The regulation of TXNIP gene expression

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
THEREGULATIONOF
TXNIPGENEEXPRESSION
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
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
FAXINGYU
(B.Sc.(Hons.),NUS)
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ATHESISSUBMITTED
FORTHEDEGREEOFDOCTOROFPHILOSOPHY
DEPARTMENTOFBIOCHEMISTRY
NATIONALUNIVERSITYOFSINGAPORE
2009
Acknowledgments

Ifirstthankmysupervisor,Dr.YanLuo,forhisvaluablecommentsonhowto
designandperformexperiments,forhisgenerousgivingoffreedomtoexplorenew
fields,forhisenthusiasticencouragementwhenIencountereddifficulties,andforhis
criticalread ingandsuggestionsonmymanuscripts andthesis.Itisimpossibleto
havethis thesis presented herewithout Dr. Luo’s  patient guidance throughout my
graduatestudy.
I wish to thank Drs. Edwin Cheung and Xinmin Cao, my thesis advisory
committeemembers,forsharingtheirknowledgeandwisdom.Ialsowishtothank
Drs. Thilo Hagen and Ruping Dai for their constructive comments and friendly

discussionsonmyprojects.
IwouldalsoliketothankpastandpresentmembersofLuolab; allofushave
constructedaharmoniousandproductiveworkingenvironment.Specialthanksto
ShuangruGoh,shehasexperimentallycontributedtoaportionofresultsdescribed
inthisthesis,andDrs.HongpengHe,LilingZhengandMingjiJin,whohavegiven
mewonderfulsuggestionsonthesiswriting.
Finally,Ithankmylovingwife,myparents,mysiblings,mygrandpa,andother
familymembersfortheirunderstanding,supportsandsacrificesintheseyears.
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I
TableofContents
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 page
 
Acknowledgments I
 
TableofContents II
 
Summary IX
 
ListofTables XI
 
ListofFigures XII
 
Abbreviations XVII
 
Chapter1Introduction 1
 

1.1AGeneralOverviewofEukaryoticGeneTranscription 1
 
1.1.1RNAPolymerases 1
 
1.1.2CorePromotersandGeneralTranscriptionFactors(GTF) 2
 
1.1.3Sequence‐specificTranscriptionFactors 3
 
1.1.4Cofactors 4
 
1.1.5OtherRegulatoryMechanisms 6
 
1.2ExtracellularSignalsRegulateGeneTranscription 7
 
1.2.1InternalSensorsRegulateTranscriptionFactors 7
 
1.2.2CellPermeableLigandsRegulateTranscriptionFactors 9
 
1.2.3PlasmaMembraneReceptorsRegulateNuclearTranscription
Factors
9
 
1.2.4PlasmaMembraneReceptorsRegulateCytoplasmicTranscription
Factors
10
 
II
1.3ThioredoxinInteractingProtein(Txnip) 10
 
1.4TxnipFunctions 14

 
1.4.1TxnipandRedoxState 14
 
1.4.2TxnipandCellProliferationandCellDeath 15
 
1.4.3TxnipandCellDifferentiation 18
 
1.4.4TxnipandCellularMetabolism 18
 
1.5TxnipExpressioninResponsetoDifferentSignals 20
 
1.6Objectives 23
 
Chapter2MaterialsandMethods 25
 
2.1ChemicalsandBuffers 25
 
2.2PlasmidConstructs 25
 
2.3PurificationofBacteriallyExpressedRecombinantProteins 26
 
2.4MammalianCellCulture 27
 
2.5SodiumDodecylSulfatePolyacrylamideGelElectrophoresis
(SDS‐PAGE)
27
 
2.6Immuno‐Blot 27
 
2.7RNAExtraction,RT(ReverseTranscription)‐PCRandReal‐TimePCR 28

 
2.8GenomicDNAExtra ction  29
 
2.9SmallInterferingRNAs(siRNAs)andRNAInterferenceAssays 29
 
2.10Transfection 30
 
2.11PromoterActivity(Reporter)Assays 30
 
2.12Immunocytometry 30


2.13Fluorescence‐activatedCellSorting(FACS) 31
III
 
2.14ElectrophoresisMobilityShiftAssays(EMSA) 31
 
2.15ChromatinImmunoprecipitation(ChIP)Assays 34
 
2.16ThioredoxinActivityAssays 35
 
2.17GlucoseTransportAssays 35
 
2.18StatisticalAnalyses 36
 
Chapter3IdentificationofMoleculesModulatingTxnipExpression 37
 
3.1Preface 37
 
3.2Results 38


 
3.2.1Adenosine‐containingMoleculesInduceTxnipExpression 38
 
3.2.1.1NAD(H)andATPStimulateTxnipExpression 38

3.2.1.2MoleculesContainingAdenosineGroupInduceTxnip
Expression
40

3.2.1.3AdenosineisNecessaryandSufficientforInducingTxnip
Expression
40

3.2.1.4Adenosine‐containingMoleculesInduceTxnipExpression
inaDose‐dependentManner
43

3.2.1.5LongTermEffectofAdenosine‐containingMoleculeson
TxnipExpression
44

3.2.1.6Adenosine‐containingMoleculesInduceTxnipExpression
attheTranscriptionalLevel
45

3.2.1.7Adenosine‐containingMoleculesInduceTxnipExpression
IsMediatedbyanEarlierDefinedChoRE
47


3.2.1.8TheMLX/MondoAComplexMediatestheInductionof
TxnipExpressionbyAdenosine‐containingMolecules
49

3.2.1.9Adenosine‐containingMoleculesFacilitateMondoA
NuclearTranslocation
52
IV

3.2.1.10GlucoseIsRequiredfortheInductionofTxnipExpression
byAdenosine‐containingMolecules
54

3.2.1.11GlucoseInducedTxnipExpressionIsAmplifiedby
Adenosine‐containingMolecules
56

3.2.1.12PotentialPlasmaMembraneTarget(s)ofAdenosine‐
containingMolecules
57

3.2.1.12.1PurinergicReceptorsAreNotRequiredforthe
InductionofTxnipExpression
59

3.2.1.12.2Adenosine‐containingMoleculesMayTarget
AdenosineTransporters
59

3.2.1.13SignalingPathway(s)EvokedbyAdenosine‐containing

MoleculesforRegulatingTxnipExpression
62

3.2.1.13.1TheInductionofTxnipExpressionRequires
IntracellularCa
2+

62

3.2.1.13.2TheInductionofTxnipExpressionDoesNot
RequirecAMP
64

3.2.1.13.3TheInvolvementofMAPKintheInductionof
TxnipExpression
64

3.2.1.13.4Non‐involvementofAMPKintheInductionof
TxnipExpression
66

3.2.1.14Adenosine‐containingMoleculesRepressThioredoxin
ActivityandGlucoseTransport
67

3.2.1.15Adenosine‐containingMoleculesAffectCellCycle
Progression
69

3.2.2EffectsofGlucoseAnalogsonTxnipExpression 70

 
3.2.2.1EffectsofSelectedMonosaccharidesandDisaccharideson
TxnipExpression
70

3.2.2.2EffectofPD169316onGlucose,2DGorMaltose/NAD
+

InducedTxnipExpression
75
V

3.2.2.3ACa
2+
ChelatorAbolishestheStimulatoryEffectofGlucose
onTxnipExpression
75

3.2.3InhibitorsofOxidationPhosphorylationRepressedTxnip
Expression
76

3.2.3.1NitricOxide(NO)andSodiumAzide(NaN
3)RepressTxnip
Expression
76

3.2.3.2InhibitionofOxidativePhosphorylationRepressesTxnip
Expression
80


3.2.3.3ACa
2+
ChelatorRescuesTxnipExpressioninthePresenceof
OxidativePhosphorylationInhibitors
82

3.3Discussion 83
 
3.3.1Adenosine‐containingMoleculesMayRemainExtracellularto
InduceTxnipExpression
83
 
3.3.2PotentialMembraneTargetsforAdenosine‐containingMolecules 84
 
3.3.3SignalingPathway(s)InvolvedintheInductionofTxnip
ExpressionbyAdenosine‐containingMolecules
85
 
3.3.4TheMondoA/MLXComplexMediatesTxnipExpression 87
 
3.3.5PhysiologicalSignificance 89
 
3.3.6TwoSignalingPathwaysEvokedbyGlucoseforInducingTxnip
Expression
91
 
3.3.7TxnipExpressionIsaSensorofOxidativePhosphorylationStatus 93
 
3.4ConclusionandPerspectives 94

 
Chapter4RegulatoryMechanismsUnderlyingtheInductionofTxnip
ExpressionbyGlucoseorAdenosine‐containingMolecules
96
 
4.1Preface 96
 
4.2Results 98
 
VI
4.2.1RegulatoryMechanismsatthePromoterLevelGoverning
GlucoseorAdenosine‐containingMoleculesInducedTxnip
Expression
98
 
4.2.1.1TxnipPromoterRegionsCriticalforExpressionInduction
byNAD
+
orGlucose
98

4.2.1.2TandemChoREsonTxnipPromoters 102

4.2.1.3TheMondoA/MLXComplexBindstoBothChoREsinvitro 105

4.2.1.4BothChoREsareRequiredforOptimalTxnipPromoter
Activity
108

4.2.1.5ChoREsAreNotSufficientfortheInductionofTxnip

Expression
111

4.2.1.6TandemNF‐YBindingSitesAreRequiredfortheInduction
ofTxnipExpression
111

4.2.1.7NF‐YMediatedInductionofTxnipExpressionbySAHA
RequiresMondoA/MLX
114

4.2.1.8TxnipPromoterRecruitsMondoA/MLXComplexinan
NF‐YDependentManner
116

4.3TheRoleofUSFsinTxnipExpression 121
 
4.3.1ExpressionandPurificationofHis‐taggedUSF1 121
 
4.3.2USF1InteractswithChoRESitesinvitro 122
 
4.3.3Down‐regulationofUSFsReducesTxnipExpression 125
 
4.3.4Over‐expressionofUSFsInducesTxnipPromoterActivity 127
 
4.3.5USFIsNotInvolvedintheTxnipInductionby
Adenosine‐containingMolecules
129
 
4.3.6USFIsNotInvolvedintheTxnipInductionbyGlucose 132

 
4.4Discussion 133
 
4.4.1TandemChoREsontheTxnipGenePromoter 133
VII
 
4.4.2FullInductionofTxnipExpressionRequiresBothChoREsand
CCAATBoxes
134
 
4.4.3NF‐YandMondoA/MLXCooperatetoStimulateTxnip
Expression
136
 
4.4.4TheRoleofUSFsonTxnipExpression 138
 
4.5ConclusionandPerspectives 140
 
References 143
 
Appendices 162
 
AppendixI,Buffers/GelsUsedinThisStudy 162
 
AppendixII,RNAIntegrity 165
 
AppendixIII,PrimerSpecificity 166
 
AppendixIV,PrimersUsedinThisStudy 167
 

AppendixV, Paper1(Abstract) 172
 
AppendixVI,Paper2(Abstract) 173









VIII
Summary

Thioredoxininteractingprotein(Txnip)isamultifunctionalproteininvolved
inregulation ofcellcycleeventsandcellularmetabolism.TheexpressionofTxnipis
induced by glucose, and this induction is mediated by a carbohydrate response
element (ChoRE) on Txnip promoter and its associated transcription factors,
MondoA and Max‐like proteinX (MLX).In this study,I havediscoveredthat the
transcription of the Txnip gene is induced by an array of adenosine‐containing
molecules, of which an intact adenosine moiety is necessary and sufficient.The
induction of Txnip expression by adenosine‐containing molecules is glucose
dependent,andMondoAandMLXhavebeenshowntoconveystimulatorysignals
from extracellularmolecules tothe Txnippromoter. Therefore,the regulatoryrole
ofadenosine‐containingmolecules isexerted viaamplifying glucose signaling,and
thissuggeststhatthesemoleculesmaymodulatethekineticsofglucosehomeostasis.
I have also studied the underlying regulatory mechanisms of glucose and
adenosine‐containingmoleculesonTxnipexpression.AnadditionalChoREonthe
promoter of Txnip gene has been identified, and this ChoRE is able to recruit

MondoA and MLXin a similar fashionas the previously identified ChoRE in vitro
and in vivo.Both ChoREs function cooperatively  to mediate optimal Txnip
expression under glucoseor adenosine‐containingmolecules treatment.However,
thesetwoChoREsarenotsufficienttomediatetheinductionofTxnipexpressionby
glucoseoradenosine‐containingmolecules,andtwoCCAATboxes,bothcanrecruit
IX
nuclearfactorY(NF‐Y)totheTxnippromoter,arealsorequiredfortheinduction.I
also found that the function of ChoREs and associated factors is contingent on
tandemCCAATbo xes,inthattheoccupancyoftheTxnippromoterbytheCCAAT
box‐associatedNF‐Yisaprerequisit eforefficaciousrecruitmentofMondoA/MLXto
ChoREs under glucose stimulation.Such a strategy suggests a synergy between
NF‐Y and MondoA/MLX in enhancing Txnip expression  presumably through
inducingdynamicchromatinchangesinresponsetodiversephysiologicalinducers.
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ListofTables

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Table1.OligonucleotidesusedforEMSA. 32
 
Table2.PrimersusedinChIPassay. 33
 
Table3.Theeffectsofdifferentmolecules onTxnipmRNAlevels. 41
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ListofFigures
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Figure1.Asimplifiedversionofeukaryotictranscriptionmachineryand
transcriptionregulatorymechanisms.
5
 
Figure2.Possiblemechanismsfortheregulationofgenetranscriptionby
extracellularmoleculesandinternalenvironmentchanges.
8
 
Figure3.AphylogenetictreeofTxniporthologsfromdifferentorganisms. 11
 
Figure4.GenestructuresofTxnip,arrestinβ2andARRDCgenes. 13
 
Figure5.ProteinprimarystructureofTxnip,arrestinβ2andARRDCs. 14
 
Figure6.SignalingpathwaysregulatingTxnipexpressionandtheir
targetingcis‐regulatoryelementsonTxnippromoter.
21
 
Figure7.NAD(H)orATPinducedTxnipmRNAlevel. 38
 
Figure8.NAD
+
inducedTxnipproteinlevel. 39
 
Figure9.NAD(H)orATPinducedTxnipexpressionindiversecelllines. 40
 
Figure10.NAD
+
andATPshareacommonadenosinegroup. 41
 

Figure11.StructureofATP ,dATP,Bz‐ATPorTnp‐ATP . 42
 
Figure12.Titrationsofadenosine‐containingmoleculesonTxnipexpression. 43
 
Figure13.Timecourseofadenosine‐containingmoleculesonTxnip
expression.
44
 
Figure14.Adenosine‐containingmoleculesinduceTxnippromoteractivity. 45
 
Figure15.Adenosine‐containingmoleculesdidnotinduceTxnipexpression 
inthepresenceofactinomycinD.
46
 
Figure16.NAD
+
orATPdidnotinduceactivityofChoRE‐mutatedTxnip
promoters.
48
 
XII
Figure17.NAD
+
orATPcouldnotinduceTxnippromoteractivityinthe
presenceofdominantnegativeMLX.
48
 
Figure18.EffectsofectopicMLXand/orMondoonTxnippromoteractivity. 50
 
Figure19.EffectsofsiRNAsagainstMLXorMondoAonTxnipexpression. 51

 
Figure20.LocalizationofHA‐MondoAinL6cellsundercontrolor2DG
treatment.
53
 
Figure21.MondoAnucleartranslocationwasfacilitatedbyadenosine‐
containingmolecules.
53
 
Figure22.Glucose‐dependentinductionofTxnipexpressionbyNAD
+
orATP. 55
 
Figure23.NAD
+
inducedTxnipexpressionina widetitrationofglucose. 56
 
Figure24.TheinductionofTxnipexpressionwasnotabolishedbyARL
67156.
57
 
Figure25.Inhibitorsforpurinergicreceptorsdid notinhibittheinductionof
TxnipexpressionbyNAD
+
orATP .
59
 
Figure26.StructuresofAdenosine,NBTI,DipyridamoleorDilazep. 60
 
Figure27.TheeffectofNAD

+
orATPonTxnipexpressionwasblockedby
inhibitorsofadenosinetransporters.
61
 
Figure28.EffectofCa
2+
chelatorsonTxnipexpression. 62
 
Figure29.cAMPsignalingpathwaydidnotmediatetheinductionofTxnip
expressionbyNAD
+
.
63
 
Figure30.TheeffectofMAPKinhibitorsonTxnipexpressionanditsresponse
toNAD
+
orATPinU2OScells.
65
 
Figure31.TheeffectofMAPKinhibitorsonTxnipexpressionanditsresponse
toNAD
+
orATPinHeLacells.
66
 
Figure32.AMPKwasnotinvolvedintheinductionofTxnipexpressionby
adenosine‐containingmolecules.
67

 
Figure33.Adenosine‐containingMoleculesRepressedThioredoxinActivity. 68
 
XIII
Figure34.Adenosine‐containingMoleculesInhibitedGlucoseTransport. 68
 
Figure35.NAD(H)treatmentrepressedcellcycleprogression. 69
 
Figure36.NAD(H)treatmentselevatedp21
cip1
expressionlevel. 70
 
Figure37.Structuresofglucoseanalogsandtheirfateincellularmetabolism. 71
 
Figure38.TheeffectofglucoseandglucosehomologsonTxnipexpressionin
theabsenceorpresenceofNAD
+
.
72
 
Figure39.TheeffectofPD169316onTxnipinductionbydifferent
carbohydratesand/orNAD
+
.
74
 
Figure40.GlucoseinducedTxnipexpressionwasrepressedbyBAPTA‐AM. 76
 
Figure41.EffectsofNOdonorsandGCinhibitoronTxnipexpression. 77
 

Figure42.TheeffectofNOonTxnipexpressionwasnotmediatedbyGC. 78
 
Figure43.TheeffectofNaN
3onTxnipmRNALevels. 79
 
Figure44.Asimplifiedrepresentationofoxidativephosphorylation. 80
 
Figure45.EffectsofoxidativephosphorylationinhibitorsonTxniporH2B
expression.
81
 
Figure46.OxidativephosphorylationinhibitorsdidnotrepressTxnip
expressioninthepresenceBAPTA‐AM.
82
 
Figure47.Anegativefeed‐backloopforglucoseuptake. 90
 
Figure48.AschematicrepresentationoftruncatedTxnippromoters. 99
 
Figure49.ResponsesofTxnippromoterstoNAD
+
. 99
 
Figure50.ResponsesofTxnippromoterstoNAD
+
orGlucose. 100
 
Figure51.TheminimalTxnipprom otersequencerequiredformediatingthe
stimulatoryeffectofNAD
+

.
101
 
Figure52.cis‐regulatoryelementsonTxnippromoter. 102
 
Figure53.AlignmentofTxnippromotersfromdifferentspecies. 103
XIV
 
Figure54.PhylogenetictreeofTxnippromoters. 104
 
Figure55.SequencealignmentoffishandfrogTxnippromoterswiththe
humanTxnippromoter. 
105
 
Figure56.EMSAsusingtwoTxnippromotersegmentscontainingChoRE‐a
orChoRE‐b.
106
 
Figure57.Responsesofwild‐type(WT)orChoREmutantTxnippromotersto
NAD
+
orglucose.
109
 
Figure58.EffectofsiRNAsagainstMLXorMondoAonTxnippromoter
activities.
110
 
Figure59.TheresponseofhybridTxnippromoterstoNAD
+

orglucose. 112
 
Figure60.Responsesofwild‐type(WT)orCCAATboxmutantTxnip
promoterstoNAD
+
orglucose.
113
 
Figure61.TheeffectofSAHA. 115
 
Figure62.StablecelllinesforChIPassays. 117
 
Figure63.RecruitmentofMLXandNF‐Ytorespectivetargets(ChoREsand
CCAATboxes)assessedbyChIPassays.
118
 
Figure64.MLXisnotrecruitedontoCCAATboxes‐mutatedTxnippromoter. 119
 
Figure65.Theinteraction betweenMLXandChoREisglucosedependent. 121
 
Figure66.BacterialexpressionofUSF1. 121
 
Figure67.His‐USF1interacts with ChoRE‐aorChoRE‐b. 122
 
Figure68.ChoRE‐containingprobesinteractwithaprotein(s)inHeLanuclear
extract(NE).
124
 
Figure69.BandsshiftbyUSFantibodies. 125
 

Figure70.Theeffects ofUSF1‐orUSF2‐specificsiRNAsonTxnipexpression. 126
 
Figure71.TheinductionofTxnippromoteractivitybyover‐expressionof
USFs.
127
XV
 
Figure72.TheresponseofTxnippromotertoNAD
+
orATPunderUSF
over‐expression.
128
 
Figure73.EffectsofUSFover‐expressionontheactivitiesoftruncatedor
mutantTxnippromoters.
130
 
Figure74.A‐USFdidnotrepresstheinductionofTxnippromoteractivityby
NAD
+
orATP.
131
 
Figure75.A‐USFdidnotrepresstheinductionofTxnippromoteractivityby
glucose.
132
 
Figure76.AmodelforthetranscriptionalregulationoftheTxnipgene
promoterbyNF‐Y,MondoA/MLXandother(co)factors.
137

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XVI
Abbreviations

2DG 2‐deoxy‐glucose
3OMG 3‐O‐Methylglucose
5‐aza‐CdR 5‐AZA‐2ʹ‐deoxycytidine
AC Adenylylcyclase
AICAR 5‐aminoimidazole‐4‐carboxamide

ribonucleoside
AP‐1 Activatorprotein1
ARRDC Arrestindomaincontaining
ASK‐1 Apoptosissignal‐regulatingkinase1
BrdU Bromodeoxyuridine
cAMP Cyclicadenosinemonophosphate

CDK4 Cyclin‐dependentkinase4
cDNA ComplementaryDNA
ChIP Chromatinimmunoprecipitation
ChoRE Carbohydrateresponseelement
ERK Extracellularsignal‐regulatedkinases
EMSA Electrophoresismobilityshiftassay
FACS Fluorescence‐activatedcellsorting
FCHL Familialcombinedhyperlipidemia
FOXO1 ForkheadboxO1
G6P Glucose‐6‐phosphate
XVII
GC Guanylatecyclase
Glut Glucosetransporter
GPCR G‐proteincoupledreceptors
GR Glucocorticoidreceptor
GRE Glucocorticoidresponseelement
GTF Generaltranscriptionfactors
HA Hemagglutinin
HAT Histoneacetyltransferases
HDAC Histonedeacetylases
HDMT Histonedemethylases
HIF‐1 Hypoxia‐inducingfactor1
HMT Histonemethyltransferases
HRE HIF‐responsiveelement
HSE Heatshockresponseelements
HSF‐1 Heatshockfactor1
HSP Heatshockproteins
HXT Hexosetransporter
IP Immunoprecipitation
IPTG Isopropyl‐beta‐D‐thiogalactopyranoside

Jab‐1 Junactivationdomain‐bindingprotein1
JAK Januskinase
JNK c‐JunN‐terminalkinases
XVIII
MAPK Mitogen‐activated

proteinkinase
MLX Max‐likeproteinX
mRNA MessengerRNA
mTOR Mammaliantargetofrapamycin
NE Nuclearextract
NF‐κB Kappa‐light‐chain ‐enhancerofactivatedBcells
NF‐Y NuclearfactorY
NO Nitricoxide
Oct‐1 Octamer‐1
PIC Pre‐initiationcomplex
PKA ProteinkinaseA
PKB/AKT ProteinkinaseB
PKG ProteinkinaseG
PolII RNApolymeraseII
PP2A Proteinphosphatase2A
PPAR Peroxisomeproliferator‐activatedreceptor
PPRE Peroxisomeproliferatorhormoneresponseelement
PTEN Phosphataseandtensinhomolog
ROS Reactiveoxygenspecies
RTK Receptortyrosinekinases
SAHA Suberoylanilidehydroxamicacid
SDS‐PAGE Sodiumdodecylsulfatepolyacrylamidegelelectrophoresis
XIX
SGLT Na

+
/glucosesymporter
siRNA SmallinterferenceRNA
STAT Signaltransducersandactivatoroftranscription
TBP‐2 Thioredoxin‐bindingprotein2
TGF‐β Transforminggrowthfactorbeta
Txnip ThioredoxinInteractingProtein
USF Upstreamstimulatoryfactor
VDRE VitaminDresponseelement
VDUP1 VitaminD
3up‐regulatedprotein1
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XX
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Chapter1


Introduction
Chapter1
1.1AGeneralOverviewofEukaryoticGeneTranscription
GeneticinformationisstoredintheformofDNA.Toexerttheirfunctions,
genes are required to be expressed to RNA or proteins.Transcription, or RNA
synthesis,istheprocessoftranscribingsequenceinformationfromDNAtoRNAina
complementarymanner.RNAinturncanbeusedastemplateforproteinsynthesis,
thisprocessiscalledtranslation.
1.1.1RNAPolymerases
Transcription is a biochemical reaction catalyzed by RNA polymerases.In
eukaryotes,three typesof RNApolymerases have been identifiedand respectively
dubbed as RNA polymerase I, II and III (Roeder and Rutter, 1969).Among these
polymerases,RNApolymeraseIisresponsibleforthesynthesisofribosomalRNAs
(rRNA);RNApolymeraseII(PolII,orRNAPII)mediatesthesynthesisofmessenger
RNAs(mRNA),somesmallnuclearRNAs(snRNA),smallnucleolarRNAs(snoRNA)
andmicroRNAs;andRNApolymeraseIIIisresponsibleforthesynthesisoftransfer
RNAs(tRNA),5SrRNAandsomesnRNAs(RoederandRutter,1970;Leeetal.,2004).
Novel RNA polymerases have also been discovered recently that are involved in
synthesis of some specific RNAs (e.g., synthesis of certain small interfering RNAs
[siRNA]inplants[Herretal.,2005]).
 PolIIisaproteincomplexcontaining12subunitsencodedbydifferentgenes;
among these subunits, some are Pol II‐specific while others are shared with other
RNApolymerases(Sklaretal.,1975).Thesynthesisofprotein‐codingmRNAsbyPol
II is a complicated process facilitated by many other factors, and is controlled by
multipleregulatorypathwaystomeetthespatialandtemporalrequirementforthe
expressionofspecificgenes.Thelastfourdecadeshavewitnessedextensivestudies
1
Chapter1
onthestructure,functionandregulationofPolIIusingbiochemical,structuraland
geneticapproaches(reviewedinRoeder,2003;LevineandTjian,2003;andKornberg,

2007).
1.1.2CorePromotersandGeneralTranscriptionFactors(GTF)
 ForagivenmRNAencodinggene,acorepromoterisdefinedastheminimal
DNA sequence required for accurate transcription initiation.Core promoters are
typicallypositionedaroundthetranscriptionstartsite(~35nucleotidesupstreamor
downstream)andcontainsignatureelementsincluding,tonameafew,TATAbox,
initiator(Inr)anddownstreamcorepromoterelement(DPE)(ButlerandKadonaga,
2002).Thereappears to be no universal core promoterelements; forinstance, few
housekeepinggenes,suchastheoneencodinghistone1,containaTATAboxinthe
promoters (Cooper et al., 2006; Isogai et al., 2007).I am working on a TATA box‐
containinggene,thereforebelowIwillfocusontheintroductionofTATA‐regulated
promoters/genes.
Although RNA polymerases are enzymes responsible for RNA synthesis,
theyaloneareunabletoinitiateaccuratetranscription(Weiletal.,1979).Sixancillary
protein or proteincomplexes, namely TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH
(respectively,transcriptionfactorsA,B,D,E,FandHforPolII),havebeenisolated
andcharacterizedfrom wholecellornuclearextracts;they(withatotalof32distinct
polypeptides) are collectivelycalled general transcription (initiation)factors (GTFs,
Figure 1; Matsui et al., 1980; Samuels et al., 1982; Reinbergand Roeder, 1987a;
ReinbergandRoeder,1987b;Floresetal.,1990;Floresetal.,1992).PolIIandGTFs
canformapre‐initiationcomplex(PIC)importantforaccuratetranscriptioninitiation
fromacorepromoteratabasallevel(VanDykeetal.,1988;Buratowskietal.,1989).
2
Chapter1
GTFsarewidelyexpressedandrequiredfortranscriptionofmajority,ifnot
all, mRNA genes; however, a subset of cellular genes may require a subset of the
GTFsthatalongwithcertainspecialized(co)factorsbringsabouttissue‐and/orgene‐
specifictranscriptionalactivation(reviewedinGreen,2000;Berk,2000).
1.1.3Sequence‐specificTranscriptionFactors
 ManyevolutionaryconservedshortDNAsequenceshavebeenidentifiedon

promoter region of most genes, and these short DNA elements (cis‐regulatory
elements)areabletomediatetranscriptionregulationbyrecruitingsequence‐specific
transcription factors.The proximal sequencesupstream (~250) ofthe transcription
startsiteusually containprimaryregulatoryelements, andthese regionsarecalled
proximal promoters.Less frequently, some elements are located proximally or
distally downstream, or at distal regions upstream, of the transcription start  site.
Theseelementsare knownas distal promoterorenhancerelements.Among these
cis‐regulatory elements, some can recruit  positive regulatory factors (transcription
activators); on  the other hand, some can  recruit negative regulatory factors
(transcription repressors).For convenience, these two types of cis‐regulatory
elementsaredubbed,respectively,enhancerandrepressor(Figure1).
Sequence‐specific transcription factors occupy enhancers or repressors via
multiple interactions, such as hydrogen bonds, ionic bonds and hydrophobic
interactions.Thesetranscriptionfactorsusuallycontainstructures(withmultipleα
helices, loops orβsheets) which can bind to the major groove of DNA.Such
structures include Helix‐Turn‐Helix motifs, Zinc Fingers, Leucine Zippers, Helix‐
Loop‐Helix motifs and possibly others (Alberts et al., 2002).For instances, the
homeodomain of octamer‐1 (Oct‐1) is a Helix‐Turn‐Heli x motif, and this motif is
3