Characterization of the CD83 promoter, enhancer complex [Elektronische Ressource] / Marcello Francesco Stein. Betreuer: Lars Nitschke

De
Characterization of the CD83 promoter/enhancer complex Charakterisierung des CD83 Promotor/Enhancer Komplexes Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades Dr. rer. nat vorgelegt von Marcello Francesco Stein aus Schweinfurt am Main Als Dissertation genehmigt von den Naturwissenschaftlichen Fakultäten der Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 26.09.2011 Vorsitzender der Promotionskommission: Prof. Dr. Rainer Fink Erstberichterstatter: Prof. Dr. Lars Nitschke Zweitberichterstatter: Prof. Dr. Eckhardt Kämpgen Table of contents Abbreviations and Symbols 1. Summary/Zusammenfassung ..................................................................... 1 - English/Deutsch 2. Introduction....................................................................................................... 5 2.1. The biology of dendritic cells ... 6 2.1.1. DC subsets .............................................................................................. 6 2.1.2. DCs as immunological sentinels 9 2.1.2.1 Antigen capture ....................... 9 2.1.2.2 Antigen processing .................................................................................. 9 2.1.2.3. Activation of DCs ................... 11 2.1.2.3.1.
Publié le : samedi 1 janvier 2011
Lecture(s) : 56
Source : D-NB.INFO/1016377185/34
Nombre de pages : 222
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Characterization of the
CD83 promoter/enhancer complex

Charakterisierung des
CD83 Promotor/Enhancer Komplexes






Der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur
Erlangung des Doktorgrades Dr. rer. nat







vorgelegt von
Marcello Francesco Stein
aus Schweinfurt am Main









Als Dissertation genehmigt von den Naturwissenschaftlichen Fakultäten der
Universität Erlangen-Nürnberg
















Tag der mündlichen Prüfung: 26.09.2011

Vorsitzender der Promotionskommission: Prof. Dr. Rainer Fink

Erstberichterstatter: Prof. Dr. Lars Nitschke

Zweitberichterstatter: Prof. Dr. Eckhardt Kämpgen Table of contents

Abbreviations and Symbols

1. Summary/Zusammenfassung ..................................................................... 1
- English/Deutsch

2. Introduction....................................................................................................... 5

2.1. The biology of dendritic cells ... 6
2.1.1. DC subsets .............................................................................................. 6
2.1.2. DCs as immunological sentinels 9
2.1.2.1 Antigen capture ....................... 9
2.1.2.2 Antigen processing .................................................................................. 9
2.1.2.3. Activation of DCs ................... 11
2.1.2.3.1. Innate immunity activation signals ......................... 11
2.1.2.3.2. Adaptive immunity activation signals ..................................................... 14
2.1.2.4. Maturation of DCs ................................................. 14
2.1.2.5. Migration of DCs .................... 15
2.1.2.6. Control of different T cell responses by DCs ......................................... 15
2.1.3. The surface molecule CD83 .................................. 17
2.1.3.1. Structure of CD83 gene and protein ...................... 18
2.1.3.2. Functions of membrane bound and soluble forms of CD83 ................... 19
2.1.3.3. CD83 as a target for viral immune escape mechanisms ....................... 20
2.1.3.4. The CD83 promoter ............................................................................... 21
2.1.4. Adenoviruses and gene therapy ............................ 22
2.2. Genetic regulation and expression mechanisms ................................... 24
2.2.1. Structural modifications of DNA ............................................................. 24
2.2.2. DNA Methylation ................................................... 24
2.2.3. Histone modification and chromatin remodeling .................................... 27
2.2.4. Histone methylation ............... 27
2.2.5. Histone acetylation ................................................................................ 28
2.2.6. Regulation of transcription ..... 29
2.2.7. Regulatory DNA elements ..................................................................... 29 2.2.7.1. Promoters .............................................................................................. 30
2.2.7.2. Upstream regulatory elements 31
2.2.7.3. Enhancers ............................. 32
2.2.7.4. Silencers ................................................................................................ 33
2.2.7.5. Insulators ............................... 33
2.2.8. DNA binding proteins............................................................................. 34
2.2.8.1. The role of transcription factors during regulation of transcription ......... 34
2.2.9. The transcription factor SP1 .................................................................. 35
2.2.10. NF B-family of transcription factors....................... 36
2.2.10.1. Regulation of NF B ............................................................................... 37
2.2.10.2. NF B-pathways ..................... 38
2.2.10.3. The role of NF B-transcription factors in the development of DCs ........ 40
2.2.11. Interferon regulatory factors .................................................................. 41
2.2.11.1. Activation and regulation of IRF-pathways ............ 42
2.2.11.2. IRFs in development and function of DCs ............................................. 44
2.2.11.3. The role of IRFs in DC activation ........................... 47

3. Tasks ................................................................................................................. 51

4. Material and Methods .................. 53

4.1. Material ................................................................................................. 53
4.1.1. Chemicals .............................. 53
4.1.2. DNA modifying enzymes ....... 54
4.1.3. Buffers and reagents ............................................................................. 55
4.1.3.1. General reagents and buffers 55
4.1.3.2. Buffers and reagents for DEAE-Dextran cell transfection ...................... 55
4.1.3.3. Buffer and reagents for SDS- polyacrylamide gel electrophoresis
(PAGE) and Western blot analyses ....................................................... 56
4.1.3.4. Reagents for chemical-competent E.coli and transformation by heat .... 57
4.1.3.5. Buffers and reagents for electrophoretic mobility shift assay (EMSA) ... 57
4.1.3.6. Buffers and reagents for DNA electrophoresis ...................................... 58
4.1.3.7. Ladders and markers............................................. 58
4.1.4. Cell culture ............................................................................................ 59 4.1.4.1. Cell lines and cell culture media ............................................................ 59
4.1.4.2. Additional cell culture media .................................. 60
4.1.4.3. General cell culture reagents . 61
4.1.4.1. General cell culture dishes and plastic ware ......... 61
4.1.4. Equipment and instruments ................................................................... 61
4.1.4.1 Software and websites .......... 63
4.1.5. Antibodies .............................................................. 63
4.1.5.1. Antibodies used for FACS ..................................................................... 63
4.1.5.2. Antibodies used for EMSAs ... 64
4.1.5.3. Antibodies used for Western blot analyses ............ 65
4.1.6. DNA Oligos ............................................................................................ 66
4.1.6.1. Primers .................................. 66
4.1.6.1.1. Sequencing primers............................................... 66
4.1.6.1.2. Cloning primers for transcription factors ................................................ 67
4.1.6.1.3. Cloning primers for luciferase reporter constructs ................................. 67
4.1.6.1.4. Cloning primers for the mutated 185 bp enhancer 68
4.1.6.2. Oligos for EMSA .................................................................................... 69
4.1.6.2.1. Oligos NF B-sites.................. 69
4.1.6.2.2. Oligos IRF-sites ..................... 69
4.1.7. Plasmid vectors ................................................................ 70
4.1.7.1. Donated or purchased vector ................................ 70
4.1.7.2. Cloned vectors ...................... 71
4.1.7.2.1. Luciferase reporter constructs ............................................................... 71
4.1.7.2.2. Luciferase reporter constructs with mutated IRF-sites........................... 73
4.1.7.2.3. Schematic depiction of CD83 Intron 2 fragments for luciferase assay .. 75
4.1.8. Adenoviruses ......................................................................................... 76
4.1.9. Human cytokines and maturation agents .............. 76
4.1.10. Bacteria ................................. 77
4.1.11. Purchased Kits ...................................................................................... 77
4.2. Methods 78
4.2.1. General molecular biology methods ...................... 78
4.2.1.1. Generation of chemical-competent E.coli and transformation by heat .. 78
4.2.2. DNA based molecular biology methods................................................. 78
4.2.2.1. Isolation of DNA .................................................... 78 4.2.2.1.1. Isolation of small plasmids (< 12 kb) ..................................................... 78
4.2.2.1.2. Isolation of large plasmids (> 12 kb) ...................... 79
4.2.2.1.3. Isolation of human genomic DNA from cells by QIAamp DNA Mini Kit .. 80
4.2.2.1.4. Preparation of DNA fragments from PCR reactions and enzymatic
digestions .............................................................................................. 80
4.2.2.1.5. Preparation of DNA fragments from gel electrophoresis ....................... 80
4.2.2.2. Nucleic acid quantification ..................................... 80
4.2.2.3. Separation of DNA or RNA using agarose gel electrophoresis ............. 80
4.2.2.4. Polymerase chain reaction (PCR) based methods 81
4.2.2.4.1. Single-step PCRs .................................................................................. 81
4.2.2.5. Hybridization of DNA oligos for elctrophoretic mobility shift assay
(EMSA) .................................................................................................. 82
4.2.2.6. Radioactive labeling of DNA oligos for EMSA ....... 83
4.2.2.7. Cloning of DNA fragments or PCR products ......... 84
4.2.2.7.1. Dephosphorylation of cleaved DNA ....................................................... 84
4.2.2.7.2. Conversion of DNA overhangs .............................. 84
4.2.2.7.3. Ligation .................................................................................................. 85
4.2.2.7.4. Sequencing of DNA ............... 85
4.2.2.7.5. Synthesis of DNA sequences 85
4.2.3. Protein biochemistry methods ............................................................... 85
4.2.3.1. Generation of cell lysates ...................................... 85
4.2.3.1.1. Preparation of whole cell extracts for SDS-polyacrylamide gel
electrophoresis (PAGE) ......................................... 85
4.2.3.1.2. Preparation of nuclear extracts for EMSA and SDS-PAGE ................... 86
4.2.3.1.3. BCA Protein Assay Reagent (bicinchoninic acid) .................................. 86
4.2.3.2. EMSA .................................................................... 87
4.2.3.2.1. EMSA bandshift and supershift reaction ................................................ 87
4.2.3.2.2. EMSA non denaturizing polyacrylamide gel run and signal detection ... 88
4.2.3.3. SDS-PAGE: Laemmli method ............................................................... 89
4.2.3.4. Western blot analyses ........................................... 89
4.2.4. Cell culture ............................................................ 90
4.2.4.1. Generation of dendritic cells (DC) ......................................................... 90
4.2.4.2. Cryopreservation of primary cells .......................... 90
4.2.4.3. Cryopreservation of cell lines ................................ 91
4.2.4.4. Heat inactivation of fetal calf serum (FCS) and human autologous ....... 91 4.2.5. Flow cytometric analysis (FACS) ........................................................... 91
4.2.6. Transient transfection methods and luciferase reporter assay .............. 92
4.2.6.1. DNA-transfection using the DEAE-Dextran method .............................. 92
4.2.6.2. Electroporation of Raji and Jurkat cell lines ........................................... 92
TM TM4.2.6.3. Lipofection of DNA with Lipofectamine LTX and PLUS reagent ..... 93
4.2.6.4. Electroporation of DCs with DNA using AMAXA technology ................. 94
4.2.6.5. Luciferase reporter assay ...................................................................... 94
4.2.7. Recombinant adenoviruses ... 95
4.2.7.1. Cloning of plasmids containing the recombinant adenoviral genome .... 95
4.2.7.2. Preparation of recombinant adenoviruses ............................................. 95
4.2.7.3. Determination of the physical particle concentration ............................. 96
4.2.7.4. Determination of the infectious particle concentration ........................... 96
4.2.7.5. Adenoviral transduction of cells ................................ 97
4.3. Statistical analysis ................................................. 97

5. Results .............................................................................. 98

5.1. Previous approaches to identify the human CD83 promoter ................. 98
5.1.1. CD83 is not expressed in HFF cells, weakly in iDCs and strongly in
mDCs..................................................................................................... 98
5.1.2. A 6 kB region of CD83 intron 2 is specifically H3K9 acetylated in
mDCs... 100
5.2. Analyses of the acetylated region of intron 2 within the CD83 gene by
luciferase reporter assays .................................................................... 103
5.2.1 CD83 Intron 2 Fragment C shows an enhancing activity on MP -261
in the DC-like cell lineXS52 . 105
5.2.2. Narrowing down the putative enhancer sequence: A 185 bp sequence
within fragment C shows enhancer function ........................................ 107
5.2.3. The CD83 intron 2 fragment C and the 185 bp enhancer induce the
MP -261 specifically in human DCs ..................................................... 110
5.2.4. CD83 intron 2 fragment C from and the 185 bp enhancer do not
induce the MP -261 in B and T cell lines ............. 112
5.3. Biocomputational analyses and modeling of the human
CD83 promoter .................................................................................... 114
5.4. Functional characterization of the predicted UpP and the spacer
sequence S1 within the pGL3/MP -261 reporter plasmids .................. 116 5.4.1. Neither the spacer sequence S1, nor the UpP do significantly
affect the induction of the CD83 promoter in cell lines ......................... 117
5.4.2. The spacer sequence S1 does not significantly affect the induction
of the CD83 promoter in mDCs. .......................................................... 119
5.5. Adenoviral transduction confirms the cell type- and status-specific
interaction of UpP, MP -261 and 185 bp enhancer .............................. 121
5.5.1. The proposed ternary complex of UpP, MP -261 and the 185 bp
enhancer forms in mDCs ..................................................................... 122
5.5.2. The proposed ternary complex does not form in Raji, Jurkat and
JCAM cell lines .................................................................................... 124
5.6. Analyses of the predicted transcription factor binding sites by
electrophoretic mobility shift assays (EMSA) ....................................... 127
5.6.1. Bandshift analyses of the predicted NF B- and IRF-sites ................... 127
5.6.2. Supershift analyses for the transcription factors of the NF B-family ... 129
5.6.2.1. The statistical evaluation confirms the results of the EMSAs
for the NF B- sites ............................................................................... 135
5.6.3. Supershift analyses for the transcription factors of the IRF-family ....... 138
5.6.3.1. The statistical evaluation confirms the results of the EMSAs
for the IRF-sites ................................................................................... 141
5.6.4. Summary of the EMSAs ...... 143
5.7. Functional analyses of the three IRF-sites using mutagenesis and
luciferase assays ................................................................................. 144
5.7.1. Mutation of individual IRF-sites results in a significantly reduced
luciferase expression in XS52 cells and mDCs ................................... 146
5.8. Verification of the functionality of the NF B-sites in the UpP and
the MP -261 by cotransfection experiments in 293T cells ................... 150
5.8.1. IRF-5, p50, p65 and cRel can be overexpressed in 293T cells ........... 151
5.8.2. IRF-5, p50, p65 and cRel induce the MP -261 in 293T cells ............... 152
5.8.3. The combination of p65 and IRF-5 induces the UpP in 293T cells ...... 155
5.9. The transcription factors IRF-1, IRF-2, IRF-5, p50, p65 and cRel
are differentially expressed in iDCs, mDCs and HFF cells .................. 158
5.10. The DNA in the CD83 promoter region is not differentially
CpG methylated in iDCs, mDCs and HFF cells ................................... 160


6. Discussion ..................................................................................................... 164

6.1. Full characterization of the human CD83 promoter ............................. 164
6.2. A 185 bp sequence within CD83 intron 2 has mDC-specific enhancer
function ................................................................................................ 164
6.3. A ternary complex consisting of MP -261, 185 bp enhancer and UpP
regulates mDC-specific CD83 expression ........... 166
6.4. The formation of the ternary complex is mediated by NF B- and IRF-
transcription factors ............................................................................. 169
6.4.1. Verification of the NF B-transcription factor binding sites ................... 170
6.4.2. Verification of the IRF-transcription factor binding sites....................... 174
6.5. Summary and future prospects ........................................................... 176

7. References .................................................................... 180

Acknowledgements/Danksagung

Patents, Publications, Presentations, Participations and Commitments


















Abbreviations and Symbols

µg Microgram mCD83 Membrane bound CD83
µl Microliter SP1 Specificity factor 1
Ad Adenovirus mg Milligram
APC Antigen presenting cell L-DC Lymphoid dendritic cell
as Antisense n.s. Not significant
Transcription factor
bp Basepair TFBS
binding site
CDS Coding sequence HAT Histone acetyltransferase
ChIP Chromatin immuno precipitation IL Interleukine
Pattern recognition
CTL Cytotoxic T cell PRR
receptor
d Day vp Virus particle
DC Dendritic cell LPS Lipopolysaccharide
E Exon BS Binding site
E.as Enhancer in antisense orientaion °C Degree Celsius
E.s Enhancer in sense orientaion RT Room temperature
EMSA Electrophoretic mobility shift assay RNA Ribonucleic acid
Enh. Enhancer cpm Counts per minute
Real time polymerase
FACS Fluorescence assisted cell sorting RT-PCR
chain reaction
Fig. Figure Treg Regulatory T cell
h Hour TLR Toll like receptor
GM- Granulocyte macrophage
H3K9 Acetylation of lysine 9 of histone 3
CSF colony-stimulating factor
Polymerase chain
HFF Human foreskin fibroblast PCR
reaction
HSV Herpex simplex virus HCMV Human Cytomegalovirus
I Intron Ac Acetylation

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