MECHANISMS OF SSX GENE EXPRESSION REGULATION AT THE PROMOTER LEVEL A THESIS SUBMITTED TO THE DEPARTMENT OF MOLECULAR BIOLOGY AND GENETICS AND THE INSTITUTE OF ENGINEERING AND SCIENCE OF BILKENT UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE BY DERYA DÖNERTAŞ JULY 2009 I I certify that I have read this thesis and that in my opinion it is fully adequate, in scope and in quality, as a thesis for the degree of Master of Science. ____________________________ Dr. Sreeparna Banerjee I certify that I have read this thesis and that in my opinion it is fully adequate, in scope and in quality, as a thesis for the degree of Master of Science. ____________________________ Dr. Cengiz Yakıcıer I certify that I have read this thesis and that in my opinion it is fully adequate, in scope and in quality, as a thesis for the degree of Master of Science. ____________________________ Dr. Ali O. Güre Approved for the Institute of Engineering and Science ____________________________ Director of Institute of Engineering and Science Prof. Mehmet Baray II ABSTRACT MECHANISMS OF SSX GENE EXPRESSION REGULATION AT THE PROMOTER LEVEL Derya Dönertaş M.S. in Molecular Biology and Genetics Supervisor: Dr. Ali O. Güre July 2009, 101 pages Cancer Testis (CT) Antigen Genes are not transcribed in any of the adult tissues except spermatogonia, oogonia and trophoblasts. This tight regulation of expression is reversed resulting in the reactivation of CT transcription in a wide variety of cancers. CT genes are coordinately expressed and known to be regulated epigenetically. CT genes are reactivated in cancers by a mechanism that leads to the specific hypomethylation of their promoter-proximal sequences. The mechanisms leading to this phenomenon are unknown. The main objective of this thesis was to further unravel the mechanisms regulating CT gene expression at the promoter level. For this purpose, SSX4 ,a typical CT-X gene known to be under the control of a bidirectional promoter, was chosen as a model. We characterized the minimal critical sequences controlling the sense and antisense promoter and discovered a bidirectional promoter with overlapping promoter activities within a 40 bp region. To study how the antisense promoter could mediate sense promoter repression and vice versa, we used two different reporter genes for each of the promoters in a single construct and found that measurable antisense promoter activity was dramatically reduced upon the introduction of a reporter for the sense promoter. The SSX4 antisense promoter is capable of producing a noncoding transcript from the neighboring ornitine aminotransferase-like pseudogene in vivo. This, however, wasn’t confirmed in this study. The possibility of transcriptional interference or the production of a small dsRNA that could affect the regulation of SSX4 gene expression is discussed in the context of the data. Results from experiments where the down-regulation of DICER was studied as a mechanism that could influence CT gene expression are also discussed III ÖZET SSX GENİ İFADESİNİN DÜZENLENMESİNDE ROL ALAN PROMOTOR MERKEZLİ MEKANİZMALAR Derya Dönertaş Moleküler Biyoloji ve Genetik Yüksek Lisansı Tez Yöneticisi: Dr. Ali O. Güre Temmuz 2009, 101 sayfa Kanser Testis (KT) antijen genleri spermatogonya, oogonya ve trofoblast haricindeki yetişkin dokularında ifade edilmeyen genlerdir. İfade edilimdeki bu sıkı düzenleme birçok kanser türünde tersine dönerek KT genlerinin yeniden ifade edilmesine yol açar. KT genleri eşgüdümlü olarak ifade edilir ve ifadeleri epigenetik etmenler tarafından düzenlenir. KT genlerinin kanserlerde yeniden ifade edilmesinin altında yatan neden promotor bölgelerinin özel olarak hipometilasyona uğramasıdır. Bu olaya yol açan mekanizmalar bilinmemektedir. Bu tezin asıl amacı KT geni ifadesinin düzenlenmesinde rol alan promotor merkezli mekanizmaları açığa çıkartmaktı. Bu amaçla tipik bir KT geni olan ve çift taraflı bir promotora sahip olduğu bilinen SSX4 geni model olarak seçildi. Bu bağlamda, anlamlı (sense) ve anlamsız (antisense) promotor aktivitelerinden sorumlu en küçük DNA dizilerini karakterize ettik ve 40 baz çiftini kapsayan bir bölgede çift yönlü ve örtüşen bir promotor ortaya çıkardık. Anlamsız promotor aktivitesinin anlamlı promotor aktivitesini nasıl baskıladığını anlayabilmek için, bir vektör yapısında her iki promotor için farklı birer haberci gen kullandık. Ölçülebilen anlamsız promotor aktivitesinin, vektör yapısına anlamlı promotorun aktivitesini ölçen haberci genini eklediğimizde önemli ölçüde azaldığını gözlemledik. Canlı dokularda SSX4 geninin anlamsız promotor aktivitesi, komşu ornitine aminotransferaza benzer yalancı-gen bölgesi üzerinden bir kodlamayan transkript ifade edebilme kapasitesine sahiptir fakat bu transkript ifadesi bu çalışmada onaylanamamıştır. Yazılım karışması ya da küçük çift dizili RNA (dsRNA) oluşumunun SSX4 geni ifadesinin düzenlenmesindeki muhtemel etkileri, bulunan veriler bağlamında ele alınmış ve tartışılmıştır. Ayrıca, DICER geninin bağlı olduğu mekanizmaların KT genleri ifadesinin düzenlenmesine etkisi, DICER geninin azaltarak düzenlendiği deneylerin sonuçları üzerinden tartışılmıştır. IV To My Family… V ACKNOWLEDGEMENTS First of all I would like to thank my supervisor Dr. Ali O. Güre for his supervision, patience, encouragement and support throughout this study. He always helped me through the difficulties that I have experienced during this study and always looked on the bright side, unlike me. I would like to thank AOG group members for their support and friendship. I also would like to thank all of my friends in the department for technical support and for their friendship especially to Sinan, Tamer, Fırat, Onur, Nilufer, Gurbet and Atıl. I would like to thank Dr. Mayda Gürsel for her experimental support. Lastly my deepest thanks are to my family. They have supported me in every decision I made and I am more than grateful to have them in my life. Throughout my M.S. study, I was personally supported by TÜBİTAK-BİDEB scholarship 2228. This work was also supported by grants from TÜBİTAK and European Commission to Dr. Ali O. Güre. VI TABLE OF CONTENTS SIGNATURE PAGE ..................................................................................................................................... II ABSTRACT ................................................................................................................................................III ÖZET ....................................................................................................................................................... IV DEDICATION PAGE ................................................................................................................................... V ACKNOWLEDGEMENTS .......................................................................................................................... VI TABLE OF CONTENTS ............................................................................................................................. VII LIST OF TABLES ....................................................................................................................................... XI LIST OF FIGURES .................................................................................................................................... XII ABBREVIATIONS..................................................................................................................................... XV 1. INTRODUCTION 1.1 Epigenetic Regulation of Gene Expression ...........................................................................1 1.1.1 DNA Methylation .....................................................................................................1 1.1.2 Histone Modifications ..............................................................................................1 1.1.3 Chromatin Remodeling ............................................................................................2 1.2 Cancer Epigenomics ..............................................................................................................3 1.3 Eukaryotic Promoters ...........................................................................................................4 1.3.1 General Information ................................................................................................4 1.3.2 ENCODE Findings and Their Importance .................................................................4 1.3.3 Bidirectional Promoters ...........................................................................................5 1.4 Cancer Testis Antigens ..........................................................................................................5 1.4.1 General Information ................................................................................................5 1.4.2 Function and Conservation ......................................................................................6 1.4.3 Regulation of Expression .........................................................................................7 1.4.4 Promoters of Cancer Testis Antigens .......................................................................8 1.4.5 SSX Gene Family .......................................................................................................9 1.5 Small RNA Mediated Epigenetic Regulation ...................................................................... 10 1.5.1 miRNAs .................................................................................................................