNLPVDDSK QDLQEVINQR 722 767 H.sap. FQSDAMFEPD MQEANFGGSP RRAYPTTYSPP EDPEEEEVEK EGNATV---- ---------- C.fam. FQSDAMFEPD MQEANFGGSP RRAYPTTYSPS EEPEEEEAEK EGNATV---- ---------- M.mus. FQSDAMFEPD MQEANFGGSP RKAYPSYSPP EEPEEEEEEK EGNATV---- ---------- G.gal. MKNNPGFDSD PDCALFGGSL HGSAALAKPG MFPSRRPLEH EGCLQSEHHS GVEAAGREPQ D.rer. YPQSPPRSQA VKEGVPRIRG QVQESSLFAP CADSVLNPQV IETTRPQPQS DDQGSLRPSR Figure S2, continued

768 813 H.sap. ---------- -SFSQALVEF TSNGNLFSSM SCS---SDSD SSFTQNLPEL PPMVTFDIADC.fam. ---------- -SFSQALVEF TSSGNLFSSM SCS---SDSD SSFAQNLPEL PPMVTFDIADM.mus. ---------- -SFSQALVEF TSNGNLFTTSM SYS---SDSD SSFTQNLPEL PPMVTFDIADG.gal. ADSECEPEHA MSFSQALVEF TSSGTTLFSSL SESLGSSDSG SSFTQNLPVL PTMVTFDIT VDD.rer. GCSQSQEELM VCFSQALVDF TKT NTRLYRNS TESLDGSESS SPFGPSLRAL PAIVTFDVVD 814 864 H.sap. VERDGEGKCE ENPEFHNDED LAASLEAFEL G-YYHKHAFN NYHSRFYQGL P--------WC.fam. VEREGEGKCE EHPEFHN-ED LAASLEAFEL G-YYQKHTTFN NYHSRFYQGL P--------WM.mus. VERDGEGKCE ENPEFNNDED LTTASLEAFEL G-YYHKHAFN SYHSRFYQGL P--------WG.gal. VEQEGEGECE QHLEMNADED MAASFEAFDE S-YVQKESFA ECDERMFLGY PQGSFQSCNWD.rer. MENEGECEQQ TDLAEEEEEL ASPYEPFEDD GCYLQQDAFA ECDQRTFDAY EQSLLLSNAW 865 915 H.sap. GVSSLPRYLG LPGLHPRPPP AAMALNRRSR SLDTAETTLEM E--------- LSNSHLVQGYC.fam. GVSSLPRYLG LPGMHPRPPP AAMALNRRSR SLDTAETTLEL E--------- LSNSRPAQGYM.mus. GVSSLPRYLG LPGVHPRPPP AAMALNRRSR SLDNAESLEL E--------- LSSSHLAQGYG.gal. GVASLPRHLH LHGLSPAMP- APLSLNRRSR SLDTESLEFE LADLQVSKNG LKPCQLWSKW D.rer. GIASLPRHLS LGRPCP-PVP APLALNRRSR SLDTDSLEFQ TSEIYTSVTK YDSK------ 916 975 H.sap. LESDELQAQQ EDSDEEDEEE EEGEWSRDSP LSLYTEPPGA YDWPAWAPCP LPVGPGPAWI C.fam. IVSDELQAQQ EDSNEEEEEE EWG---RDSP LSLYTEPPGA YDWSAWAPCP LPVGPGPAWI M.mus. MESDELQAHQ EDSDEEGEEE E-GEWGRDSP LSLYTEPPGV YDWPPWAHCP LPVGPGLAWM G.gal. DKKYSDGARR SRSKEEAELS APEGGETNGV LSWPGLQHLQ YDAELAPGGI KHWGFAPAAG D.rer. ---------- ---------- ---------- ---------- ---------- -----GTTAFS 976 1033 H.sap. SPNQLDRPSS QSPYRQATCC IPPMTMSISL SVP--ESRAP GESGPQLARP SHLHLPMGPCC.fam. SPSQLDGSSS QSPYEQTACC IPPVAMSMLL SVPGPEPRVP GISRPQLSRP SYLPLPMVSCM.mus. SPNQLYEPFN QSSYVQATCC VPPVAMPVSV PG-----RTTP GDSVSQLARP SHLPLPMGPCG.gal. MPSSWEPSER SDADPSFFPL SRSCARETLE RQPQDPEPNR LLVRPSNLPL QPDTRQPQEA D.rer. QSRTVDCNDM DFPRQPCRIT VDSWRRGYRQ NFDSSNASQQ ELKLPHLSQS TVRPSHLPLK 1034 1093 H.sap. YNLQPQASQS MRARPRDVLL PVDEPSCSSS SGGFSPSPLP QAKPVGITHG IPQLPRVRPEC.fam. YNLQPQAFQS VRARPQDMLL PIDEPNCFSS SGGFSSSPLS QAKPVGITHG IPQLPRVQPEM.mus. YNLQSQASQS GRAKPRDVLL PVDEPSCSSI SGANSQS--- QAKPVGITHG IPQLPRVRPEG.gal. SGSYRYHGEA PAKKLARVLP LGEPEPPQSI SFAQSPEKPA KCKPVGVTQT G MPQCHDSDDT D.rer. NNCRSRNLPA ATRVDGEGEI LFGGGDALYP CSYPPMGTQW KNRPVGVTQT G VPHLRSEQSA 1094 1135 H.sap. HPQPQPTTHYG PSSLDLSKER AEQGASLAT- --SYS-STAM NGNLAKC.fam. PQQPQPIHCK VSSLDLSKEM AEQGASLPL- --PTSYSTAV NGKLAE M.mus. PFQLQPNHYR ASNLDLSKER GEQGASLST- --SYS-STAM NGNLAKG.gal. ETLKSSACFA ERYGSTSKEL LKARAAPGNV LPSGCLSTAV NVMGAKD.rer. DHQEITMKNR RGELKGGFTP APPKL Figure S2, continued

Wilms tumor T33

Wilms tumor T36

X centromereWTX

Telomeric Xq

(female) Matched normal tissue 33

(female) Matched normal tissue 36

Figure S3. Single copy deletions of WTX in females. FISH for two representative female Wilms tumor and matched normal pairs is shown. A centromeric X chromosome probe (blue), a telomeric Xq probe (green) and a WTX probe (red) were used. In both cases, one chromosome lacks a red WTX signal in tumor cells. Scale bars, 3 μm.

Vector WTX

Cle

aved

cas

pase

-3D

AP

I

Figure S4. Induction of apoptosis in HEK-293 cells 48 hours after transient transfection with WTX but not vector control . Cleaved caspase-3 (green, upper panels) and DAPI staining of nuclei (lower panels) are shown. Scale bars, 100 μm.

Table S1. Wilms tumor samples with WTX deletions and point mutations

Sample ID Nucleotide change Predicted effect Matched (N=82) on protein control

Male cases

T13 Deletion Absent Wildtype

T15 Deletion Absent (0/35) **

T19 Frameshift, 1648DelC 561Ter (0/269) *




T50 Missense, 876 G>C Lys292Asn Wildtype

T58 Frameshift, 439InsT 157Ter Wildtype

T82 Nonsense, 1072 C>T Arg358Ter Wildtype

Female cases

T7 Nonsense, 1000 G>T Glu334Ter (0/269) * (Active X)

T29 Deletion Absent Wildtype(Active X)






T51 Frameshift, 1069DelA 363Ter (0/269) *

T65 Nonsense, 1072 C>T Arg358Ter Wildtype

Mutations in female cases that were verified to reside in the active X-chromosome are indicated. To determine that mutations were somatic, matched normal tissues were used when available. When matched samples were not available, population controls were used (the amplicon containing each mutation was sequenced). The control samples were derived from healthy blood donors in the Boston metropolitan area.Two synonymous SNPs were identified: 1137C>T (1/82), 3057A>G (16/82). One known non-synomous SNP was also detected: 477T>G (Phe159Leu, 9/82, ref SNP ID: rs34677493).

* Point mutation not detected in 269 normal individuals by sequencing ** Deletion not detected in 35 normal tissues by FISH.

Table S2. Mutations in WT1 and β-catenin

Wilms tumor WT1 β-catenin

T2 313Ter * Ser45Phe T8 Arg301Ter Thr41Ala

T10 Arg433Cys WildtypeT24 Wildtype Ser45delT37 Wildtype Gly38-Pro44dupT55 Wildtype Ser45delT57 Wildtype His36ProT73 Wildtype Thr41AlaT74 532Ter ** Ser45PheT76 Wildtype Ser45del

* Frameshift in exon 7** Insertion within exon 10 (leading to a frameshift)

Table S3. Primers for amplification and sequencing of WTX from genomic DNA

Designation Primer sequence (5’-3’) Primer Orientation

WTX.AF CCAGACCCCACTGTGATGCTTCCTG SenseWTX.BF CAGCTCCAAGAAAGGTCTCAGCAAG SenseWTX.CF GTCAGAGCCAGGCCTCATGAGCACG SenseWTX.DF GGGATGTGACATCCCTGAAAAGCTTTG SenseWTX.EF CAATATGAACCTGGGCTACCATCCCAC SenseWTX.FF CCTCCATGGTCGAAGCTCTGAGATG SenseWTX.GF GAGTATCAGATGAGGCCCTTAGG SenseWTX.HF GGAATGCCACTGTGAGTTTCTCACAGG SenseWTX.HFa GATGCCATGTTTGAGCCAGACATGC SenseWTX.IF GTGAGCAGCCTCCCTCGATACTTGG SenseWTX.JF CCAACCAGTTGGACAGGCCTTCCAG SenseWTX.KF CTTCCAGTTCTGGAGGCTTCAGC SenseWTX.AR CAGCCACAGATGTCTTACATCTGGAGC AntisenseWTX.BR CTGGATCTTTACAGGCCATTTTCTC AntisenseWTX.CR CATCATCATCTGGCAAGGCCATCTC AntisenseWTX.DR CCTCACCTGAATCATCCTCAAATCC AntisenseWTX.ER CTGGCATGAGCTTCTCGGGCACGTG AntisenseWTX.FR CATAACGCTTCTCCAGAGGACGGAAG AntisenseWTX.GR CACATCAGCGATGTCAAAGGTCACC AntisenseWTX.HR GTCCTCCTCATCTGAATCTTCCTGC AntisenseWTX.IR ACGAGCTAGTTGAGGCCCAGATTC AntisenseWTX.JR CTAGGTTTCCATTCATGGCAGTGGAG AntisenseWTX.KR CCATAGATGGCAGAAGAGGCAAGTGG AntisenseWTX.KRa GCAGATGCACTTGAGTTGAACGTGG Antisense

Table S4. Primers for amplification and sequencing of WT1 from genomic DNA

Designation Primer sequence (5’-3’) Primer Orientation

WT1-1F CTTCCACGTGTGTCCCGGAGCCGGCGTCTC SenseWT1-EXON2F TGGCTGGTTCAGACCCACTGCCCCGTCTTG SenseWT1-EXON3F CAGTGCCCCAGGCTCAGGATCTCGTGTCTC SenseWT1-EXON4F CATTCTGGAAAATGTGGAGGCTTGCACTTTC SenseWT1-EXON5F TAGCTAGCTTGATGTCTCTGTGTATTGAGG SenseWT1-EXON6F CCATCATTCCCTCCTGATTGCAGATAAGC SenseWT1-EXON7F CTTTGGATATACTCCAGTGCTCACTCTCC SenseWT1-EXON8F GAAGTGCCTTAGGCATTTTGGGATCTGTTC SenseWT1-EXON9F CAGACATTGCAGGCATGGCAGGAAATGCTG SenseWT1-EX10-F CACTCGGGCCTTGATAGTTGAACTTGTGC SenseWT1-1R GATTGCGAATAGCGGGCTGGCTCTCGAGG AntisenseWT1-EXON2R CCTAATTTGCTGTGGGTTAGGAATTCCTGG AntisenseWT1-EXON3R AGGCGTCTCGTGCCTCCAAGACCCTGCATG AntisenseWT1-EXON4R CACAGAGAGCTTTGCCCTTTCTTCTAAAAC AntisenseWT1-EXON5R TTCCCATCCACCAAATGCTACCCTGATTAC AntisenseWT1-EXON6R AGTGAAGAAGAGGCTGCCAGGGCCAAAGAG AntisenseWT1-EXON7R CAGTGCTTACTTTCCATCCTGGAAATAACC AntisenseWT1-EXON8R CATGGCTGACTCTCTCATTCATATTCAAC AntisenseWT1-EXON9R CTTTTACACTAGTCTTTTCCAATCCCTCTC AntisenseWT1-EX10-R GGTATCTTGTCTTGGAAGTTGGATG Antisense


Supporting references 1. C. Brennan et al., Cancer. Res. 64, 4744 (2004). 2. E. Heard, F. Mongelard, D. Arnaud, P. Avner, Mol. Cell. Biol. 19, 3156 (1999). 3. F. Corpet, Nucleic Acids Res. 16, 10881 (1988). 4. W. J. Kent, Genome Res. 12, 656 (2002). 5. R. E. Palmer et al., Cancer Cell 2, 497 (2002).

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