Wayne State University Wayne State University Dissertations 1-1-2016 Whsc1l1 Regulates Estrogen Receptor Activity In Sum44 Breast Cancer Cells Jonathan Curtis Irish Wayne State University, Follow this and additional works at: https://digitalcommons.wayne.edu/oa_dissertations Part of the Oncology Commons Recommended Citation Irish, Jonathan Curtis, "Whsc1l1 Regulates Estrogen Receptor Activity In Sum44 Breast Cancer Cells" (2016). Wayne State University Dissertations. 1641. https://digitalcommons.wayne.edu/oa_dissertations/1641 This Open Access Dissertation is brought to you for free and open access by DigitalCommons@WayneState. It has been accepted for inclusion in Wayne State University Dissertations by an authorized administrator of DigitalCommons@WayneState. WHSC1L1 REGULATES ESTROGEN RECEPTOR ACTIVITY IN SUM44 BREAST CANCER CELLS by JONATHAN CURTIS IRISH DISSERTATION Submitted to the Graduate School of Wayne State University, Detroit, Michigan in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY 2016 MAJOR: Cancer Biology Approved By: Advisor Date © COPYRIGHT BY JONATHAN CURTIS IRISH 2016 All Rights Reserved DEDICATION I dedicate this work to my loving, patient wife Cesira. To my parents, Lori and Sean, who have been unwavering in their support of me and my family during my temporally and geographically long educational journey. Finally, to my grandparents and aunt, Curtis, Norma, and Linda, for motivating me to study cancer. ii ACKNOWLEDGEMENTS I would like to thank my mentor, Dr. Ethier, for providing an environment where I could learn think critically and become proficient at applying the scientific method to problems in biology. I owe my future in science in no small part to the time spent with Dr. Ethier and all of the members of the Ethier lab from whom I was able to learn so much. Thank you also to Dr. Wilson and the MUSC sequencing core for their patience and expertise during my struggles with my next-gen sequencing experiments. I also would like to thank Dr. Yang for his co-mentorship at Wayne State, and his support in helping me with my understanding of my work in the context of previous work performed by him and others in the Ethier lab, and especially with his help in organizing and structuring my writing for my dissertation. Thank you to Dr. Sheng for giving me my first chance in the lab, and for allowing me to develop my passion for developing the technical skills required to perform challenging experiments soundly. To my committee, particularly Dr. Matherly, and Dr. Brush, thank you so much for your guidance and patience. I could not have done this without your support. iii TABLE OF CONTENTS DEDICATION ................................................................................................................ ii ACKNOWLEDGEMENTS ............................................................................................... iii LIST OF TABLES .......................................................................................................... viii LIST OF FIGURES ......................................................................................................... ix LIST OF ABBREVIATIONS ............................................................................................. xi CHAPTER 1 INTRODUCTION ...................................................................................... 1 1.1 General Introduction ..................................................................................................... 1 1.2 Genomic basis of cancer ................................................................................................ 4 1.3 Breast Cancer Oncogenomics ......................................................................................... 7 1.4 Epigenetic Regulation .................................................................................................... 8 1.4.1 DNA Methylation ........................................................................................................... 9 1.4.2 DNA methylation in cancer .......................................................................................... 11 1.4.3 Histone Modification ................................................................................................... 12 1.4.4 Histone Variants .......................................................................................................... 14 1.4.5 Histone Tail Modification ............................................................................................. 14 1.4.6 Histone Acetylation ..................................................................................................... 15 1.4.7 Histone Acetyltransferases (HATs) .............................................................................. 16 1.4.8 Histone Phosphorylation ............................................................................................. 17 1.5 Histone Methylation ..................................................................................................... 19 1.5.1 Histone lysine demethylases ....................................................................................... 21 1.5.2 Histone H3 methylation on lysine 4 ............................................................................. 22 1.5.3 H3K9 methylation ........................................................................................................ 24 iv 1.5.4 H3K27 methylation ...................................................................................................... 26 1.5.5 H3K79 methylation ...................................................................................................... 27 1.6 NSD family of histone lysine methyltransferases .......................................................... 29 1.7 Epigenetic Dysregulation in Breast Cancer .................................................................... 32 1.7.1 WHSC1L1 is amplified in 12-15% of Breast Cancers .................................................... 34 1.7.2 Breast cancer sub-types ............................................................................................... 37 1.7.3 Histological Subtyping of Breast Cancer ...................................................................... 38 CHAPTER 2 KNOCKDOWN OF WHSC1L IN SUM44 CELLS ALTERS CELL PROLIFERATION AND GENE EXPRESSION ............................................................................................. 44 2.1 Introduction ................................................................................................................. 44 2.2 Results .......................................................................................................................... 47 2.2.1 WHSC1L1-short is the major expressed isoform in breast epithelial cells .................. 47 2.2.2 WHSC1L1-short knockdown reduces cell growth ........................................................ 50 2.2.3 Knockdown of WHSC1L1-short alters expression of seVeral genes actiVe in breast cancer ................................................................................................................................... 55 2.2.4 SUM44 cells respond negatiVely to estrogen .............................................................. 60 2.2.5 Gene expression profiling of SUM44 cells after treatment with estrogen .................. 66 2.3 Discussion .................................................................................................................... 70 2.4 Materials and Methods ................................................................................................ 74 2.4.1 shRNA Vectors .............................................................................................................. 74 2.4.2 LentiViral infection protocol ........................................................................................ 74 2.4.3 Antibodies .................................................................................................................... 74 2.4.4 Cell culture ................................................................................................................... 75 2.4.5 Proliferation assays ...................................................................................................... 75 2.4.6 Western blotting .......................................................................................................... 76 2.4.7 RNA expression profiling ............................................................................................. 76 v 2.4.8 Bioinformatics and Statistical analysis ......................................................................... 77 CHAPTER 3 ESTROGEN RECEPTOR ALPHA BINDS CHROMATIN IN THE ABSENCE OF ESTROGEN IN SUM44 CELLS ....................................................................................... 78 3.1 Introduction ................................................................................................................. 78 3.2 Results .......................................................................................................................... 79 3.2.1 ERa binds chromatin in the absence of estrogen in SUM44 cells ................................ 79 3.2.2 Motif enrichment at ERa binding sites ........................................................................ 84 3.2.3 ERa binding decreases upon WHSC1L1-short knockdown .......................................... 87 3.3 Discussion ...................................................................................................................