Abstract

AnalysisandConclusions

Introduc2on

References

Acknowledgements

TheBurs2ngofAc2va2on-InducedCy2dineDeaminasemRNADuringBCellAc2va2on

AarjavJoshi,Yi(Joy)Zhou,Ph.D.,CornelisMurre,Ph.D.TheDepartmentofMolecularBiology,UniversityofCaliforniaSanDiego,9500GilmanDrive,LaJolla,California

Methods

Results

During B lymphocyte acJvaJon and differenJaJon in the germinal center of secondarylymphoid organs, two processes known as SomaJc HypermutaJon (SHM) and Class SwitchRecombinaJon (CSR) are uJlized to opJmize the anJbody response to anJgens. SHM allows forbasepairmutaJonstoresultinimprovedaffiniJesforanJgens,whileCSRprecipitatestheexcisionofconstantregions(CH)thatareundesired,generaJngadesiredheavychainconstantregion,orIgisotype, for the immunoglobulin anJbody that targets an anJgen. These two processes areexecuted by the enzyme AcJvaJon-Induced CyJdine Deaminase (AID). The transcripJon of thegene for AID, on a cellular level, occurs in transcripJonal bursts, (i.e. intense periods of acJvitydispersedamongstlongperiodsofinacJvity).WesJmulatedthenaïve(unexposedtoananJgen)Bcellswith lipopolysaccharide (LPS),anendotoxin foundonGram-negaJvebacteria,andaYerLPS-exposed incubaJon,we used the RNA Fluorescent In Situ HybridizaJon (RNA FISH) technique tocheck the singlemolecule levelofAIDmRNA.WeuJlized thismethod to specifically localizeandquanJfytheAIDmRNA,allowingustoidenJfywhenthebursJngofAIDtranscripJonoccursandtowhatextentitwasacJvatedoverJme.

Inthesecondary(peripheral)lymphoidorgans,naïve mature B lymphocytes (or Follicular B cells)undergo SHM in the dark zone of the lymphaJcgerminal center and are referred to as centroblasts(Fig. 1). As a result of SHM, centroblasts undergomutaJons and experience either higher affinity orlower affinity for the anJgen. These centroblastsenter the light zone of the germinal center and arereferred to as centrocytes. The centrocytes with animproved affinity for the anJgen undergo CSR toproduce various isotypes of immunoglobulin heavychain constants (Ig isotypes), necessary for theimmuneresponse,whilethecentrocyteswithaloweraffinity for the anJgen undergo apoptosis. Theselected centrocytes, aYer CSR, differenJate intoplasmacellsandmemoryBcells.

Figure1.Bcellac2va2onanddifferen2a2oninthegerminalcenterof

asecondary(peripheral)lymphoidorgan,includingsoma2chypermuta2on

andclassswitchrecombina2on

DuringSHMandCSR,theenzymeAIDplaysacrucialroleinsJmulaJngimprovedaffiniJesforanJgensandmoresuitableisotypesforimmunoglobulins.InSHM,AIDdeaminatesCytosineinG:C base pairs into Uracil (i.e. G:U) on the variable segments of immunoglobulin genes, whichallowsforG:NmutaJons,A:TmutaJons,mutaJonsinneighboringA:Tbasepairs,ornormalrepair.This results differences in anJbody protein structure, and consequently, improved or loweraffiniJesfortheanJgen.InCSR,AIDdeaminatesdCnucleoJdesinthetopandbo`omstrandsoftheswitch(S)regionslocatedupstreamofeachconstantregion(CH)intheimmunoglobulinheavychaingene,producingdouble-strandedDNAbreaks(DSB’s)intheSregionaheadoftheCμheavychain constant (Sμ) and the S region ahead of the desired constant CH region (Fig. 2). ThisprecipitatestheexcisionofundesiredCHregions,prompJngtranscripJonforthedesiredanJbodyheavychain.TheDSB’smadeinCSRbyAIDallowfortheproducJonofdifferentimmunoglobulinheavy chain constant isotypes (classes), each with various funcJons in execuJng the efficientimmuneresponse.

ThetranscripJonofmRNA, includingAIDmRNA,occurs intranscripJonalburstsorpulses,whichmaybearesultofclosed/openchromaJnformaJon,transcripJonfactors,cellcycleeffects,cell size extracellular signaling, etc. (Fig. 3). TranscripJon factors regulate mRNA acJvaJon byalternaJngbetweenarepressive,inacJvestate,andapermissive,acJvestate,generaJngsurgesofintense transcripJon.ThestochasJcnatureof thisoccurrenceproducesvariaJon inquanJtyandlocaJon of mRNA within Jssues. Single molecule Fluorescent In Situ HybridizaJon (smFISH) canidenJfybothmatureandpre-mRNAtranscriptsofendogenousgenes.ThismethodprovidesuswithmoreinsightsontheacJvaJonofAIDtranscripJonanditstranscripJonalbursJng.

Figure2.Classswitchrecombina2onwithac2va2on-inducedcy2dinedeaminasein

heavychaingene

Figure3.Factorsaffec2ngburstsizeandburstfrequencyintranscrip2onalburs2ng

1. Use slides’ rough edges to mash a mouse (Musmusculus)spleentoyieldaspleencellsuspension

2.Uponfilteringthesuspension,add2μgAnJ-CD23BioJnAnJbodiesand30μLAnJ-BioJnMicroBeads

3.PassthemixturethroughamagneJcfilterwith12mLofMACSbuffer

4.SucJonoutthemixturea`achedtothemagneJcfilter, yielding naïve B cells, and add 100 μg/mLlipopolysaccharide(LPS)

5.Incubatein37.0°Cand5.0%CO2for17hours

6.Put50μLofcellsonacoverslipandincubatefor20minutes

7.Placecoverslipsin2mLPBS,then1mL4%PFAforfixaJon, then PBS 2 mL thrice, then 2 mL 70%Ethanol

8.Storeovernightin4°C

9.Washwith 2mL10%Formamide/2xSSCwashingbuffer,thenadd2pLmRNAprobes

10.Hybridizefor12hoursin37degreesCelsius

11.Washwith2mL10%Formamide/2xSSC, then2mL2xSSC, then stainnucleuswithDAPIfluorescentstain

12. Image under wide-field epifluorescencemicroscope

Figure4a.Wide-fieldepifluorescencemicroscopyimagingofBcellsincubatedfor17hoursLPSs2mula2on.MinimalmRNAtranscrip2onalburs2ngac2vitywasdetected,indicatedbylowpresenceofmaturemRNA(green)andpre-

mRNA(red/yellow)

13.Repeat steps5-12withB cells incubated for24hoursbetweenLPSsJmulaJonandfixaJon,aswellas48hours

Figure4b.Wide-fieldepifluorescencemicroscopicimagingofBcellsincubatedfor24hoursLPSs2mula2on.MildmRNAtranscrip2onal

burs2ngac2vitywasdetected,indicatedbymoderate

presenceofmaturemRNA(green)andpre-mRNA(red/

yellow)

DAPI/MaturemRNA/Pre-mRNA

Figure4c.Wide-fieldepifluorescencemicroscopicimagingofBcellsincubatedfor48hoursLPSs2mula2on.HighmRNAtranscrip2onal

burs2ngac2vitywasdetected,indicatedbyhighpresenceofmaturemRNA(green)andpre-

mRNA(red/yellow)

17hr 24hr 48hr

AID#mRNA

Fig.5a Fig.5b Fig5c.

Figure5a.CellcountsperAID#mRNA17hoursa[erLPSs2mula2on

Fig.5d

Figure5b.CellcountsperAID#mRNA24hoursa[erLPSs2mula2on

Figure5c.CellcountsperAID#mRNA48hoursa[erLPSs2mula2onFigure5d.BoxplotofAID#mRNAwith17,24,and48hours2mula2on,respec2vely

Analyzingtheimages,weidenJfiedanincreaseinAIDmRNAquanJJesfromthe17hourLPSsJmulatedtothe48hourLPSsJmulatedcells,illustratedbythesurgeinbrightgreenpoints(maturemRNA).Inthe17hoursLPSsJmulatedcells,74%(37outof50)ofcellsexhibitedzeroAIDmRNAmolecules,andanother16%exhibitedonlyoneAIDmRNAmolecules.Incontrast,inthe24hoursLPSsJmulaJoncells,approximately89%(44outof56)exhibitedAIDmRNAmolecules,whileover5moleculesofAIDmRNAweredetectedin23%ofcells.Usingthewide-fieldepifluorescencemicroscopy,wewereabletovisualizeBcelltranscripJonalbursJngofAIDmRNAasaphenomenonthatissignificantlyacJvatedmorethan24hoursaYerLPSsJmulaJon.AYer48hoursofLPSsJmulaJon,astaggeringmajorityofcellsexperiencedintensetranscripJonalbursJngacJvity,with38%ofcells(19outof50)displayingAIDmRNAmoleculequanJJesgreaterthan10.Theexperimentalresultsillustratedatwo-state,repressiveandpermissivetranscripJonalbursJngmodel,inwhichatanygivenpointinJme,theAIDmRNAquanJtywouldexhibitsignificantstandarddeviaJon,withlargedifferencesbetweenthemeanandmaximum.Thiswasdemonstrated,asthestandarddeviaJonforthe17,24,and48hourLPSsJmulaJoncellswasapproximately1.99,3.75,and12.35,withasignificantdifferencebetweenthemeanandtheupperlimitofthethirdquarJle,letaloneoutliers.ThestrongdegreeofvariabilityinAIDmRNAtranscripJonalbursJngacJvaJon,asseeninthetwo-statetranscripJonalbursJngmodel,isaresultoftheinnatevariabilityofindividualcellsinbursJngacJvaJon.Thediscrepanciesbetweencellsincellsize,cellcyclestage,extracellularsignals,transcripJonfactors,andvariedchromaJnconfiguraJoninfluencetheacJvaJonofmRNAtranscripJon.ItiswellestablishedthatAIDmRNAexpressionlevelincreaseswithBcellacJvaJon,duetotheneedforAIDenzymetoexecuteSHMandCSR.InaddiJontothisknowledge,wewereabletoidenJfythatsignificantacJvaJonforAID,andconsequently,significantmRNAtranscripJon,occursmorethan24hoursaYeranJgen(LPS)sJmulaJon.Inthefuture,wecanbe`erunderstandthemechanismofAIDacJvaJoninBcellsbyobservinglive-imagingtranscripJonalbursJng.ThepurposeofthisresearchistogainmoreinsightonBcellbiology.

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Stavnezer,J.,Guikema,J.E.J.,Schrader,C.E.(2008).MechanismandregulaJonofclassswitchrecombinaJon.Annualreviewofimmunology,26,261-292.doi:10.1146/annurev.immunol.26.021607.090248

Firstandforemost,IwouldliketothankDr.KomivesfordedicaJngherJme,efforts,energy,andlovetokindlingourpassionfortruescienceandresearchandmakingthisincrediblyinspiringprogramareality. Next, I would like to thank Dr. Murre for welcoming me into his lab, for the Murre Lab forsupporJngmeinmyendeavors,andJoyZhouformentoringmeandguidingmethroughthelabanditsunique experiments. AddiJonally, thank you to Eduardo Ramirez, Ms. Dong and the AcademicConnecJonsstaffforprovidingmewiththisamazingopportunity.

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*©LaurieO’Keefe