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ABERRANT DNA METHYLATIONOF RUN DOMAIN CONTAINING 3B in LYMPHOID MALIGNANCIES a Thesis Pr ABERRANT DNA METHYLATIONOF RUN DOMAIN CONTAINING 3B IN LYMPHOID MALIGNANCIES _______________________________________ A Thesis presented to the Faculty of the Graduate School at the University of Missouri-Columbia _______________________________________________________ In Partial Fulfillment of the Requirements for the Degree Master of Science _____________________________________________________ by DANE BURMEISTER Dr. Kristen H Taylor, Thesis Supervisor JULY 2013 The undersigned, appointed by the Dean of the Graduate School, have examined the thesis entitled ABERRANT DNA METHYLATIONOF RUNDOMAIN CONTAINING 3B IN LYMPHOID MALIGNANCIES Presented by DANE BURMEISTER A candidate for the degree of Master of Science And hereby certify that, in their opinion, it is worthy of acceptance. ____________________________________________ Kristen Taylor, Ph.D. ____________________________________________ Wade Davis, Ph.D. ____________________________________________ Gerald Arthur, M.D. M.S. ACKNOWLEDGEMENTS First and foremost I would like to acknowledge my academic advisor Dr. Kristen H. Taylor. I am sure it is not easy to select your first graduate student and I am very grateful for the opportunity to be hers. Dr. Taylor’s patience and guidance is the reason this project is now completed. I have to give her a great deal of credit for investing so much time and effort in a student with little research experience while always approaching my development with positivity and encouragement. I would also like to thank the other members of my thesis committee: Dr. Wade Davis and Dr. Gerald Arthur. Both of these men were nothing but helpful and accommodating. Their contributions to my project would have been sorely missed had they not agreed to be involved. I would also like to acknowledge the many people I’ve worked with in our lab. Thank you to Dr. Stephanie McKay, Jie Ning, and Jennifer Schnabel who all, at one point or another, helped facilitate my transition into a functional scientist. Another big thank you goes out to Dan Miller, Melanie Spoor, and Allison Tegge for their guidance and advice on being a graduate student. Thank you to the undergraduate students who worked in our lab, RiyaSohel, Brad Collins, and Annette Van Swaay for supporting my growth as a mentor. Finally, a great big thank you goes to MdAlmamun. I sat next to Mamun everyday through this entire process and he was always the first person I turned to; not just because of the proximity of our desks. To conclude the acknowledgements I would like to thank my parents and close family. They are the most supportive people I know and instilled the values in me that allowed me to complete this project. The biggest thank you of all goes to Stephanie Loria; her ambition and work ethic are a constant source of inspiration. ii TABLE OF CONTENTS Acknowledgements...................................................................................................................ii List of Figures……….…………………………………………………………………..…….v List of Tables………………………………………………………………………………....vi Nomenclature………………………………………………………………………………...vii Preface……………..…………………………………………………………………………..1 Literature Review…………..……………………………………………………………….…2 Lymphoid Malignancies…………………………………………………………...…2 DNA Methylation…….…………………………………………………………..…..3 Cancer and Epigentics...................................................................................................5 RUNDC3B………………..………………………………………………………......7 ABCB1…………….…………………………………………………………………..9 Canonical Wnt Signaling in Cancer and Lymphoid Tissues…………..…………….10 Summary of RUNDC3B Research and Rationale for Thesis………...……….……………...10 Experimental Aims and Hypothesis………………………….……………………………...12 Materials and Methods ……………………………………………………………………...14 Cell Line Selection……………………………………………………………….....14 DNA Quality Check……………………………………………………………..….15 SssI Treatment………………………….………………………………………..….16 Bisulfite Treatment………………….……………………………………………....16 Primer Design………………………….……………………………………….…...17 COBRA & MSP….……………….……………………………..…………….…....17 RT-PCR…………………………….…………………………………………….....18 iii Statistical Methods…………………………………………………………….…….20 RUNDC3B DNA Sequence Analysis………..…………………………………........21 Results……………………………………………………………………………………..…22 Methylation Assays………………………………………………………………....22 Expression Assays…………………………………………………………………..24 RUNDC3B DNA Sequence Analysis………..……………………………..…….....27 Alternate Pathway Analysis…………….…………………………………………...29 Preliminary Patient Data………………...………………………………………….29 Discussion…………………………………………………………………………………..43 Bibliography………………………………………………………………………………...50 VITA…………………………………...…………………………………………………...54 Appendix……………………………………………………………………………………55 iv LIST OF FIGURES Figure.1 Promoter region of RUNDC3B and the location of primers used in methylation....33 Figure.2 Workflow of methylation assays COBRA and MSP………………………….......34 Figure .3 A representative sample of COBRA results…….…………………………...........35 Figure.4 A representative sample of MSP results………….………………………………..35 Figure.5 Results of methylation assays………………….…………………………………..36 Figure.6 Summary of methylation and expression assays…….…………….…………........38 Figure.7 RUNDC3B CGI methylation and the effect on neighboring gene expression..…...39 Figure.8 Regulatory elements of the RUNDC3B promoter region and conservation of putative TFBS across species………………………………………………………………...41 Figure.9 RUNDC3B promoter methylation in phases of B cell development……..…….….42 Figure.10 Aberrant silencing of RUNDC3B may lead to increased expression of Wnt target genes…………………………………………………………………………………………42 v LIST OF TABLES Table.1 Summary of cell lines and donor samples …………………………………..……..31 Table.2 SssI treatment reaction components and concentrations…………………………...32 Table.3 RUNDC3B CGI primer information……………………………………...…….…..34 Table.4COBRA, MSP, and Bst-U1 enzyme digestreaction components and concentrations………………………………………………………………………………..32 Table.5RT-PCR reaction components and concentrations………………………….……..32 Table.6Odds Ratio Illustration………………………………………………………..….....33 Table.7 Methylation across the RUNDC3B CGI is associated with expression of RUNDC3B…………….………………………………………………………………………......37 Table.8 Odds Ratio resultsfor associations between RUNDC3B CGI methylation and RUNDC3B expression ……………………………………………………..…………..…....37 Table.9 Spearman’s correlation results for associations between RUNDC3BCGImethylation and RUNDC3B expression………………………………...…………………………………39 Table.10Comparison of location of putative EGR-3 TFBS across species………………...41 vi NOMENCLATURE 5-AZA 5’-azacytosine ABCB1 ATP-binding cassette sub-family B member 1 ALL Acute Lymphoblastic Leukemia AP-2 Activating Protein-2 BL Burkitt’s Lymphoma BLAST Basic Local Alignment Search Tool CD Cluster of Differentiation CGI CpG Island CLL Chronic Lymphoblastic Leukemia COBRA Combined Bisulfite Restriction Analysis CpG Cytosine-phospho-group-Guanine CSNK1G1/2 Casein Kinase 1 Gamma 1/2 Ct Cycle Threshold CT conversion reagent Cytosine-Thymine conversion reagent DLBCL Diffuse Large B cell Lymphoma DMR Differentially Methylated Region DNA Deoxyribonucleic acid DNMT1 DNA methyltransferase 1 EGR-3 Early Growth Response-3 FL Follicular Lymphoma G9a A histone methyltransferase vii GAPDH Glyceraldehyde 3-phosphate dehydrogenase HSC Hematopoietic Stem Cells MAPK Mitogen Activated Protein Kinase MBD Methyl Binding Domain MCL Mantle Cell Lymphoma MRD Minimal Residual Disease MSP Methylation Specific Polymerase Chain Reaction NCBI National Center for Biotechnology Information NHL Non-Hodgkin’s Lymphoma PCR Polymerase Chain Reaction RNA Ribonucleic Acid RT-PCR Real Time Polymerase Chain Reaction RUNDC3A RUN domain containing protein 3A RUNDC3B RUN domain containing protein 3B siRNA Small Interfering Ribonucleic Acid TFBS Transcription Factor Binding Site UCSC Genome Brower University of California Santa Cruz Genome Browser UTR Untranscribed Region viii PREFACE The goal of this research proposal is to investigate the epigenetic modification known as DNA methylation and how it can contribute to the development of lymphoid malignancies. Specifically, how the aberrant methylation of the RUNDC3Bgene can, in part, induce tumorigenesis. By analyzing the methylation status of the promoter region of RUNDC3B in lymphomas, leukemia, myelomas, solid tumors and unaffected samples the prevalence of aberrant methylation was determined. Epigenetic regulation wasestablishedbyanalyzing the methylation data in conjunction with RUNDC3B expression data. Finally, by determining possible links between RUNDC3Bsilencing and the physiology of cancer a novel hypothesis for the role of RUNDC3B in lymphoma and leukemia pathogenesis was formulated. Our experiments revealed that aberrant DNA methylation of the RUNDC3BCGI is more prevalent in lymphoid malignancies. The samples displaying this epigenetic misregulationalso exhibited downregulation of the gene.Further analysis of this differentially methylated region (DMR) revealed that it contains evolutionarily conserved regulatory elements, providing evidence that this region may have an important regulatory function in the vertebrate genome. The experimental findings suggest that RUNDC3Bshould be studied further as an understanding of its physiological role could yield important pathogenic insights. 1 LITERATURE REVIEW Lymphoid Malignancies At times the number of distinct cancers that can occur in lymphoid tissues is daunting. Malignancies can develop during every phase of B cell development and this can occur across a wide range of ages. The variations of the morphological
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