Transcriptional Regulation of and by NFATc1 in Lymphocytes This thesis submitted in partial fulfillment of the requirements for the award of the degree Doctor rerum naturalium of the Julius-Maximilians-University Würzburg Submitted by Ronald Rudolf from Stuttgart, Baden-Württemberg Würzburg 2013 Eingereicht am: ....................................................................................................................... Mitglieder der Promotionskommission: Vorsitzender: Prof. Dr. W. Rössler......................................................................................... 1. Gutachter: Prof. Dr. E. Serfling.…...................................................................................... 2. Gutachter: Prof. Dr. T. Müller............................................................................................. Tag des Promotionskolloquiums: ............................................................................................. Doktorurkunde ausgehändigt am: ............................................................................................ Affidavit I hearby confirm that my thesis entitled “Transcriptional Regulation of and by NFATc1 in Lymphocytes” is the result of my own work. I did not receive any help or support from commercial consultants. All sources and/or materials applied are listed and specified in the thesis. Furthermore, I confirm that this thesis has not yet been submitted as part of another examination process neither in identical nor in similar form. Place, Date..................................................................... Signature........................................................................ Eidesstattliche Erklärung Hiermit erkläre ich an Eides statt, die Dissertation „Transcriptional Regulation of an by NFATc1 in Lymphocytes“ eigenständig, d.h. insbesondere selbstständig und ohne Hilfe eines kommerziellen Promotionsberaters, angefertigt und keine anderen als die von mir angegebenen Quellen und Hilfsmittel verwendet zu haben. Ich erkläre außerdem, dass die Dissertation weder in gleicher noch in ähnlicher Form bereits in einem anderen Prüfungsverfahren vorgelegen hat. Ort, Datum................................................................... Unterschrift................................................................. I. INTRODUCTION .................................................................................................................. 6 1.1 The adaptive immune system ........................................................................................... 6 1.2 Development and function of T-lymphocytes .................................................................. 8 1.2.1 T-cell development .................................................................................................... 8 1.2.2 T-cell activation ....................................................................................................... 12 1.3 Development and function of B-lymphocytes ............................................................... 15 1.3.1 B-cell development ................................................................................................. 15 1.3.2 B-cell activation and function ................................................................................. 18 1.4 B-cell lymphomas .......................................................................................................... 20 1.4.1 B-cell lymphoma classification ............................................................................... 20 1.4.2 WEHI-231 B-lymphoma cells ................................................................................. 22 1.5 Cell death ........................................................................................................................ 23 1.5.1 Apoptosis and Activation Induced Cell Death ........................................................ 23 1.6 NFATc proteins .............................................................................................................. 25 1.6.1 NFAT activation ...................................................................................................... 26 1.6.2 The NFAT family .................................................................................................... 29 1.6.3 NFATc1 isoforms .................................................................................................... 31 1.7 NFAT in non-lymphoid cells ......................................................................................... 33 1.7.1 NFAT in bone development .................................................................................... 33 1.7.3 NFAT in heart-valve development .......................................................................... 34 1.8 Goals of the experimental work ..................................................................................... 35 CONTENT II. MATERIAL AND METHODS ........................................................................................... 36 2.1 Material .......................................................................................................................... 36 2.1.1 Mouse strains ........................................................................................................... 36 2.1.2 Cell culture .............................................................................................................. 37 2.1.3 Bacteria .................................................................................................................... 37 2.1.4 Retroviral expression vectors .................................................................................. 39 2.1.5 Primers .................................................................................................................... 40 2.1.6 Chemicals ................................................................................................................ 41 2.1.7 Buffers and solutions ............................................................................................... 43 2.1.8 Antibodies ............................................................................................................... 44 2.1.9 Technical equipment ............................................................................................... 45 2.2 Methods .......................................................................................................................... 46 2.2.1 DNA purification ..................................................................................................... 46 2.2.2 DNA quantification ................................................................................................. 46 2.2.3 DNA electrophoresis ............................................................................................... 47 2.2.4 Isolation of DNA fragments .................................................................................... 48 2.2.5 DNA Cloning .......................................................................................................... 48 2.2.6 Retroviral infection of cell-lines and primary B-cells: ............................................ 52 2.2.7 Chromatin Immunoprecipitation ............................................................................. 53 2.2.8 Next Generation Sequencing (performed by TRON, Translat. Oncology, Univ. Mainz) .............................................................................................................................. 57 1 CONTENT 2.2.9 Protein (Co-)Immunoprecipitation (Co-IP) ............................................................. 58 2.2.10 Flow cytometry ..................................................................................................... 58 2.2.11 Real-time PCR (qRT-PCR) ................................................................................... 59 2.2.12 Confocal-microscopy: ........................................................................................... 60 III. RESULTS ........................................................................................................................... 61 3.1 Nfatc1 induction in lymphocytes .................................................................................... 61 3.1.1 Nfatc1 induction in peripheral T-cells ..................................................................... 61 3.1.2 Nfatc1 induction in peripheral B-cells .................................................................... 63 3.2 Regulation of Nfatc1 transcription in lymphocytes ........................................................ 66 3.2.1 Characterization of two regulatory elements within the Nfatc1 locus .................... 66 3.2.2 Regulation of Nfatc1 expression by the two intronic elements E1 and E2 in primary lymphocytes ..................................................................................................................... 68 3.2 NFATc1-avidin fusion proteins ..................................................................................... 71 3.2.1 Cloning of expression plasmid pEGZ-NFATc1/A-avi ......................................... 71 3.2.2 Cloning of expression plasmids pEGZ-NFATc1/C-avi and pEGZ-NFATc1/C- aviFl .................................................................................................................................. 73 3.3 Bacterial Artificial Chromosome Nfatc1 Ex.9-avidin .................................................... 77 3.3.1 Cloning of the shuttle-vector pLD53.SC2