Dissertation der Fakultät für Biologie der Ludwig-Maximilians-Universität München Foxc1 regulates Pecam-1 Expression in embryonic Endothelial Progenitor Cells Eingereicht von Mathias Lamparter München, 15. Januar 2008 Angefertigt am Institut für Klinische Molekularbiologie und Tumorgenetik, Helmholtz Zentrum München – Deutsches Forschungszentrum für Gesundheit und Umwelt, und Division of Cardiovascular Medicine, Vanderbilt University Medical Center, Nashville, TN, USA Erster Gutachter: Prof. Dr. Dirk Eick Zweiter Gutachter: Prof. Dr. Manfred Schliwa Tag der mündlichen Prüfung: 02. Juli 2008 SUMMARY ................................................................................................................. 1 1. INTRODUCTION .................................................................................................... 3 1.1 The Vascular System........................................................................................ 3 1.2 De novo formation of the vasculature – Vasculogenesis................................... 3 1.3 Expansion and maturation of the vasculature – Angiogenesis.......................... 4 1.4 The vasculature in disease states..................................................................... 5 1.5 Characteristics of adult EPCs ........................................................................... 5 1.6 EPC promote neovascularization...................................................................... 6 1.7 Molecular control of blood vessel formation ...................................................... 6 1.7.1 VEGF and VEGF-Receptors ...................................................................... 7 1.7.2 Angiopoietins and Tie-Receptors................................................................ 8 1.7.3 Ephrins and Eph Receptors........................................................................ 8 1.7.4 Extracellular Matrix and Cell Adhesion Molecules...................................... 8 1.7.5 Other signaling pathways involved in vascular development...................... 9 1.7.6 Cytokines.................................................................................................. 10 1.7.7 Angiogenic inhibitors ................................................................................ 10 1.7.8 Haemodynamic forces.............................................................................. 10 1.8 Transcriptional control of vascular formation................................................... 10 1.8.1 Ets transcription factors................................................................................ 11 1.8.2 Basic Helix-Loop-Helix Transcription Factors........................................... 11 1.8.3 Homeobox Transcription Factors.............................................................. 12 1.8.4 Further Transcription Factors ................................................................... 12 1.8.5 Hypoxia and HIF ααα .................................................................................... 12 1.9 Embryonic endothelial progenitor cells (eEPCs) as a model system .............. 13 1.10 Foxc1 and Foxc2 are induced during eEPC in vitro differentiation................ 14 1.11 Forkhead (Fox) Transcription Factors........................................................... 14 1.11.1 General characteristics of Fox Genes .................................................... 14 1.11.2 Nomenclature of Fox Genes................................................................... 15 1.11.3 Chromosomal organization of Fox genes ............................................... 15 1.11.4 Fox Genes in Development .................................................................... 16 1.11.5 Fox Genes in Signaling Pathways.......................................................... 17 1.11.6 Fox Genes in Human Diseases.............................................................. 18 1.11.7 Fox Genes in the Adult Organism........................................................... 19 1.12 The FoxC subfamily - Foxc1 and Foxc2 ........................................................ 19 1.12.1 Expression Patterns ............................................................................... 19 1.12.2 Abnormalities in FoxC mutant embryos.................................................. 20 1.12.3 Foxc1 and Foxc2 - signaling pathways and target gene activation......... 21 1.13 Aims of the Ph.D. Project.............................................................................. 22 2. MATERIAL and METHODS.................................................................................. 23 2.1 Tissue Culture................................................................................................. 23 2.1.1 Cell lines................................................................................................... 23 2.1.2 Tissue culture media ................................................................................ 24 2.1.3 Passage of cell cultures............................................................................ 24 2.1.4 Freezing and thawing of cell lines............................................................. 25 2.1.5 Transfection of cell lines ........................................................................... 25 2.2 Molecular biology techniques.......................................................................... 26 2.2.1 Total RNA isolation................................................................................... 26 2.2.2 Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) ............... 27 2.2.3 Polymerase Chain Reaction (PCR) .......................................................... 28 2.2.4 PCR Primer Design .................................................................................. 29 2.2.5 DNA Sequencing...................................................................................... 29 2.2.6 Plasmid DNA preparation ......................................................................... 31 2.2.7 Transformation of CaCl 2-competent DH5 ααα E.coli ..................................... 32 2.2.8 DNA restriction digests ............................................................................. 33 2.2.9 DNA ligation ............................................................................................. 34 2.2.10 Plasmid Constructs................................................................................. 34 2.2.11 Agarose gel electrophoresis ................................................................... 37 2.2.12 Ethanol Precipitation of DNA .................................................................. 38 2.3 Quantitative Real-Time Amplification (qPCR) ................................................. 38 2.3.1 LightCycler Real-Time PCR System......................................................... 38 2.3.2 iQ5 Real-Time PCR System..................................................................... 40 2.4 Fluorescence Activated Cell Sorting (FACS) Analysis .................................... 42 2.5 Microscopy and Fluorescence Microscopy ..................................................... 43 2.6 Immunofluorescence....................................................................................... 43 2.7 Promoter-Luciferase Assays ........................................................................... 44 2.8 Western Blot ................................................................................................... 46 2.9 Chromatin Immunoprecipiation ....................................................................... 48 2.10 Online Databases and Bioinformatics Programs........................................... 51 3. RESULTS............................................................................................................. 53 3.1 Expression of Fox genes in eEPCs................................................................. 53 3.2 Foxc1 and Foxc2 regulate expression of Pecam-1 in eEPCs ......................... 56 3.3 Pecam-1 as an endothelial target gene of Foxc1............................................ 60 3.4 Cis-regulatory areas of the Pecam-1 promoter ............................................... 61 3.5 Pecam-1 promoter and enhancer analysis in different cell lines ..................... 62 3.6 Foxc1 activates transcription through the 5’-flanking 3.5kb-fragment ............. 65 3.7 The distal 5’-flanking 3.5kb-fragment responds specifically to Foxc1 ............. 66 3.8 Foxc1 activates transcription specifically in eEPCs ........................................ 68 3.9 Endogenous Pecam-1 RNA analysis matches promoter activation studies.... 70 3.10 Dose-dependent activation of the Pecam-1 promoter by Foxc1 ................... 71 3.11 Activation of the Pecam-1 promoter takes place in multiple, independently isolated eEPC clones............................................................................................ 72 3.12 Pecam-1 promoter activity – Summary ......................................................... 73 3.13 Localization of Foxc1-responsive sites.......................................................... 74 3.14 Analysis of the Pecam-1 promoter using Bioinformatic Tools ....................... 76 3.15 The (TTTGT) n motif is found at the human PECAM-1 locus ......................... 78 3.16 Deletion of the repeat motif abolishes promoter activity................................ 81 3.17 Foxc1 binds the (TTTGT) n motif on native chromatin.................................... 83 3.18 Results – Summary......................................................................................