Characterization of the MIR23A Cluster in Diffuse Large B Cell Lymphoma Regulation and Targetome Identification Doctoral Thesis In partial fulfillment of the requirements for the degree “Doctor rerum naturalium (Dr. rer. nat.)“ in the Molecular Medicine Study Program at the Georg-August University Göttingen submitted by Natalie Veronika Freytag born in Zabrze, Poland Göttingen 2017 Members of the Thesis Committee: Prof. Dr. Dieter Kube, Supervisor Department of Haematology and Oncology University Medical Center Göttingen Prof. Dr. Peter Burfeind, Reviewer Department of Human Genetics University Medical Center Göttingen Halyna Shcherbata, PhD, Reviewer Max Planck Research Group for Gene Expression and Signaling Max Planck Institute for Biophysical Chemistry Göttingen Date of oral examination: 3.02.2017 Affidavit Here I declare that my doctoral thesis entitled “Characterization of the MIR23A Cluster in Diffuse Large B Cell Lymphoma - Regulation and Targetome Identification” has been writ- ten independently with no other sources and aids than quoted. Göttingen, October 2016 iii List of publications Annica Vlad-Fiegen, Natalie Veronika Freytag, Susanne Dorn, Oliver Müller, Sonja Eberth “The Wnt Pathway Target Gene CCND1 Changes Mitochondrial Localization and Decreases Mitochondrial Activity in Colorectal Cancer Cell Line SW480” Journal of Biosciences and Medicines, 2016, 4, 132-143 DOI: 10.4236/jbm.2016.412017 Natalie V. Freytag, Claudia Pommerenke, Yvonne Merkhoffer, Hilmar Quentmeier, Hans G. Drexler, Sonja Eberth “microRNAs encoded by the MIR23A-cluster on chr. 19 primarily target genes from the same chromosome” Abstract and Poster Transcription and Chromatin EMBL Conference, Heidelberg, 2016 Sonja Eberth, Natalie Klytta, Theres Lusansky, Andreas Rosenwald, Reiner Siebert, Hans G. Drexler, Hilmar Quentmeier “Out of epigenetic control: Regulation of the miR-23a~27a~24-2 in B cell non-Hodgkin Lymphoma and classical Hodgkin Lymphoma” Abstract Haematologica 2015: 760-760 20th European Hematology Association (EHA), Vienna, 2015 Natalie Klytta, Claudia Pommerenke, Dieter Kube, Lorenz Trümper, Michael Haid, Jessica Höll, Hilmar Quentmeier, Hans G. Drexler, Sonja Eberth ”The Role of microRNA Cluster MIR23A~27A~24-2 in the Development of Aggressive B-cell Lymphoma” FEBS Journal 2015 (282): 160-160 Poster and oral presentation at the 40th Federation of European Biochemical Societies (FEBS) Congress, Berlin, 2015 v Contents Contents vii List of Figures . xi List of Tables . xv Abbreviations xvii Abstract xxi 1 Introduction 1 1.1 B cell development, maturation and malignant transformation . 2 1.2 Signaling pathways involved in B cell activation . 5 1.2.1 B cell receptor signaling . 5 1.2.2 CD40 signaling . 8 1.2.3 NFkB signaling . 9 1.2.4 MAPK/ERK signaling . 9 1.3 B cell non Hodgkin Lymphoma (B-NHL) . 10 1.4 MicroRNAs . 13 1.4.1 MicroRNA biogenesis . 14 1.4.2 Mechanism of translational inhibition by microRNAs . 16 1.4.3 MiRNAs in tumorigenesis . 17 1.5 The MIR23A cluster . 18 1.6 Aims . 22 2 Materials and Methods 23 2.1 Biological Material . 23 2.1.1 Primary Material and Data . 23 2.1.2 Cell Lines . 23 2.1.3 Bacteria . 23 2.2 Chemicals, Buffers & Consumables . 24 2.2.1 Chemicals . 24 2.2.2 Buffers and Solutions . 25 2.2.3 Inhibitors . 26 vii viii Contents 2.2.4 Consumables . 27 2.3 Equipment . 27 2.4 Stimulants . 28 2.5 Antibodies . 29 2.6 Oligonucleotides . 30 2.6.1 Primer . 30 2.6.2 siRNAs . 31 2.7 Ready to use reaction systems . 31 2.8 Software . 31 2.9 Eukaryotic expression vectors . 32 2.10 Cell Biology . 37 2.10.1 Cell culture . 37 2.10.2 Isolation of CD77+ GCB cells from primary pediatric tonsills . 38 2.10.3 Nucleofection of cells . 39 2.10.4 Inhibitor treatment . 39 2.10.5 MTT Assay . 39 2.10.6 Stimulation of cells . 39 2.10.7 Generation of stable transduced U2932 R1 cells . 40 2.10.8 Flow cytometry . 42 2.11 Protein biochemistry . 42 2.11.1 Whole cell lysates and cell fractionation . 42 2.11.2 Determination of protein concentration by Bradford assay . 43 2.11.3 SDS PAGE and Western Blotting . 43 2.11.4 Ago2-RNA immunoprecipitation . 44 2.12 Molecular Biology . 45 2.12.1 Total RNA isolation . 45 2.12.2 Reverse transcription . 45 2.12.3 Quantitative real-time polymerase chain reaction (qRT-PCR) . 46 2.12.4 Conventional PCR . 47 2.12.5 DNA restriction digestion . 47 2.12.6 Agarose gel electrophoresis . 48 2.12.7 DNA fragment extraction . 48 2.12.8 Determination of DNA and RNA concentration . 48 2.12.9 Ligation of DNA fragments . 48 2.12.10Transformation . 49 2.12.11Cultivation of Bacteria . 49 2.12.12Plasmid Isolation . 49 Contents ix 3 Results 51 3.1 Identification of signaling pathways regulating the MIR23A cluster . 51 3.1.1 CD40L signaling does not change MIR23A cluster expression in B-NHL 51 3.1.2 LPS does not change MIR23A cluster expression . 53 3.1.3 BCR signaling activates the MIR23A cluster in BL and DLBCL . 55 3.1.3.1 BCR signaling activates the MIR23A cluster in BL cell line BL-2 . 55 3.1.3.2 BCR signaling activates the MIR23A cluster in DLBCL cell line U2932 R1 . 57 3.1.3.3 BCR dependent MIR23A activation is a general mechanism in DLBCL, but not in healthy germinal center B cells . 58 3.1.3.4 Inhibition of protein de-novo synthesis does not affect the MIR23A activation upon BCR stimulation . 59 3.1.3.5 BTK/MEK/ERK signaling activates the MIR23A cluster . 61 3.1.3.6 c-MYC as a potential activator of MIR23A cluster in DLBCL . 64 3.1.3.7 ELK1 as a potential activator of MIR23A in response to BCR . 69 3.2 Identification of the MIR23A targetome . 72 3.2.1 Experimental setup for MIR23A targetome identification . 72 3.2.2 Cloning of pre-miR-23a and pre-miR-27a into the transient expression vector pSG5 for miRNA overexpression . 74 3.2.3 Cloning of pre-miR-23a and pre-miR-27a into the lentiviral vector pGIPZ 74 3.2.4 Generation of stable miR-23a and miR-27a overexpressing clones . 75 3.2.5 Characterization of stable miR-23a and miR-27a overexpressing clones . 76 3.2.6 Establishment of an Ago2-RNA immunoprecipitation assay for miRNA targe- tome identification in DLBCL . 76 3.2.7 RNA sequencing & analysis . 79 3.2.8 MiR-23a and miR-27a targetome in DLBCL . 81 3.2.8.1 Clustering of miR-23a and miR-27a target genes on chromosome 19................................... 88 3.2.9 Validation of Ago2-RIP targets . 91 3.2.9.1 VRK3 protein is not regulated by miR-23a . 91 3.2.9.2 LIMK1 protein is downregulated by miR-27a . 92 3.2.9.3 PUMA protein can not be induced in miR-27a overexpressing cells 94 3.3 MIR23A function . 95 3.3.1 Global affected processes by miR-23a or miR-27a overexpression . 95 3.3.2 miR-27a attenuates the sensitivity of DLBCL cells to undergo apoptosis . 97 3.4 The MIR23A cluster in BL and DLBCL patients . 99 3.4.1 Expression of newly identified and validated miR-23a and miR-27a targets in BL and DLBCL patients . 100 x Contents 4 Discussion 103 4.1 Regulation of the MIR23A cluster . 103 4.1.1 BCR signaling activates the MIR23A cluster . 103 4.1.2 BCR downstream transcription factors . 105 4.2 MiR-23a & miR-27a targetome identification in DLBCL . 108 4.2.1 Ago2-RIP assay . 108 4.2.2 Direct miR-23a and miR-27a targets in DLBCL . 109 4.2.2.1 PUMA and apoptosis . 109 4.2.2.2 LIMK1 and migration . 111 4.2.2.3 VRK3 and MEK/ERK signaling . 112 4.2.2.4 Zinc finger proteins . 113 4.2.3 Enrichment of targets on chromosome 19 . 114 4.3 Biological function & global effects of MIR23A cluster in DLBCL . 115 5 Summary and Conclusion 117 Bibliography 119 A Supplementals 141 Curriculum Vitae 147 List of Figures 1.1 Germinal center reaction . 4 1.2 Signaling cascades activated upon BCR activation . 7 1.3 MiRNA biogenesis pathway . 15 1.4 MIR23A cluster . 18 2.1 Lentiviral vector pGIPZ . ..