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Transcription Factors in Hematopoiesis

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Transcription Factors in Hematopoiesis Role of NFAT (Nuclear Factor of Activated T Cells) Transcription Factors in Hematopoiesis DISSERTATION zur Erlangung des akademischen Grades Doctor of Philosophy (PhD) vorgelegt der Fakultät Mathematik und Naturwissenschaften der Technischen Universität Dresden von Laleh Sadat Arabanian, MSc Geboren am 05.03.1979 in Tehran, Iran Eingereicht am Juli 2012, Dresden Die Dissertation wurde in der Zeit von März 2008 bis Juni 2012 in der Medizinische Klinik (Universitätsklinikum Carl Gustav Carus) an der Technischen Universität Dresden angefertigt. To my grandpa, though no longer with us, remains the compass of my life… 1 Reviewers: Prof. Gerhard Rödel Institute of Genetics, Department of Biology, Faculty of Mathematics and Natural Sciences, Dresden University of Technology, Dresden, Germany PD Dr. Alexander Kiani Department of Medicine I, Faculty of Medicine, Dresden University of Technology, Dresden, Germany Prof. Gerhard Ehninger Department of Medicine I, Faculty of Medicine, Dresden University of Technology, Dresden, Germany 2 Table of contents 1 Abstract ............................................................................................................................................................ 6 2 Introduction ................................................................................................................................................ 9 2.1 Nuclear Factor of Activated T cells ............................................................................................ 9 2.1.1 NFAT transcription factors .................................................................................................. 9 2.1.2 Calcium‐calcineurin‐NFAT signaling ............................................................................ 10 2.1.3 Transcriptional partners of NFAT proteins ............................................................... 11 2.1.4 Inhibition of NFAT activation .......................................................................................... 12 2.2 Hematopoiesis ................................................................................................................................. 14 2.2.1 NFAT in hematopoiesis ...................................................................................................... 15 2.2.2 HSC transplantation: A tool to investigate the regulation of hematopoiesis. ........................................................................................................................................................16 2.3 Mouse model of NFATc2 deficiency ....................................................................................... 17 2.4 Megakaryocytes and megakaryopoiesis .............................................................................. 18 2.4.1 Transcriptional regulation of megakaryopoiesis .................................................... 20 2.4.2 Transcriptional regulation by NFAT in megakaryocytes ..................................... 21 2.4.2.1 Fas Ligand ............................................................................................................................ 21 2.4.2.2 Fas/Fas Ligand‐induced apoptosis ........................................................................... 21 2.5 RNA sequencing .............................................................................................................................. 22 3 Aim of the study ...................................................................................................................................... 24 4 Materials and methods ........................................................................................................................ 26 4.1 Materials ............................................................................................................................................ 26 4.1.1 Reagents, stimulators, inhibitors and growth factors ........................................... 26 4.1.2 Antibodies ................................................................................................................................ 27 4.1.3 KITs ............................................................................................................................................. 28 4.1.4 Primers ...................................................................................................................................... 29 4.2 Equipment ......................................................................................................................................... 29 4.2.1 Instruments ............................................................................................................................. 29 4.2.2 Software .................................................................................................................................... 30 4.3 Cell culture ........................................................................................................................................ 31 3 4.3.1 Co‐culture experiments ........................................................................................................ 33 4.4 Mice ...................................................................................................................................................... 34 4.5 Protein analysis .............................................................................................................................. 36 4.6 Histological analysis ..................................................................................................................... 37 4.7 RNA sequencing .............................................................................................................................. 38 4.8 Statistical analysis ......................................................................................................................... 39 5 Results .......................................................................................................................................................... 41 5.1 Role of NFATc2 in hematopoietic progenitor cells.......................................................... 41 5.1.1 Reciprocal transplantation experiments: effects on peripheral blood parameters. ............................................................................................................................................... 42 5.1.2 Reciprocal transplantation experiments: effects on bone marrow parameters. ............................................................................................................................................... 45 5.1.3 Reciprocal transplantation experiments: effects on extramedullary hematopoiesis. ......................................................................................................................................... 49 5.1.4 Homing of NFATc2‐deficient HSCs into the bone marrow ................................. 51 5.2 Role of NFATc2 in mature megakaryocytes ....................................................................... 54 5.2.1 Calcium ionophore stimulation leads to megakaryocyte death ....................... 54 5.2.2 Activation of the calcineurin pathway induces apoptosis in megakaryocytes. ........................................................................................................................................................56 5.2.3 Apoptosis induction in megakaryocytes is dependent on NFAT activation 58 5.2.4 NFAT induces expression of FASLG protein in megakaryocytes ..................... 60 5.2.5 FASLG is located on the cell surface .............................................................................. 62 5.2.6 The Fas/FASLG pathway is involved in calcineurin/ NFAT‐mediated apoptosis induction in megakaryocytes ....................................................................................... 65 5.2.7 Calcineurin/NFATc2‐dependent FASLG expression in megakaryocytes induces apoptosis in (Fas‐positive) bystander cells ............................................................... 67 5.3 NFATc2 target genes in megakaryocytes ............................................................................ 71 5.3.1 Generation of ex vivo pure and intact murine megakaryocytes ........................ 71 5.3.2 RNA‐sequencing (next generation sequencing) experimental design .......... 72 5.3.3 Primary analysis: Bioinformatics and statistical analysis (performed by bioinformaticians) ................................................................................................................................. 73 4 5.3.4 Secondary analysis: Genes of interest (significantly differentially expressed NFATc2 target genes). .......................................................................................................................... 76 5.3.5 Functional annotation (performed by Miltenyi Biotec Company) ..................... 81 6 Discussion .................................................................................................................................................. 85 6.1 Lack of the transcription factor NFATc2 in hematopoietic stem cells results in profound hematological abnormalities in mice ............................................................................. 85 6.1.1 HSC transplantation ............................................................................................................. 85 6.1.2 NFATc2 deficiency in HSCs leads to hematological and stroma abnormalities
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