Regulation of pluripotency in male germline stem cells by Dmrt1Supplementaldata.docx
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RegulationofpluripotencyinmalegermlinestemcellsbyDmrt1Supplementaldata
Supplementalinformationfor
RegulationofpluripotencyinmalegermlinestemcellsbyDmrt1
SeijiTakashima,MichikoHirose,NarumiOgonuki,MikiEbisuya,KimikoInoue,MitoKanatsu-Shinohara,TakashiTanaka,EisukeNishida,AtsuoOgura,TakashiShinohara
Supplementalinformationforthispapercontainsmaterialsshownbelow.
SupplementalFigureS1-10
SupplementalTableS1-5
Supplementalexperimentalprocedures
Supplementalreferences
SupplementalFigureS1.ConfirmationofKDbyrealtimePCR.（A）RealtimePCRanalysisofDnmt1inp53KOGScells7daysaftertransfection（n=3）.（B,C）RealtimePCRanalysisofp53（B）andDnmt1（C）inWTKOGScells7daysaftertransfection（n=3）.pSicoRwasusedasacontrol.
SupplementalFigureS2.ExpressionofGCTcandidategeneOE/KDinGScells.（A）ConfirmationofKDbyrealtimePCR.p53KOGScellswereinfectedwiththeindicatedlentivirusandgeneexpressionwasanalyzedbyrealtimePCR7daysaftertransfection（n=3）.WTGScellswereusedforBaxKD.pLKO.1EGFPwasusedasacontrol.（B）ConfirmationofOEbyRT-PCR.p53KOGScellswereinfectedwiththeindicatedlentivirusandgeneexpressionwasanalyzedbyRT-PCR7daysaftertransfectionusingtransgene-specific（cDNA-IRES2）primerpair.AlentivirusvectorcontainingtheappropriatecDNA-IRES2sequencewasusedasapositivecontrol.CyclinE1wasco-transfectedwithcyclinD2topromoteitstumorigenicity（Leeetal.2009）.CSII-EF1α-IRES2-hKO1wasusedasacontrolforHras,whereasCSII-EF1α-IRES2-Venuswasusedasacontrolfortherestofthegenes.（C）ConfirmationofOEbyrealtimePCR.p53KOGScellswereinfectedwiththeindicatedlentivirusandgeneexpressionwasanalyzedbyrealtimePCR7daysaftertransfection（n=3）.CSII-EF1α-IRES2-Venuswasusedasacontrol.moi,multiplicityofinfection.
SupplementalFigureS3.DerivationofDmrt1-mGScells.（A）Developmentofepiblast-likecoloniesduringreprogrammingthatoccurredinGScellsderivedfromatransgeniclinethatexpressesEGFPunderthecontrolofaNanogpromoter.（B）DerivationofDmrt1-mGScellsfromadultWTDBA/2GScells.Bar=200μm（A）,100μm（B）.
SupplementalFigureS4.CharacterizationofDnmt1-andDmrt1-mGS.（A）RT-PCRanalysis.（B）Flowcytometricanalysis.（C）Nanogimmunostaining.（D）BisulfitesequencinganalysisoftheDMRsofH19andIgf2r.Opencircles,unmethylatedCpG;closedcircles,methylatedCpG.（E）Designofthep53KDandDnmt1KDvectors.EGFPandtheshRNAoligoareflankedbyloxPsites（yellowtriangles）,whichcanberemovedbycre-mediatedrecombination.5’LTR,5’longterminalrepeat;Psi,packagingsignal;cPPT,centralpolypurinetract;CMV,cytomegaloviruspromoter;WPRE,woodchuckhepatitisvirusposttranscriptionalregulatoryelement;3’LTR∆U3,3’LTRwithdeletionoftheenhancerandpromotersequencesintheU3region.（F）Dnmt1-mGScellsinducedfromROSAGScells.Cre-mediatedrecombinationwasconfirmedbyextinctionofEGFPfluorescence.（G）Sectionofteratomasundertheskin,showingtissuesofthreegermlayers.（H）RT-PCRanalysisofspermatogoniamarkerexpressioninDnmt1-andDmrt1-mGScells.（I）COBRAoftailDNAfromoffspringofDmrt-mGSchimera.PCRproductsweredigestedwiththeindicatedenzymes.Percentmethylationisshownbelowthegels.U,non-cleaved;C,cleaved.Bar=100μm（C,G）,50μm（F）.
SupplementalFigureS5.RealtimePCRanalysisofDmrt1targetgenesinWT-GScellsafterDmrt1KD（n=3）.pLKO.1EGFPwasusedasacontrol.Allexperimentswerecarriedoutwithconcurrentp53KD.
SupplementalFigureS6.ApoptosisofGScellsbytransfection.（A,B）Suppressionofapoptosisbyp53deficiencyafterSox2（A）orOct4（B）OE.Foreachcelltype,atleast82cellswerecounted7daysaftertransfection（n=5）.CSII-EF1α-IRES2-hKO1andCSII-EF1α-IRES2-VenuswereusedascontrolsforSox2andOct4OE,respectively.（C）Suppressionofapoptosisbyp53deficiencyafterOctKD.Foreachcelltype,atleast100cellswerecounted7daysaftertransfection（n=5）.pLKO.1EGFPwasusedasacontrol.
SupplementalFigureS7.WesternblotanalysisofOct4andSox2expressioninp53KOGScells4daysafterSox2OE.CSII-EF1α-IRES2-hKO1wasusedasacontrol.
SupplementalFigureS8.RealtimePCRanalysisofOct4targetgeneexpressioninp53KOGScellsafterOct1OE（A）,Oct1KD（B）,Oct4OE（C）andOct4KD（D）14daysaftertransfection.CSII-EF1α-IRES2-VenusandpLKO.1EGFPwasusedascontrolsforOEandKDexperiments,respectively.ExpressionofDax1,Fgf4,Lefty1,Pml,andNanogwasnotdetectableinGScells.
SupplementalFigureS9.CharacterizationofSox2-andOct4-mGS.（A）RT-PCRanalysis.（B）Flowcytometricanalysis.（C）Nanogimmunostaining.（D）Invitrodifferentiation.（E）BisulfitesequencinganalysisoftheDMRsofH19andIgf2r.Opencircles,unmethylatedCpG;closedcircles,methylatedCpG.（F）DesignoftheSox2/Oct4removableexpressionvector.ThecDNAinsertisflankedbyloxPsites（yellowtriangles）,whichcanberemovedbycre-mediatedrecombination.DeletionofthecDNAinsertinitiatesDsRedexpression.5’LTR,5’longterminalrepeat;Psi,packagingsignal;cPPT,centralpolypurinetract;CMV,cytomegaloviruspromoter;WPRE,woodchuckhepatitisvirusposttranscriptionalregulatoryelement;3’LTR∆U3,3’LTRwithdeletionoftheenhancerandpromotersequencesintheU3region.（G）Sox2-mGSandOct4-mGSinducedfromgreenGScells.Cellswerethensubjectedtocre-mediateddeletionoftheSox2/Oct4transgene.Cre-mediateddeletionwasconfirmedbyexpressionofDsRedfluorescence.Bar=100μm（C,D）,50μm（G）.
SupplementalFigureS10.Scatterplotcomparisonoftheglobalgeneexpressionprofiles.Blacklinesindicateboundariesof2-folddifferenceingeneexpression.
SupplementalTableS1.SummaryofmGScellinduction
GScell
Genetransduction
Multiplicityofinfection
No.ofwells
cultured
No.ofwellswithmGScells
Daycoloniesexamined
p53KO
Dnmt1KD
2
3
2
28
Dmrt1KD
2
3
3
Dnd1KD
4.7
3
1
PtenKD
0.9
3
0
Cdk4OE
3.4
3
0
CyclinD2OE
CyclinE1OE
9.5
0.97
3
0
AktOE
0.3
3
0
β-CateninOE
1.7
3
0
HRasOE
0.8
3
0
Bcl6bOE
4.9
3
0
Fgfr3OE
4.8
3
0
Lats2KD
1.7
3
0
Sprouty4OE
3.2
3
0
KitLOE
4.7
3
0
TertOE
0.7
3
0
WT
pSicoR
20
6
0
pLKO.1EGFP
2
4
0
p53KD
10
22
0
Dnmt1KD
2
4
0
Dmrt1KD
2
7
0
Dnmt1KD
p53KD
2
10
15
3
Dnmt1KD
p53KD
10
10
12
9
Dmrt1KD
p53KD
2
10
30
21
Dnd1KD
p53KD
10
10
7
0
Sox2OE
p53KD
10
10
22
7
21
UTFOE
p53KD
10
10
12
0
p19KD
p53KD
10
10
20
0
p18KD
p53KD
10
10
12
0
Oct4OE
p53KD
10
10
31
12
Oct1KD
p53KD
10
10
12
3
Oct4OE
Oct1KD
p53KD
10
10
10
15
8
Oct4OE
Oct1OE
p53KD
10
10
10
8
3
Oct6KD
p53KD
10
10
12
0
Cells（5105）wereculturedin6-wellplates.
SupplementalTableS2.ContributionofmGScellstoembryonicdevelopment
Donorcell
Blastocyst
No.ofembryostransferred
No.ofembryosimplanted（%）
No.ofpupsborn（%）
Chimera
Male
Female
Dnmt1-mGS
B6
ICR
153
30
38（24.8）
NA
1（0.0）
10（33.3）
NA
1/4（25.0）
NA
0（0）
Dmrt1-mGS
B6
214
38（17.8）
2（0.9）
1/1（100.0）
0（0）
Sox2-mGS
B6
42
14（33.3）
9（21.4）
3/5（60.0）
1/3（33.3）
ICR
72
20（27.8）
9（12.5）
1/3（33.3）
2/5（40.0）
Oct4-mGS
B6
52
25（48.1）
3（5.8）
NA
NA
ICR
54
NA
21（38.9）
8/12（66.7）
4/8（50.0）
NA,notapplicable.Cannibalizedpupsarenotincludedinchimeraanalysis.
SupplementalTableS3.pLKO.1lentivirusvectorsusedinKDexperiments
Gene
TRCIDNumber
Bax
TRCN0000009668,TRCN0000009669,TRCN0000009670,TRCN0000009672
Dmrt1
TRCN0000084388,TRCN0000084389,TRCN0000084390,TRCN0000084391,TRCN0000084392
Dnd1
TRCN0000247049,TRCN0000247050,TRCN0000247051,TRCN0000257069
EGFP
TRCLentiviralEGFPshRNAPositiveControl
Lats2
TRCN0000022705,TRCN0000022706,TRCN0000022707
Oct1
TRCN0000175063,TRCN0000175090,TRCN0000240638
Oct4
TRCN0000009611,TRCN0000009612,TRCN0000009613,TRCN0000009614,TRCN0000009615
Oct6
TRCN0000075418,TRCN0000075419,TRCN0000075420,TRCN0000075421,TRCN0000075422
p18
TRCN0000085033,TRCN0000085034,TRCN0000085037
Pten
TRCN0000028990,TRCN0000028992,TRCN0000028993
Sox2
TRCN0000085748,TRCN0000085749,TRCN0000085750,TRCN0000085751,TRCN0000085752
SupplementalTableS4.PrimersforPCRandbisulfitesequencing
Gene
Forwardprimer
Reverseprimer
Akt
TTCCTCTCAAGAACGATGG
CTCTGTCTTCATCAGCTGGC
Akt-IRES2
AGATGATCACCATCACGCC
CGACATTCAACAGACCTTGC
β-catenin
AGCAAGCTCATCATTCTGGC
TGAACAAGTCGCTGACTTGG
β-catenin-IRES2
TTCACTCTGGTGGATACGGC
CTTATTCCAAGCGGCTTCG
Baf155
GATGTGTGTGCTGTGATGAGG
GAGGTGATCGAAGATGCAGG
Bax
AGGATGATTGCTGACGTGG
TGATGGTTCTGATCAGCTCG
Bcl6b
CCCGGGCTCAAGAGACTTC
TTCCTGGGCGGTGGATTAGC
Bcl6b-IRES2
GCAGCAGTGAAGAAGGAACC
CTTCGGCCAGTAACGTTAGG
c-myc
TGCAGGACCTCACCGC
CTTCTTGCTCTTCTTCAGAGTCG
Cdk4
TGCAGTCTACATACGCAACACC
TCGTCTTCTGGAGGCAATCC
Cdk4-IRES2
TAAGCCAATCTCTGCCTTCC
CGACATTCAACAGACCTTGC
Cripto
GGTCTATCCTCCATGGCACC
CATCACGTGACCATCACAGC
CyclinD2
TTCATTGAGCACATCCTTCG
TTCATCATCCTGCTGAAGCC
CyclinD2-IRES2
ATGATTGCAACTGGAAGCG
CTTCGGCCAGTAACGTTAGG
CyclinE1-IRES2
CTCAGGTTATCAGTGGTGCG
CTTCGGCCAGTAACGTTAGG
Dax1
ATCTGGAAGCAGGGCAAGTA
TCCTGTACCGCAGCTATGTG
Dmrt1
ATGAAGACCTCAGAGAGCCG
CAAGCCAGAATCTTGACTGC
Dnd1
AGTTCAGTACGCACCGAGC
GTTGACACAGCAGTTGCAGG
Dnmt1
ACTGGAAGAGGTAACAGCGG
GCCTCAATGATAGCTCTCTGG
Eras
TCAGATCCGCCTACTGCC
TTACCAACACCACTTGCACC
Esrrb
ACCATTCAAGGCAACATCG
CACACCTTCCTTCAGCATCC
Etv5
AACTTGGTGCTTCATGCTCC
ACTTAGCACCAAGAGCCTGC
Fbxo15
AGAAGCGTTGCTCTTCCTCC
AATGCAGCTGCGTACTTCC
Fgf4
GAGGCGTGGTGAGCATCTT
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