GLOBAL DNA METHYLATION AND GENE EXPRESSION ANALYSIS IN PRE-B CELL ACUTE LYMPHOBLASTIC LEUKEMIA A Dissertation presented to the Faculty of the Graduate School at the University of Missouri-Columbia In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy by MD ALMAMUN Dr. Kristen H. Taylor, Dissertation Supervisor JULY 2015 © Copyright by Md Almamun 2015 All Rights Reserved The undersigned, appointed by the dean of the Graduate School, have examined the Dissertation entitled GLOBAL DNA METHYLATION AND GENE EXPRESSION ANALYSIS IN PRE-B CELL ACUTE LYMPHOBLASTIC LEUKEMIA Presented by Md Almamun A candidate for the degree of Doctor of Philosophy And hereby certify that, in their opinion, it is worthy of acceptance. Kristen H. Taylor James M. Amos-Landgraf Gerald L. Arthur Mark A. Daniels J. Wade Davis DEDICATION This work is dedicated to my parents, Abdul Aziz & Khosh Nahar Begum, my inspiration. ACKNOWLEDGMENTS I would first like to acknowledge my advisor, Dr. Kristen H. Taylor for her guidance and patience throughout this research project. I was able to gain knowledge, research passion and complete this project with her tremendous helps and encouragement. Thank you for being so positive and for everything you did for me. I would also like to thank my degree committee members, Dr. J. Wade Davis, Dr. Gerald L. Arthur, Dr. James M. Amos-Landgraf, and Dr. Mark A. Daniels for their advice and guidance in this endeavor. I would like to special thank Dr. J. Wade Davis, Benjamin T. Levinson, and Nathan T. Johnson for analyzing MIRA-seq and RNA-seq data, and Annette C. van Swaay for helping me during data analysis. Thank to Dr. Stephanie D. McKay for her advice during method development. I would like to thank the members of the Taylor Lab, specifically Ambima H. Buzhyason, Alexei J. Stuckel, and Darren C. Hawkins for being so nice to me and for your suggestions in different aspects of my research works. I have truly enjoyed every day because of the great people in Dr. Taylor’s Lab. Finally, I would like to thank my parents, my wife Fauzia Huq Nur, and other family members for always supporting me. I could not have done any of this work without their support, love and encouragement. ii TABLE OF CONTENTS ACKNOWLEDGMENTS……………………………………………………………………...…ii LIST OF TABLES …………………………………………………………………………......viii LIST OF FIGURES………………………………………………………………………………ix LIST OF ABBREVIATIONS…………………………………………………………………...xii ABSTRACT…………………………………………………………………………………….xiii Chapter Page CHAPTER 1: INTRODUCTION ………………………………………………………………...1 TABLES AND FIGURES ………………………………………………………………..9 REFERENCES ………………………………………………………………………….11 CHAPTER 2: BACKGROUND…………………………………………………………………15 2.1 ACUTE LYMPHOBLASTIC LEUKEMIA (ALL)…………………………………15 2.1.1 ALL induction mechanisms ……………………………………………….15 2.1.2 ALL classifications ………………………………………………………..16 2.1.3 ALL treatments and prognosis …………………………………………….18 2.1.3.1 Diagnosis ………………………………………………………...18 2.1.3.2 Treatment ………………………………………………………..20 2.1.3.3 Prognosis ………………………………………………………...23 2.2 NORMAL B-CELL DEVELOPMENT ……………………………………………..24 2.3 EPIGENETICS ……………………………………………………………………...27 2.4 DNA METHYLATION ……………………………………………………………..28 2.4.1 DNA methyltransferases …………………………………………………..30 2.4.2 DNA methylation in normal development including B-cell developments.34 2.4.3 DNA methylation in cancer ……………………………………………….36 iii 2.4.4 DNA methylation as a biomarkers for diagnosis ………………………….39 2.4.5 DNA methylation inhibitors for cancer treatment ………………………...42 2.4.6 Methods for DNA methylation studies ……………………………………44 2.5 HISTONE MODIFICATIONS ……………………………………………………...46 2.6 LINC-RNA ………………………………………………………………………….47 2.7 MICRO-RNA ………………………………………………………………………..49 2.7.1 MicroRNA in B-cell development ………………………………………...49 2.7.2 MicroRNA in cancer ………………………………………………………50 2.8 PSEUDOGENES ……………………………………………………………………52 2.9 GENOMIC REGULATORY REGIONS …………………………………………...55 2.9.1 Promoter regions …………………………………………………………..56 2.9.2 Enhancer regions …………………………………………………………..56 2.9.3 Dnase I hypersensitive site (DHS) ………………………………………...59 2.9.4 Transposable elements …………………………………………………….60 2.9.5 CpG islands ………………………………………………………………..62 TABLES AND FIGURES ………………………………………………………………63 REFERENCES ………………………………………………………………………….67 CHAPTER 3: ISOLATION OF PRECURSOR-B CELL SUBSETS FROM HUMAN UMBILICAL CORD BLOOD ………………………………………………………………….88 3.1 ABSTRACT.………………………………………………………………………...88 3.2 INTRODUCTION …………………………………………………………………..89 3.3 PROTOCOL ………………………………………………………………………...91 3.3.1 Isolation of mononuclear cells from umbilical cord blood…………….......91 iv 3.3.2 Modified B-cell isolation procedure from mononuclear cells using MACS separation ………………………………………………………………..92 3.3.3 Antibody labeling and preparation for cell sorting ………………………..94 3.3.4 Cell sorting using the MoFlo XDP flow cytometer ……………………….94 3.4 REPRESENTATIVE RESULTS ……………………………………………………96 3.5 DISCUSSION ……………………………………………………………………….96 TABLES AND FIGURES ……………………………………………………………..100 REFERENCES ………………………………………………………………………...106 CHAPTER 4: GENOME-WIDE DNA METHYLATION ANALYSIS IN PRECURSOR B-CELLS ………………………………………………………………………………………108 4.1 ABSTRACT ……………………………………………………………………….108 4.2 INTRODUCTION …………………………………………………………………109 4.3 MATERIALS AND METHODS …………………………………………………..111 4.3.1 Isolation of precursor B-cell subsets ……………………………………..111 4.3.2 DNA isolation and MIRA-seq library preparation ………………………111 4.3.3 Data processing, alignment, and peak identification …………………….113 4.3.4 Annotation and enhancer prediction ……………………………………..114 4.3.5 Differentially methylated regions of interest …………………………….114 4.4 RESULTS ………………………………………………………………………….116 4.4.1 Genomic distribution of DNA methylation during B-cell development…116 4.4.2 Differentially methylated regions in precursor B-cell differentiation……117 4.5 DISCUSSION ……………………………………………………………………...119 TABLES AND FIGURES ……………………………………………………………..122 REFERENCES ………………………………………………………………………...131 v CHAPTER 5: INTEGRATED METHYLOME AND TRANSCRIPTOME ANALYSIS REVEALS NOVEL REGULATORY ELEMENTS IN PEDIATRIC ACUTE LYMPHOBLASTIC LEUKEMIA …………………………………………………………….134 5.1 ABSTRACT ………………………………………………………………………..134 5.2 INTRODUCTION …………………………………………………………………135 5.3 MATERIALS AND METHODS …………………………………………………..136 5.3.1 Patient samples …………………………………………………………...136 5.3.2 Antibodies ………………………………………………………………..137 5.3.2 MIRA-seq library preparation ……………………………………………137 5.3.3 Identification of methylated peaks and differentially methylated regions in ALL ……………………………………………………………138 5.3.4 Annotation and enhancer prediction ……………………………………..139 5.3.5 RNA-seq library preparation and data analysis ………………………….140 5.4 RESULTS ………………………………………………………………………….141 5.4.1 Genome-wide DNA methylation profiles ………………………………..141 5.4.2 Differentially methylated regions in ALL ……………………………….141 5.4.3 B-cell development genes and epigenetic modifiers are aberrantly expressed in ALL …………………………………………………………145 5.4.4 Differential expression of transcripts with epigenetic regulatory functions ………………………………………………………………….146 5.5 DISCUSSION ……………………………………………………………………...148 TABLES AND FIGURES …………………………….……………………………….152 REFERENCES ……………………………………………………………………..167 vi CHAPTER 6: TRANSCRIPTIONAL INACTIVATION OF REGULATORY ENHANCERS ASSOCIATED WITH THE MIS-REGULATION OF NEIGHBORING GENES……………171 6.1 ABSTRACT ………………………………………………………………………..171 6.2 INTRODUCTION …………………………………………………………………172 6.2 MATERIALS AND METHODS …………………………………………………..175 6.2.1 ChIP-seq data …………………………………………………………….175 6.3.2 RNA-seq data …………………………………………………………….175 6.3.3 Differentially methylated regions between ALL and healthy pre-B cell...176 6.3 RESULTS ………………………………………………………………………….176 6.3.1 Enhancer identification in normal B lymphocytes (GM12878)………….176 6.3.2 Alteration of Enhancer methylation in ALL ……………………………..177 6.3.3 Enhancer transcript (eRNA) associated with the expression of neighboring genes ………………………………………………………...179 6.4 DISCUSSION ……………………………………………………………………...183 TABLES AND FIGURES …………………………………………………………188 REFERENCES …………………………………………………………………….201 CHAPTER 7: SUMMARY AND FUTURE DIRECTIONS ………………………………….206 REFERENCES…………………………………………………………………………217 VITA …………………………………………………………………………………………..220 vii LIST OF TABLES Table Page 2.1: Epigenetic aberrations among different tumor types………………………………………..64 3.1: Representative cell sort statistics…………………………………………………………..100 3.2: Sorted subsets of B-cell from human umbilical cord blood……………………………….104 4.1: Read and alignment statistics for individual samples ……………………………………123 4.2: Differentially methylated enhancer target genes involved in leukocyte activation and BCR signaling pathway ……………………………………………………………….130 5.1: Pre-B ALL patients characteristics ………………………………………………………..152 5.2: Illumina sequencing data for MIRA-seq ……………………………………………...…..153 5.3: Gene promoter hypermethylation associated with significant decreased expression in ALL …………………………………………………………………………159 5.4: Gene promoter hypomethylation associated with significant increased expression in ALL …………………………………………………………………………160 5.5: Differentially expressed genes in ALL involved in B-cell development and epigenetic modifications …………………………………………………………………..162 5.6: Up-regulated lincRNA in ALL ……………………………………………………………164 5.7: Down-regulated lincRNA in ALL ………………………………………………………...165 6.1: Significantly up-regulated genes related with the hypomethylation of poised enhancers in ALL …………………………………………………………………………………….192 6.2: Significantly up-regulated genes related with the hypomethylation of poised enhancers in ALL …………………………………………………………………………193