From the Department of Laboratory Medicine Karolinska Institutet, Stockholm, Sweden CONSERVED STRUCTURE-FUNCTION RELATIONSHIPS IN THE MEDIATOR COMPLEX Tomas Linder Stockholm 2007 Published and printed by 2007 GårdsvägenPublished and 4, printed 169 70 by Solna Stockholm, Sweden ©Tomas Linder, 2007 ISBN 978-91-7357-096-1 Tillägnas min morfar ABSTRACT The Mediator complex is an essential multiprotein coregulator of RNA polymerase II (pol II)- dependent transcription in fungi and metazoans. Mediator interacts directly with both pol II and sequence-specific transcriptional regulators and thus acting as a bridge between the two. We have performed a comparative biochemical and functional characterisation the Mediator in the two ascomycete yeasts S. cerevisiae and S. pombe to address to what degree subunit architecture and specific regulatory functions have been conserved between these two species. In Paper I we identified the Med31 protein as a stable subunit of Mediator in S. cerevisiae and S. pombe. We defined a highly conserved central motif in the Med31 protein that appeared to be required but not sufficient for the association of Med31 with the rest of the Mediator complex. In Paper II we investigated the structural basis for the global shutdown of pol II- dependent transcription in the S. cerevisiae MED17 temperature sensitive allele srb4-138. We found that the srb4-138 allele disrupts the interaction between the head and middle domains both in vitro and in vivo. This suggested that the global shutdown of transcription in srb4-138 cells at the non-permissive temperature is caused by destabilisation of the core Mediator complex. In Paper III we characterised the architecture of the S. pombe Cdk8 module by biochemical means and addressed its relationship to the non-essential Med1 subunit. We found that the Med1 subunit is closely linked both structurally and functionally with the Cdk8 module. Electron microscopy of wildtype and �med1 Mediator resolved the position of the Med1 subunit as proximal to the Cdk8 module within the pol II-binding cleft of the core Mediator complex. Expression profiling of �med1 cells showed significant overlap with the changes seen in cells lacking either Med12 or Med13. In Paper IV we carried out a comprehensive functional characterisation of the S. pombe Mediator complex and identified two distinct morphological pathways that require different parts of the Mediator for their proper regulation. Head domain-linked mutants tended to grow as hyphae due to their inability to express genes required for proper cell separation after mitosis. Mutants of the Cdk8 module aggregated into large clumps due to impaired repression of genes that code for cell surface agglutinins. Comparison with similar studies done in S. cerevisiae revealed that both regulatory roles for these subcomplexes are conserved. LIST OF PUBLICATIONS I. Tomas Linder & Claes M. Gustafsson (2004) The Soh1/MED31 protein is an ancient component of Schizosaccharomyces pombe and Saccharomyces cerevisiae Mediator. J Biol Chem, 2004, 279, 49455-9. II. Tomas Linder, Xuefeng Zhu, Vera Baraznenok & Claes M. Gustafsson. (2006) The classical srb4-138 mutant allele causes dissociation of yeast Mediator. Biochem Biophys Res Commun, 349, 948-53. III. Vera Baraznenok, Hans Elmlund, Olga Khorosjutina, Martin Lindahl, Camilla O. Samuelsen, Nina N. Rasmussen, Philip Koeck, Tomas Linder, Steen Holmberg, Hans Herbert & Claes M. Gustafsson (2007) Functional and structural interactions between Med1 and the Cdk8 module in fission yeast Mediator. J Biol Chem (submitted) IV. Tomas Linder, Nina N. Rasmussen, Camilla O. Samuelsen, Vera Baraznenok, Jenny Beve, Peter Henriksen, Claes M. Gustafsson & Steen Holmberg. (2007) Two evolutionary conserved functional modules of Schizosaccharomyces pombe Mediator regulate distinct cellular pathways. Manuscript. All published papers and the following figures are reproduced with permission from the copyright owners: Fig 3A – “Reprinted with permission from Asturias et al., Science, 1999. Copyright 1999 AAAS” Fig 3B – “Reprinted with permission from Dotson et al., Proc Natl Acad Sci U S A, 2000. Copyright 2000 National Academy of Sciences U.S.A.” Fig 6 – “Reprinted with permission from Elmlund et al., Proc Natl Acad Sci U S A, 2006. Copyright 2006 National Academy of Sciences U.S.A” TABLE OF CONTENTS Abstract...............................................................................................................................5 Introduction ........................................................................................................................1 1.1 The need for gene regulation...................................................................... 1 1.2 Initiation of transcription at coding genes in eukaryotic cells ..................... 4 1.3 Discovery of Mediator ............................................................................... 7 1.4 Architecture of yeast Mediator complexes ............................................... 14 1.4.1 The head domain.............................................................................. 15 1.4.2 The middle domain .......................................................................... 19 1.4.3 The tail domain ................................................................................ 21 1.4.4 The Cdk8 module............................................................................. 23 Functional states of Mediator .............................................................................. 27 1.6 Repressive functions of Mediator............................................................. 31 1.7 A revised model of pol II dependent initiation cycle ................................ 37 1.7.1 Order and mechanism of coregulator recruitment ............................. 40 1.7.2 Occupancy versus gene activity and post-recruitment mechanisms of activation ....................................................................................................... 42 1.8 The role of metazoan Mediator in embryonic development and cancer..... 45 1.9 Evolutionary conservation of Mediator .................................................... 47 1.10 Future perspectives .................................................................................. 50 Aims of this thesis............................................................................................................53 Results and discussion......................................................................................................54 PAPER I ............................................................................................................. 54 PAPER II ............................................................................................................ 55 PAPER III........................................................................................................... 57 PAPER IV........................................................................................................... 59 Summary............................................................................................................. 65 Acknowledgements..........................................................................................................68 References ........................................................................................................................70 LIST OF ABBREVIATIONS bp base pair Cdk cyclin-dependent kinase ChIP chromatin immunoprecipitation CTD C-terminal domain CycC Cyclin C DBD DNA binding domain EM electron microscopy or electron micrograph ESP eukaryotic signature protein EST expressed sequence tag GTF general transcription factor HAT histone acetyl-transferase HDAC histone deacetylase kDa kilo Dalton (103 Da) MDa mega Dalton (106 Da) mRNA messenger RNA PIC pre-initiation complex pol II RNA polymerase II RNAi RNA interference Rpb RNA polymerase B (RNA polymerase II) TAD transcriptional activation domain TAF TBP-associated factor TAP tandem affinity purification TBP TATA-binding protein TFIIB transcription factor II B TFIID transcription factor II D TFIIE transcription factor II E TFIIF transcription factor II F TFIIH transcription factor II H TFIIS transcription factor II S ts temperature sensitive allele UAS upstream activating sequence(s) INTRODUCTION 1.1 THE NEED FOR GENE REGULATION “The cell is a clever beast.” Prof. Steve Yeaman Each of the billions of cells in our bodies contains the identical set of some 30,000 genes that make up our genome (Venter et al., 2001). Despite this basic genetic circuitry our cells display a remarkable variation in appearance as well as in function – from the bone forming osteoblasts in our skeleton to the free ranging lymphocytes that patrol our bodies in the search of microscopic intruders. The astounding complexity of the human body originates from a single diploid cell, which has to undergo an extremely intricate program of multiplication and differentiation in order to produce a fully developed organism. We are only beginning to understand this process but we now know that it requires a highly regulated process of activating some genes and repressing others at specific times and in specific cells and tissues in the developing embryo. Thus the cells of our bodies are not distinguished by the basic genetic material of the genome but rather which components of the genome are active in each individual cell. In addition our cells must constantly adjust the activity of their genes from one moment to the next depending on