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(Cxxc5) a Thesis Submitted to the Graduate STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF THE CXXC-TYPE ZINC FINGER PROTEIN 5 (CXXC5) A THESIS SUBMITTED TO THE GRADUATE SCHOOL OF NATURAL AND APPLIED SCIENCES OF MIDDLE EAST TECHNICAL UNIVERSITY BY GAMZE AYAZ ŞEN IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BIOLOGY NOVEMBER 2018 Approval of the thesis: STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF THE CXXC-TYPE ZINC FINGER PROTEIN 5 (CXXC5) submitted by GAMZE AYAZ ŞEN in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biology Department, Middle East Technical University by, Prof. Dr. Halil Kalıpçılar Dean, Graduate School of Natural and Applied Sciences Prof. Dr. Orhan Adalı Head of Department, Biological Sciences Prof. Dr. Mesut Muyan Supervisor, Biological Sciences Dept., METU Examining Committee Members: Prof. Dr. A. Elif Erson Bensan Biological Sciences Dept., METU Prof. Dr. Mesut Muyan Biological Sciences Dept., METU Asst. Prof. Dr. Murat Alper Cevher Molecular Biology and Genetics Dept., Bilkent University Assoc. Prof. Dr. Nurcan Tunçbağ Informatics Institute, METU Asst. Prof. Dr. Onur Çizmecioğlu Molecular Biology and Genetics Dept., Bilkent University Date: 16.11.2018 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: Gamze Ayaz Şen Signature: iv To All Women Who Pursue Their Dreams In Science v ABSTRACT STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF THE CXXC-TYPE ZINC FINGER PROTEIN 5 (CXXC5) Ayaz Şen, Gamze Ph.D., Biology Department Supervisor: Prof. Dr. Mesut Muyan November 2018, 89 pages Estrogen hormones, particularly 17β-estradiol (E2), are involved in the regulation of physiological and pathophysiological functions of many organs and tissues including breast tissue. The expression of CXXC type zinc finger protein 5 (CXXC5) gene is regulated by E2 through estrogen receptor α. Due to a highly conserved zinc-finger CXXC domain (ZF-CXXC), CXXC5 is considered to be a member of ZF-CXXC family, which binds to non-methylated CpG dinucleotides of transcriptionally active DNA regions. This binding is thought to play critical roles in epigenetic modulation of transcription through the prevention of cytosine methylation and the recruitment of DNA modifying enzymes. The structure and function of CXXC5 and its role in cellular events are yet unclear. However, accumulating evidence is suggesting that CXXC5 is involved in transcriptions as a transcription factor, co-regulator and/or epigenetic factor. In this PhD thesis, I successfully expressed and purified the full-length CXXC5 protein, with which I showed that CXXC5 is a non-methylated CpG DNA binding protein and that the ZF-CXXC domain is indeed responsible for the ability of the protein to interact with DNA. Since proteins exert their functions in the context of dynamically changing interacting protein network, I envisioned vi that identification of interacting protein partners of CXXC5 would be a critical step in the elucidation of cellular function of the protein. To address this issue, I performed proximity dependent biotinylation assay (BioID) in a cell line model derived from breast adenocarcinoma. Of the identified proteins by Liquid chromatography-tandem mass spectrometry (LC-MS/MS), I validated that CXXC5 protein interacts with MeCP2 (MethylCpG binding protein 2), MAZ (Myc-associated Zinc Finger Protein) and EMD (Emerin) proteins by co- immunoprecipitation assay. I found that the zinc finger domain of CXXC5 is necessary for protein interaction as well. The findings of this study could provide important insights into the mechanism of CXXC5 actions in E2- mediated cellular events. Keywords: estrogen signaling, CXXC domain, non-methylated CpG binding protein, BioID, protein interaction vii ÖZ CXXC-TİPİ ÇİNKO PARMAK 5 PROTEİNİN (CXXC5) YAPISAL VE FONKSİYONEL KARAKTERİZASYONU Ayaz Şen, Gamze Doktora, Biyoloji Bölümü Danışman: Prof. Dr. Mesut Muyan Kasım 2018, 89 sayfa Östrojen hormonları, özellikle 17β-estradiol (E2), meme dokusu dahil olmak üzere birçok dokunun fizyolojisi ve patofizyolojisinde rol oynamaktadır. CXXC tipi çinko parmak protein 5 (CXXC5)’in ekspresyonunu östrojen reseptör α aracılığıyla E2 ile regüle edilmektedir. CXXC5’in yapısal ve fonksiyonel özelliği ve hücre içindeki rolü henüz bilinmemektedir. Yüksek derecede korunmuş bir çinko parmak-CXXC domaini (ZF-CXXC) nedeniyle, CXXC5 transkripsiyonel olarak aktif olan DNA bölgelerindeki metile olmamış CpG’lere bağlanan ZF- CXXC protein ailesinin bir üyesi kabul edilmektedir. Bu bağlanmanın, sitosin metilasyonunu engelleyerek ve DNA’yı modifiye eden enzimlerin bu bölgede yoğunlaşmasını sağlayarak, epigenetik bir rol oynadığı düşünülmektedir. CXXC5 proteinin yapısal ve fonksiyonel özelliği ve hücre içerisindeki rolü henüz bilinmemektedir. Ancak son yıllarda CXXC5 üzerine yapılan çalışmaların sayısı artmaktadır. Tüm bu çalışmalar, çeşitli sinyal yolakları tarafından düzenlenen hücre çoğalması, farklılaşması, metabolizması ve/veya ölümünde CXXC5’in epigenetik faktör ve/veya kromatin düzenleyici komplekslerin bir protein partneri olarak transkripsiyonlarda rol oynadığı öne sürülmüştür. viii Bu tez çalışmasında, tam uzunluktaki (full-length) CXXC5 proteini rekombinant olarak üretildi ve saflaştırıldı. Saflaştırılmış CXXC5 proteininin metile olmamış DNA’ya bağlanan bir protein olduğu gösterildi. Etkileşim gösteren proteinleri belirleyebilmek için, insan meme kanser hücrelerinden türemiş bir hücre modelinde olası protein partnerleri, yakınlığa-bağlı biyotin tanımlaması ve mass spektrometri analizi (BioID-MS) ile tarandı. Eş-immünçökeltme yöntemi, in situ protein etkileşimlerini doğrulamak için kullanıldı. Sıvı kromatografisi-ardışık kütle spektrometresi (LC-MS/MS) analizi sonucu elde edilen proteinler arasından MeCP2, MAZ ve EMD proteinlerin CXXC5 ile etkileşim gösterdiği doğrulandı. Çinko parmak CXXC domainin bu bağlanma için gerekli olduğu gösterildi. Bu sonuçlar, CXXC5’in E2 ile düzenlenen hücre çoğalma mekanizmalarındaki rolünün anlaşılmasına katkılar sağlayabilir. Anahtar Kelimeler: östrojen sinyali, CXXC domain, metile olmamış CpG DNA bağlanan protein, BioID, protein etkileşimleri ix ACKNOWLEDGEMENTS I must first and foremost thank to my advisor Mesut Muyan who has always supported and encouraged me during my PhD studies. He provided me with the scientific support and guidance: I learned how to think critically and do my scientific research independently. I would like to thank my thesis committee members; Prof. Dr. A. Elif Erson Bensan, Asst. Prof. Murat Cevher, Assoc. Prof. Nurcan Tunçbağ and Asst. Prof. Onur Çizmecioğlu for their time and efforts to share their critical comments on my PhD thesis. My special thanks are extended to my labmates. I want to express my very great appreciation to Pelin Yaşar, Sırma Damla User, Gizem Güpür, Çağla Ece Olgun, Burcu Karakaya, Gizem Kars, Negin Razizadeh, Gizem Turan and Kerim Yavuz individually for their friendship and kind support all the past years. In addition, I want to thank all the members of Erson-Bensan Lab and Banerjee Lab for their support and help. I also would like to thank Prof. Dr. Rengül Atalay and her lab members for their help in using fluorescent microscope. I would like to express my very great appreciation to Dr. Jinrong Min and his lab members to host me for a year in University of Toronto and doing a fruitful collaboration for my PhD study. I do not know how to express my gratitude to my beloved husband Bilgecan, my parents Güler and Turan and my lovely sister Tuğba. Without them, none of the work I have done and will have any real meaning. I gratefully thank TUBITAK whose generous support throughout my PhD education. I was supported by TUBITAK 2211-A and TUBITAK 2214-A scholarships and TUBITAK-KBAG 315S045 grant. This PhD work was supported by TUBITAK-KBAG 114Z243 grant. x TABLE OF CONTENTS ABSTRACT ................................................................................................................ vi ÖZ ............................................................................................................................. viii ACKNOWLEDGEMENTS ......................................................................................... x TABLE OF CONTENTS ............................................................................................ xi LIST OF FIGURES ................................................................................................... xiii LIST OF TABLES .................................................................................................... xvi CHAPTERS 1. INTRODUCTION ................................................................................................ 1 1.1. CXXC5 as an E2-ERα responsive gene in breast cancer ............................ 1 1.2. CXXC type Zinc Finger Protein 5 (CXXC5) .............................................. 2 1.3. The Zinc Finger CXXC Protein Family ...................................................... 5 1.4. CpG Islands and ZF-CXXC Proteins .......................................................... 7 1.5. The aim of study .......................................................................................... 8 2. MATERIALS AND METHODS ....................................................................... 11 PART I: TESTING THE DNA BINDING ABILITY OF THE FULL-
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