EXPRESSION PROFILING OF THERMOPLASMA VOLCANIUM GSS1 UNDER STRESS CONDITIONS WITH SPECIFIC EMPHASIS ON PROTEASOME ASSOCIATED REGULATORY VAT GENES A THESIS SUBMITTED TO THE GRADUATE SCHOOL OF NATURAL AND APPLIED SCIENCES OF MIDDLE EAST TECHNICAL UNIVERSITY BY TÜLAY YILMAZ IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN BIOLOGY MAY 2013 i ii Approval of the thesis: EXPRESSION PROFILING OF THERMOPLASMA VOLCANIUM GSS1 UNDER STRESS CONDITIONS WITH SPECIFIC EMPHASIS ON PROTEASOME ASSOCIATED REGULATORY VAT GENES submitted by TÜLAY YILMAZ in partial fulfillment of the requirements of the degree of Master of Science in Biology Department, Middle East Technical University by, Prof. Dr. Canan Özgen Dean, Graduate School of Natural and Applied Sciences Prof. Dr. Gülay Özcengiz Head of Department, Biology Prof. Dr. Semra Kocabıyık Supervisor, Biology Department, METU Examining Committee Members: Prof. Dr. Emel Arınç Biology Dept., METU Prof. Dr. Semra Kocabıyık Biology Dept., METU Prof. Dr. Fatih İzgü Biology Dept., METU Assoc. Prof. Dr. Tülin Yanık Biology Dept., METU Assoc. Prof. Dr. Ayşen Tezcaner Engineering Dept., METU Date: 02.05.2013 iii 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: Tülay YILMAZ Signature: iv ABSTRACT EXPRESSION PROFILING OF THERMOPLASMA VOLCANIUM GSS1 UNDER STRESS CONDITIONS WITH SPECIFIC EMPHASIS ON PROTEASOME ASSOCIATED REGULATORY VAT GENES Yılmaz, Tülay M.Sc., Department of Biology Supervisor: Prof. Dr. Semra Kocabıyık May 2013, 150 pages In this study differential expression of the two proteasome associated VAT genes (TVNO382 and TVN0947) of Thermoplasma volcanium GSS1 (Tpv) cells challenged by extracellular stresses, i.e., pH, heat-shock and hydrogen peroxide were investigated using quantitative RT- PCR and Western blotting/hybridization techniques. We also performed a comprehensive transcriptome analysis of the Tpv as response to environmental stresses by genome-wide expression arrays. Quantitative RT-PCR analyses revealed that VAT genes elicited specific response under heat-shock (at 65ºC and 70ºC), pH stress (at pH 4.0) and oxidative stress (at 0.02 mM H2O2) over 120 min. Western hybridization corroborated the qRT-PCR results at protein level. These results suggested a critical role for VAT proteins possibly in association with the 20S proteasome in Tpv cells for adaptation to external stress, especially heat-shock. Microarray analyses revealed that Tpv cells’ gene expression over 60 min stress exposure was essentially down-regulated. Within this group of genes those functionally linked to energy production and conversion, transcription and translation were significantly and extensively inhibited. Response to heat stress (at 65ºC) could be linked to elevated expression of genes encoding heat-shock related genes (e.g. GrpE) whereas the stress response to pH and oxidative stresses was characterized by up-regulation of membrane and transport related genes. Among the stress responsive genes sugar permease (TVN1145) and GrpE (TVN0489) genes were selected for verification of the microarray data by qRT-PCR technique. The results demonstrated a consistent trend in the expression patterns of the selected genes by two techniques. Collectively, our results suggest that the transcription and translation are retarded to prevent error prone synthesis of proteins and to avoid energy requiring synthetic processes. The energy balance may be maintained by activation of carbohydrate transport and metabolism under all stress conditions. Tpv also modulates stress specific genes (e.g., GrpE-Heat-shock, redoxins-Oxidative stress, transporters-pH stress) as part of its stress defense mechanism(s). Key words: Archaea, heat/pH/oxidative stress, Thermoplasma volcanium, VAT, microarray v ÖZ THERMOPLASMA VOLCANİUM GSS1’İN STRES KOŞULLARI ALTINDA PROTEAZOMA İLİŞKİN DÜZENLEYİCİ VAT GENLERİ AĞIRLIKLI OLMAK ÜZERE GEN ANLATIM PROFİLİNİN BELİRLENMESİ Yılmaz, Tülay Yüksek Lisans, Biyoloji Bölümü Tez yöneticisi: Prof. Dr. Semra Kocabıyık Mayıs 2013, 150 Sayfa Thermoplasma volcanium (Tpv) GSS1’in dış stres etkenleri olan; pH, ısı-şoku ve hidrojen peroksit ile tetiklendiğinde proteasoma ilişkin iki VAT geninin anlatımında oluşan farklılıklar nicel RT-PCR ve Western aktarım ve hibritleme teknikleri ile incelenmiştir. Ayrıca genom boyutunda mikrodizinler aracılığı ile Tpv’nin çevresel streslere tepkisinin geniş kapsamlı transkriptom düzeyinde analizi yapılmıştır. Nicel RT-PCR analizleri göstermiştir ki, VAT genleri ısı şoku (65ºC ve 70ºC), pH stresi (pH 4.0) ve oksidatif strese (0.02 mM H2O2) 120 dakika boyunca maruz bırakıldıklarında özgün stres yanıtı oluşturmuşlardır. Western hibritleme qRT-PCR sonuçlarını doğrulamıştır. Bu sonuçlar Tpv hücrelerinde VAT proteininin muhtemelen proteasomla birlikte çevresel strese özellikle ısı- şokuna uyumda, kritik bir role sahip olduğunu göstermektedir. Mikroarray analizi sonuçları Tpv hücrelerinde genlerin strese maruz kaldıkları 60 dakika boyunca çoğunlukla anlatımlarının düştüğünü ortaya çıkartmıştır. Bu grup genler içinde işlevsel olarak enerji üretimi ve dönüşümü, transkripsiyon ve translasyon ile bağlantılı olanlar geniş kapsamlı ve önemli ölçüde inhibe olmuşlardır. Isı-şoku yanıtı, ısı-şoku bağlantılı genlerin (örneğin, GrpE) anlatımının artması ile ilişkilendirilebilirken, pH ve oksidatif strese yanıt membran ve transport ile bağlantılı genlerin anlatımının artması ile karakterize edilmişlerdir. Strese tepki gösteren genler arasından seçilen şeker permeaz ve GrpE genleri mikrodizin verisini doğrulamak için qRT-PCR ile analiz edilmişlerdir. Bu sonuçlar seçilen genlerin her iki teknikle belirlenen anlatım örüntülerinin tutarlı olduğunu göstermiştir. Toplu olarak bu sonuçlar öneriyor ki, tüm stres koşullarında hata eğilimli protein sentezini önlemek ve enerji gerektiren sentetik süreçlerden kaçınmak için transkripsiyon ve translasyonun engellenmektedir. Tüm stres koşullarında enerji dengesinin sürdürülebilirliği karbonhidrat taşıma ve metabolizmasının aktivasyonu ile sağlanmıştır. Tpv ayrıca stresten korunma mekanizmasının bir parçası olarak strese özgü genleri de (ör, GrpE-Isı-şoku, redoksinler- Oksidatif stres, Taşıyıcılar-pH stres) düzenlemektedir. Anahtar kelimeler: Arkea, ısı/pH/oksidatif stres, Thermoplasma volcanium, VAT, mikroarray vi To my family, vii ACKNOWLEDGEMENTS Foremost, I would like to express my great appreciation to my supervisor Prof. Dr. Semra Kocabıyık for her valuable and constructive suggestions during the planning and development of this research work. Her willingness to give her time so generously has been very much appreciated. Her guidance helped me in all steps of this thesis. It would have not been possible to finish without her help. Secondly, I want to thank dear professors Prof. Dr. Emel Arınç, Prof. Dr. Fatih İzgü, Assoc. Prof. Dr. Tülin Yanık and Assoc. Prof. Dr. Ayşen Tezcaner for their valuable contributions. I wish to thank to my lab partners, Ilir Sheraj and Esra Yetişgin for their help. I would like to thank to my beloved, Paul J. Swenson, for his unequivocal support, and great patience at all times. I’m thankful to my great friends Yiğit Ozan Çolak, Erkan Arslan, Güler Bozok, Negar Taghavi and Kaveh Dehganian for sharing all my happy and distressed moments. I also thank to Gül Açıkgöz Çakmak, for reminding me the potential in me whenever I felt down. I’m lucky to meet you all in different periods of my life. And the last but not least I would like to thank to my mother, İsminaz Yılmaz for her peerless support in every stage of my life, I would be nothing without her help. I also thank to my father Faruk Yılmaz and brother Tümay Evren Yılmaz for being there whenever I needed. I would like to thank for the financial support for the project and student fellowship of ODTÜ-BAP-07-02-2012-101 and TUBİTAK/TBAG109T946, respectively. viii TABLE OF CONTENTS ABSTRACT .............................................................................................................................. v ÖZ ............................................................................................................................................ vi ACKNOWLEDGEMENTS .................................................................................................. viii TABLE OF CONTENTS ........................................................................................................ ix LIST OF TABLES ................................................................................................................ xiii LIST OF FIGURES ................................................................................................................. xv LIST OF ABBREVIATIONS ............................................................................................... xix CHAPTERS 1. INTRODUCTION .............................................................................................................. 1 1.1 Life in Extreme Conditions .......................................................................................... 1 1.2 Benefits of Studying with Archaea .............................................................................. 2 1.3 Stress and Stress Response ........................................................................................... 2 1.4 Proteasomes