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.