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Numerousautoimmunedisordersexhibitastrongfemalebias, includingSystemicLupusErythematosus (SLE) where 85% ofpatientsarefemale. WhilefemalecellshavetwoXchromosomes, maleshaveanXandY. TheX-chromosomehasbeenimplicatedinthedevelopmentofautoimmunity, asindividualswithmultipleXs (XXY; XXX) havegreaterriskfordevelopingSLE. TheX-chromosomecontainsmanygenesinvolvedinimmunefunction. Female, butnotmale, SLEpatientsexhibitabnormalover-expressionofseveralofthesegenesinlymphocytes, yetthemechanismsresponsibleareunknown. NormallyfemalecellswillinactivateallbutoneoftheirXchromosomesthroughXchromosomeinactivation (XCI). ThisisdonethroughalargeRNAmoleculeknownasXist, whichbindstotheinactiveX (Xi), andrecruitssilencingcompounds. However, recentworkfromtheAngueralabfoundthatfemalelymphocyteshaveanunusualformofXCImaintenance: naïve (unstimulated) quiescentBandTcellslackXistRNAandsilencingmarks, andthesefeaturesarepresentontheXiinactivatedlymphocytes. Fromthisobservation, wequestionedwhetherallquiescentcellswouldlackXistRNAontheinactiveX.

ThroughthePennUndergraduateMentorshipProgram,IhadtheopportunitytoworkintheAngueralabandperformprofilingoftheinactiveXusingthreedistinctquiescentcellpopulations: theHopx+ intestinalstemcellpopulation, naivemonocytes (atypeofimmunecell) andtheBeta-4±lungairwayepithelialcells. Toprofilecells, IusedRNAFISHtovisualizeXistRNAandratetheextentofitslocalization. Inaddition, IdeterminedthetimepointforpeaklocalizationofXistRNAtotheinactiveXduringTcellactivation.

Inordertoprofilethesedifferentcelltypes, Ihadtobecomeproficientinisolating and culturing T cells, RT-PCRandRNAFISH.

Bytheendofthesummer, IfoundthatbothBeta-4 negativecellsexhibitweakerXistRNAlocalizationthanBeta-4 positivecells, despitebothproducingXistRNAatsimilarlevels, whileHopx+ cellshadweakerXistRNAlocalizationthantheHopx- fraction. IdeterminedthatmurineTcellsachievepeakXistRNAlocalizationbetween two and three dayspoststimulation. ItappearedthatdifferentsubsetsofTcells (Treg and Th1 cells) haddifferentpeaktimepoints. ProfilingofnaivemonocytesrevealedthattheyalsosharedthecharacteristiclackofXistRNAlocalization. Uponstimulatingthemonocytes,IwasabletoobserveXistRNAlocalizingtotheXi. These results further reveal a relationship between XCI and quiescent cells that I’ll be doing further research on this fall.

ThroughoutthesummerIgainedvaluableexperiencenotonlypresentingmyworkandfindingsatweeklylabmeetings, butalsocollaboratingwithotherlabs. Additionally, Ihadtolearntoorganizemyexperimentsandtimewisely. Thereweremanymovingpartstomyprojectandkeepingeverythingrunningsmoothlyforcedmetobecomeamoredisciplinedandpracticalexperimentalist. Asanaspiringphysician-scientisttheseareskillsIhopetoutilizeinthefuture.

Numerousautoimmunedisordersexhibitastrongfemalebias, includingSystemicLupusErythematosus (SLE) where 85% ofpatientsarefemale. WhilefemalecellshavetwoXchromosomes, maleshaveanXandY. TheX-chromosomehasbeenimplicatedinthedevelopmentofautoimmunity, asindividualswithmultipleXs (XXY; XXX) havegreaterriskfordevelopingSLE. TheX-chromosomecontainsmanygenesinvolvedinimmunefunction. Female, butnotmale, SLEpatientsexhibitabnormalover-expressionofseveralofthesegenesinlymphocytes, yetthemechanismsresponsibleareunknown. NormallyfemalecellswillinactivateallbutoneoftheirXchromosomesthroughXchromosomeinactivation (XCI). ThisisdonethroughalargeRNAmoleculeknownasXist, whichbindstotheinactiveX (Xi), andrecruitssilencingcompounds. However, recentworkfromtheAngueralabfoundthatfemalelymphocyteshaveanunusualformofXCImaintenance: naïve (unstimulated) quiescentBandTcellslackXistRNAandsilencingmarks, andthesefeaturesarepresentontheXiinactivatedlymphocytes. Fromthisobservation, wequestionedwhetherallquiescentcellswouldlackXistRNAontheinactiveX.

ThroughthePennUndergraduateMentorshipProgram,IhadtheopportunitytoworkintheAngueralabandperformprofilingoftheinactiveXusingthreedistinctquiescentcellpopulations: theHopx+ intestinalstemcellpopulation, naivemonocytes (atypeofimmunecell) andtheBeta-4±lungairwayepithelialcells. Toprofilecells, IusedRNAFISHtovisualizeXistRNAandratetheextentofitslocalization. Inaddition, IdeterminedthetimepointforpeaklocalizationofXistRNAtotheinactiveXduringTcellactivation.

Inordertoprofilethesedifferentcelltypes, Ihadtobecomeproficientinisolating and culturing T cells, RT-PCRandRNAFISH.

Bytheendofthesummer, IfoundthatbothBeta-4 negativecellsexhibitweakerXistRNAlocalizationthanBeta-4 positivecells, despitebothproducingXistRNAatsimilarlevels, whileHopx+ cellshadweakerXistRNAlocalizationthantheHopx- fraction. IdeterminedthatmurineTcellsachievepeakXistRNAlocalizationbetween two and three dayspoststimulation. ItappearedthatdifferentsubsetsofTcells (Treg and Th1 cells) haddifferentpeaktimepoints. ProfilingofnaivemonocytesrevealedthattheyalsosharedthecharacteristiclackofXistRNAlocalization. Uponstimulatingthemonocytes,IwasabletoobserveXistRNAlocalizingtotheXi. These results further reveal a relationship between XCI and quiescent cells that I’ll be doing further research on this fall.

ThroughoutthesummerIgainedvaluableexperiencenotonlypresentingmyworkandfindingsatweeklylabmeetings, butalsocollaboratingwithotherlabs. Additionally, Ihadtolearntoorganizemyexperimentsandtimewisely. Thereweremanymovingpartstomyprojectandkeepingeverythingrunningsmoothlyforcedmetobecomeamoredisciplinedandpracticalexperimentalist. Asanaspiringphysician-scientisttheseareskillsIhopetoutilizeinthefuture.