promoter mutation in ccna2 reveals novel functions of the protein

Promoter Mutation in Ccna2 Reveals Novel Functions of the Protein in Spermatogenesis Manuel Torres, Lindsey N. Kent, Maria Cuitino, Yannis Hadjiannis, Jim Dowdle and Gustavo Leone

INTRODUCTIONThecyclinsareasetofproteinsthatplayakeyroleinregula4ngthecellcyclebyac4va4ngtheCDKs,whichinpart

coordinate the events that lead to cellular division (both mito4c and meio4c). Cyclin A2 (CCNA2) is the regulatorysubunitofCDK1and2;together,theyphosphorylatespecificproteinsattheSphaseandduringtheG2/Mtransi4on.1

During spermatogenesis (Fig 3C), germ cells called Spermatogonial Stem Cells (SSCs) undergo subsequentdifferen4a4oneventsun4lspermatozoaareproduced.SSCsare themaincell typethatexpressesCcna2,andare thesourceofcon4nuousproduc4onofsperm.2 Interes4ngly,duringdifferen4a4on,Ccna2expression isdownregulatedinorderforsubsequentmeio4ceventstooccur.3

This temporal specificity of Ccna2 expression in the cell cycle is mediated mostly by the E2Fs, a family oftranscrip4onfactorsthatregulatetheexpressionofmanycellcyclerelatedgenes.4Therelevanceoftheprecise4mingandexpressionlevelofE2F-drivenCcna2expressionhasnotbeenthoroughlystudiedinvivo.

Toevaluate the roll of E2F-mediated regula4onofCcna2 in vivo,wehavegeneratedmicewith amutatedE2FbindingsiteinthepromoterofCcna2(Ccna2m).Althoughmiceareviableandappearhealthy,weobservedinfer4lityandtes4cularatrophyinmaleshomozygousforthepromotermuta4on(Fig2B).

METHODS •  AmouselinewithanullE2Fbindingsiteatthe

Ccna2locuswasgenerated(Fig1)•  Mouseviabilityandfer4litywastracked

throughoutlife.•  Micewerecollectedat2,3,4,6and12weeksof

age.Tes4swereweighedandthenfixed.•  Tes4cularatrophywasassessedviahistological

analysisandcomparingtestes:bodyweightra4osbetweencohorts.

•  Sampleswerefixedwitheitherbouin’sfixa4veforhistologicalanalysisandformalinformarkeranalysisviaimmunohistochemistry(IHC)

RESULTS Analysis of breeding revealed that mutants are completely infer4le, and that one copy of the func4onal

promotersiteissufficientforfer4lity(Fig2A).Histologicalanalysisrevealedadelayed1stwaveofspermatogenesisat 2 weeks followed by progressive degenera4on of the seminiferous tubules; degenera4on peaks at 4 weeks.Surprisingly,theseminiferoustubulesbegintorecovertheircellpopula4onsastheyreachadulthood(Fig3A).IHCconfirmed thepresenceofCCNA2 inmutantSSC,which indicates that thepromotermuta4ondoesnotpreventCCNA2expression(Fig3B).

ACKNOWLEDGEMENTS Funding provided by The Ohio State’s Undergraduate Research Office: Research Scholarship Award and Undergraduate Summer Research Fellowship.

College of Arts and Sciences / Department of Molecular Genetics

Fig1:Diagramofmouselines.ThewildtypelocusofCcna2(Ccna2+)hasanE2F site 11 base pairs from the transcrip4on start site. The mouse linegenerated(Ccna2m)hasfourbasepairsmutatedatthebindingsiteanda5xMYCtaginserteda]erthetransla4onstartsite.

Ccna2+

Ccna2m TAGTCTTAAG ATCAGAATTC

MutatedE2FSite

TAGTCGCGGG ATCAGCGCCC

WildtypeE2FSite

MYC

Bibliographies 1.Wolgemuth,D.J.,Manterola,M.,&Vasileva,A.(2013).Roleofcyclinsincontrollingprogressionofmammalianspermatogenesis.Int.J.Dev.Biol.TheInterna9onalJournal

ofDevelopmentalBiology,57(2-3-4),159-1682.Hermo,L.,Pelle4er,R.,Cyr,D.G.,&Smith,C.E.(2009).Surfingthewave,cycle,lifehistory,andgenes/proteinsexpressedbytes4culargermcells.Part1:Backgroundto

spermatogenesis,spermatogonia,andspermatocytes.MicroscopyResearchandTechniqueMicrosc.Res.Tech.,73(4),241-278.3.Peckham,M.,&Knibbs,A.(2003,January).HistologyGuide|Male.RetrievedFebruary13,2016,fromwww.histology.leeds.ac.uk/male/sertoli_cells4.Henglein,B.,Chenivesse,X.,Wang,J.,Eick,D.,&Brechot,C.(1994).Structureandcellcycle-regulatedtranscrip4onofthehumancyclinAgene.Proceedingsofthe

Na9onalAcademyofSciences,91(12),5490-5494.

CONCLUSIONS/FUTURE PROJECTIONSThedatasuggeststhatE2Fregula4onofCcna2isessen4alforasuccessful1stwaveofspermatogenesis.Since

cellpopula4onsseemtorecover,furtherbreedingstudieswillbeconductedonoldermicetoiden4fyifeventuallythereisacompleterecoveryoffer4lity(upto24weeksofage).Themolecularpathwaycausingthisphenotypeiscurrently being studied through sperma4d squashprepara4ons andmarker analysis via quan4ta4vePCR (qPCR)andIHC.PreliminarydatasuggeststhatthephenotyperesemblesthoseofCdk2mutantmice,butfurthertestsarerequiredinordertoconfirmthis.

Fig2:AdultCcna2m/mmalemiceareinfer7leandhavetes7cularatrophy.(A) Adult male mice of each group were placed with females and theirfer4litywasevaluated.Ccna2m/mmalesmatedsuccessfullyasmeasuredbythepresenceofacopula4onplug,butnolilerswereobserved.Analysisofthemalereproduc4ontractrevealedextremetes4cularatrophyinCcna2m/

madultmiceasmeasuredby thebody : testesweight ra4o.BothCcna2+/+andCcna2+/mmicepresentednormaltes4culardevelopment,butCcna2m/mhada three-fold reduc4onof tes4cularmass. (B)Representa4veexamplesof6week-oldCcna2+/+andCcna2m/mtestesoutlinedinred.

Genotype Fer7lemales(%)

Testes:bodyweightra7o(%)

Ccna2+/+ 100%(n=5) 0.62%(n=5)

Ccna2+/m 100%(n=8) 0.58%(n=7)

Ccna2m/m 0%(n=4) 0.19%(n=4)

A B

Ccna2+/+

Ccna2m/m

Fig3:Ccna2m/mmaleshaveadefec7ve1stwaveofspermatogenesis(A)H&Estainedsec4onsofbouin’s-fixedtestes (10x).At2weeks, thewild-type’sseminiferous tubuleshaveZygotenespermatocytes(blackarrow),whilethemutantiss4llatthepre-Leptotenestage.At3weeks,Ccna2+/+maleshavesecondarymeiocytes(redarrow)whilethetubulesinthemutantbegintodegenerate;thisdegenera4onpeaksat4weeks.However,tubulesbegintorecovertheircellpopula4ons as they reach adulthood. No viable sperm was found in the epididymis (6->12weeks).(B)IHCusinganan4bodyagainstCCNA2todetectexpressionintestesfrom4week-oldmice (4x). CCNA2-posi4ve cells are dividing SSCs. (C) Stages of spermatogenesis andCcna2’stemporalloca4oninthedifferen4a4onprocess.

Ccna2

+/+

Ccna2

m/m

2 wk 3 wk 4 wk 6 wk 12 wkA

B

AdaptedfromWolgemuthet.al.(2013)

C

Ccna2

+/+

Ccna2

m/m

Ccna2