Functional studies on the galectin-4 promoter and its use for establishing a transcription factors array assay [Elektronische Ressource] / presented by Reham Helwa

Helwa

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2010
• Nombre de lectures: 26
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Dissertation

submitted to

The Combined Faculties for the Natural Sciences
and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany


for the degree of
Doctor of Natural Sciences



Presented by
Master Sci. Reham Helwa
born in Cairo, Egypt




Oral examination:








Functional studies on the galectin-4 promoter and its use for
establishing a transcription factors array assay













Refrees: PD. Dr. Stefan Wiemann
Prof. Dr. Ruediger Hell

































To my wonderful parent who were always behind me from my childhood till this
moment.


To my great love, Egypt.






















Table of contents

Abbreviations ................................................................................................................................. I
Abstract ....... III
Zusammenfassung ....................... V
Part I: Expression Profiling Analysis of Colorectal Cancer Cell Lines: Reveals a Twin SNPs in
Galectin-4 Promoter Associated with its Upregulation ................................................................ 1
Introduction .............................................................................................. 2
1.1. Colorectal cancer (CRC) .......................... 3
1.1.1. Molecular carcinogenesis ....................................................... 3
1.1.2. Risk factors ............................................................................. 4
1.1.3. Colorectal cancer screening .................... 4
1.1.4. Colorectal Cancer staging ....................... 6
1.1.4.1. Duke’s classification ........................................................................................ 6
1.1.4.2. TNM staging .................................... 6
1.1.5. Prognosis ................................................ 6
1.2. Galectins ........................ 7
1.2.1. Galectins expression in normal alimentary canal ................................................... 8
1.2.2. Galectins in colorectal cancer ................................................. 8
1.2.3. Galectin-4 ............................................... 9
Material and Methods ............. 11
2.1. Materials ...................................................... 11
2.1.1. Chemicals ............................................. 11
2.1.2. Enzymes ................................................ 12
2.1.3. Kits ........................ 12
2.1.4. Buffers .................................................. 13
2.1.5. Consumables ......................................... 13
2.1.6. Equipment ............................................. 14
2.1.7. Antibodies and electrophoresis ladders ................................ 14
2.1.8. Primers .................................................................................. 14
2.2. Methodology ................ 16
2.2.1. Cell culture ........................................... 16
2.2.2. mRNA Expression Profiling ................. 16
2.2.2.1. Microarray production ................................................................................... 16
2.2.2.2. Sample preparation and hybridization ........................... 16
2.2.2.3. Detection and analysis 16
2.2.3. Galectin-4 validation by qRT-PCR ...................................................................... 17
2.2.4. Western blotting for galectin-4 ............. 17
2.2.5. Galectin-4 promoter analysis ................ 18
2.2.5.1. Promoter sequencing ..................................................................................... 18
2.2.5.2. Luciferase reporter assay ............... 18
2.2.5.3. Pull down of the binding proteins .................................. 19
2.2.5.4. Methylation status .......................... 19
2.2.5.5. Promoter genotyping in colorectal cancer patient samples ........................... 19
3. Results ................................................................................................ 20
3.1. Expression profiling classifies colorectal cancer cell lines to bad and good prognosis
rather than tumor stages ...................................... 20
3.2. Galectin-4 is significantly upregulated in LT97 and KM20L2 ... 22
3.3. A twin SNPs in the regulatory region are associated with galectin-4 upregulation in
LT97 and KM20L2 ............................................................................. 22

3.4. Effect of the two SNPs activity on promoter ............................................................... 23
3.5. The two SNPs are affecting the protein binding sites ................. 25
3.6. The expression of the binding proteins in different cell lines ..................................... 30
3.7. Methylation status ........................................................................ 33
3.8. Two SNPs are shown together in patient samples ....................................................... 34
3.9. Galectin-4 is upregulated in colorectal cancer patients ............... 38
3.10. galectin-4 upregulation is associated with promoter methylation in the colorectal
cancer patients .................................................................................... 39
Discussion ............................................................... 41
Outlook 46
Part II: Setting up Transcription Factor Protein Array Detecting DNA-Protein Interactions .... 50
1. Introduction ........................................................................................ 51
1.1. DNA-protein interaction .............................................................. 52
1.2. Analysis of DNA-protein interactions; from nitrocellulose filter-binding assays to
microarray studies ............................................................................................................... 53
1.2.1. Nitrocellulose filter-binding assay ........ 53
1.2.2. DNase I fingerprinting .......................... 53
1.2.3. Dimethyl sulphate (DMS) protection fingerprinting ............ 54
1.2.4. Electrophoretic mobility shift assay (EMSA) ...................................................... 54
1.2.5. Methylation interference assay ............................................. 55
1.2.6. Chromatin immunoprecipitation (ChIP) ............................... 55
1.2.7. DNA adenine methyltransferase identification (DamID) ..................................... 56
1.2.8. Surface plasmon resonance (SPR) measurement ................. 57
1.2.9. Systematic evolution of ligands by exponential enrichment (SELEX) ................ 57
1.2.10. Yeast one-hybrid system .................................................................................... 57
1.2.11. Proximity ligation ............................... 58
1.2.12. Microarray-based assays ..................... 59
2. Material and methods ......................................................................................................... 62
2.1. Materials ...................... 62
2.1.1. Equipment ............. 62
2.1.2. Chemicals 62
2.1.3. Kits ........................................................................................................................ 63
2.1.4. Buffers and mediums ............................ 63
2.1.5. Labware ................ 64
2.1.6. Enzymes, vectors, bacterial strains ....................................................................... 65
2.1.7. Software and web pages ....................................................................................... 65
2.2. Methodology ................................................ 65
Setting up TFs-chip ........ 65
2.2.1. DNA-binding protein expression and purification ............... 66
2.2.1.1. Protein expression .......................................................................................... 66
2.2.1.2. Protein detection and purification .................................. 66
2.2.2. Protein spotting and immobilization . 67
2.2.3. On-chip DNA-protein interactions ................................... 67
2.2.4. Electrophoretic Mobility Shift Assay (EMSA) ................................................ 67
2.2.6. Verifying Transfac database consensus sequences using DNA-microarray .... 68
2.2.7. Applying oligos, PCR products of promoter regions, and genomic DNA to
TFs-chip ...................................................................................... 69
3. Results ................................................................ 70
3.1. Protein expression and purification ............. 70
3.2. Protein spotting and immobilization ............ 74
3.3. On-chip DNA-protein interaction ................................................................................ 80

3.3.1. DNA-protein interaction and not Fluorochrome-protein interaction ................... 80
3.3.2. TFs-array vali