KLF6 Cooperates with NUR77 and SF1 to Activate the Human INSL3 Promoter in Mouse MA-10 Leydig Cells

56:3

m a tremblay and others KLF6 regulates human INSL3 56:3 163–173 Research transcription

KLF6 cooperates with NUR77 and SF1 to activate the human INSL3 promoter in mouse MA-10 leydig cells

Maxime A Tremblay1, Raifish E Mendoza-Villarroel1, Nicholas M Robert1, Francis Bergeron1 and Jacques J Tremblay1,2 Correspondence 1Reproduction, Mother and Child Health, Centre de recherche du centre hospitalier universitaire de should be addressed Québec, Québec City, Québec, Canada to J J Tremblay 2Centre for Research in Reproduction, Development and Intergenerational Health, Email Department of Obstetrics, Gynecology, and Reproduction, Faculty of Medicine, Université Laval, Jacques-J.Tremblay@ Québec City, Québec, Canada crchudequebec.ulaval.ca

Abstract

Insulin-like 3 (INSL3), a Leydig cell-specific hormone, is essential for testis descent during Key Words foetal life and bone metabolism in adults. Despite its essential roles in male reproductive ff insulin-like 3 and bone health, very little is known regarding its transcriptional regulation in Leydig ff transcription cells. To date, few transcription factors have been shown to activate INSL3 promoter ff KLF activity: the nuclear receptors AR, NUR77, COUP-TFII and SF1. To identify additional ff cooperation regulators, we have isolated and performed a detailed analysis of a 1.1 kb human INSL3 ff nuclear receptor promoter fragment. Through 5′ progressive deletions and site-directed mutagenesis, ff leydig cells we have mapped a 10 bp element responsible for about 80% of INSL3 promoter activity ff testis in Leydig cells. This element is identical to the CPE element of the placental-specific

Journal of Molecular Endocrinology glycoprotein-5 (PSG5) promoter that is recognized by the developmental regulator Krüppel-like factor 6 (KLF6). Using PCR and western blotting, we found that KLF6 is expressed in several Leydig and Sertoli cell lines. Furthermore, immunohistochemistry on adult mouse testis revealed the presence of KLF6 in the nuclei of both Leydig and Sertoli cells. KLF6 binds to the 10 bp KLF element at −108 bp and activates the −1.1 kb human, but not the mouse, INSL3 promoter. KLF6-mediated activation of the human INSL3 promoter required an intact KLF element as well as Leydig/Sertoli-enriched factors because KLF6 did not stimulate the human INSL3 promoter activity in CV-1 fibroblast cells. Consistent with this, we found that KLF6 transcriptionally cooperates with

NUR77 and SF1. Collectively, our results identify KLF6 as a regulator of human INSL3 Journal of Molecular transcription. Endocrinology (2016) 56, 163–173

Introduction

Insulin-like 3 (INSL3), also known as Leydig-insulin-like expressed in a sexually dimorphic pattern and produced (Ley-I-L) and relaxin-like factor (RLF), is a small peptide almost exclusively by Leydig cells. During foetal life, INSL3 hormone (20 aa) belonging to the insulin–IGF–relaxin was found to be a critical regulator of testicular descent. family of growth factors and hormones (Adham et al. Insl3-deficient mice exhibit bilateral undescended testes 1993, Pusch et al. 1996). During development, INSL3 is located high in the abdominal cavity (Nef & Parada 1999,

http://jme.endocrinology-journals.org © 2016 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/JME-15-0139 Printed in Great Britain Downloaded from Bioscientifica.com at 09/25/2021 12:41:07PM via free access

10.1530/JME-15-0139 Journal of Molecular Endocrinology ). mammalian species(reviewedinPearsonetal.2008). far, sixteenKLFfactors(KLF1-16)havebeenisolatedin scription factorsthatbelongtotheSP1/KLFfamily. So Zimmermann etal.1999).InInsl3 recently COUP-TFII(NR2F2)(Mendoza-Villarroel etal.2014). ( testosterone-activated androgen receptor (AR, NR3C4) NR4A1) (Tremblay &Robert2005,etal.2006),the et al.2003,Sadeghian2005),NUR77(NGFI-B, ( ity invariousspecies.TheseincludeSF1(Ad4BP, NR5A1) receptor familyareinvolvedinINSL3promoteractiv- shown thattranscriptionfactorsbelongingtothenuclear ing itstranscriptionalregulation.We andothershave littleisknownregard- isolated fromvariousspecies,very functional status(Forestaetal.2004,Ivell2013). highly specific markerof Leydig celldifferentiation and to itsimportantfunctionalroles,INSL3constitutesa metabolism inmales(Ferlinetal.2008,2013).Inaddition 1999). Duringadultlife,INSL3wasfoundtoregulatebone magnitude higherthaninadultfemales(Bullesbachetal. et al.1998),albeitexpressioninadultmalesisanorderof 1993, Puschetal.1996,Zimmermann1997,Balvers cells) andfemales(thecaluteal(Adhametal. 2003). Inadults,Insl3isexpressedinbothmales(Leydig had descendedovaries(Adhametal.2002,Koskimies is thefactthatfemaletransgenicmiceexpressingINSL3 INSL3 asafundamentalregulatorofgonadalpositioning β-cells (Adhametal.2002).Furthersupportforarole descent wasrestoredbyexpressingINSL3inpancreatic Laguë Zimmermann etal.1998,Koskimies2002,Truong DOI: 10.1530/JME-15-0139 http://jme.endocrinology-journals.org Research Krü Despite the fact that the &Tremblay 2008,Tremblay et al. 2009) and more ppel-like factors(KLF)areC promoter has been INSL3 promoter has been m

tremblay 2 H −/ 2 zincfingertran- mice,testicular Printed inGreatBritain andothers

human INSL3promoteractivity. the nuclearreceptors NUR77 andSF1tofurther stimulate more, we report that KLF6 functionally cooperates with the nuclei of somatic cells of the mouse testis. Further- the KLF6 transcription factor, which we have located in found thatthiselementisrecognizedandactivatedby element specifically in thehuman or RT-PCR. 2000) weredetectedinadulttestisbyNorthernblotting 1993, Inuzukaetal.1999), et al.1995), Godmann etal.2008)whereasmRNAforKlf2( to germ cells and Sertoli cells (Behr & Kaestner 2002, in thetestis.Sofar, KLF4hasbeenimmunolocalized littleisknownregardingtheirexpressionandroles very Although KLFshavebeendetectedinnumeroustissues, differentiation andtumorigenesis(Pearsonetal.2008). tissue-specific geneexpression,apoptosis,cellgrowth, diverse biologicalprocessesincludingcellproliferation, a widevarietyoftissuesandhavebeenimplicatedin of targetgenes(Pearsonetal.2008).KLFsarefoundin CA-rich sequenceCACCCpresentinthepromoterregion vators, repressors,orbothbybindingtotheconsensus Members of the KLF family act as transcriptional acti- The Plasmids Materials andmethods human transcription KLF6 regulateshumanINSL3 Published byBioscientifica Ltd. In thisstudy, wehaveidentifiedakeyregulatory − 1137, INSL3 promoterfragmentand the − Klf5 ( − 920, Sogawa etal.1993), − of the− difference. letter indicatesastatisticallysignificant MA-10 cells.Foreachcellline,adifferent ( previously characterizedNBRE/SF1 construct harbouringamutationinthe indicated ontheleftofgraph)ora (the 5′ constructs ofthehumanINSL3promoter co-transfected withvarious5′ and CV-1 fibroblasts(rightpanel)were promoter. MA-10Leydigcells(leftpanel) region intheproximalhumanINSL3 Localization ofanessentialregulatory Figure 1 activity (± and ARelements.Resultsareshownas% position oftheNBRE/SF1,SF1,COUP-TF the promoterdiagramsindicate depicted byalargeX.Thicklinesbelow Robert etal.2006).Themutationis 656, Downloaded fromBioscientifica.com at09/25/202112:41:07PM -end pointofeachconstructis 1137 − Klf13 andKlf14(

322 and− s . e . m bp wild-typereporterin . ) relativetotheactivity INSL3 promoter and 56 Klf6 ( 93 : 3 bp to+ deletion Sogawa etal. Scohy etal. Anderson

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Journal of Molecular Endocrinology into pcDNA3(Invitrogen)themouseKlf6cDNAwas KLF6 expressionvectorwasgeneratedbysubcloning reporter construct( constructs weresubclonedinamodifiedpXP1Luciferase aaC TTGACCTTTTTCCTGGGC-3′ TTT TTCCTG-3′ 5 CTG GCACTA ACaaCACCCTTGACCTTTTTC-3′ GGG CGGGTCCTGAAGAAT G-3′ GGT CC-3′ 5 in thestudyby ACC aac CCC TTG ACC TTT TTC CTG GGC G-3′ CCA CCCTTGACC-3′ M2 5′ TGG GAGAAAttaTCTGGCACTAACCCCACCC-3′ the mutationsareinlowercase: M15′ sequence ofthe sense oligonucleotide is shown),where along withthefollowingoligonucleotides(only using theQuikChangeXLMutagenesisKit(Stratagene) the contextof− trinucleotide anddinucleotidemutantconstructsin wild-type reporterinMA-10Leydig cells. Different lettersindicate statisticallysignificantdifferences. constructs to− described previously( containing amutationintheNBREat− indicate thepositionofNBRE/SF1, SF1,COUP-TFandARelements.Resultsareshownas%activity(± binding site( dinucleotide mutatedconstructs(M1 –M8; themutationsareinlowercase). TheunderlinedsequencecorrespondstothepreviouslydescribedNBRE/SF1 fibroblast cells)weretransfectedwith various−1137 bphumanINSL3promoterconstructs:awild-type andaseriesoftrinucleotideor Fine mappingofthe40 Figure 2 ′ ′ http://jme.endocrinology-journals.org DOI: 10.1530/JME-15-0139 Research -AAG GCTCTGGCACTA ACCCacCCCTTG -GGC ACTAACCCCACCTTtcaCTTTTTCCTGGGC -CCT GGGAGAAAGGCTCTGGacaTA ACC Robert etal.2006,Tremblay &Robert2005,Tremblay etal.2009 , M55′ 132 and− , M85′

bp elementintheproximalhumanINSL3 promoter. Severalcelllines(MA-10andMLTC-1 Leydigcells,MSC-1SertoliCV-1 Laguë -CCC CACCCTGACCTTTTgaaT Tremblay &Viger 1999 1137 Robert etal.2006 , M35′ -GCT CTGGCACTA ACCCCA &Tremblay (2008) 85 bp reporterweregenerated -AAG GCTCTGGCACTA bp havebeendescribed m

tremblay , M65′ -CCC TGGCCC 95 ). Thedeletion Printed inGreatBritain . Allreporter andothers bp havebeen ). Themouse -AAG GCT . Various , M4 , M7 ,

). TheCA-richsequenceisboxed.Thick linesbelowthepromoterdiagrams Sertoli celllineMSC-1( obtained fromATCC (Manassas,VA, USA).Themouse The mouseLeydigcelllinesMLTC-1, LC540andTM3were Cell culture andtransfections City, Canada). green monkeykidneyCV-1 fibroblastcellswereobtained medium supplementedwith15%horseserum.African of Iowa,IowaCity, IA,USA),weregrowninWaymouth’s (Ascoli 1981),providedbyDrMario(University University, Pullman,WA, USA).MouseMA-10Leydigcells provided byDrMichaelDGriswold(Washington State g All plasmids were verified bysequencing(Centrede Recherches CliniquesdeMontré (Laboratoire deGé et al.1997 Viger 2001 plasmid hasbeendescribedpreviously( TGC CTCTTCATG TGC-3′ cloning siteunderlined)5-CGGGAT CCTCAGAGG TGA AACTTTCACCTGCGCTCC-3′ (XbaIcloningsiteunderlined)5′ forward obtained byRT-PCR usingthefollowingprimers: transcription KLF6 regulateshumanINSL3 é Published byBioscientifica Ltd. nomique deQué ) waskindlyprovidedbyDrJacquesDrouin ). ExpressionvectorforNUR77( bec, CHULResearch Centre,Qué s . e n . m é McGuinness etal.1994 . ) relativetotheactivityof−1137 bp tique Molé Downloaded fromBioscientifica.com at09/25/202112:41:07PM . ThemouseSF1expression al, Montré culaire, Institutde 56 HI ; reverse(BamHI : 3 -GCT CTA GAA Tremblay & al, Canada). ) waskindly Philips 165 bec via freeaccess

Journal of Molecular Endocrinology binding. used inFig.2)andaroundtheCA-rich element.n.s.,non-specific that containmutations(M3,M4;numbers correspondtotheconstructs corresponding tothewild-type−108 bp element(wt)oroligonucleotides triangles; molarexcessesof2×and5 ×) ofunlabelledoligonucleotides Binding oftheproteinwasthenchallenged byincreasingdoses(black oligonucleotide correspondingtotheCA-richelementat−108 bp. MA-10 cells(MA-10N.E.)toadouble-stranded element. EMSAwasusedtoassessthebindingofnuclearextractsfrom A nuclearproteinfromMA-10cellsspecificallybindstotheCA-rich Figure 3 using the Superscript III Reverse Transcriptase System were synthesizedfroma5µ RNeasy Plus Extraction Kit (Qiagen). First-strand cDNAs Total RNAwasisolatedfromMA-10Leydigcellsusingthe RNA isolation,reverse transcriptionandPCR DNA preparations,eachperformedinduplicate. average ofatleastfourexperiments (n of aRSV-Luciferase reporter. Datareportedrepresentthe MSC-1 andCV-1 cellswerestandardizedtotheactivity Scientific). LuciferaseactivitiesinMA-10,MLTC-1, mega) and the Luminoskan Ascent luminometer (Thermo ties measuredusingtheDualLuciferaseAssaySystem(Pro- et al.2006).Thecellswerethenlysedandluciferaseactivi- as describedpreviously(Tremblay &Viger 2001,Robert plates usingthecalciumphosphateprecipitationmethod newborn calfserum.Alltransfectionsweredonein24-well from ATCC and grown in DMEM supplemented with 10% DOI: 10.1530/JME-15-0139 http://jme.endocrinology-journals.org Research g aliquot of the various RNAs g aliquotofthevariousRNAs m

tremblay 32 P-labelled ≥ 4) usingdifferent Printed inGreatBritain andothers

Recombinant KLF6proteinwasinvitro translatedusingtheT Electromobility shiftassays (1:5000, Vector Laboratories,Ontario,Canada). peroxidase-conjugated anti-goat antibody a secondary (C-20, 1:500dilution;SantaCruzBiotechnologies)and performed usingagoatpolyclonalanti-LMNB1antiserum Ontario, Canada).DetectionofLAMINB(LMNB1)was anti-rabbit antibody(1:5000dilution,Vector Laboratories, biotinylated Santa Cruz Biotechnologies) and a secondary KLF6 rabbitpolyclonalantiserum(R-173,1:200dilution, Detection ofKLF6wasperformedcarriedoutusingananti- instructions (Vector Laboratories,Ontario,Canada). avidin–biotin approach,accordingtothemanufacturer’s (Millipore). Immunodetection was performed using an by SDS–PAGE andtransferredontoPVDFmembrane boiled 10 control (Life Technologies). Nuclear proteins (15 siRNAs) directedagainstKLF6orscrambledsiRNAas 400 instruction (PolyPlus-transfection,Illkirch, France),with using jetPRIME as recommended in the manufacturer’s experiments, mouseMA-10Leydigcellsweretransfected estimated usingstandardBradfordassay. Inknockdown by Nuclear extractswerepreparedbytheprocedureoutlined Protein preparation blottings andwestern agarose gelelectrophoresisandethidiumbromidestaining. step of5 54° C-3′ 5 CCT GCTAC-3′ oligonucleotides used as probe were (the KLF element is oligonucleotides usedasprobe were(theKLFelementis ( or 3 were performedusingeither 3 Quick CoupledTnTSystem(Promega). DNAbindingassays 30 cyclesofdenaturation(45 under thefollowingconditions:5 DNA polymerase(NewEnglandBiolabs,Beverly, MA,USA) thermocycler (Biometra,Gö PCRs forKlf6andTuba1a wereperformedonaT previously (Moisanetal.2008).Semi-quantitative underlined) 5′ following (Life Technologies). PCRswereperformedusingthe transcription KLF6 regulateshumanINSL3 Martin &Tremblay 2005).The ′ Published byBioscientifica Ltd. -GGG GTA CCAGCCCCATAG TTGAGATTC C) andextension(45 Schreiber . Theprimersforα -translated protein as described previously μL ofinvitro-translated proteinasdescribed previously nM ofsiRNA(amix133 min at72° min inadenaturingloadingbuffer, fractionated I cloning site (XbaIcloningsite Klf6-specific primers:forward t al.(1989)Proteinconcentrationswere et -GCT CTA GA I cloning site underlined): ; reverse(KpnIcloningsiteunderlined): C. PCR products were then analysed by C. PCRproductswerethenanalysedby Tubulin ( Downloaded fromBioscientifica.com at09/25/202112:41:07PM s at72° ttingen, Germany)usingVent T ATC TTCAGAGTGAGC s at95° μ 32 g of MA-10 nuclear extracts g ofMA-10nuclearextracts P-labelled double-stranded P-labelled double-stranded C) with a final extension C) witha final extension Tuba1a ) weredescribed min at95° nM ofthreedifferent C), annealing(45 56 : 3 C followed by C followedby µg) were 166 gradient s at s at via freeaccess 7

Journal of Molecular Endocrinology was replaced with normal rabbit IgG (SC2027, 4 the sameprocedureexceptthatanti-KLF6antibody Cruz Biotechnologies).Negativecontrolcorrespondsto KLF6 rabbitpolyclonalantiserum(R-173,4 KLF6 proteinlocalizationwasassessedusingananti- ABC reagent(Vector Laboratories,Ontario,Canada). to themanufacturer’s instructionsfortheVectastain Elite was performedusinganavidin– and antisense5′ underlined): sense5′ 0.5% goatseruminPBSfor1 Following paraffinremoval,tissueswereblockedwith xylene, embeddedinparaffinandcutinto5 Tissues werethendehydrated withethanol,substituted fixed withice-cold4%paraformaldehyde(w/v)for24 Adult CD-1mice(~ Immunohistochemistry were addedtothebindingreaction. anti-KLF6 antiserum(R-173,SantaCruzBiotechnologies) 3 or5 added to the reaction. For supershift/blocking experiments, as describedaboveforthepromotermutagenesis)were double-stranded oligonucleotides (wild-type or mutated the competitionexperiments,2-or5-foldmolarexcessof killed byCO http://jme.endocrinology-journals.org DOI: 10.1530/JME-15-0139 Research μ g of normal rabbit IgG or a commercially available g ofnormalrabbitIgGoracommercially available 2 inhalation. The testes were harvested and and inhalation.Thetesteswereharvested -AGG TCAAGGGTG 40 -ACT AACCCCACCTTGACCT-3′ day old) were obtained on site and day old)wereobtainedonsiteand h at25° biotin approach according biotin approachaccording m

tremblay C. Immunodetection C. Immunodetection G GG TTA GT-3′ Printed inGreatBritain andothers μ µ g/mL; Santa g/mL; Santa M sections. M sections. μ g/mL, g/mL, . For . For h. h.

San Jose,CA,USA). using SigmaStatsoftwarepackage(SystatSoftwareInc, statistically significant.Allstatisticalanalysesweredone For allstatisticalanalyses,P met) followed by a Student–NewmanKeuls post test. ANOVA orANOVA onranks (when parameterswerenot Statistical analyseswerecarriedoutusingone-way Statistical analyses Ethics CommitteeofLavalUniversity(protocol#2009011). Care and have been approved by the Animal Care and conducted according tothe Canadian Council for Animal Mount-Royal, Qué with hematoxylinGillno.1(VWRInternational, as substrateandthesectionswerecounterstained using AEC(3-amino-9-ethylcarbazolefromSigma-Aldrich) Santa CruzBiotechnologies).Finaldetectionwasdone the well-characterizedMA-10celllinemodel.To locate several mouseLeydigcelllineswereused,including As thereiscurrentlynohumanLeydigcelllineavailable, proximal humanINSL3promoter Mapping ofanimportantregulatory elementinthe Results transcription KLF6 regulateshumanINSL3 Published byBioscientifica Ltd. org/10.1530/JME-15-0139. version ofthisfigureisavailableathttp://dx.doi. control (inset).Magnifications:400× Omission oftheprimaryantibodyserved asnegative (arrowheads) cellsarelabelled(red-orange staining). antiserum. ThenucleiofLeydig(arrows)andSertoli on adultmousetestissectionsusingananti-KLF6 somatic cells.Immunohistochemistrywasperformed (D) Withinthetestis,KLF6isspecificallyexpressedin cells; LAMINB(LMNB1)wasusedasaloadingcontrol. confirm KLF6proteinlevelsinknockdownMA-10 (siRNA KLF6)andwesternblottingswereusedto scrambled siRNA(siRNACtl)oragainstKLF6 (C) MouseMA-10Leydigcellstransfectedwith LAMIN B(LMNB1)wasusedasaloadingcontrol. or anexpressionvectorforKLF6(MA-10+KLF6). expression vector (MA-10+Ctl) empty an with fected (CV-1), aswellfromMA-10Leydigcellstrans- MLTC-1, LC540,TM3),Sertoli(MSC-1),fibroblast including Leydig (MA-10, lines cell various from blottings wereperformedusingnuclearextracts control. (B)FordetectionofKLF6protein,western (MSC-1) celllines;α Leydig (MA-10,MLTC-1, LC540,TM3)andSertoli Klf6 wasusedtodetectthepresenceofmRNAin Semi-quantitative RT-PCR usingprimersspecificfor KLF6 isexpressedintesticularsomaticcells.(A) Figure 4 bec, Canada). All experiments were bec, Canada).Allexperimentswere Downloaded fromBioscientifica.com at09/25/202112:41:07PM < 0.05 wasconsideredtobe Tubulin ( 56 Tuba1a ) asused : 3 . A full colour . Afullcolour 167 via freeaccess

Journal of Molecular Endocrinology a mutationat− a and/or COUP-TFIIelement,cellsweretransfectedwith − possibility thatthelossinpromoteractivitywith promoter (Mendoza-Villarroel etal.2014).To testthe ( site forthebindingofnuclearreceptorCOUP-TFII & Robert2005,etal.2006),aswellaDR0 NUR77 thatcanalsoberecognizedbySF1(Tremblay NBRE/SF1 bindingsiteat− − a elementsarelocatedwithin that importantregulatory 15– decrease in human INSL3promoteractivity, with only ( − et al.2005)andourunpublisheddata).Deletionto express with thefactthatMA-10Leydigcellsendogenously compared with CV-1 fibroblast cells,which isconsistent reporter constructwashigherinMA-10Leydigcells fibroblasts. As shown inFig. 1, theactivity of a transfected inmouseMA-10LeydigcellsandCV-1 promoter, various5′ elements in the human important regulatory − Fig. 1).Furtherdeletionto− DOI: 10.1530/JME-15-0139 http://jme.endocrinology-journals.org 93 93 132 − ~ Research 91/− 40 1137 humanINSL3 17% ofactivityremaining(Fig.1).Thisindicates bp constructmightbeduetoremovaloftheNBRE/SF1 bp. Thisregioncontainsapreviouslycharacterized bp didnotsignificantlyaffectpromoteractivity bp regionlocatedbetweennucleotides− 103 Insl3 whereasCV-1 cellsdonot((Sadeghian bp) originallyidentifiedinthemouseInsl3 97 progressivedeletionconstructswere bp knowntoabolishbindingof reporter constructthatharbours 95 bp for the nuclearreceptor 93 m

tremblay Printed inGreatBritain andothers − 132 and 1137 INSL3 bp bp

within the conferring nearly80%ofhumanINSL3promoteractivity binding siteforthenuclearreceptors. − that themajorityofactivitywithin− decrease of~ et al.2014).Asshownin et al. 2006) aswell as COUP-TFII(Mendoza-Villarroel NUR77 and SF1 (Tremblay & Robert2005, Robert of three nucleotidesinaCA-richsequenceresultedloss In MA-10andMLTC-1 Leydigcells,mutantM3thatchanges Leydig cell lines compared with Sertoli and fibroblast cells. human and CV-1 fibroblasts.AsshowninFig.2,activityofthe lines, MA-10andMLTC-1, aswellinSertoliMSC-1cells promoter weregeneratedandtransfectedintwoLeydigcell reporter constructsinthecontextof− promoter activity None ofthemutationsdecreasedINSL3promoteractivity cells, albeitlessthanthetrinucleotidemutantM3(Fig.2). led todecreasedhumanINSL3promoteractivityinLeydig nucleotides intheCA-richregion(M6,M7andM8)also 2).Additionalmutationsthatchangetwo and M5)(Fig. than M3 or did not reduce INSL3 promoter activity (M2 and M4) but to a lesser extent either sideofthisCA-richsequencealsodecreased transcription KLF6 regulateshumanINSL3 93 Published byBioscientifica Ltd. ~ ). Mutations on 75% inINSL3promoteractivity(Fig.2).Mutationson To furtherdelineatethesequencesresponsiblefor bp region is conferred by element(s)other than the promoter is significantly higher in both INSL3 promoterissignificantlyhigherinboth − 18– 132 to 20% ofpromoteractivity. Thisindicates produced KLF6 to a double-stranded, vitro-produced KLF6toadouble-stranded, element. EMSAwasusedtoassessthebindingofin produced KLF6specificallybindstotheCA-rich MA-10 Leydigcells(MA-10N.E.).(B)Invitro- reticulocytes (TNTCtl)andnuclearextractsfrom vitro-produced KLF6(TNTKLF6),unprogrammed the humanINSL3promoteralongwithin corresponding totheCA-richelementat− CA-rich elementat− IgG wasusedascontrol. 5 (++)gofaKLF6antiserum(α KLF6 bindingwasblockedbyaddition of3(+)and in MA-10LeydigcellstotheCA-richelement. The determine thebindingofKLF6protein present binds totheCA-richelement.EMSA was usedto element. (C)KLF6fromMA-10nuclearextracts mutations within(M3)andoutside(M4)theCA-rich (wt) ortwooligonucleotidesthatharbour corresponding tothewild-type− 2 increasing doses(blacktriangles;molarexcessesof promoter. KLF6bindingwasthenchallengedby 32 a double-stranded, MA-10 nuclearextracts.EMSAwasperformedusing vitro-produced KLF6co-migrateswithaproteinin KLF6 bindstotheCA-richelement.(A)In Figure 5 × P-labelled oligonucleotide corresponding to the P-labelled oligonucleotidecorrespondingtothe and5× − 93 Downloaded fromBioscientifica.com at09/25/202112:41:07PM bp regions, a series of mutated ) of unlabelled oligonucleotides ) ofunlabelledoligonucleotides Fig. 1, this mutationledtoa promoter activity (M1 INSL3 promoter activity (M1 32 108 P-labelled oligonucleotide P-labelled oligonucleotide bp inthehumanINSL3 56 : 3 KLF6). Normal rabbit KLF6). Normalrabbit 108 bp element bp element 1137 108 132 to 168 bp in bp in

bp bp via freeaccess

Journal of Molecular Endocrinology in CA-rich sequence in the human Leydig cell nuclear extracts could specifically bind to the Next, weusedEMSAtodeterminewhetherprotein(s)from Binding ofaspecificprotein totheCA-richsequence maximal humanINSL3promoteractivityinLeydigcells. previously characterized in thehumanplacental-specific ingly similar to the CPE element (CTGACCCCACCCATG) human Sequence analysisrevealedthattheCA-richelementof KLF6 isexpressed inthe mousetestis the CA-richsequenceofhumanINSL3promoter. MA-10 Leydigcellnuclearextractsspecificallybindsto the binding( limited effectonpromoteractivity, M4 (mutationoutsidetheCA-richsequencewhichhas and 6), whereas oligonucleotides corresponding to mutant could notdisplacethebindingcomplex( CA-rich sequencethatbluntspromoteractivity, otides correspondingtomutantM3(mutationwithinthe for promoteractivity. Asshownin containing the same mutations as those shown in petition experimentsusingunlabelledoligonucleotides lanes 3and4).Bindingspecificitywasassessedbycom- of unlabelledwild-type(WT)oligonucleotides( be competedwithincreasingmolarexcess(2-and5-fold) located between− together, theseresultsindicatethataCA-richsequence in SertoliMSC-1andCV-1 fibroblastcells( activation overcontrol(± cells (middlepanel)andMSC-1Sertoli cells(rightpanel).ThepositionoftheKLFelementisrepresentedbyablack rectangle.Resultsareshownasfold expression vector(− wild-type orharbouringamutation (depicted byalargeX)intheCA-richelementthatpreventsKLF6binding (M3in KLF6 activatesthehumanINSL3promoter. MA-10Leydigcellsweretransientlytransfectedwith two− Figure 6 http://jme.endocrinology-journals.org DOI: 10.1530/JME-15-0139 Research Fig. 3 , abindingwasdetected( INSL3 promoter(CTAACCCCACCCTTG) isstrik- Fig. 3 , openbars)oranexpressionplasmid encodingKLF6(+,blackbars)weretransfectedinMA-10Leydigcells(left panel),CV-1 fibroblast , lanes7and8).Thus,aproteinfrom

112 and− s . e . m . ). Anasteriskindicatesastatisticallysignificant difference from control. 105 INSL3 promoter. As shown m Fig. 3

Fig. 2 bp is important for bp isimportantfor tremblay Fig. 3 , lane2)thatcould ) couldcompete Printed inGreatBritain Fig. 3 andothers , oligonucle- Fig. 2 , lanes5 Fig. 3 Fig. 2 Fig. 2 ). All ). All ) ,

mouse testis, KLF6 is present in the nuclei of somatic cells. antibody was omitted ( whentheprimary specific sincenosignalwasobserved et al.1999 Sertoli (arrowheads in protein levelswhilecontrolsiRNAhadnoeffect( in MA-10cellsleadtoasignificantreductionKLF6 Conversely, transfectionofsiRNAsdirectedagainstKLF6 KLF6. Asshownin empty expression vector or an expression vector encoding ( (MSC-1) celllinesbutnotintheCV-1 fibroblastcellline Leydig (MA-10,MLTC-1, LC540andTM3)Sertoli KLF6 proteinwasdetectedbywesternblottingsinboth At theproteinlevel,abandof45 shown in and aSertolicellline(MSC-1)wasfirstperformed.As ous Leydigcelllines(MA-10,MLTC-1, LC540andTM3) expressed inLeydigcells.RT-PCR usingcDNAsfromvari- detected inthewholetestisbyNorthernblotting( cially KLF6 ( bers oftheKLFfamilytranscriptionfactors,andespe- This elementinthePSG5promoterisrecognizedbymem- glycoprotein 5(PSG5)promoter staining) inthenucleiofLeydig(arrows ting results,theKLF6proteinwasdetected(red-orange sections. InagreementwiththeRT-PCR andwesternblot- onadultmousetestis performed immunohistochemistry further confirmKLF6expressioninLeydigcellsvivo,we data validatethespecificityofanti-KLF6antibody. To Both theoverexpression( serum intheKLF6-overexpressingMA-10cells( was detectedbywesternblottingwiththeanti-KLF6anti- transcription KLF6 regulateshumanINSL3 Fig. 4 Published byBioscientifica Ltd. B). MA-10Leydigcellswerealsotransfectedwithan Fig. 4 ), wesoughttodeterminewhetherKLF6was Koritschoner et al. 1997 A, 1137 Klf6 mRNA was detected in all cell lines. Fig. 4 reporter constructs either bp humanINSL3reporterconstructseither Fig. 4 Fig. 4 Downloaded fromBioscientifica.com at09/25/202112:41:07PM Fig. 4 B, abandofincreasedintensity Figs 3 D, inset). Thus, within the B) andknockdown( D) cells. This labelling is and kDa correspondingtothe Koritschoner etal.1997 4 ) along with an empty ) alongwithanempty ). As 56 : 3 Klf6 has been Fig. 4 Fig. 4 Fig. 4 Inuzuka Fig. 4 D) and 169 C). B). C) via freeaccess ).

Journal of Molecular Endocrinology Different lettersindicatestatisticallysignificantdifferences. and KLFelements.Resultsareshown as foldactivationovercontrol(± promoter diagramsindicatetheposition oftheNBRE/SF1,SF1,COUP-TF, AR (open bars)orinthepresenceofKLF6 ( expression vector(CTL)or vectors forSF1andNUR77eitheralone was transientlytransfectedinMA-10 Leydig cellsalongwithanempty construct containinga− blottings. LAMINB(LMNB1)wasusedasaloadingcontrol.(B)Areporter cells, MSC-1SertolicellsandCV-1 fibroblastcellswasdeterminedbywestern INSL3 promoter. (A)ProteinlevelsofKLF6,NUR77andSF1inMA-10Leydig KLF6 cooperateswiththenuclearreceptorsSF1andNUR77onhuman Figure 7 DOI: 10.1530/JME-15-0139 http://jme.endocrinology-journals.org Research 1137 promoter bp fragmentofthehumanINSL3promoter solid bars). Thick lines above the solid bars).Thicklinesabovethe m

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. ). ). the factthatKLF6bindsto− Sertoli cellsbutnotinCV-1 fibroblasts.Consistentwith INSL3 reporterconstructinMA-10LeydigandMSC-1 of KLF6ledtoincreasedactivitythe− and MSC-1Sertolicells.AsshowninFig.6,expression transfections inMA-10Leydigcells,CV-1 fibroblasts INSL3 promoteractivity, weperformedtransient To determineifKLF6coulddirectlyregulatehuman KLF6 activatesthehumanINSL3promoter −108 bp inthehumanINSL3promoter. MA-10 LeydigcellscanbindtotheCA-richelementat Thus endogenousKLF6presentinnuclearextractsfrom tion ofan anti-KLF6 antiserum(Fig.5C, lanes 4and6). in MA-10 nuclearextracts could be competed by the addi- KLF6 binding(Fig.5B).Furthermore,thecomplexpresent in the CA-rich sequence was not as efficient at competing while theM3oligonucleotidethatcontainsamutation and M4(mutationoutsidetheCA-box)oligonucleotides, assays revealedthatKLF6bindingwasdisplacedbyWT from MA-10Leydigcells.Furthermore,competition co-migrates withaproteinpresentinnuclearextracts by EMSA.AsshowninFig.5A, to theCA-richsequenceofhumanINSL3promoter We nexttestedwhetherinvitro-produced KLF6 could bind INSL3 promoter KLF6 bindstotheCA-richsequenceofhuman human KLF6. Asexpected,bothSF1 andNUR77activatedthe transcription factors might functionally cooperate with CV-1 cells(Fig.7A),wetestedthepossibility thatthese SF1 areexpressedinLeydig andSertolicellsbutnotin elements areadjacent(Fig.2),andsinceNUR77 from CV-1 fibroblasts.Because theKLFandNBRE/SF1 are common to both Leydig and Sertoli cells but absent indicates thatKLF6likelyrequiresotherfactor(s) INSL3 promoteractivityinheterologousCV-1 cells(Fig. 6) The factthatKLF6wasunabletostimulatethehuman on thehumanINSL3promoter KLF6 transcriptionallycooperateswithNUR77andSF1 requires anintactKLFelementat− activation of thehumanINSL3promoterby KLF6 ). Thus,maximal Leydig andMSC-1Sertolicells(Fig. 6 was nolongersignificantlyactivatedbyKLF6inMA-10 construct harbouringamutationintheKLFelement that wenownametheKLFelement,a− transcription KLF6 regulateshumanINSL3 Published byBioscientifica Ltd. INSL3 promoter2.2-and3.7-fold, respectively, in Downloaded fromBioscientifica.com at09/25/202112:41:07PM 108 in vitro-produced KLF6 bp CA-richsequence 108 56 : 1137 3 bp. 1137 bp reporter bp human 170 via freeaccess

Journal of Molecular Endocrinology CACC motif(M6andM7) hadminimalornoeffect. an intact cell lines, whereas mutations thatpreserved promoter activityto~ or CAAAC)resultedina dramatic decreaseinINSL3 that increased the number of As (e.g. CACto CAAC cell lines,MA-10andMLTC-1. Mutations(M3andM8) core CACtrinucleotideswhenanalysedintwoLeydig CA-rich sequence(CCCCACCC) and particularlythe the Mutation of 2 or3ntin this regioninthecontextof region, more precisely between INSL3 promoterarelocatedwithinthe− elements ofthehuman revealed thatthemainregulatory conferring Leydigcell-specificactivity. Deletion analyses elements INSL3 promoterfragmentcontainsregulatory in Leydigcells.Thisindicatesthatthe1.1 this promoterfragmentwasmoreactiveby~ heterologous (CV-1 fibroblasts) cell lines revealed that promoter inahomologous(MA-10Leydigcells)and Comparison oftheactivitya− A CA-richsequenceessentialforINSL3promoter activity human in Leydigcellswhereitcontributestotheactivityof found thattheKrü Tremblay 2008,Tremblay etal.2009).Inthisstudy, we 2005, Tremblay &Robert2005,et al. 2006,Laguë Koskimies etal.2002,Truong etal.2003,Sadeghian al.1998, (NR4A1) andAR(NR3C1)(Zimmermannet activated bythenuclearreceptorsSF1(NR5A1),NUR77 et al.2005) et al. 1998, Koskimies et al. 2002) and rat (Sadeghian sufficient toconferactivitythemouse(Zimmermann upstream ofthetranscriptionstartsitewerefoundtobe the MA-10Leydigcelllineasamodel,first200 within arelativelyshort5′ expression inLeydigcellsarebelievedtobecomprised motifsdrivingINSL3 gene structure,theregulatory intron (mouse) of the immediately downstream (human) or within the last Depending onthespecies,INSL3geneislocatedeither Discussion the humanINSL3promoter. receptors NUR77andSF1transcriptionallycooperateon the SF1 orwithNUR77ledtoastrongeractivation(~7-fold)of MA-10 Leydigcells(Fig.7B).CombinationofKLF6with http://jme.endocrinology-journals.org DOI: 10.1530/JME-15-0139 Research INSL3 promoter (Fig. 7B). Thus KLF6 and the nuclear − 1.1 INSL3 promoterbutnotthemouseInsl3promoter. kb reporteruncoveredtheimportanceofa Insl3 promoter. Thisregionisalsoknowntobe ppel familymemberKLF6isexpressed Jak3 gene. Because of this unusual 25% ofwildtypeinboth Leydig flankingsequence.Using m

1.1 tremblay − 132 and kb humanINSL3 132 Printed inGreatBritain andothers bp proximal kb human − 93 500% bp. bp bp &

in endogenousINSL3transcriptionremainsto be reporter constructinLeydig celllines,itsinvolvement gene transcriptioninLeydig cells. other KLF family members could also be involved in of KLF6,wecannotformallyexcludethepossibilitythat which KLF6binds, was mutated.Apartfromtheimplication transactivation wasabrogatedwhentheCA-richmotif, to INSL3 promoter in mouse MA-10 Leydig cells, and this tissue/testis). Furthermore,KLF6canactivatethehuman ( cells, albeitwithmoderateintensityandlowfrequency revealed thepresenceofKLF6innucleusLeydig testis. In humans, data from the Human Protein Atlas project the nucleiofbothLeydigandSertolicellsinadultmouse Leydig andSertolicelllines tested. KLF6 was alsodetected in was detectedatthemRNAandproteinlevelinallmouse to furthercharacterizethistranscriptionfactor. KLF6 have mappedinthehumanINSL3promoter, wechose regionwe essentially identicaltothecriticalregulatory in Leydig cells. Because KLF6 binds to asequence that is 2002, Godmannetal.2008),nonehavebeenreported Inuzuka etal.1999,Scohy2000,Behr&Kaestner in thetestis(Sogawaetal.1993,Anderson1995, et al.1997).AlthoughsomeKLFfactorshavebeendetected to thisCA-richmotifintheCPEelement(Koritschoner promoter. TheKLFfamilymemberKLF6wasfoundbound lined) totheCPEelementidentifiedinhumanPSG5 nearly identical(13/15nt;mismatchesareunder- the human The CCCCACCCmotifandsurroundingsequencesof KLF6 isanovelactivatorofthehumanINSL3promoter rat, porcine, bovineand canine(datanotshown). promoter of all other species analysed including mouse, human the CA-richsequence(CCCCACCC)isuniqueto Koskimies etal.2002;andourunpublishedobservations), et al.2006)andSF1at− ( ( such astestosterone/ARresponsivenessat− elements and mouseINSL3promotersshareconserved promoter activityinLeydigcells.Althoughthehuman the CACCmotifisthusindispensableforhumanINSL3 (MSC-1 Sertoli and CV-1 fibroblast cells). The integrity of in celllinesthatdonotendogenouslyexpressInsl3 None of the mutations reduced ( transcription KLF6 regulateshumanINSL3 Zimmermann etal.1998,Koskimies2002,Robert Mendoza-Villarroel etal.2014 ), NBRE/SF1at− Laguë http://www.proteinatlas.org/ENSG00000067082-KLF6/ Published byBioscientifica Ltd. Although KLF6canregulate ahumanINSL3 &Tremblay 2008),COUP-TFIIat− INSL3 promoterandwasnotfoundintheInsl3 INSL3 promoter(CTAACCCCACCCTTG) are 45 Downloaded fromBioscientifica.com at09/25/202112:41:07PM bp (Zimmermannetal.1998, INSL3 promoteractivity 56 : 3 103 to− 118 to− 95 INSL3 91 171 92 bp bp bp bp bp bp via freeaccess

Journal of Molecular Endocrinology Leydig cells. that directs human the combinatorialcode oftranscriptionfactors in Leydigcells.Morework isrequiredtofullydecipher INSL3 promoterissignificantly lowerinSertolithan is supportedbythefactthatactivityofhuman involved, asINSL3isnotexpressedinSertolicells.This in LeydigcellsbutnotSertolimustalsobe promoter activity. Despitethis,otherfactor(s) present with bothSF1andNUR77inregulatinghumanINSL3 this, wefoundthatKLF6functionallycooperates SF1 andNUR77(Robertetal.2006).Consistentwith characterized bindingsiteforthenuclearreceptors human Tremblay 2010).Interestingly, theKLFelementin White 2010)andNUR77(reviewedinMartin& the nuclearreceptorsSF1(reviewedinSchimmer& finger factorGATA4 (reviewedinViger etal.2008)and MADS-box factors MEF2 (Daems et al. 2014), the zinc present inbothSertoliandLeydigcellsincludethe from fibroblasts.Transcription factorsknowntobe are commontoLeydigandSertolicellsbutabsent KLF6 requiresthepresenceofadditionalfactor(s)that indicates thatinadditiontobindingitselement, cell lineMSC-1butnotinCV-1 fibroblastcells.This activate thehumanINSL3promoterinSertoli In additiontoMA-10Leydigcells,KLF6couldalso NUR77 andSF1onthehumanINSL3promoter KLF6 functionallycooperateswiththenuclearreceptors to theactivityofhumanINSL3promoter. regulation ofmouseInsl3geneexpressionbutcontributes together, thesedataindicatethatKLF6isnotinvolvedinthe lacks aCCCCACCCmotifforthebindingofKLF6.Taken consistent withthefact that themouseInsl3promoter MLTC1 Leydigcells(datanotshown).Alltheseresultsare the mouseInsl3promoter(1.1 Insl3 promoterregion.Finally, KLF6failedtoactivate onthemouse but norecruitmentofKLF6wasobserved (data not shown).ChIP experiments were also performed endogenous Conversely, overexpressionofKLF6didnotincrease Insl3 mRNA levels were not decreased (data not shown). In KLF6-depleted(usingsiRNA)mouseMA-10Leydigcells, transcription endogenouslyinmouseMA-10Leydigcells. order todeterminewhetherKLF6couldregulateInsl3 cell lineavailable,weturnedtothemousesystemin humans andsincetherearecurrentlynohumanLeydig demonstrated. Sincethiscannotbeeasilyaddressedin DOI: 10.1530/JME-15-0139 http://jme.endocrinology-journals.org Research INSL3 promoterisadjacenttoapreviously Insl3 mRNAlevelsinmouseMA-10Leydigcells INSL3 expression exclusively in kb fragment)inMA-10and m

tremblay Printed inGreatBritain andothers

plasmids usedinthisstudy. de recherchescliniquesMontréal,Canada)forprovidingcelllinesand Griswold (Washington State University, USA),JacquesDrouin(Institut The authorsthankDrsMarioAscoli(UniversityofIowa,USA),Michael Acknowledgements and wrotethepaper. N MR,FBandJTanalysedthedata.designedresearchstudy M AT, REMV, NMRandFBperformedtheresearch.AT, REMV, Author contribution 81387) toJT. Canadian InstitutesofHealthResearch(fundingreferencenumberMOP- recherche duQuébec—Sant.Thisworkwassupportedbyagrantfromthe J TholdsaChercheur-Boursier SéniorScholarshipfromtheFondsdela Funding perceived asprejudicingtheimpartialityofresearchreported. 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38. 13 13 via freeaccess