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Open Abigailharrisbecker Thesis.Pdf The Pennsylvania State University The Graduate School College of Medicine EPIGENETIC AND GENE EXPRESSION CHANGES MEDIATED BY HISTONE H3 METHYLATION IN ACUTE MYELOID LEUKEMIA A Thesis in Biomedical Sciences by Abigail Harris Becker 2018 Abigail Harris Becker Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science May 2018 The thesis of Abigail Harris Becker was reviewed and approved* by the following: Sergei Grigoryev Professor of Biochemistry and Molecular Biology Thesis Advisor Kristin Eckert Professor of Pathology, and Biochemistry and Molecular Biology Gregory S. Yochum Associate Professor of Biochemistry and Molecular Biology and Surgery Ralph L. Keil Associate Professor of Biochemistry and Molecular Biology Chair of Biomedical Sciences Graduate Program *Signatures are on file in the Graduate School iii ABSTRACT Histone modifications are the principal regulators of chromatin dynamics, mediating the extent of chromatin compaction which in turn alters DNA accessibility for gene regulation and repair. Large-scale chromatin remodeling, manifested by the progressive condensation of chromatin marked by the repressive histone modification H3K9me2, is essential to establish cell-specific gene expression patterns during lineage commitment and cellular differentiation. Dysregulation of this large-scale regulatory mechanism has been linked to cellular transformation and oncogenesis in cancer cell lines, diverse solid tumors and hematopoietic cancers. Acute myeloid leukemia (AML) is a highly malignant blood cancer and many of the recurrent gene fusions and driver mutations associated with AML are directly related to epigenetic regulation and chromatin higher order structure, suggesting broad genetic and epigenetic disruption in the pathogenesis of AML. We conducted Chromatin Immunoprecipitation followed by massively parallel sequencing (ChIP-seq) to analyze genome-wide H3K9me2 distribution in primary AML patient samples, CD34+ hematopoietic progenitors, and mature granulocytes, and identified distinct genomic regions marked by significant bidirectional changes of H3K9me2, including domains persistently altered in AML patient samples. In addition, we showed that targeting the histone methyltransferase G9a, which is responsible for the majority of euchromatic H3K9me2, with a pharmacologically active inhibitor, reverses some of the changes observed in AML cells. We performed ChIP-qPCR and transcriptional analysis of genes within the specific regions and in iv this thesis present evidence in support of the ChIP-seq analysis. We propose that the bidirectional changes in H3K9me2 domains (i.e. loss or gain of H3K9me2, rearrangement in domain size or location) results in coordinated activation or silencing of gene clusters, which significantly contributes to the development of AML. v TABLE OF CONTENTS List of Figures .............................................................................................................. vii List of Tables ................................................................................................................ viii List of Abbreviations .................................................................................................... ix Acknowledgements ...................................................................................................... x Chapter 1: Introduction ............................................................................................... 1 1.1 Epigenetic mechanisms mediated by Histone H3 modifications .......................... 1 1.1.1 Regulation of heritable changes in chromatin state .............................. 1 1.1.2 Role of Histone H3 K9 dimethylation in cellular differentiation ............ 2 1.1.3 The histone methyltransferases G9a and GLP ........................................ 3 1.2 Epigenetic dysregulation in Acute Myeloid Leukemia ........................................... 5 1.2.1 Evidence of broad disruption of epigenetic mechanisms in AML .......... 5 1.2.2 H3K9 dimethylation domains and oncogenesis ..................................... 8 1.3 Characterization of H3K9me2 domains in primary AML cells, CD34+ cells and granulocytes ......................................................................................................... 9 1.3.1 Distinguishing characteristics associated with H3K9me2 domains ........ 13 1.3.2 Deregulation of H3K9me2 domains and gene expression in AML ......... 18 1.3.3 Inhibiting the histone methyltransferases G9a and GLP in K562 cells ... 23 Chapter 2: Materials and Methods .............................................................................. 25 2.1 Cells and cell culture .............................................................................................. 25 2.1.1 Human blood samples ................................................................................ 25 2.1.2 Bone marrow CD34+ cells ........................................................................... 25 2.1.3 K562 cells..................................................................................................... 25 2.1.4 G9a/GLP inhibition in K562 cells by UNC0638 ........................................... 25 2.2 Antibodies .............................................................................................................. 26 2.3 SDS-PAGE and Western blot ................................................................................ 26 2.4 Chromatin immunoprecipitation ......................................................................... 27 2.4.1 Nuclei isolation ............................................................................................ 27 2.4.2 Immunoprecipitation .................................................................................. 28 2.5 Library preparation and Next-Generation sequencing ....................................... 29 2.6 Quantitative PCR .................................................................................................. 30 2.6.1 Instrumentation .......................................................................................... 30 2.6.2 Primer sequences ........................................................................................ 30 2.6.3 ChIP-qPCR .................................................................................................... 34 2.6.4 RNA extraction and mRNA expression analysis ......................................... 34 vi Chapter 3: Domain-specific changes in H3K9 dimethylation in AML patient samples 35 3.1 Global H3 Lysine 9 methylation in AML ................................................................. 35 3.2 Evaluating H3K9me2 levels at selected dLOCKs by ChIP-qPCR ............................. 38 Chapter 4: Domain-specific inhibition of G9a/GLP in K562 cells and coordinate effects on H3K9me2 domains and gene expression ........................................... 41 4.1 Global H3K9 di- and tri-methylation in K562 cells after pharmacological inhibition of G9a/GLP ............................................................................................................ 41 4.2 Quantitative PCR evaluation of H3K9me2 levels and gene expression following G9a inhibition ....................................................................................................... 44 Chapter 5: Discussion ................................................................................................... 47 References ................................................................................................................... 53 Appendix: Protocol, ChIP-seq of histone modifications and chromatin-binding proteins 59 vii LIST OF FIGURES Figure 1: H3K9me2 domain in granulocytes, CD34+ progenitors and K562 cells. ...... 12 Figure 2: Genome-wide correlation analysis of H3K9me2 domains in granulocytes, CD34+ and K562 cells ............................................................................................ 14 Figure 3: H3K9me2 domain correlation analysis separates AML samples into two epigenetically distinct clusters. ............................................................................ 17 Figure 4: Genome-wide analysis of H3K9me2 domains reveals sites recurrently deregulated in AML. ............................................................................................. 19 Figure 5: Gene regulation within AML-associated H3K9me2 domains ....................... 22 Figure 6: Analysis of K562 cells following G9a inhibition with the small molecule UNC0638. .............................................................................................................. 24 Figure 7: qPCR assay validation using standard curve and melt peak analysis. .......... 33 Figure 8: Analysis of global histone H3K9 methylation in AML samples and K562 cells. ...................................................................................................................... 37 Figure 9: Comparing ChIP-seq and ChIP-qPCR analysis in AML samples and granulocytes. ........................................................................................................ 39 Figure 10: Treatment with UNC0638 results in selective and reversible reduction of global histone H3K9me2 but not H3K9me3 in K562 cells. ................................... 43 Figure 11: ChIP- and RT-qPCR analysis of K562 cells following G9a inhibition............ 45 viii LIST OF TABLES Table 1. Quantitative PCR cycling program ................................................................. 30 Table 2.
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