The Role of Histone Demethylases JMJD1C in the Pathopyhsiology Of
Total Page：16
File Type：pdf, Size：1020Kb
Aus dem Department Innere Medizin Klinik für Innere Medizin I: Hämatologie, Onkologie und Stammzellentransplantation des Universitätsklinikums Freiburg im Breisgau The Role of Histone Demathylase JMJD1C in the Pathophysiology of Myeloproliferative Neoplasms Inaugural-Dissertation zur Erlangung des Medizinischen Doktorgrades der Medizinischen Fakultät der Albert-Ludwigs-Universität Freiburg im Breisgau Vorgelegt 2019 von Elias Johannes Louis Potomac Schoenwandt geboren in Gießen Dekan Prof. Dr. Norbert Südkamp 1. Gutachterin Prof. Dr. H. L. Pahl 2. Gutachterin Prof. Dr. Dr. K. Domschke Jahr der Promotion 2020 Contents Contents 1 List of AbbreviationsV 2 Summary XVIII 3 Introduction1 3.1 Hematopoiesis..................................1 3.2 Myeloproliferative Neoplasms.........................9 3.2.1 Current Understanding of Etiopathogenesis............. 11 3.2.2 Polycythemia vera........................... 20 3.2.3 Essential Thrombocythemia...................... 23 3.2.4 Primary Myelofibrosis......................... 27 3.3 Epigenetics................................... 31 3.3.1 Histone Demethylases......................... 33 3.3.2 Role of Epigenetics in MPN...................... 36 3.4 Aim....................................... 42 4 Materials and Methods 43 4.1 Cloning..................................... 43 4.1.1 Restriction digest............................ 43 4.1.2 Removal of single stranded overhangs................ 45 4.1.3 Vector dephosphorylation....................... 45 4.1.4 Ligation................................. 45 4.2 Design of shRNA................................ 47 4.3 Bacterial transformation............................ 49 4.4 Agarose gel electrophoresis.......................... 51 4.5 Gel purification and extraction........................ 53 4.6 Nucleic acid quantification........................... 53 4.7 Plasmid DNA preparation........................... 53 4.7.1 Mini preparation of plasmid DNA.................. 54 4.7.2 Maxi preparation............................ 54 4.8 RNA extraction................................. 55 4.9 cDNA synthesis................................. 55 4.10 Polymerase Chain Reaction.......................... 56 4.10.1 Colony PCR.............................. 57 4.10.2 Quantitative real-time PCR...................... 58 III Contents 4.11 Protein Techniques............................... 61 4.11.1 Protein Extraction........................... 61 4.11.2 Protein Quantification......................... 63 4.11.3 SDS-PAGE............................... 63 4.11.4 Coomassie Staining........................... 65 4.11.5 Western blot.............................. 66 4.11.6 Antibodies............................... 66 4.11.7 Reprobing of blots........................... 67 4.12 Cell Culture................................... 69 4.12.1 Transient Transfection......................... 70 4.12.2 Isolation and Handling of CD34+ cells................ 72 4.12.3 Freezing and Thawing of Cells.................... 76 4.13 Virological Methods.............................. 76 4.13.1 pLeGo-iG-hU6............................. 77 4.13.2 Production of Viral Particles..................... 78 4.13.3 Titration of Viral Yield........................ 79 4.14 Fluorescence Activated Cell Sorting...................... 80 5 Results 81 5.1 Cloning of JMJD1C .............................. 81 5.1.1 Human JMJD1C ............................ 81 5.1.2 Murine JMJD1C ............................ 82 5.1.3 Introduction of myc-tag........................ 84 5.2 Characterization of western blot antibodies................. 87 5.3 Characterization of final shRNAs....................... 88 5.4 Verification of knockdown efficiency...................... 90 5.4.1 Verification by qRT-PCR....................... 90 5.4.2 Verification by western blot...................... 92 5.5 Lentiviral transduction............................. 94 5.5.1 Virus production............................ 95 5.6 JMJD1C knock down in healthy donor CD34+ cells............ 98 6 Discussion 100 6.1 JMJD1C’s Demethylase activity....................... 100 6.2 JMJD1C’s Role in Stemness and Differentiation............... 104 6.3 Outlook..................................... 113 IV 1 List of Abbreviations 1 List of Abbreviations µ micro 5-FU 5-Fluorouracil 5-LOX 5-Lipoxygenase 12/15-LOX 12/15-Lipoxygenase A Adenine A Absorbance A Alanine A Ampère aa Amino Acid aaDIPSS age-adjusted DIPSS ABL1 Abelson murine leukemia viral oncogene homolog 1 ACD Asymetric Cell Division ADSC Adipose derived stem cells AEV Avian erythroblastosis virus AF9 Myeloid/lymphoid or mixed-lineage leukemia; translocated to 3(MLLT3) AGM Aorta-gonad-mesonephros AKML Acute megakaryoblastic leukemia ARCH Age-related Clonal Hematopoiesis AKT Proteinkinase B alloSCT Allogenic stem cell transplantation APS Ammonium persulfate ASA Acetylsalicylic acid V 1 List of Abbreviations ASXL1 Additional Sex Combs like 1 ATM Ataxia Telangiectasia Mutated serine/threonine kinase ATR Ataxia Telangiectasia and Rad3-related protein avWS Acquired von Willebrand syndrome B2M β2 microglobulin BCR Breakpoint Cluster Region BES Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid BFU-E Burst Forming Unit - Erythroid BM Bone marrow bp Base pairs BRCA1 Breast Cancer 1 BSA Bovine Serum Albumin C Carbon C Celsius C Cysteine C Cytosine c centi Ca Calcium CALR Calreticulin CD Cluster of Differentiation CCD Charge-coupled Device CDS Coding DNA Sequece cDNA complementary DNA CFBβ Core-binding factor subunit beta VI 1 List of Abbreviations CFU Colony Forming Unit CFU-C Colony Forming Unit - Cytokine (Precursor of Granulocytes and Macrophages CFU-E Colony Forming Unit - Erythroid CFU-G Colony Forming Unit - Granulocytic CFU-GEMM Colony formimg unit - Granulocyte, erythrocyte, monocyte, megakaryocyte ChIP Chromatin Immunoprecipitation CIP Calf Intestine Phosphatase Cl Chlorine CLP Common Lymphoid Progenitor CML Chronic Myelogenous Leukemia CMP Common Myeloid Progenitor CMP-WHO Clinical Molecular and Pathological Criteria – World Health Organization CoIP Coimmunoprecipitation CoREST RE1-Silencing Transcription factor corepressor 1 CXCL4 C-X-C chemokine ligand 4 CXCR4 C-X-C chemokine receptor type 4 C- Carboxy c- Cellular protooncogene C/EBPα CCAAT-enhancer-binding proteinα c-myb MYB proto-oncogene D Aspartate d deci VII 1 List of Abbreviations Da Dalton dd double destilled DAMP Danger Associated Molecular Pattern DGCR8 DiGeorge syndrome chromosomal region 8 DIPSS Dynamic International Prognostic Scoring System DMEM Dulbecco’s Minimally Enhanced Medium DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid dNTP Deoxynucleoside triphosphate DRED Direct Repeat Erythroid-Definitive DSB Double Strand Breakage DTT Dithiothreitol d-HSC Dormant hematopoietic stem cell E Glutamate E2A Transcription Factor 3 (TCF3) ECL Electrochemiluminescence EDTA Ethylenediaminetetraacetic acid EEC Endogenous Erythroid Colonies eGFP Enhanced Green Fluorescent Protein EPO Erythropoietin EPOR Erythropoietin receptor ER Endoplasmic reticulum ESC Embryonic Stem Cell EMH Extramedullary Hematopoiesis VIII 1 List of Abbreviations ET Essential Thrombocythemia EZH2 Enhancer of zeste homolog 2 F Phenylalanine FACS Fluorescence Activated Cell Sorting FAM Fluorescein amidite FCS Fetal Calf Serum Fe Iron FGF5 Fibroblast Growth Factor 5 Flt3 FMS-like kinase 3 FRET Förster resonance energy transfer G Glycine G Guanine g gram g Earth’s gravitational acceleration; m/sec2 GATA1 GATA-binding factor 1 GBX2 Gastrulation Brain Homeobox 2 GCSFR Granulocyte-colony stimulating factor receptor GFP Green Fluorescent Protein GIPSS Genetically Inspired Prognostic Scoring System GLI1 Glioma-associated oncogene GMP Granulocyte-macrophage progenitor GRN Genetic Regulatory Network gRNA guide RNA G-CSF Granulocyte-colony stimulating factor IX 1 List of Abbreviations H Histone H Histidine H Hydrogen h hour HA Hemaglutinin Hb Hemoglobin Hct Hematocrit HDM Histone Demethylase HMR High Molecular Risk HEB Transcription Factor 12 (TCF12) HEL Human Erythroleukemia HETE 15-(S)-Hydroxyeicosatetraenoic Acid HIF Hypoxia Inducible Factor HODE 13-(S)-Hydroxyoctadecadienoic Acid HOXA9 Homeobox protein Hox-A9 HP1α Heterochromatin Protein 1α HRP Horse Raddish Peroxidase HSA Human Serum Albumin HSC Hematopoietic Stem Cell HU Hydroxyurea I Isoleucine IDH Isocitrate dehydrogenase IgG Immunoglobulin G IL-1β Interleukin 1β X 1 List of Abbreviations IL-6 Interleukin 6 IL-7R Interleukin 7 receptor IL-8 Interleukin 8 IL-33 Interleukin 33 IPSET-thrombosis International Prognostic Score of Thrombosis in World Health Organization – Essential Thrombocythemia IPSS International Prognostic Scoring System IRES Internal Ribosome Entry Site IMDM Iscove’s Modified Dulbecco’s Medium JAK1 Janus Kinase 1 JAK2 Janus Kinase 2 JmjC Jumonji C domain JMJD1C Jumonji domain containing 1C K Lysine K Potassium kb Kilobase; 1000 bp L Leucine l liter LB Lysogeny Broth LCR Locus Control Region Ldb1 LIM domain-binding protein 1 LDLR Low Density Lipoprotein Receptor LepR Leptin Receptor Lin Lineage XI 1 List of Abbreviations Lmo2 LIM domain only 2 LMPP Lymphoidprimed multipotent progenitor LOH Loss of heterozygosity LSD1 Lysine-specific histone demethylase 1A LSK Lin−Sca1+Kit− LT-HSC Long Term Hematopoietic Stem Cell LTRA Long Term Repopulation Ability Lyl1 Lymphoblastic leukemia associated hematopoiesis regulator 1 휆 average replication rate of a hematopoietic cell M Molar m meter m milli MAPK Mitogen-activated protein kinase MCS Multiple Cloning Site MDC1 Mediator of DNA damage checkpoint protein 1 MDS Myelodysplastic syndrome MegE Megakaryocytic-erythroid