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PPAR-γ-Peroxisomeproliferator-ac4vatedreceptorgamma
PPAR-γ-Peroxisomeproliferator-ac4vatedreceptorgamma
C/EBPalpha-CCAAT/enhancer-bindingproteinalpha
C/EBPαcontains:- afunc)onallyrelatedleucinezipperdimeriza)ondomain(LZ)atitsC-terminusandanadjacenthighlyconservedbasicregion(BR)thatmediatessequence-specificDNAbinding.
- atransac)va)ondomains(TADs)andaregulatorydomain(RD)locatedintheirN-terminalregions.
Cell differentiation
WNTpathway
Chroma4nstructureandgeneexpression
MurineAdherentFibroblast
invitroosteogenesismodelbyexposingC3H10T1/2cellstobonemorphogene4cprotein2(BMP2)
PPAR-γmRNAexpressionprofileduringC3H10T1/2osteogenesisbyreal-4mePCRandwesternbloOng
AdenovirusvectorexpressingsiRNAagainstPPAR-γtodown-regulatePPARcexpression
alsousedG3335,aspecificantagonistofPPAR-γ,toblockitsac4vity
PPAR-γcontributedtoC/EBPaexpressionduringC3H10T1/2osteogenesisatleastinpart
up-regula4onofPPAR-γandC/EBPaexpression
ThesedatasuggestthatPPAR-γac4vatesC/EBPaexpressioninC3H10T1/2cells
Figura21:MappacircolaredelveUorepGL3-basic.La regione del promotore -1785/-20 del 5’ del gene SLC25A1 è stato inserito, mediante diges<one con le endonucleasi di restrizione BglII e HindIII, nella regione a monte del gene reporter per la luciferasi.
CLONING-TRANSFECTION-LUCIFERASEASSAY
CLONING-TRANSFECTION-LUCIFERASEASSAY
CLONING-TRANSFECTION-LUCIFERASEASSAY
BindingofPPAR-γtothe1286bp/1065bppromoterregiontoac4vateC/EBPaexpression
a transient reporter assay was performed in C3H10T1/2 cells treated with BMP2,rosiglitazoneand/orG3335for72h
ChIPexperiments
ThesedataconfirmbindingofPPAR-γtothe1286bp/1065bpregionoftheC/EBPapromoter.
BindingofPPAR-γtothe1286bp/1065bppromoterregiontoac4vateC/EBPaexpression
ChIPexperimentsperformedinC3H10T1/2cellstreatedwithBMP2,usingPPARgan4body
PlasmidInvitromethyla4on:SAMtreatment
DNAmethyla4onandPPAR-γ-associatedrepressionofHDAC1inthe1286bp/1065bpregionoftheC/EBPa
promoterInapreviousreport[2],DNAhypermethyla)oninthe1286bp/1065bpregionoftheC/EBPapromoterwasobservedattheterminalstage(21days)ofosteogenesisofC3H10T1/2cells
TheresultsshowedthatDNAmethyla4oncountersPPAR-γbindingtothe1286bp/1065bpregion
DNAmethyla4onandPPAR-γ-associatedrepressionofHDAC1inthe1286bp/1065bpregionoftheC/EBPapromoter
Theacetyla4onlevelofhistones3and4wasreducedandHDAC1bindingwassignificantlyenhancedinthe1286bp/1065bpregionattheterminalstageof
osteogenesiscomparedwiththeearlystage
Inaddi)ontoDNAhypermethyla)on,hypoacetyla)onofhistones3and4inthe1286bp/1065bpregionoftheC/EBPapromoterwasalsoobservedattheterminalstageofosteogenesis[2].
Ac4va4onofC/EBPaexpressionbyPPAR-γduringadipogenesisofC3H10T1/2cells
C3H10T1/2cellswereinducedtoundergoadipogenesisbyinsulin,fetalbovineserum,methylisobutylxanthineanddexamethasone(IFMDprotocol)
Luciferasereportervectorinwhichexpressionofluciferasewasdrivenbythe1286bp/1065bpregionoftheC/EBPapromoter.A^erinvitromethyla)on,thevectorsweretransfectedintoC3H10T1/2cells,andChIPwasperformedinIFMDandcontrolcultures
PPAR-γandHDAC1bindingandtheDNAmethyla4onandhistoneacetyla4onstatusinthe1286bp/1065bpregionoftheC/EBPapromoterduringinvitro
osteogenesisofmouseBMSC(BonemarrowfromBALB/cmice)
PPAR-γandHDAC1bindingandtheDNAmethyla4onandhistoneacetyla4onstatusinthe1286bp/1065bpregionoftheC/EBPapromoterduringinvitro
osteogenesisofmouseBMSC(BonemarrowfromBALB/cmice)
BalancebetweenosteogenesisandadipogenesisofBMSCbymodula4onofDNAmethyla4onandhistoneacetyla4on
Theadipogenesispoten4al,whichdecreasedattheterminalstageofosteogenesis,wasrecoveredby5ʹ-aza
andTSAtreatment
ModelforC/EBPaexpressionregulatedbyPPAR-γattheearlyandterminalstagesofosteogenesis
Theresultsofthatstudywereinteres4ng:DNAhypermethyla4onandhypoacetyla4onofhistones3and4inthe1286bp/1065bpregionoftheC/EBPapromoterdownregulatedC/EBPatranscrip4onattheterminalstageofosteogenesis,andhypoacetyla4onofhistones3and4dependedonDNAhypermethyla4onthroughunknownmolecularmechanisms.Ques4onsremainopenregardingthebalancebetweenosteogenesisandadipogenesisregulatedbyC/EBPathatrequireelucida4oninmorestringent,mechanis4cterms.
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