the burs2ng of ac2va2on-induced cy2dine deaminase mrna ...€¦ · abstract analysis and...
TRANSCRIPT
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.
Alberts,B.,Bray,D.,Lewis,J.,Raff,M.,Roberts,K.,Watson,J.D.(1994).Theimmunesystem.InM.Robertson,R.Adams,D.Goertzen,P.Bessas(Eds.),Molecularbiologyofthecell(pp.1195-1254).NewYork,NY:GarlandPublishing,Inc.
Halpern,K.B.,Tanami,S.,Landen,S.,Chapal,M.,Szlak,L.,Hutzler,A.,Nizherbg,A.,Itzxovitz,S.(2015).Burstygeneexpressionintheintactmammalianliver.Molecularcell,58,147-156.h`p://dx.doi.org/10.1016/j.molcel.2015.01.027
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