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Assessment of 17Beta-Estradiol-Estrogen Receptor Alpha Complex-Mediated Changes in Genome-Wide Methylation and Gene Expression Profiles

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Assessment of 17Beta-Estradiol-Estrogen Receptor Alpha Complex-Mediated Changes in Genome-Wide Methylation and Gene Expression Profiles ASSESSMENT OF 17BETA-ESTRADIOL-ESTROGEN RECEPTOR ALPHA COMPLEX-MEDIATED CHANGES IN GENOME-WIDE METHYLATION AND GENE EXPRESSION PROFILES A THESIS SUBMITTED TO THE GRADUATE SCHOOL OF NATURAL AND APPLIED SCIENCES OF MIDDLE EAST TECHNICAL UNIVERSITY BY SIRMA DAMLA USER IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN BIOLOGY MAY 2016 Approval of the Thesis: ASSESSMENT OF 17β-ESTRADIOL-ESTROGEN RECEPTOR ALPHA COMPLEX-MEDIATED CHANGES IN GENOME-WIDE METHYLATION AND GENE EXPRESSION PROFILES submitted by SIRMA DAMLA USER in partial fulfillment of the requirements for the degree of Master of Science in Biology Department, Middle East Technical University by, Prof. Dr. M. Gülbin Dural Ünver Dean, Graduate School of Natural and Applied Sciences Prof. Dr. Orhan Adalı Head of Department, Biology Prof. Dr. Mesut Muyan Supervisor, Biology Dept., METU Examining Committee Members: Assoc. Prof. Dr. A. Elif Erson-Bensan Biology Dept., METU Prof. Dr. Mesut Muyan Biology Dept., METU Assoc. Prof. Dr. Tolga Can Computer Engineering Dept., METU Assoc. Prof. Dr. Çağdaş Devrim Son Biology Dept., METU Assist. Prof. Dr. Bala Gür Dedeoğlu Biotechnology Institute, Ankara University Date: 30.05.2016 I hereby declare that all information in this document has been obtained and presented in accordance with academic rules and ethical conduct. I also declare that, as required by these rules and conduct, I have fully cited and referenced all material and results that are not original to this work. Name, Last name: Sırma Damla User Signature : iv ABSTRACT ASSESSMENT OF 17BETA-ESTRADIOL-ESTROGEN RECEPTOR ALPHA COMPLEX-MEDIATED CHANGES IN GENOME-WIDE METHYLATION AND GENE EXPRESSION PROFILES User, Sırma Damla M.S., Biology Department Supervisor: Prof. Dr. Mesut Muyan May 2016, 142 pages 17β-estradiol (E2), the most potent estrogen hormone, induces cellular responses primarily through Estrogen Receptor-alpha (ERα), which is a transcription factor. Interfering E2 signaling indicates that E2 is mitogenic for cells, exemplified by MCF7 cells derived from breast adenocarcinoma, synthesizing ERα endogenously. Studies used exogenous expression of ERα in ERα-negative cell lines to examine structural/functional properties of the receptor. What was unexpected from these studies is the observation that E2 treatment represses cellular proliferation. However, mechanism(s) of this paradoxical phenomenon remains unknown. Methylation is an important epigenetic DNA modification. Changes in methylation alter gene expressions critical for cellular proliferation/differentiation, embryonic development, genomic imprinting and cancer. We therefore hypothesize that distinct methylation statuses of responsive genes’ regulatory regions underlie differential gene expressions, and hence, proliferative and anti-proliferative effects of E2 in cell models. To test this prediction, we generated a cell model stably expressing ERα in MDAMB231 breast cancer cell line. Of the monoclones synthesizing ERα, the MDA-ERα5, based on expected ERα functions, was selected as the cell model to v comparatively assess the E2 effects on changes in methylome and transcriptome profiles to those observed in MCF7 cells. Our studies suggest that cell models have cell-specific methylation patterns for the same genomic region at which E2 induces distinct alterations and differentially modulates gene expressions. However, due to the existence of variations among experimental replicates, establishing a correlation between the methylation statuses to gene expression profile of cell lines appears to be immature. An increase in sample size could circumvent this issue. Keywords: estrogen, estrogen receptor, methylation, gene expression vi ÖZ 17BETA-ÖSTRADİOL-ÖSTROJEN RESEPTÖR ALFA KOMPLEKSİ ARACILIĞI İLE GENOM ÇAPINDA OLUŞAN METİLASYON VE GEN İFADESİ DEĞİŞİKLERİNİN BELİRLENMESİ User, Sırma Damla Yüksek Lisans, Biyoloji Bölümü Tez Yönetcisi: Prof. Dr. Mesut Muyan Mayıs 2016, 142 sayfa 17β-östradiol (E2), en güçlü östrojen hormonudur ve transkripsiyon faktörü olan Östrojen Reseptörü-alfa (ERα) aracılığıyla hücrede birçok yanıtın oluşmasına neden olur. E2 sinyal yolağına müdahale ile yapılan çalışmalarda, MCF7 gibi ERα’yı endojen olarak sentezleyen hücrelerde E2’nin hücresel çoğalma için gerekli olduğu gösterilmiştir. ERα-negatif hücre hatlarında, ERα’nın harici olarak sentezlenmesiyle reseptörün yapısal/fonksiyonel özellikleri çalışılmıştır. Beklenmedik olan ise E2’nin hücresel çoğalmayı bu hücre hatlarında baskılamasıdır. Bu çelişkili gözlemlerin sebebi ise belirsizliğini korumaktadır. Metilasyona önemli bir epigenetik modifikasyondur. Metilasyon sonucu oluşan gen ifadelerindeki değişiklikler, embryonik gelişim, genomik imprinting, kök hücre başkalaşımı ve kanser gibi birçok moleküler işlemde gereklidir. Bu nedenle, genlerin düzenleyici bölgelerindeki farklı metilasyon durumlarının, farklı gen ifadelerinin, bu nedenle de E2’nin endojen ya da harici olarak ERα sentezleyen hücrelerde, sırasıyla, hücre çoğalmasını destekleyen ya da engelleyen etkisinin sebebi olabileceğini varsayımında bulunuyoruz. vii Farklı metilasyon durumlarını çalışabilmek adına MDAMB231 meme kanseri hücre hattında stabil olarak ERα sentezleyen bir model hücre hattı geliştirdik. Harici olarak hücreye verilen reseptörün işlevliğini kontrol etmek için birçok fonksiyonel tarama yaptıktan sonra MDA-ERα5 monoklon hücre hattının, genom çapında metilasyon farklılıklarını çalışmak için uygun olduğuna karar verdik. Ayrıca, metilasyonun gen ifadesi ile arasında bir bağıntı kurmak adına tüm transkriptomik profil analizi gerçekleştirdik. Genom çapında yapılan metilasyon profillemesi, aynı bölgeler için, her hücre hattının kendine özgü metilasyon desenleri olduğunu gösterdi. Ek olarak, E2 muamelesi ile bu değişiklikler daha da artmıştır. Fakat, transkriptomik analizlerdeki varyasyonlar nedeniyle, sonuçlarımız metilasyon ve gen ifadesi arasında bir bağıntı kurmak için yetersiz kalmıştır. Anahtar kelimeler: östrojen, östrojen reseptörü, metilasyon, gen ifadesi viii ACKNOWLEDGEMENTS I would like to thank my supervisor Prof. Dr. Mesut Muyan for, first of all, teaching me how to think critically and to question every single result. Also, for teaching me to mentally plan subsequent steps and anticipate the possible results. I have learned the beauty of scientific world thanks to him. There is no way to express my gratitude for all the guidance he has provided and the patience he has shown. I am grateful to Assoc. Prof. Dr. A. Elif Erson-Bensan for her support and valuable inputs in this thesis work. I would like to thank Assoc. Prof. Dr. Tolga Can for his guidance and enormous input in analyzing the data. I would like to thank all my thesis committee members; Prof. Dr. Mesut Muyan, Assoc. Prof. Dr. A. Elif Erson-Bensan, Assoc. Prof. Dr. Tolga Can, Assoc. Prof. Dr. Çağdaş Devrim Son, and Assist. Prof. Dr. Bala Gür Dedeoğlu once again for being here to provide critical feedbacks to my work. I want to express my sincere thanks to my lab members Gamze Ayaz, Pelin Yaşar, Çağla Ece Olgun, Burcu Karakaya, and Gizem Kars as well as the former lab member Gizem Güpür for their endless help and comforting when I needed the most. Also, I would like to thank Bilge Aşkın for her help in numerous experiments. In addition, I want to thank all the members of Erson-Bensan Lab for their support and help. I would like to thank Bilgi Güngör and Ersin Gül for their invaluable help in Flow Cytometry. I also would like to thank Prof. Dr. Rengül Çetin Atalay and her lab members for their help in fluorescent microscope use. My special thanks go to Berfin Karakeçili, Ece Öztürk, Ege Tuna, Gülce Çakman, Nazlı Ünal, and Pınar Boz for being the source of joy in my life. I would like to thank Ece Zıraman, Nermin Uğur, Onur Demir, and Tilbe Mutluay for their wonderful friendship. I want to thank Aylin Kömez, Beliz Bediz, and Neşe Erdem for their support and friendship. ix There are no words to express how much I owe to my dearest friend Armağan Hergüner for his endless support. I cannot express my heartfelt thanks to my sweetheart sisters Berna User and Rana User-Smits together with Kerem Göçen and Kenny Smits. They were, are, and will always be there to support me. I also would like to thank my beloved parents, who loved and supported me unconditionally throughout my life. I owe them everything I have. Finally, I would like to thank TUBITAK for supporting this study through 212T031 and 114Z243 projects. I also express my gratitude to METU BAP for supporting this work through BAP-08-11-2015-019. x TABLE OF CONTENTS ABSTRACT.............................................................................................................v ÖZ ..........................................................................................................................vii ACKNOWLEDGEMENTS ................................................................................... ix TABLE OF CONTENTS ....................................................................................... xi LIST OF TABLES ............................................................................................... xiv LIST OF FIGURES ............................................................................................. xvi CHAPTERS 1. INTRODUCTION ............................................................................................... 1 1.1. Breast Cancer ................................................................................................ 1 1.1.1. Breast Cancer Cell Lines ......................................................................
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