Screening for nuclear reprogramming factors and analysis of DNA demethylation during in vitro myoblasts differentiation [Elektronische Ressource] / presented by Suresh Kumar Swaminathan

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Screening for nuclear reprogramming factors and analysis of DNA demethylation duringin vitro myoblasts differentiation Examiners: Prof. Dr. Christof Niehrs Prof. Dr.

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Published 01 January 2007
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Screening for nuclear reprogramming factors
and analysis of DNA demethylation during
in vitro myoblasts differentiation





















Examiners: Prof. Dr. Christof Niehrs
Prof. Dr. Werner Müller






Dissertation

submittedtothe
CombinedFacultiesfortheNaturalSciencesandforMathematics
oftheRupertoCarolaUniversityofHeidelberg,Germany
forthedegreeof
DoctorofNaturalSciences








presentedby

MasterofScience(Biotechnology)
SureshKumarSwaminathan
borninVellore,India


Oralexamination:
























±ý¦Àü§È¡Õì̺Á÷À½õ
Dedicated to my parents
Abbreviations

Ab Antibody
ATP Adenosinetriphosphate
BA Betaactin
BCA BicinchonicAcid
BER Baseexcisionrepair
BMP Bonemorphogeneticproteins
bp basepairs
BSA Bovineserumalbumin
cDNA ComplementaryDNA
COBRA CombinedBisulphiteRestrictionAnalysis
DE Distalenhancer
DEPC Diethylpyrocarbonate
DMEM Dulbecco'sModifiedEagle'sMedium
dn Dominantnegative
DNA Deoxyribonucleicacid
DNase Deoxyribonuclease
Dnmt DNAmethyltransferase
dNTP Deoxynucleotides
DTT Dithiothreitol
ECcells Embryoniccarcinomacells
EDTA Ethylenediaminetetraacetate
EGcells Embryonicgermcells
EGFP EnhancedGreenFluorescentprotein
EGTA Ethyleneglycoltetraacetate
EScells Embryonicstemcells
FBP FUSEbindingprotein
FCS Fetalcalfserum
FGF Fibroblastgrowthfactor
FUSE FarUpstreamElement
Gadd45 GrowtharrestandDNAdamage45
GFP GreenFluorescentprotein
HEPES Hydroxylpipperazineethanesulphonicacid
HRE Hormoneresponsiveelement
HTS Highthroughputscreening
ICM Innercellmass
IL6 Interleukin6
JAK Janusactivatedkinases
kb Kilobasepairs
kDa Kilodaltons
LIF LeukemiaInhibitoryFactor
LIFR LIFreceptor
Luc Luciferase
M Molar
MAPK Mitogenactivatedproteinkinase
MEF Mouseembryonicfibroblasts
MHC Majorhistocompatibilitycomplex
MSPCR MethylationsensitivePCR
NER Nucleotideexcisionrepair
nm Nanometer
No. Number
PBS PhosphatebufferedSaline
PcG Polycombgroup
PE Proximalenhancer
PEG Polyethyleneglycol
PGC Primordialgermcells
POU PitOctUnc
RA RetinoicAcid
RNA RibonucleicAcid
RT Reversetranscription
RTPCR Reversetranscriptionpolymerasechainreaction
SDS Sodiumdodecylsulphate
siRNA SmallinterferingRNA
STAT3 Signaltransducerandactivatoroftranscription
TGF Transforminggrowthfactor
v/v Volume/volume














TABLE OF CONTENTS

1.1 SUMMARY ................................................................................................1
1.2 ZUSAMMENFASSUNG.............................................................................2
2 INTRODUCTION ..........................................................................................3
2.1 Biology of Stem cells.........................................................................................................................3
2.1.1Characteristicpropertiesofstemcells ........................................................................................3
2.1.2Derivationofstemcells..............................................................................................................5
2.2 Determinants of pluripotency..........................................................................................................6
2.2.1Signaltransductionpathways .....................................................................................................6
a)LIFSTAT3pathway ..................................................................................................................6
b)basicFGFpathway.....................................................................................................................7
2.2.2Transcriptionfactors...................................................................................................................7
a)Oct4 ............................................................................................................................................7
b)Sox2..........................................................................................................................................11
c)Nanog .......................................................................................................................................12
d)Transcriptionalnetworkinpluripotency ..................................................................................13
2.2.3Epigeneticmechanisms ............................................................................................................13
a)Chromatinfeatures....................................................................................................................14
b)DNAmethylation .....................................................................................................................15
2.3 Nuclear reprogramming of somatic cells......................................................................................16
2.3.1Nuclearreprogrammingbynucleartransfer .............................................................................16
2.3.2Nuclearreprogrammingbycellfusion .....................................................................................18
2.3.3Nuclearreprogrammingbycellextracts...................................................................................20
2.3.4Nuclearreprogrammingbydefinedreagents............................................................................21
2.4 Epigenetics and cell differentiation...............................................................................................24
2.4.1CpGmethylationandgenesilencing........................................................................................24
2.4.2MediatorsofDNAmethylationanddemethylation..................................................................25
2.4.3DNAmethylationanddemethylationindevelopment .............................................................28
2.5 Experimental aims and strategies .................................................................................................30
3 RESULTS ...................................................................................................32
Part I: Screening for reprogramming factors....................................................................................32
3.1 Expression screening of Xenopus tropicalis cDNA libraries .......................................................32
3.1.1ScreeningbyusingpDETKEGFPreporter ............................................................................33
3.1.2ScreeningbyusingprimaryfibroblastsfromOct4/EGFPtransgenicmice ..............................41
3.1.3Screeningbyusing293TcellswithendogenousOct4readout ................................................42
3.1.4Overviewofscreenings ............................................................................................................43
3.2 Screening of small molecules library ............................................................................................44
3.2.1ScreeningofthesmallmoleculeslibraryusingOG2fibroblasts..............................................44
3.2.2Screeningofthesmallmoleculeslibraryusingaluciferasereporter .......................................49


Part II: Gadd45 mediated DNA demethylation.................................................................................55
-/-3.3 Gadd45 as a mediator of active DNA demethylation in Dnmt1 cells.......................................55
3.4 Role of Gadd45 in muscle differentiation.....................................................................................57
4 DISCUSSION .............................................................................................63
Part I: Screening for reprogramming factors....................................................................................63
4.1 Screening of the Xenopus cDNA libraries ....................................................................................63
4.2 Screening of the small molecules library......................................................................................64
4.3 ‘SWOT’ analysis of the screening for reprogramming factors ..................................................65
Part II: Gadd45 mediated DNA demethylation.................................................................................68
-/- 4.4 Gadd45alpha in DNA demethylation of Dnmt1 cells ................................................................68
4.5 Role of Gadd45 in muscle differentiation.....................................................................................69
5 MATERIALS AND METHODS ...................................................................74
5.1 Equipments and materials.............................................................................................................74
5.1.1Equipments...............................................................................................................................74
5.1.2Materials...................................................................................................................................74
a)Reagentsandconsumables .......................................................................................................74
b)Enzymesandspecialkits..........................................................................................................75
c)Celllines...................................................................................................................................75
d)Antibodies ................................................................................................................................75
e)Libraries....................................................................................................................................76
f)Constructs..................................................................................................................................76
g)siRNA.......................................................................................................................................77
5.2 General molecular methods...........................................................................................................77
5.3 Screening of the libraries ...............................................................................................................77
5.3.1ScreeningoftheXenopusoocytescDNAlibrary .....................................................................77
5.3.2ScreeningoftheXtSt1030cDNAexpressionlibrary..............................................................79
5.3.3Screeningofthesmallmoleculeslibrary..................................................................................79
5.4 Reprogramming by using Xenopus laevis egg extracts................................................................79
5.4.1PreparationofXenopus laeviseggextracts ..............................................................................79
5.4.2Reprogrammingexperiment .....................................................................................................80
5.5 Cell based assays.............................................................................................................................80
5.5.1Cellfusion ................................................................................................................................80
5.5.2DNAtransfections ....................................................................................................................81
5.5.3EstablishmentofOG2fibroblastsculture.................................................................................81
5.5.4DifferentiationofC2C12cells..................................................................................................82
5.5.5siRNAtransfectioninC2C12cells...........................................................................................82
5.5.6HpaII/MSmultiplexPCR .........................................................................................................83
5.5.7CombinedBisulphiteRestrictionAnalysis(COBRA)..............................................................84
5.5.8ReverseTranscriptionPolymeraseChainReaction .................................................................86
5.5.9Luciferaseassay........................................................................................................................91
5.5.10Betagalactosidasestaining.....................................................................................................91
5.6 Protein methods..............................................................................................................................92
5.6.1Celllysis ...................................................................................................................................92
5.6.2Proteinconcentrationestimation(BCAMethod) .....................................................................92
5.6.3SDSPAGEandWesternBlotting ............................................................................................92
5.6.4Immunostaining........................................................................................................................92
5.7 Bioinformatic tools .........................................................................................................................93
6 LITERATURE .............................................................................................94
7 PUBLICATION .........................................................................................105
ACKNOWLEDGMENTS .............................................................................106
































Summary
1.1 Summary
Theprimary objective ofthisthesis was to identify nuclear reprogrammingfactors
that would convert somatic cells back into pluripotency. Two broad screening
strategiesusingtheexpressionofstemcellmarkerOct4asamolecularreadoutwere
employed.
i) Expression screening of Xenopus egg cDNA library:Xenopuseggsaretotipotent
astheyhavethecapacitytogiverisetowholeorganism.XenopuseggcDNAlibraries
werescreenedbyoverexpressioninvariouscelllinestoisolatethegenesthatwould
upregulatetheOct4expression.
ii) Chemical Genomics screen:Alibraryofabout3000smallmoleculesavailableat
theDKFZEMBLchemicalgenomicscorefacilitywasscreenedinaHEK293stable
celllinethatharboursaluciferasereporterunderthecontroloftheOct4promoterand
primary fibroblasts obtained from the mouse harbouring a GFP reporter under the
controloftheOct4promoter.
Icouldnotisolateanygeneorsmallmoleculethatcouldspecificallyupregulatethe
Oct4expressionbytheabovementionedscreens.
The second objective of this thesis concerns epigenetic changes during cellular
differentiation.SeveralreportshaveindicatedthatDNAdemethylationaccompanies
cellular differentiation.As a model case when C2C12 myoblasts were induced to
differentiateintomyotubes,thepromoterofthemusclespecifictranscriptionfactor
Myogenin gene is demethylated and thereby facilitating its expression. Our group
recently showed that Gadd45alpha mediates active DNA demethylation by a
nucleotide excision repair mechanism. I tested if Gadd45 may mediate the
demethylation observed during the C2C12 differentiation by knocking down the
different isoforms of Gadd45. Gadd45beta knockdown blocked the Myogenin
promoterdemethylationasanalysedbyMSPCRandCOBRAassays.Moreover,the
Myogenin expression was significantly reduced. In conclusion, Gadd45 is indeed
required to relieve gene silencing during the differentiation of myoblasts into
myotubes.
1Summary
1.2 Zusammenfassung
Das grundlegende Ziel dieser Arbeit war die Identifizierung von nukleären,
reprogrammierenden Faktoren mit der Fähigkeit, somatische Zellen in pluripotente
Zellenzukonvertieren.HierfürwurdenzweiumfassendeStrategienangewandt,indenen
dieExpressiondesStammzellmarkersOct4alsmolekularerReadoutverwendetwurde.
i) Expressionsanalysen unter Verwendung von Xenopus cDNA Bibiliotheken:
Xenopus Eier sind totipotent, d.h. sie können Ausgangspunkt für einen kompletten
Organismus sein. Eine cDNA Bibiliothek aus Xenopus Eiern wurde in verschiedenen
Zelllinienüberexprimiert,umGenezuisolieren,dieOct4Expressioninduzieren.
ii) Chemische genomweite Analysen: Eine Bibliothek von 3000 kleinen chemischen
Verbindungen der „DKFZEMBL chemical genomics core facility“ wurde für die
Analyse in einer HEK293 stabilen Zelllinie verwendet, die ein Luziferase
Reporterplasmid unter der Kontrolle des Oct4 Promotors trägt. Die Bibiolothek wurde
ebenfalls in primären Fibroblasten einer transgenen Maus getestet, die einen GFP
ReporterunterderKontrolledesOct4Promotorstragen.
Ich konnte in den durchgeführten Screens kein Gen oder Molekül isolieren, welches
spezifischdieOct4Expressionhochreguliert.
Die zweite Fragestellung dieser Arbeit befasst sich mit epigenetischen Veränderungen
während der zellulären Differenzierung. Mehrere Untersuchungen belegen, dass
DifferenzierungmitDNADemethylierungeinhergeht.SowirdzumBeispielwährendder
Differenzierung von C2C12 Myoblasten zu Myotuben der Promotor eines Muskel
spezifischen Transkriptionsfaktors Myogenin demethyliert und somit dessen
Transkription induziert. Kürzlich wurde in unserer Arbeitsgruppe gezeigt, dass
Gadd45alpha aktive DNA Demethylierung unter Einbeziehung von Nukleotid
Entfernungsreparaturvermittelt.Ichhabegetestet,obGadd45ebenfallseinSchlüsselrolle
bei der Demethylierung während der C2C12 Differenzierung spielt, indem ich die
Expression verschiedener Gadd45 Isoformen dezimiert habe. MSPCR und COBRA
Analysen zeigen, dass Gadd45beta Knockdown die Demethylierung des Myogenin
Promotorsblockiert.DarüberhinauswardieMyogeninExpressionsignifikantreduziert.
FolglichistGadd45inderTatnotwendig,GenStilllegungwährendderDifferenzierung
vonMyoblastenzuMyotubenaufzuheben.
2