Abstract

Tissuemanufacturingforskinreplacementtherapyisextremelyinefficient,inpartduetoanincomplete understandingofgeneexpressionmechanismsregulatingstemcellcommitment.Onesuchpoorlyunderstood mechanismischromatinlooping,whichhasbeenthoughttoserveasatemplateforgeneexpressionchanges duringcellstatetransitions.Thisknowledgegapisamajorroadblockinproducingsufficientgraftable keratinocytesfromgeneticallycorrectedpatientstemcellsfortreatmentofdebilitatingskindiseases.Thelong- termgoalofthisproposalistounderstandthemolecularmechanismsunderlyingstemcelldifferentiationto improveskinreplacementtherapy.Preliminarydatafromtheapplicant?sgroupsuggestthatretinoicacid(RA) andbonemorphogenicprotein(BMP4)morphogenscaninducestemcell-derivedgraftablekeratinocytes throughinductionofthemasterepithelialregulatorandtranscriptionfactorp63.Interestingly,p63cannot activatedownstreamgeneexpressionprogramsintheabsenceofthesemorphogens.Further,high dimensionalchromatinanalysessuggestthatmorphogenscausemajorchangesinchromatinlooping. Therefore,thecentralhypothesisisthatRAandBMP4stimulateloopingbetweenp63bindingsitesanddistal loci,aswellasalterthep63interactome,tofacilitatep63-dependentgeneexpression.Thisproposalwilltest thehypothesisbypursuingtwospecificaims.
Aim1 willdeterminewhichregulatoryregionswithinchromatin loopsarerequiredforp63-dependentgeneexpression.CRISPR/Cas9tools,whichhavebeenwellestablished intheapplicant?slaboratory,willbeusedtosequentiallyblockp63bindingsitesandthelocitowhichtheyare looped.Theeffectsofeachdeletionwillbemeasuredbydownstreamgeneexpression,loopformation,and cellulardifferentiationmarkers.TheproposedexperimentswillfocusonloopsatTFAP2CandHES1loci, whichareknowntobecrucialduringstemcellcommitment.
Aim2 willelucidatep63interactingproteinsthat arerequiredfordownstreamgeneexpression.Candidatemembersofthep63interactomewillbeidentified usinganovelproximitylabelingsystemknownasBASU.Thissystemhasbeenvalidatedintheapplicant?s laboratoryusingembryonicstemcells.Theimportanceofeachp63interactingproteinwillthenbeevaluated usingaCRISPR/Cas9lossoffunctionscreenfollowedbymeasurementsofp63-dependentgeneexpression andcellulardifferentiation.Therationalefortheproposedresearchisthatitwillprovideusefulchromatin dynamicinformationwhichcanbeusedtoimprovethecurrentstemcelldifferentiationprotocolfortissue replacementtherapy.Thisproposalisinnovative,becauseitusesnovelgeneticandproteomicstechniquesto elucidatemechanismsofchromatinloopsthatwerepreviouslyunrecognized.Thefindingswillbesignificant, becausetheywillbroadentheunderstandingofp63duringdevelopmentaswellascontributetomuchneeded improvementsforskinreplacementtherapy.Takentogether,thisknowledgewillprovidecrucialinformationon howchangesinchromatinarchitecturecangeneratediversityinmorphologicalpatterning.

Public Health Relevance

Theproposedresearchissignificanttohumanhealthbecausethechromatindynamicinformationobtained fromthesestudieswillultimatelybeusedtoimproveskintissuemanufacturingtotreatgeneticskinconditions withregenerativestemcelltherapy.Therefore,thisprojectisrelevanttoNIH?smissionbecauseitwillboth broadentheunderstandingofhumandevelopmentandreducetheburdensassociatedwithdebilitatinggenetic skindiseases.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32AR074221-02
Application #
9848433
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Tseng, Hung H
Project Start
2019-02-01
Project End
2022-01-31
Budget Start
2020-02-01
Budget End
2021-01-31
Support Year
2
Fiscal Year
2020
Total Cost
Indirect Cost

  Institution

Name
Stanford University
Department
Dermatology
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305