mice: on the role of and Prdm9 allelic · PDF filechromosomal fusions and Prdm9 allelic...

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Transcript of mice: on the role of and Prdm9 allelic · PDF filechromosomal fusions and Prdm9 allelic...

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    SupportingInformation

    GeneticrecombinationvariationinwildRobertsonianmice:ontheroleofchromosomalfusionsandPrdm9allelicbackgroundLaiaCapilla1,2,NuriaMedarde2,AlexandraAlemanySchmidt3,MariaOliverBonet3,JacintVentura2,AuroraRuizHerrera1,41InstitutdeBiotecnologiaiBiomedicina(IBB),UniversitatAutnomadeBarcelona,CampusUAB,08193,Barcelona,Spain.2DepartamentdeBiologiaAnimal,BiologiaVegetaliEcologia,UniversitatAutnomadeBarcelona,CampusUAB,08193,Barcelona,Spain.3UnitatdInvestigaci,HospitalUniversitariSonEspases,CtraValldemossa79,07010,Palma,Spain,4DepartamentdeBiologiaCellular,FisiologiaiImmunologia,UniversitatAutnomadeBarcelona,CampusUAB,Barcelona,Spain.

    1.MaterialandMethods________________________ Page22.SupplementaryTables________________________ Page53.SupplementaryFigures________________________ Page84.References__________________________________ Page18

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    1.MaterialandMethodsa)Animalsandchromosomalcharacterization

    Atotalof31wildmalemicewerelivetrappedincommensalhabitatsfrom10different

    localitiesrepresentativesoftheBarcelonaRbsystem(TableS1andFig.S1).Threemalesfrom

    thelaboratorystrainC57BL6werealsoincludedintheanalysisforcomparison.Mitotic

    metaphasespreadswereobtainedfromfibroblastculturesandbonemarrowcells.Cell

    cultureswerederivedfromfreshtissuessamplesandtheharvestingofcellsfollowedstandard

    procedures.KaryotypesweredeterminedbyanalyzingGbandedchromosomalpreparations

    [1].WhenGbandingidentificationwasnotsufficientforchromosomalidentification,weused

    commercialmousechromosomespecificpaintingprobes(Cambio,Cambridge,UK)toconfirm

    Gbandinghomologiesfollowing[2].TheEthicsCommitteeoftheUniversitatAutnomade

    Barcelonaapprovedtheanimaltreatmentprotocolsadoptedherein.

    b)Immunoflourescence

    Immunofluorescence(IF)ofspermatocytespreadswasperformedfollowing[3].Differentsets

    ofantibodieswereused:mousemonoclonalantibodyagainstMLH1(BDPharmigen)forthe

    detectionofcrossovers(COs),rabbitpolyclonalserumagainstcentralelementproteinofthe

    synaptonemalcomplex(SC)(SYCP3,Abcam),humancalcinosis,Raynaudsphenomenon,

    oesophagealdysfunction,sclerodactylyandtelangiectasia(CREST)serumforthedetectionof

    thecentromeres,mouseH2AX(Millipore),rabbitH3K9me3(Abcam)andmouseRPA(Abcam).

    Correspondingsecondaryantibodieswere:antimouseconjugatedwithFITC,antimouse

    conjugatedwithCy3,antirabbitCy3andantihumanCy5(allpurchasedfromJackson

    Immunoresearch).

    c)Fluorescenceinsituhybridization

    WiththeaimofidentifyingthechromosomesimplicatedinRbfusions,fluorescenceinsitu

    hybridization(FISH)wasperformedonpreviouslyimmunostainedpreparationsusingBacterial

    ArtificialChromosome(BAC)probesforspecificmousechromosomes(chromosomes4,9,11,

    12,13and14).BACcloneswhereselectedfromtheUCSCgenomebrowser

    (http://genome.ucsc.edu)andpurchasedfromCHORI(http://bacpac.chori.org)(TableS3).DNA

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    waspurifiedfrombacterialpelletsusingaMidiprepextractionkit(Quiagen).FISHwas

    performedonbothmetaphasechromosomesandspermatocytespreadsaspreviously

    described[2,3],withmodifications.Briefly,1goftheplasmidDNAwaslabeledwithdUTP

    digoxigeninusinganicktranslationkit(Abbot)andethanolprecipitatedwithcompetitorDNA

    (Cot1MouseDNA,Invitrogen,1mg/ml),salmonspermDNA(Invitrogen,10mg/ml)and1/10

    volumeof3mol/Lsodiumacetateovernightat20C.Theslidesweredehydratedinan

    ethanolscalecontaining70%formamideat74Cbefore,andafterthedenaturation.Theprobe

    wasprecipitatedandwashedtwicewith70%ethanolandthendilutedin15ulofhybridization

    buffer(50%deionizedformamide,10%dextransulfate,2xSSCand0.5mol/Lphosphate).

    Chromosomesweredenaturedat74atwhichpointtheprobehybridizationmixwasplaced

    directlyontheslides,whichwerecoverslippedandincubatedO/Nat37Cinahumid

    chamber.Washeswereperformedwith2Xsalinesodiumcitrate(SSC)and50%formamide

    solutionbeforeapplyingtheantidigoxigeninconjugatedwithFITC.

    d)Imageprocessinganddataanalysis

    PreparationswerevisualizedusingaZeissAxioskopepifluorescencemicroscopeequippedwith

    theappropriatefiltersandachargedcoupleddevicecamera(ProgResCS10plus,Jenoptik).

    ImageswerecapturedandproducedbytheProgRessoftware(2.7.7).Imagesforeach

    specimenweretakenblindly(i.e.,specimenswerenumericallyassignedirrespectivelyoftheir

    chromosomalcomposition)inordertoavoidagainstunintentionalbiasesinMLH1scores.

    TheMicromeasure3.3software[4]wasusedfortheanalysisofrecombinationmaps

    (basedonthedistancesbetweenadjacentMLH1andRPAfoci).Foreachchromosome

    analyzed,thepositionofeachfocus(MLH1andRPA)wasrecordedasarelativeposition

    (percentageofthesynaptonemalcomplextotallength)fromthecentromere,identifiedbythe

    CRESTsignalineachpreparation.ThepositionsofMLH1/RPAfociwerecalculatedforeach

    chromosomeusingthecentromereasreferencepoint,(i.e.,fromthecentromeretothe

    telomereintheqarm).Thusforcomparisonamongchromosomes,thepositionsofMLH1foci

    wereexpressedastherelativepositionofeachCOtothelengthofthechromosome(the

    lengthofeachSCwasdividedinto10%intervals).Thesedatawereusedtoconstruct

    cumulativefrequencydistributionplotsandwecomparedthedifferentdistributionsusinga

    KolmogorovSmirnovtest(KStest).Moreover,weanalyzedthefrequenciesofMLH1focialong

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    SCsbydividingtheminthreeregionsdependingontherelativedistancefromthecentromere:

    (i)proximal(fromthecentromereto30%oftheSC)(ii)interstitial(between30%70%ofSC)

    and(iii)distal(from70%totelomericregion).FortheanalysisofH3K9me3chromosomal

    distribution,theMicromeasuresoftwarewasusedtocalculatethesignalarea(measuredin

    micrometers)overlappingtheSCforeachchromosomalarmanalyzed.

    ThestatisticalanalysiswascarriedoutbymeansofPAWSStatistics18andJMP7

    softwarepackage.Giventhatthedistributionofcrossoversperspecimendidnotfollowa

    normaldistribution(ShapiroWilktest,pvalue0.001),weappliednonparametricanalysis,

    suchasMannWhitneyUtestorKruskalWallistestformeancomparisonandSpearmantest

    forcorrelations.

    e)Prdm9genotyping

    GenomicDNAwasextractedandpurifiedfromcellculturesderivedfrommiceoftheRb

    polymorphismzone(n=27)followingthestandardphenolchloroformprotocol.Wesequenced

    thePrmd9exon12,fromZnrepeat+3towardsthecterminaldomain.Thisexoncontainsthe

    ZnfingerdomainarraythatrecognisesandmethylatesthespecificDNAsequences[5,6].

    PRDM9zincfingerarrayswereamplifiedbyPCRusingExTaq(Takara),with4%DMSO,

    primersfl1500U20and2848L23[5]asfollows:95C(3min),30cyclesof95C(30s);56C(30

    s);72C(90s).PCRproductswererunina2%agarosegelinordertodistinguishdifferent

    haplotypes(Fig.S5).Whenmicehadallelesofthesamelength(lengthhomozygotes,n=20),

    PCRamplifiedfragmentswerepurifiedwiththeGeneJETPCRpurificationkit(Fermentas).For

    micewithallelesoftwodifferentlengths(lengthheterozygotes,n=7),bandswerecutfromthe

    agarosegelandpurifiedusingtheGeneJETGelExtractionKit(Fermentas).Inbothcases,

    purifiedgeneproductsweresubjectedtobidirectionalSangersequencingwithprimers

    fl1822U24and2848L23[6]andreadsweresubsequentlyanalyzedusingtheCLCbioMain

    Workbenchprogram,version6.8(Aarhus,DK).Forthoseanimalsfoundheterozygotesfor

    allelesofthesamelength,twodifferentsoftwareswereusedinordertodistinguishallelic

    variantsanddefinethehaplotypes:Phase

    (http://stephenslab.uchicago.edu/software.html#phase)andChampuruv1.0

    (http://www.mnhn.fr/jfflot/champuru/).Inanycase,allnewallelicvariantswereconfirmedby

    resequencingandreanalyzed.

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    SupplementaryTables

    TableS1:Listofspecimens,localities,diploidnumber(2n),chromosomalcharacteristics,meannumberofMLH1foci/cell(standarddeviation,SD),numberofcellsanalyzedintherecombinationstudy(N)andPrdm9genotypingofmicefromthechromosomalpolymorphismareaofBarcelonaincludedinthestudy.St=micewithstandardkaryotype(2n=40).Rb=micewithRobertsonianfusions.n.a.=DNAnotavailableforgenotyping.

    Specimen Location 2n FusionsMeanMLH1foci/cellSD

    N Prdm9alleles

    St1 Arbeca 40 21.652.43 55 n.a.

    St2 Vacarisses 40 21.162.23 89 n.a.

    St3 CastellfollitdelBoix 40 20.901.74 20 10A/12B

    St4 CastellfollitdelBoix 40 20.801.43 21 12B/12B

    St5 CastellfollitdelBoix 40 20.681.35 22 10A/10A

    St6 CastellfollitdelBoix 40 20.681.43 25 10A/12B

    St7 CastellfollitdelBoix 40 20.570.87 21 10A/12C

    St8 CastellfollitdelBoix 40 20.571.45 14 10A/12C

    St9 CastellfollitdelBoix 40 20.271.40 26 12B/12B

    St10 CastellfollitdelBoix 40 20.161.17 25 10A/10A

    St11SantaPerptuade

    Mogoda40 20.581.02 31 10B/11B

    Rb1 SantSadurnd'Anoia 39 Rb(12.13) 20.261.68 27 n.a.

    Rb2 SantSadurnd'Anoia 37 Rb(4.14)+2(9.11) 19.712.22 40 10A/10A

    Rb3 SantSadurnd'Anoia 38 Rb(4.14)+(9.11) 19.711.65 46 10A/10A

    Rb4 SantSadurnd'Anoia 37 Rb(4.14)+(9.11)+(12.13) 20.571.90 46 n.a.

    Rb5 SantSadurnd'Anoia 37 Rb(4.14)+(9.11)+(12.13) 20.261.86 47 10A/10A

    Rb6 ElPapiol 38 Rb(4.14)+(12.13) 19.391.19 37 10A/10A

    Rb7 ElPapiol 38 Rb(4.14)+(12.13) 19.641.27 11 10A/10A

    Rb8 AmetlladeSegarra 39 Rb(4.14) 18.930.68 16 10A/10A

    Rb9 Castelldefels 29 Rb2(4.14)+2(9.11)+2(12.13)+2(3.8)+2(5.15)+(6.10) 19.951.67 44 10A/10A

    Rb10 Castelldefels 32 Rb2(4.14)+(9.11)+2(12.13)+2(5.15)+(6.10) 19.921.59 40 10A/10A

    Rb11 Castelldefels 30 Rb2(4.14)+(9.11)+2(12.13)+2(3.8)+2(5.15)+(6.10) 19.851.61 27 10A/10A

    Rb12 Castelldefels 30 Rb2(4.14)+2(9.11)+2(12.13)+(3.8)+2(5.15)+(6.10) 19.591.53 37 10A/10A

    Rb13 Castelldefels 28 Rb2(4.14)+2(9.11)+2(12.13)+2(3.8)+2(5.15)+2(6.10) 20.231.81 18 10A/10A

    Rb14 Castelldefels 30 Rb2(4.14)+2(9.11)+2(12.13)+(3.8)+2(5.15)+(6.10) 19.000.94 19 10A/10A

    Rb15 Castelldefels 29 Rb2(4.14)+2(9.11)+2(12.13)+2(3.8)+2(5.15)+(6.10) 18.951.25 22 10A/10A

    Rb16 Castelldefels 30 Rb2(4.14)+2(9.11)+2(12.13)+(3.8)+2(5.15)+(6.10) 18.73