Abstract

Classswitchrecombination(CSR)isageneticprocesswhereaBcellswitchesantibodyisotype productionthroughsite-specificintra-chromosomalDNArearrangementstimulatedbytheformation ofDNAdouble-strandbreaks(DSBs)attheimmunoglobulinheavychain(IgH)locus.DSBsare normallyrepairedbythenon-homologousend-joining(NHEJ)andalternative-endjoining(alt-EJ) DNArepairpathways.DuringCSR,DSBformationishighlyregulatedinvolvingacomplexinterplayof transcriptionalactivation,proteinrecruitmentandchromatinreorganization.Understandingthefactors regulatingDSBformationandrepairhasahighimpactonlymphomagenesis.Rloopsarethree strandedRNA:DNAhybridstructuresformedatIgHduringCSR.WhileRloopsareimplicatedin promotingDSBformationatIgH,theirroleinclassswitchrecombinationremainsundefined.Wefind thatmicedefectiveforRloopremovalareproficientatclassswitchrecombination,howeverBcells containunrepairedbreaksandchromosomefusionsatIgH.Recurrentoncogenictranslocations involvingIgHdistinguishmanyhumanlymphoidmalignancies.Thesetranslocationsoriginatefrom mis-repairedDNAdoublestandbreaks(DSBs)generatedduringnormallymphocytedevelopment. OurgoalistodeterminehowpersistentRloopsimpedeDNArepairduringCSR,andtheroleRloop metabolismplaysinsuppressinggenomeinstabilityatIgH.WehypothesizethatpersistentRloops blockefficientDNArepairbynon-homologousendjoiningattheimmunoglobulinheavychain locusduringclassswitchrecombination,leadingtopersistent,unrepairedbreaks.Totestthis hypothesis,twomousemodelswillbeemployed:theSETXmutantlackstheSenataxin(SETX) helicasethatunwindsRloops;?andRnaseh2bisdefectivefortheRNaseH2nucleasethatspecifically digeststheRNAcomponentofRloops(RNH2B).Wewillfunctionallydissecttheconsequencesof aberrantRloopformationonDNArepairandchromosomefusionsarisingduringCSRinSETX-/-, RNH2Bf/f,andSETX-/-RNH2Bf/fcells(Aim1).TodefinetheimpactpersistentRloopshaveonNHEJ, wewillcharacterizeDNArepairproteinrecruitmentinSETX-/-,RNH2Bf/f,andSETX-/-RNH2Bf/fcells (Aim2).WewillalsoidentifygenomiclociinvolvedinIgHtranslocationsusinghigh-throughput genome-widetranslocationsequencing(HTGTS-Seq),incollaborationwithDr.Feyredoun Hormozdiari.Finally,wewilldefinethemolecularpathwaysdrivingthefrequentchromosomefusions observedinSETX-/-RNH2Bf/fcells(Aim3).OurworkwilldefinehowpersistentRloopsinterferewith classswitchrecombination,leadingtounrepairedbreaks,andwilluncoverthemolecular mechanismspromotingchromosomefusionsatIgH.EnzymesregulatingRloopmetabolismwillalso provideanattractivetargetfordevelopingnovelcancertreatment.

Public Health Relevance

Publichealthrelevance DefectiverepairofDNAdamagecausestheaccumulationofmutationsandgenomicrearrangements thatpredisposetocancerdevelopment.SetxandRNaseH2protectagainstimmunoglobulin(Ig) translocationsdrivingmanylymphoidcancers.UnderstandingtheimpactofdefectsinSenataxinand RNaseH2ontheformationofgenomicmutationsandrearrangementswillprovidenoveland importantinsightsintothemoleculareventsdrivingtumorigenesis.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM134537-01
Application #
9799813
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Janes, Daniel E
Project Start
2019-08-05
Project End
2024-07-31
Budget Start
2019-08-05
Budget End
2020-07-31
Support Year
1
Fiscal Year
2019
Total Cost
Indirect Cost

  Institution

Name
University of California Davis
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
047120084
City
Davis
State
CA
Country
United States
Zip Code
95618