Dissertation submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences presented by Diplom-Biology Olga Bogatyrova from Cherkassy, Ukraine Oral-examination: ................................................ Mutations in regulators of the epigenome and their effects on the DNA methylome Referees: Prof. Dr. Stefan Wiemann Prof. Dr. Christoph Plass Division of Epigenomics and Cancer Risk Factors Head of Division: Prof. Dr. Christoph Plass German Cancer Research Center (DKFZ) Heidelberg, Germany To all the people that helped me on the way… Contributions Parts of the introduction section of this thesis was prepared, based on the published review “Mutations in regulators of the epigenome and their connections to global chromatin patterns in cancer” by Plass et al. (2013) in the journal Nature Genetic Reviews, on which I am a co-author [1]. The text sections may therefore contain input from co-authors and figures in the respective sections are partially modified from the review, which is indicated in the caption and in accordance with the copyright policy of the publishers. The chapter 7 of this thesis was prepared for manuscript under the title “Epigenetic deregulation of miRNAs activates oncogenic pathways in prostate cancer” by Bogatyrova O*, Wuttig D.* et al. For the Results chapter 7, text phrases, selected tables, figures and the respective captions are based on the prepared manuscript and contain input from co-authors. Parts of the material and methods section concerning the Illumina HumanMethylation450 BeadChip assay and the Human Sentrix expression array analysis were conducted by the Genomics & Proteomics Core facility of the DKFZ. Data analysis and cluster performance of HumanMethylation450 BeadChip data was conducted with big help and supervision of Dr. Yassen Assenov. Declarations Declarations according to § 8 (3) b) and c) of the doctoral degree regulations: a) I hereby declare that I have written the submitted dissertation myself and in this process have used no other sources or materials than those expressly indicated, b) I hereby declare that I have not applied to be examined at any other institution, nor have I used the dissertation in this or any other form at any other institution as an examination paper, nor submitted it to any other faculty as a dissertation. Heidelberg, (Olga Bogatyrova) Table of contents |13 14 | Table of contents TABLE OF CONTENTS TABLE OF CONTENTS ................................................................................................................... 14 CHAPTER 1. SUMMARY .......................................................................................................... 18 Zusammenfassung .................................................................................................................... 20 CHAPTER2. LIST OF ABBREVIATIONS ................................................................................... 22 CHAPTER 3. INTRODUCTION ................................................................................................. 25 3.1. EPIGENETICS ....................................................................................................................... 25 3.1.1. Epigenetics and epigenetic alterations ............................................................................. 25 3.1.2. DNA modifications ........................................................................................................... 25 3.1.3. Histone modifications ....................................................................................................... 27 3.2. Major regulators of the epigenome ..................................................................................... 28 3.2.1 Complexes of epigenetic regulators in cancer .................................................................. 29 3.3. Non-coding RNAs ............................................................................................................... 29 3.4. Epigenetic alterations in cancer .......................................................................................... 33 3.4.1. Global DNA methylation alterations in cancer .................................................................. 34 3.4.2. Cancer drivers vs. passengers ........................................................................................ 37 3.4.3. Cancer driver mutations in the epigenome ....................................................................... 37 3.4.4. Integrative analysis of cancer genomes and epigenomes ................................................ 37 3.4.5. Disruption of ncRNAs in cancer ....................................................................................... 38 3.5. PanCancer studies ............................................................................................................. 41 3.6. Prostate cancer .................................................................................................................. 42 3.6.1. Deregulation of ncRNA in prostate cancer ....................................................................... 42 CHAPTER 4. AIMS OF THE THESIS ........................................................................................ 46 CHAPTER 5. MATERIAL AND METHODS ............................................................................... 48 5.1. General equipment, disposables and chemical substances ................................................ 48 5.2. Primers ............................................................................................................................... 48 5.3. Tissue samples ................................................................................................................... 48 5.4. Data sets ............................................................................................................................ 51 5.5. DNA methylation analysis ................................................................................................... 51 5.6. miRNA expression analysis ................................................................................................ 54 5.7. Data analysis ...................................................................................................................... 55 Table of contents |15 RESULTS .................................................................................................................................. 59 CHAPTER 6. Mutations in regulators of the epigenome and their effects on the DNA methylome ................................................................................................................................ 59 6.1. ALTERATIONS IN EPIGENETIC REGULATORS IN CANCER ........................................................... 61 6.1.1. Putative driver epigenetic regulators ................................................................................ 63 6.1.2. Alterations in complexes of epigenetic regulators in cancer ............................................. 69 6.2. Identification patterns/clusters of DNA methylation in cancer .............................................. 70 6.3. Correlative analysis of methylome data with defects in epigenetic regulators ..................... 75 6.4. Differently methylated regions in cancer ............................................................................. 75 6.5. Discussion of results and deliveribles ................................................................................ 75 CHAPTER 7. Global integrative analyses of deregulated miRNAs in prostate cancer ............... 81 7.1. Global deregulation of miRNA expression in early-onset prostate cancer (EO-PCA) .......... 82 7.2. Genetic and epigenetic alterations associated with miRNA downregulation in EO-PCA .... 89 7.3. Genetic and epigenetic aberrations associated with miRNA upregulation in EO-PCA........ 89 7.4. Translocations associated with miRNAs deregulation in EO-PCA ...................................... 89 7.5. Tumor-suppressive and oncogenic miRNAs in EO-PCA ..................................................... 89 7.6. Expression profile of miRNAs in different groups of EO-PCA..............................................94 7.7. Promoter DNA-methylation associated with miRNA deregulation in LO-PCA ..................... 95 7.7.1. Validation of promoter DNA-methylation associated with miRNAs deregulation in PCA...95 7.7.2. Epigenetically regulated miRNAs activate key oncogenic pathways in PCA………………97 7.8. PTEN inactivation by miRNA in PCA ............................................................................... 100 7.9. miRNAs promoter DNA-methylation in cancer ................................................................. 100 7.10. Discussion of results ....................................................................................................... 101 CHAPTER 8. Mechanisms leading to deregulation of lncRNA in prostate cancer – an integrative analysis .............................................................................................................................. 104 8.1. Expression profile of lncRNAs in EO-PCA ........................................................................ 105 8.2. Genetic aberrations associated with lncRNA deregulation in EO-PCA .............................. 108 8.3. Promoter DNA-methylation associated with lncRNA deregulation in EO-PCA .................. 108 8.4. Correlation