.....................................................................................................................................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