lecture 5: coordination of histone and dna methylation...lecture 5: coordination of histone and dna...

Lecture5:CoordinationofhistoneandDNAmethylatiON

CXXCdomainsmediatebindingtounmethylated CpGs

CXXCdomainsproteinscanbindtoun-methylatedCpGs

MLL1CXXCdomains bindswithunmethylated CpG(1,2:Znions)

DNMT1Binds tounmethylatedDNAviaitsCXXCdomainà PurposeCXXC:to

prevent aberrantmethylation ofunmethylated DNA

à InhibitionofMTasedomainatwrongsubstrate

à AthemimethylatedCpG CXXCdomainisremoved fromDNA

SET1H3K4 HKMTFigure:SET1invicinitytohistoneH3tail.Note:CFP1isaninteractingpartner ofSET1.CFP1hasCXXCdomain thatbindsunmethylatedCpGScenario: unmethylated CpG;CFP1bindsandrecruits SET1àH3K4me increases

MLL1translocation à Leukemia(involvemnt ofepigentic mechansims??H3K36de-methylationandCpG islandmethylation

CXXCdomainsmediatebindingtounmethylated CpGs:H3K36specificKDM2A/JHDM1A (K-HistoneDe-methylase)


DNMT1-/-WTEScells:chromocenters areDNAmethylated(alargeblockofmethylatedDNA:Flag-taggedJHDM1aisrandomlydistributed inthenucleusDNMT1-/- EScells:NoDNAmethylation atchromocenters =alargeblockofDNAarewithout DNAmethylation. InthiscaseKDM2A/JHDM1alocalizes tochromocenters =isattractedbyunmethylated CpGs

JHDM1A/KDM2A isahistonede-methylasethatensureslowH3K36me2/me1levelsatCpG islandsJHDM1A

Blackledge etal.Molecular Cell 2010

CXXCdomainbindsun-methylatedCpG islands

Tet1hasCXXCzincfingerdomain.However,theCXXCdomainofTET1hasnoDNAbindingactivityandisdispensableforitscatalyticactivityinvivo.OtherinteractingproteinsrecruitTet1toDNA

PRIMARYOBSERVATIONTHAT JHDM1A

ISLINKEDTODNAMETHYLATION

CXXCdomainsmediatebindingtounmethylated CpGs:H3K36specificKDM2A/JHDM1A (K-HistoneDe-methylase)


ChIP seq onEScells:JHDM1a/KDM2AConcentrateonun-methylatedCpG islands

Interaction isdependentontheCXXCdomain

H3K36me2ishighingenebodybutlowatpromoterHighlevelsofKDMA2atunmethylated CpGislandsisparalleledbylowH3K36me2levels


CXXCdomainsmediatebindingtounmethylated CpGs:KDM2A/JHDM1A Lecture5:CoordinationofhistoneandDNAmethylatiON


Invitro:UNDIFFERENTIATED Invitro:DIFFERENTIATED

Ncoa2:ON Cldn4:ON Cldn4:OFFNcoa2:ONDNAmethylationH3K36me2 LOW LOW LOW HIGH

LOW LOW LOW HIGH

KDM2A HIGH HIGH HIGH LOW

KDM2ABINDSTOUNMETHYLATED CpG ISLANDSANDSPECIFICALLYREDUCES H3K36me2LEVELSà SINGATUREOFUNMETHLATED CpG ISLANDANDACTIVEGENEEXPRESSION

à H3K4meandH3K36melevelscandefinethemethylationstatusofCpG islands

Bisulfitesequencing

H3K4de-methylationandCpG islandmethylation

TheH3K4de-methylase LSD1(KDM1A) isessentialforestablishingDNAmethylation


LSD1isaH3K4specific demethylase: oxidizesH3K4me2,1à H3K4me0

LSD1konditional knock-outmicedieearly inembryogenesis (E5.5)andshowstronglyreduced DNAmethylation

LSD+/- LSD-/-

LossofLSD1resultsinReducedDNAmethylation

Southern blotting usingCH3sensitive restrictionenzymes:aprobe fortheH19andIAPimprintedgenelocus areused.Theseareclassic loci arecontrolled byDNAmethylation

Note:LossofDNAmethylation results inEfficient restriction digest(moresmallfragment).ThismeansthatDNAmethylation isstronglyreducedSituation issimilar toDNMT1knock-outcellsRecruitmentofLSD1eliminatesH3K4me1,2resultinginH3K4me0

ThiscreatesabindingsiteforDNMT3Là thusrecruitingtheDNMT3L-DNMT3a tetramer

DNAmethylationsensitiverestrictionenzyme–doesnotcutwhenCpG ismethylated


CXXCdomainsmediatebindingtounmethylated CpGs

CXXCdomainsproteinscanbindtoun-methylatedCpGs

MLL1CXXCdomains bindswithunmethylated CpG(1,2:Znions)

DNMT1Binds tounmethylatedDNAviaitsCXXCdomainà PurposeCXXC:to

prevent aberrantmethylation ofunmethylated DNA

à InhibitionofMTasedomainatwrongsubstrate

à AthemimethylatedCpG CXXCdomainisremoved fromDNA

SET1H3K4 HKMTFigure:SET1invicinitytohistoneH3tail.Note:CFP1isaninteractingpartner ofSET1.CFP1hasCXXCdomain thatbindsunmethylatedCpGScenario: unmethylated CpG;CFP1bindsandrecruits SET1àH3K4me increases

MLL1translocation à Leukemia(involvemnt ofepigentic mechansims??H3K9methylationandCpG islandmethylation

TherelationofH3K9meandDNAmethylationLecture5:CoordinationofhistoneandDNAmethylatiON

H3K9mehigh

H3K4mehigh H3K9melow GeneON

GeneOFFH3K4melow

SET1MLLfam.

G9a/GLPSuv39H1/2

H3K9mehigh

H3K4mehigh CpG methylationlow

GeneON

GeneOFFCpG methylation???

SET1MLLfam.

G9a/GLPSuv39H1/2

Isthereadirect linkbetweenH3K9meandDNAmethylation

TheroleoftheG9a/GLPheterodimerincontrollingDNAmethylationLecture5:CoordinationofhistoneandDNAmethylatiON

G9aHMTase andGLPHMTase formdimerandmethylateH3K9m1;H3K9me2

Immunoprecipitation:- Cells transientlytransfectedwithFlag-taggedG9aandDnmt3a- IPanti—flagG9a: DNMT3ainteracts- IPanti– flagG9a:DNMT3binteracts

Epsztejn-Litman Nat. Struct. Mol. Biol. 2008

Suv39h1/2 Suv39h1/2 Suv39h1/2

H3K9methylation

G9aGLP

G9aGLP

G9aGLP


G9a– knock-out cellsindifferentiationShowalossofDNAmethylationAsdetected bybisulfitesequencingInCpG islandsofseveralgenes


Embryonic stemcells:

Self-renewingmESCs(pluripotent)

DifferentiatedmESCsOct4,Stk10,Gpr54Nanog,Dnmt3L, Tnfrsf8

ONNODNAMETHYLATION

Oct4,Stk10,Gpr54Nanog,Dnmt3L, Tnfrsf8

OFFDNAMETHYLATION

retinoicacid

G9a:Requiredforsilencingoftransposableelements,repeatelements,retroviralinsertions,imprintingcentersbutalsoingeneexpressioncontrol

Lehnertz elal.2009

H3K9me3

TheroleoftheSuv39h1/h2incontrollingDNAmethylationTheroleoftheG9a/GLPheterodimerincontrollingDNAmethylationLecture5:CoordinationofhistoneandDNAmethylatiON

LossofSuv39h1HMTases islinkedtoreducedDNAmethylationatpericentric repeats

Lehnertz elal.2003

SouthernblotusinggenomicDNAthatwasdigestedwithmethylationsensitiverestrictionenzyme.DNAonblotwashybridizedusingprobesforminorsatellite,

majorsatelliteandC-typeretroviralDNA

TheroleoftheSuv39h1/h2incontrollingDNAmethylationHistonemethylationandDNAmethylation


MLL

G9a/GLP

DNMT3a

K9

G9a/GLP

DNMT3a

K9MeK4 K4

DNMT3/Lbindun-methylatedH3K4viaADDdomain

lecture 5: coordination of histone and dna methylation...lecture 5: coordination of histone and dna...

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