W&M ScholarWorks Undergraduate Honors Theses Theses, Dissertations, & Master Projects 5-2015 The Role of Histone Modifying Enzyme SUV420H2 in Pediatric Ependymoma. Sarah Goggin College of William and Mary Follow this and additional works at: https://scholarworks.wm.edu/honorstheses Part of the Cancer Biology Commons, Genomics Commons, and the Molecular and Cellular Neuroscience Commons Recommended Citation Goggin, Sarah, "The Role of Histone Modifying Enzyme SUV420H2 in Pediatric Ependymoma." (2015). Undergraduate Honors Theses. Paper 215. https://scholarworks.wm.edu/honorstheses/215 This Honors Thesis is brought to you for free and open access by the Theses, Dissertations, & Master Projects at W&M ScholarWorks. It has been accepted for inclusion in Undergraduate Honors Theses by an authorized administrator of W&M ScholarWorks. For more information, please contact [email protected]. Table of Contents ABSTRACT:…………………………………………………………………………………………………………………….3 INTRODUCTION:…………………………………………………………………………………………………………..4 EPIGENETICS IN CANCER………………………………………………………………………………..……….5 GENE EXPRESSION PROFILING IN CANCER…………………………………………………….…………7 GENE EXPRESSION PROFILING IN PEDIATRIC EPENDYMOMA………………………………..…8 WHAT IS PEDIATRIC EPENDYMOMA?……………………………………………………………………………..9 ALTERED CHROMATIN STATES IN CANCER……………………………………………………………..12 ALTERED CHROMATIN STATES IN PEDIATRIC EPENDYMOMA…………………………………13 HISTONE MODIFYING ENZYME SUV4-20H2 EFFECT ON CHROMATIN STATE……………….13 METHODS:……………………………………………………………………………………………………………………16 ANALYSIS OF MICROARRAY DATA…………………………………………………………………………..16 REAL-TIME PCR FOR VALIDATION OF MICROARRAY DATA……………………………….….……18 ANALYSIS OF REAL-TIME PCR DATA………………………………………………………………………..19 CHROMATIN IMMUNOPRECIPITATION……………………………………………………….….………20 REAL-TIME PCR OF CHIP PULLDOWNS (AND ANALYSIS)…………………………………………..23 ChIP-SEQUENCING………………………………………………………………………………….…………….23 RESULTS:………………………………………………………………………………………………………………..……26 THERE ARE SIGNIFICANTLY ALTERED PATTERNS OF GENE EXPRESSION IN SUBTYPES OF PEDIATRIC EPENDYMOMA…………………………………………………………..……26 POOR PROGNOSIS SUBGROUP SHOWS ALTERED EXPRESSION OF GENES RELATED TO CELL CYCLE REGULATION, TUMOR SUPPRESSION, AND DNA REPLICATION AND REPAIR………………………………………………………………………………….…27 MICROARRAY ALTERED EXPRESSION VALIDATED AND REPLICATED IN VCU COHORT OF PEDIATRIC EPENDYMOMA……………………………………………………………..……29 SUV4-20H2 BINDS NEAR TERT PROMOTER…………………………………………………..………….31 H4K20me2 AND H4K20me3 MODIFICATION ARE FOUND NEAR TERT PROMOTER…………………………………………………………………………………………………………..33 ChIP-SEQUENCING EXPERIMENTS……………………………………………………………….…………34 DISCUSSION:……………………………………………………………………………………………………………….36 REFERENCES:………………………………………………………………………………………………………..……41 TABLES:…………………………………………………………………………………………………………………..…..45 Table 1: ChIP-qPCR RESULTS WITH anti-SUV4-20H2 ……………….…………..………………………….……58 Table 2: ChIP-qPCR RESULTS WITH anti-H4K20me2 …………………………………………………..………..61 Table 3: ChIP-qPCR RESULTS WITH anti-H4K20me3 …………………………………..……….……………….62 FIGURES:……………………………………………………………………………………………………………..………45 Figure 1: EPIGENETIC MODIFICATIONS AND CHROMATIN STATE …………………………………….…..45 !1 Figure 2: DIFFERENTIAL EXPRESSION PATTERNS IN GOOD PROGNOSIS AND POOR PROGNOSIS SUBGROUPS OF PE ……………………………………………………………………………46 Figure 3: ALTERED EXPRESSION OF GENES RELATED TO CELL CYCLE REGULATION, TUMOR SUPPRESSION, AS WELL AS DNA REPLICATION AND REPAIR IN POOR PROGNOSIS SUBGROUP………………………………………………………………………………………………………….48 Figure 4: MODEL OF SUV4-20H2 IN CANCER ……………………………………………………………………….50 Figure 5: VCU PE SAMPLES SHOW DIFFERENTIAL EXPRESSION OF SUV4-20H2 AND PROTO- ONCOGENES …………………………………………………………………………………………..………….51 Figure 6: qPCR IN VCU SAMPLES VALIDATES MICROARRAY ALTERED EXPRESSION ……………….53 Figure 7: ANTIBODIES ARE EFFECTIVE FOR USE IN CHROMATIN IMMUNOPRECIPITATION ……55 Figure 8: SCHEMATIC SHOWING GENERAL BINDING REGIONS OF SUV4-20H2 AND OTHER CHROMATIN MODIFIERS ………………………………………………………………………………..……56 Figure 9: SUV4-20H2 BINDS TO CHROMATIN NEAR TERT PROMOTER……………………………………57 Figure 10: H4K20me2 AND H4K20me3 MODIFICATIONS ARE PRESENT IN CHROMATIN NEAR TERT PROMOTER …………………………………………………………………………………………..…59 Figure 11: MODEL FOR DYSREGULATION OF TERT BY SUV4-20H2 LEADING TO PE …………………63 SUPPLEMENTARY MATERIALS: ………………………………………………………………………………65 Supplementary Material-1: PLIER METHOD….…………………………………………………………………….…65 Supplementary Material-2: PERMUTATION TESTING PARAMETERS AND CALCULATIONS..…..……65 Supplementary Material-3: GSEA METHOD OVERVIEW……….……………………………………………….…66 Supplementary Material-4: SAM METHODOLOGY- PRINCIPAL CALCULATIONS…..………………….….66 Supplementary Material-5: AGILENT 2100 BIOANALYZER- RNA 6000 PICO KIT PROTOCOL….….….67 Supplementary Material-6: NANODROP PROTOCOL………………………………………………………………………69 Supplementary Material-7: CHROMATIN IMMUNOPRECIPITATION- SPECIFIC VOLUMES…..………70 Supplementary Material-8: PRIMERS DESIGNED FOR qPCR EXPERIMENTS …………….….………..….70 Supplementary Material-9: ME-DIP AND 450K METHYLATION ARRAY……………………….……………..71 !2 Abstract Cancer is the leading cause of non-accidental death in children. Pediatric ependymomas (PE) are the third most common brain tumor in children. Despite advances leading to better survival outcomes in some cancers, these tumors remain incurable in up to 45% of patients, with recurrent local relapse being the major cause of mortality[18, 19]. Surgical resection is currently the most effective treatment, but over 50% of children whose tumors have been totally resected will still experience tumor recurrence despite aggressive adjuvant therapy [22]. Key molecular events in the pathogenesis of PE have yet to be defined, and understanding these events are proving to be increasingly necessary to developing clinically-relevant applications. In particular, errors in epigenetic regulatory machinery appear to play a substantial role in the pathogenesis of numerous cancers. As such, this research used gene expression profiling data on a large cohort (n=102) of PE samples to search for potential driving mechanisms underlying recurrence and poor prognosis in this cancer. Through this, we developed a model for the role of the histone modifying enzyme SUV4-20H2 in carcinogenesis. SUV4-20H2 confers modifications that lead to chromatin compaction and resultant silencing of genes in component/surrounding DNA. We hypothesized that this enzyme acts to directly regulate proto-oncogenes and that its loss in this cancer leads to their increased expression. We find evidence to support this model for SUV4-20H2 regulation of TERT, the catalytic subunit of telomerase. This holds significant translational value as TERT is strongly associated with recurrence and has been shown to increase tumorigenicity of tumor initiating cells, and a better understanding of it's regulation could lead to development of new therapies. Additionally, these findings implicate a potential broader role of SUV4-20H2 in driving carcinogenesis and warrant further investigation. !3 Introduction Cancer epigenomics is an emerging concentration of clinical cancer research. In conjunction with the field of oncogenomics, research aims to better define genomic, transcriptomic and proteomic differences within individual tumor subtypes and between subtypes and to relate these determined molecular phenotypic patterns to differences in prognosis and treatment response. Studies of cancer genomics led to vast leaps in understanding of the nature and underlying mechanisms of cancer. These new understandings began to point toward errors in regulatory control of essential cell processes as fundamental to cancer development and progression. However, of the key cancer-causing genes identified to date, germline mutations in these genes only account for a small fraction of their observed aberrant expression. This leaves open the question of how these hallmark mutations arise. Further, findings began and continue to emerge indicating the presence of mutations in various components of epigenetic machinery in numerous cancers. Findings such as these point to a substantial role for epigenetics in driving cancer. Research in this area has also been driven by its increasing clinical relevance. One major problem in clinical cancer research to date has been connecting diagnosis to prognosis and treatment response. Significant heterogeneity in both prognosis and treatment response have been found in cancers diagnosed by histology and location, but these remain the two primary diagnostic criteria. As such, research directed at cancer types defined by these criteria is limited in scope and applicability. More recent efforts aimed at identifying classes of transcriptomic patterning have proven far better at predicting prognosis and treatment response and have the potential to reveal more regarding the mechanisms by which malignancies arise. A shift towards gene expression-profile based diagnostics should allow for huge advances in treatment and in research as we begin to be able to categorize cancers by the underlying mechanisms of their malignancy. This thesis examines the altered gene expression patterns in pediatric ependymoma !4 (PE), and subsequently investigates the role of a particular epigenetic factor in its prognosis. This introduction will (1) present an overview of our current understanding of epigenetic states and cancer, (2) identify and justify experimental aims and practices in studying epigenetics in cancer, (3) provide background on the specific malignancy being studied and explain why it is an excellent candidate for investigation, and (4) describe in