Table Of ContentMolecular mechanisms of enhanced expression
of the chemokine Interleukin 8 (CXCL8) in cystic
fibrosis (CF) airway epithelial cells
Anna Poghosyan, BM (Hons)
Thesis submitted to the University of Nottingham for the
degree of Doctor of Philosophy
November 2014
1
ABSTRACT
Cystic fibrosis (CF) is a fatal disease caused by a mutation of the CFTR gene and
severe inflammation of the lungs. The inflammatory process is characterised by
increased production of the potent neutrophil-attracting chemokine interleukin 8
(CXCL8), but the mechanism responsible is poorly understood. We tested the
hypothesis that altered epigenetic regulation is responsible for the basal and
cytokine-induced CXCL8 upregulation in CF airway epithelial cells. We found that
CXCL8 protein levels and mRNA expression were higher in CF as compared to
normal cells both basally and following cytokine stimulation. The difference in the
expression was independent of increased mRNA stability or increased transcription
factor activation and/or expression in CF cells. We found increased basal, but not
cytokine-inducedtranscriptionfactorbindingtotheCXCL8promoterin achromatin
environment in CF cells in comparison with normal cells, increased histone H3
lysine 4 trimethylation, hypomethylation of CpG sites and increased binding of
BRD3 and BRD4 to the CXCL8 promoter. Disruption of BRD4 association with
chromatin using the selective BET bromodomain inhibitor JQ1 decreased CXCL8
protein release from CF cells to the levels observed in normal cells. Our
observations suggest that epigenetic alterations are responsible for the
upregulationofCXCL8inCFandcouldbecomepotentialtargetsinthedevelopment
ofnewtherapeuticstrategies.
2
ACKNOWLEDGEMENTS
Firstly, I would like to express my sincere gratitude to Prof. Alan Knox for his
support,patience,enthusiasm, and immense knowledge. Thiswork would not have
beenpossiblewithouthisguidance,invaluableinsightsandexcellentadvice.
I would like to express my sincere appreciation to the excellent research group of
DivisionofRespiratoryMedicineandthankallthemembersforcontinuoussupport,
help and encouragement throughout my PhD. I would like to express my special
gratitudeto an extraordinaryperson,RachelClifford,for beinga constantsource of
knowledge and invaluable advices, for all her inspiration, patience and continuous
encouragement. Her faith in me and emotional support encouraged me to grow as
aresearcherandindependentthinkermakingmyPhDyearsveryenjoyable.
I must also thank the University of Nottingham for giving me an opportunity to
carryoutaresearchprojectbyfundingit.
And last, but not least, I would like to thank my parents for all the support,
encouragementandunwaveringlovethroughoutmyPhD.
3
LIST OF PUBLICATIONS
A. Poghosyan,R. L. Clifford,W. R. Coward, L.Pang, A.J.Knox.Enhancedexpression
of interleukin 8 in cystic fibrosis airway epithelial cells. Eur Respir J 2013; 42: Suppl.
57, 2107 (abstract accepted for a thematic poster presentation at the European
RespiratorySocietyAnnualCongress,Barcelona,Spain,2013).
A. Poghosyan, R. L. Clifford, W. R. Coward, L. Pang, A. J. Knox. Molecular
mechanisms of enhanced expression of Interleukin 8 in cystic fibrosis airway
epithelial cells. The Journal of Cystic Fibrosis; Vol 12, Suppl. 1, 167, 2013 (abstract
accepted for a thematic poster presentation at the 36th European Cystic Fibrosis
Conference,Lisbon,Portugal,2013).
4
LIST OF ABBREVIATIONS
aGM1 AsialoGM1receptor LPS Lipopolysaccharide
ARE AU-richelement LZ Leucinezipper
dimerizationdomain
ASL Airwaysurfaceliquid MAPK Mitogen-activated
proteinkinase
ATP Adenosinetriphosphate MBD Methyl-CpG-binding
domain
β -M Beta -microglobulin MBP MethylCpGbinding
2 2
protein
BAL Bronchoalveolar MBT Malignantbraintumour
BCA Bicinchoninicacidprotein MDB Membranedesalting
assay buffer
BCC Burkholderiacepacia MEM Minimumessential
complex mediumEagle
BET Bromodomainsandextra- mg Milligram
terminal
BR Basicregion µl Microliter
BRD Bromodomain mL Millilitre
BSA Bovineserumalbumin mM mmol
Ca2+ Calcium mM/L Mmol/Litre
cAMP Cyclicadenosine M-MLVRT Moloneymurine
monophosphate leukaemiavirusreverse
transcriptase
CARM1 Co-activator-associated MMP Matrix
argininemethyltransferase1 metalloproteinase
CBP cAMPresponseelement mRNA Messengerribonucleic
bindingprotein(CREB) acid
bindingprotein
cDNA Complimentary MTT 3-(4,5-dimethythiazol-2-
deoxyribonucleicacid yl)-2,5-diphenyl
tetrazoliumbromide
C/EBPβ CCAAT/enhancerbinding Na+ Sodium
proteinbeta
CF Cysticfibrosis NaCl- Sodiumchloride
CFF CysticFibrosisFoundation NE Neutrophilelastase
CFTR Cysticfibrosis NF-ĸB NuclearfactorkappaB
transmembrane
conductanceregulator
CG Cytosine-guanine ng Nanogram
ChIP Chromatin NLS Nuclearlocalisation
immunoprecipitation signal
Cl- Chloride nm Nanometre
cm2 Squarecentimetre NO Nitricoxide
Co-IP Co-Immunoprecipitation NPD Nasalpotential
difference
COPD Chronicobstructive NRE Negativeregulatory
pulmonarydisease element
CT Computedtomography NRF NF-ĸB-repressingfactor
CXCL8 Interleukin8 Oct-1 Octamer1
ddH O Doubledistilledwater OligoDT Oligodeoxythymidylic
2
5
acid
DEPC Diethylpyrocarbonate P Openprobability
o
DMSO Dimethylsulfoxide P.aeruginosa Pseudomonas
aeruginosa
DNMT DNAmethyltransferase PAMP Pathogen-associated
molecularpattern
DNA Deoxyribonucleicacid PBS Phosphatebuffered
saline
dNTP Deoxynucleoside PCAF p300-CBPassociated
triphosphate factor
DTT Dithiothreitol PCR Polymerasechain
reaction
ECLTM WesternLightning™ pg Picogram
Chemiluminescence
Reagent
EDTA Ethylenediaminetetra-acetic PGE2 ProstaglandinE2
acid
ELISA Enzyme-linked PIC Proteinaseinhibitor
Immunosorbentassay cocktail
ENaC Amiloride-sensitiveepithelial PMN Polymorphonuclear
sodiumchannel neutrophil
FAD Flavinadeninedinucleotide PMSF Phenylmethylsulfonyl
fluoride
FCS Foetalcalfserum PolII PolymeraseII
g Relativecentrifugalforce PRMT Arginine
methyltransferase
GNAT Gcn5-relatedN- PRR Patternrecognition
acetyltransferase receptor
H Histone PVDF Polyvinylidene
difluoride
H O Hydrogenperoxide qPCR Quantitativereal-time
2 2
polymerasechain
reaction
H2A Histone2A R Arginine
H2B Histone2B RAW1,2 Washbuffer1and2
H3K4 Histone3lysine4 RCF Relativecentrifugal
force
H3K4me Histone3lysine4 RD Regulatorydomain
trimethylation
H3K9me Histone3lysine9 rDNase Recombinant
trimethylation deoxyribonuclease
H3K27 Histone3lysine27 Re-ChIP Re-Chromatin
immunoprecipitation
precipitation
H3K27me3 Histone3lysine27 RHD Relhomologydomain
trimethylation
H3K36 Histone3lysine36 RNA Ribonucleicacid
H4K20 Histone4lysine20 RNAase Ribonuclease
H4R3 Histone4arginine3 RNA-seA RinobucleaseA
HAT Histoneacetyltransferase ROS Reactiveoxygenspecies
HCO Bicarbonate RPM Rotationsperminute
3-
6
HDAC Histonedeacetylase RT Reversetranscription
HDM Histonedemethylase RT-QPCR Real-timequantitative
polymerasechain
reaction
H.influenza Haemophilus S Serine
influenzae
HMT Histonemethyltransferase S.aureus Staphylococcusaureus
IFN-γ Interferongamma SDS Sodiumdodecyl
sulphate
IgG ImmunoglobulinG SDS-PAGE Sodiumdodecyl
sulphatepolyacrylamide
gelelectrophoresis
IKK IĸB kinase SEM Standarderrorofthe
mean
IL Interleukin SET Suppressorof
variegation-Enhancerof
zeste-Trithorax
IL1-β Interleukin1beta SRC-1 Steroidreceptor
coactivator-1
IL-6 Interleukin6 Streptavidin- Streptavidin-
HRP horseradish-peroxidase
IL-10 Interleukin10 SUMO SmallUbiquitin-related
MOdifierprotein
IP Immunoprecipitation TBS-T Trisbufferedsalineplus
Tween-20
JmjC Jumonji TD/TAD Transactivationdomain
JNK JunN-terminalkinase TF Transcriptionfactor
K Lysine TGF-ß Transforminggrowth
factor-beta
K+ Potassium TIF-2 Transcriptional
intermediaryfactor-2
Kac Epsilon-N-acetyllysine TNF-α Tumournecrosisfactor-
alpha
kb Kilobase TNFR Tumournecrosisfactor
receptor
KC Keratinocyte USF Upstreamstimulatory
chemoattractant factors
kDa KiloDalton UV Ultraviolet
LARII LuciferaseassayreagentII V Volt
LF Lipofectamine WT Wildtype
7
TABLE OF CONTENTS
ABSTRACT.................................................................................................................................2
ACKNOWLEDGEMENTS............................................................................................................3
LISTOFPUBLICATIONS.............................................................................................................4
LISTOFABBREVIATIONS..........................................................................................................5
1 INTRODUCTION.................................................................................................................14
1.1 Overviewofcysticfibrosis...........................................................................................15
1.1.1 Aetiology......................................................................................................................15
1.1.2 Symptomsanddiagnosis.............................................................................................16
1.1.3 Therapy........................................................................................................................19
1.2 Inflammationincysticfibrosis.....................................................................................21
1.2.1 CFTRdeficiencyandlungpathology............................................................................21
1.2.2 Bacterialpresenceinthelungs....................................................................................24
1.3 Inflammatoryresponse...............................................................................................25
1.3.1 Overviewofaninflammatoryprocess.........................................................................25
1.3.2 Inflammatorychemokines...........................................................................................27
1.4 CXCL8anditsroleinCFinflammation.........................................................................28
1.4.1 ExcessiveinflammationinCF.......................................................................................28
1.4.2 CXCL8andotherpathologies.......................................................................................30
1.4.3 CXCL8structure...........................................................................................................34
1.4.4 CXCL8functions...........................................................................................................35
1.5 NF‐κB ............................................................................................................................ 38
1.5.2 OthertranscriptionfactorsinvolvedinCXCL8transcription.......................................43
1.6 Epigeneticregulationofgenetranscription................................................................48
1.6.1 Conceptofepigeneticsandepigeneticmodifications.................................................48
1.6.2 Chromatinremodelling................................................................................................49
1.6.3 Epigeneticmodifications.............................................................................................51
1.6.4 EpigeneticregulationoftheCXCL8gene.....................................................................64
1.6.5 Diseaseepigenetics.....................................................................................................65
1.6.6 EpigeneticsofCF..........................................................................................................66
1.7 Summary......................................................................................................................68
2 HYPOTHESISANDAIMS....................................................................................................69
3 MATERIALSANDMETHODS..............................................................................................71
8
3.1 Celllines.......................................................................................................................72
3.1.1 Cellculture...................................................................................................................73
3.1.2 Cellcounting................................................................................................................73
3.1.3 Cellfreezing.................................................................................................................74
3.2 HumanCXCL8enzyme-linkedimmunosorbentassay(ELISA).....................................74
3.3 Bicinchoninicacid(BCA)proteinassay........................................................................76
3.4 Real-timepolymerasechainreaction(qPCR)..............................................................76
3.4.1 TotalRNAisolation......................................................................................................77
3.4.2 Reversetranscription...................................................................................................78
3.4.3 Quantitativereal-timepolymerasechainreaction(qPCR)..........................................78
3.5 Transfections...............................................................................................................79
3.6 Cellviabilityandproliferationassay............................................................................80
3.7 Chromatinimmunoprecipitation(ChIP)......................................................................81
3.7.1 Cellfixation..................................................................................................................81
3.7.2 Sonication....................................................................................................................82
3.7.3 Immunoprecipitation...................................................................................................82
3.7.4 QPCR….........................................................................................................................84
3.8 Co-Immunoprecipitation(Co-IP).................................................................................84
3.8.1 Isolationofnuclearandcytoplasmicproteins.............................................................84
3.8.2 Co-immunoprecipitation(Co-IP)..................................................................................86
3.8.3 Westernblotting..........................................................................................................86
3.9 Bisulphitesequencing..................................................................................................89
3.9.1 GenomicDNAextraction.............................................................................................89
3.9.2 Bisulphiteconversion..................................................................................................90
3.9.3 PCRofbisulphiteconvertedDNA................................................................................90
3.9.4 Pyrosequencing...........................................................................................................91
3.10 Statistics.......................................................................................................................92
4 DIFFERENCESINEXPRESSIONANDPRODUCTIONOFCXCL8INCFANDNON-CFAIRWAY
EPITHELIALCELLS...................................................................................................................93
4.1 Introduction.................................................................................................................94
4.2 Aims.............................................................................................................................95
4.3 Methods.......................................................................................................................95
4.3.1 Concentrationresponseandtimecourseexperiments..............................................95
4.3.2 CXCL8mRNAexpression..............................................................................................96
9
4.3.3 CXCL8mRNAstabilityexperiments.............................................................................96
4.4 Results..........................................................................................................................97
4.4.1 IL-1ß stimulates increased CXCL8 protein production from CF airway epithelial
cells…………………………………………………………………………………………………………………………………97
4.4.2 IL-1ßinducesincreasedCXCL8mRNAexpressioninCFairwayepithelialcells.........103
4.4.3 The effect of transcription inhibitor Actinomycin D on basal CXCL8 mRNA
expression….........................................................................................................................107
4.5 Discussion..................................................................................................................109
5 NF‐ĸB, AP‐1 AND C/EBPß TRANSCRIPTION FACTORS ARE INVOLVED IN CXCL8
EXPRESSIONINCFAIRWAYEPITHELIALCELLS.....................................................................112
5.1 Introduction...............................................................................................................113
5.2 Aims...........................................................................................................................114
5.3 Methods.....................................................................................................................115
5.4 Results........................................................................................................................116
5.4.1 IL‐1β‐induced CXCL8 promoter activation requires C/EBPß, NF‐ĸB and AP‐1
transcriptionfactors.............................................................................................................116
5.4.2 Increased basal binding of NF‐κB p65 transcription factor to the CXCL8 promoter . 120
5.5 Discussion..................................................................................................................124
6 NF‐κB, HISTONE ACETYLATION/METHYLATION AND DNA METHYLATION AT THE CXCL8
PROMOTERINCFAIRWAYEPITHELIALCELLS......................................................................128
6.1 Introduction...............................................................................................................129
6.2 Aims...........................................................................................................................131
6.3 Methods.....................................................................................................................132
6.4 Results........................................................................................................................133
6.4.1 IncreasedhistoneH3lysine4trimethylation(H3K4me3)attheCXCL8promoterinCF
airwayepithelialcells...........................................................................................................133
6.4.2 HistoneacetylationattheCXCL8promoterinCFairwayepithelialcells..................134
6.4.3 NF‐κB acetylation at the CXCL8 promoter in CF airway epithelial cells .................... 135
6.4.4 P300bindingtotheCXCL8promoterinCFairwayepithelialcells............................136
6.4.5 DNAmethylationattheCXCL8promoterinCFairwayepithelialcells.....................138
6.5 Discussion..................................................................................................................141
7 BETPROTEININHIBITORSABOLISHCXCL8EXPRESSIONINCFAIRWAYEPITHELIAL
CELLS….................................................................................................................................146
7.1 Introduction...............................................................................................................147
7.2 Aims...........................................................................................................................149
7.3 Methods.....................................................................................................................149
10
