Previous page: Each group of four dots represents a colony of yeast cells with a different gene deletion. They grow on a substrate that is a mixture of agar and the drug fenpropimorph. Because fenpropimorph is normally toxic to yeast cells, most of the colonies do not grow to a large size. One of the exceptions, highlighted in red, is the colony in which the gene encoding the target of fenpropimorph (erg2) has been deleted.

The role of small molecules in

cell regulation

This dissertation is submitted for the

degree of Doctor of Philosophy

September 2009

Arthur Wüster

University of Cambridge,

MRC Laboratory of Molecular Biology and

St John’s College, Cambridge

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Declaration of originality This dissertation describes work I carried out at the Medical Research Council Laboratory of Molecular Biology in Cambridge between October 2006 and September 2009. The contents are my original work, although much has been influenced by the collaborations in which I took part. I have not submitted the work in this dissertation for any other degree or qualification at any other university. Arthur Wüster Cambridge, United Kingdom

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Acknowledgements I am more grateful than I can express to have had Madan as my PhD advisor. Only his inspirational guidance, extensive knowledge and good humour have made this thesis possible. From him, I have learnt more than I would have thought possible. Thank you, Madan. I especially thank the following friends and colleagues for their help during my doctoral studies: Boris Adryan (Cambridge) for the Glühwein; Hannes Braberg (Cambridge, San Francisco), without whose help and friendship my stay in San Francisco would have been impossible; Michael Bremang (Cambridge) for the fun times at Language Society; Lindell Bromham (Sussex, Canberra) for supervising my Bachelor’s project; Gerard Cagney (San Francisco, Dublin) for having style; Varodom Charoensawan (Cambridge) for being fun and sensible at the same time; Cyrus Chothia (Cambridge) for demonstrating that with great experience comes great wisdom; Subhajyoti De (Cambridge) for, amongst so many other things, helping me to get started with this thesis; Jörg Gsponer (Cambridge, Vancouver) for his generosity, and for companionship in California; Jung-Hoon Han (Cambridge, London) for enthusiasm and friendship; Daniel Hebenstreit (Cambridge) for making the model room a much more entertaining place; Nevan Krogan (San Francisco) for hosting me at UCSF; Sarath Chandra Janga (Cambridge) for entertaining put-downs; Rekin’s Janky (Cambridge) for philosophical insights; Benjamin Lang (Cambridge, Heidelberg) for ketchup battles (don’t ask); Emmanuel Levy (Cambridge, Montréal) because he is a smooth player; Siarhei Maslau (Cambridge) for barbecues that left a burning memory; Nitish Mittal (Cambridge, NIPER) for his patience; Joel Peck (Sussex) for being the most inspiring lecturer; Tina Perica (Cambridge) because she understands why Darth Vader may be ticklish; Dhruva Raman (Cambridge) for his excellent comments on this thesis; Gebhard Schertler (Cambridge) without whom chapter 2 would not have been possible; Laura Schuresko-Kapitzky (San Francisco) for making chapter 4 possible; Jing Su (Cambridge) for advice on girls; Sarah Teichmann (Cambridge) for being a fantastic second supervisor; Derek Wilson (Cambridge) for putting up with me and my frequent requests to re-install Statistics::R; and Jiewei Xu (San Francisco) for company during hours of queuing in front of the California Academy of Sciences. I would also like to thank the following friends and family for support: Jesus Aguirre, Sophie Beeren, Jan Černek, Winsome Cheung, David Delamore, Jakob Demus, Margit Demus, Laura Gardner, Alfred and Katharina Graf, Carsten Gram Hansen, Paul Harrison, Rumina Hassam, Hugo Herrmann and family, Kathy and Lynn Holland, Andrea Kaiser, Shahid Mahmood, Anna Matheson, Emanuel Mauthe, Daniel Nicholson, David Padgett and family, Tom Phillips, Matias and Kaisa Piipari, Maria Pontes, Jawaid Quraishi, Ingmar Schäfer, Florian Schnitzer, Julia Schnitzer, Juliana and Matt Smart, Alexandra Stichler-Knez, Piotr Szpunar, Raj Towfique, Kim and Mickey Trudelle, Kate Withers, Eugen Wüster, Thiele Wüster, Jennie Yang, Roberto Zanchi, and Wenting Zhang. The following organisations have made this thesis possible by providing infrastructure and funding: MRC Laboratory of Molecular Biology, St John’s College (Cambridge), UK Medical Research Council, University of California (San Francisco).

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Summary In order to construct a complete model of the cell and hence fulfil one of the promises of systems biology, we need to include data on cellular components that are not directly encoded in the genome. This includes small molecules. The chapters of this dissertation consider interactions between cells and small molecules at different levels of cellular organisation. After the introductory chapter, Chapter 2 considers small molecule-cell interactions at the atomic level, Chapter 3 at the pathway level, Chapter 4 at the proteome level, and Chapter 5 at the whole-cell level. Chapter 1: An introduction to the concepts related to small molecule-cell interactions. Chapter 2: I introduce Spial (Specificity in alignments), a tool for the computational analysis of subtype-specific features in multiple sequence alignments of proteins. I show that it can be used to gain understanding of both protein-small molecule interactions and protein-protein interactions. Chapter 3: The topic of this chapter is quorum sensing, or cell-to-cell communication between unicellular organisms. I use a combination of computational methods to identify quorum sensing systems in sequenced bacterial genomes. Furthermore, I establish a framework for the identification of transcription factors involved in the quorum sensing response in the yeast Saccharomyces cerevisiae. Chapter 4: This chapter provides a broader view of small molecule-cell interactions. It is about chemogenomics, a field of high-throughput biology that probes the reactions of cells to a large number of different small molecules. I develop computational methods for the analysis of chemogenomic data that can assist in the identification of leads for drug development. Chapter 5: Recent years have seen a rapid increase in the number of high-throughput experiments in the fields of genomics, cell and evolutionary biology, pharmacology, epigenetics, and functional genomics. I assembled more than 250 gene-centric descriptors from such high-throughput experiments in S. cerevisiae and characterised the global set of relationships between them. By assembling a large number of diverse descriptors of various aspects of the cell, the methods I outline allow an unbiased integrative investigation of a wide variety of data types. I also report several novel associations between cellular descriptors that were not known before. Chapter 6: The dissertation ends with a chapter that highlights the important aspects of the findings presented and their practical implications. Overall, this dissertation presents a framework for the investigation of the interactions between small molecules and cells at different levels of resolution.

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Table of contents

Chapter 1 Contents of chapter 1.................................................................................................. 1 Defending small molecules ......................................................................................... 2

Defining small molecules......................................................................................... 2 Chemical space, a final frontier ............................................................................... 3 Types of small molecule-cell interaction.................................................................. 4

Small molecule-nucleic acid interactions ............................................................. 5 Small molecule-chromatin interactions ................................................................ 6

Small molecule-protein interactions......................................................................... 6 The effect of small molecules on protein-protein interactions.............................. 6 Small molecules affecting active sites ................................................................. 8 What binding is .................................................................................................... 8 Quantifying small molecule-protein interactions .................................................. 9

Evolved versus non-evolved small molecule-cell interactions............................... 11 Small molecule-cell interactions at different levels of detail .................................. 11 What is it good for?................................................................................................ 12 A systems biology approach to small molecule-cell interactions........................... 13

References................................................................................................................ 14

Chapter 2 Contents of chapter 2.................................................................................................. 1 Summary of chapter 2................................................................................................. 2 Introduction ................................................................................................................. 3

Underlying assumptions .......................................................................................... 4 On the shoulders of giants....................................................................................... 4

Scorecons............................................................................................................ 5 Sequence logos ................................................................................................... 5 ConSurf................................................................................................................ 6 The evolutionary trace method ............................................................................ 6 Pairwise HMM logos ............................................................................................ 6 Two Sample Logo................................................................................................ 6

The Spial algorithm ..................................................................................................... 7 Examples of use.......................................................................................................... 9

Example 1: The dimerisation interface of STAT5a ................................................ 10 Example 2: Differences in co-ordination of retinal between vertebrate and cephalopod rhodopsin ........................................................................................... 11

Conclusions............................................................................................................... 12 References................................................................................................................ 13

Chapter 3 Contents of chapter 3.................................................................................................. 1 Summary of chapter 3................................................................................................. 3 Introduction to quorum sensing................................................................................... 3

What quorum sensing is .......................................................................................... 4 Cell-to-cell communication in bacteria..................................................................... 6

Acyl homoserine lactones.................................................................................... 7 Autoinducer-2....................................................................................................... 9 Processed oligopeptides.................................................................................... 10 Other systems.................................................................................................... 11 Quorum quenching ............................................................................................ 12

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Cross-genome interactions ................................................................................ 12 Cell-to-cell communication in yeast ....................................................................... 14

Farnesol ............................................................................................................. 14 Aromatic alcohol derivatives .............................................................................. 15

Tools and Techniques ........................................................................................... 15 Chemical ............................................................................................................ 15 Genetic............................................................................................................... 15 Computational.................................................................................................... 16

A survey of quorum sensing protein domains ........................................................... 16 Results and discussion.......................................................................................... 16

A γ-butyrolactone quorum sensing system in Rhodococcus? ........................... 18 Materials and methods .......................................................................................... 19

The agr quorum sensing system in firmicutes........................................................... 20 Results and discussion.......................................................................................... 21

The agr system is widespread among firmicutes............................................... 23 New agr-like systems......................................................................................... 25 Multiple paralogous copies of AgrB in Clostridia genomes................................ 26 A novel gene associated with the agr system in Clostridia ................................ 27 RNAIII and the agr system................................................................................. 27 agr-regulated genes........................................................................................... 28 Concluding remarks ........................................................................................... 29

Materials and methods .......................................................................................... 29 Homologue identification.................................................................................... 30 Tree building ...................................................................................................... 30 RNAIII identification ........................................................................................... 31 Gene expression data analysis.......................................................................... 31 Detection of orthologues.................................................................................... 32

The Saccharomyces cerevisiae quorum sensing system ......................................... 32 Results and discussion.......................................................................................... 34

Attributing differential expression to transcription factors .................................. 35 Quorum sensing and entry into stationary phase .............................................. 37 Genomic organization and nucleosome modification of the differentially expressed genes................................................................................................ 38 Conservation of quorum sensing components in fungal genomes .................... 40

Materials and methods .......................................................................................... 42 Gene expression data........................................................................................ 42 Identification of key transcription factors............................................................ 43 Genomic distribution of differentially expressed genes...................................... 44 Orthologue detection.......................................................................................... 44 Epigenetic modifications .................................................................................... 45

References................................................................................................................ 46

Chapter 4 Contents of chapter 4.................................................................................................. 1 Summary of chapter 4................................................................................................. 2 Introduction ................................................................................................................. 2

Chemogenomic experiments................................................................................... 4 Heterozygous deletions ....................................................................................... 5 Homozygous deletions......................................................................................... 5 Overexpression.................................................................................................... 6 Beyond deletion and overexpression libraries ..................................................... 7

Detection methods................................................................................................... 7 Non-competitive arrays ........................................................................................ 7 Competitive mutant pools .................................................................................... 7

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Data interpretation and analysis .............................................................................. 8 Clustering............................................................................................................. 8 Matrix operations ................................................................................................. 9

Applications ........................................................................................................... 10 Chemogenomic networks.......................................................................................... 12

Results and discussion.......................................................................................... 13 Interpretation of a chemogenomic network........................................................ 14 Properties of the chemogenomic network.......................................................... 15

Materials and methods .......................................................................................... 16 Data acquisition ................................................................................................. 16 Construction of the chemogenomic network...................................................... 16 Computation of network parameters and overlap with other networks .............. 17

Linking the chemical and biological properties of small molecules ........................... 17 Results and discussion.......................................................................................... 18

What differentiates bioactive small molecules from others? .............................. 18 What differentiates small molecules that hit S. cerevisiae from those that hit Sc. pombe?.............................................................................................................. 20 Conclusions ....................................................................................................... 22

Materials and Methods .......................................................................................... 22 References................................................................................................................ 23

Chapter 5 Contents of chapter 5.................................................................................................. 1 Summary of chapter 5................................................................................................. 2 Introduction ................................................................................................................. 3 Results and discussion ............................................................................................... 5

Network properties .................................................................................................. 6 One giant component........................................................................................... 7 Inter-class versus intra-class associations........................................................... 7

Novel associations between descriptors ................................................................. 9 Identifying redundancy between descriptors...................................................... 13

Conclusions........................................................................................................... 13 Material and Methods................................................................................................ 13

Assembly of the dataset ........................................................................................ 14 Interaction networks........................................................................................... 14 Evolutionary metrics........................................................................................... 16 Dosage effect..................................................................................................... 16 Membrane associated........................................................................................ 16 Gene location..................................................................................................... 16 Information flow.................................................................................................. 16 Sequence derived.............................................................................................. 17 Chemogenomic.................................................................................................. 17 Protein location .................................................................................................. 17 Epigenetic .......................................................................................................... 17 Gene expression................................................................................................ 18 Gene Ontology................................................................................................... 18

Correspondence analysis ...................................................................................... 18 Addressing multiple testing.................................................................................... 20

References................................................................................................................ 22

Chapter 6 Contents of chapter 6.................................................................................................. 1 Implications ................................................................................................................. 2

Putting together the pieces...................................................................................... 2

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Genomic location matters .................................................................................... 2 Implications for drug discovery and biotechnology.................................................. 3 Drugs to discrupt quorum sensing........................................................................... 6 A potential biotechnological use of quorum sensing ............................................... 6 Chemogenomic approaches to drug discovery ....................................................... 7 The drug-COP score ............................................................................................... 8

Towards a scoring system ................................................................................... 9 The future of the drug-COP ............................................................................... 10

Conclusion ................................................................................................................ 11 References................................................................................................................ 12

Appendix Contents of the appendix ............................................................................................ 1 Appendix 1: Quorum sensing domains ....................................................................... 2 Appendix 1: Quorum sensing domains ....................................................................... 2 Appendix 2: Transcription factors involved in yeast quorum sensing ......................... 8 Appendix 3: Full list of descriptors ............................................................................ 11

Key to classes and colour-code............................................................................. 11 Table of descriptors ............................................................................................... 11

Appendix 4: List of publications................................................................................. 18 Publications relevant to this thesis ........................................................................ 18 Other publications.................................................................................................. 18

References for the appendix ..................................................................................... 19

Abbreviations Å Ångström (1 · 10-10 m) agr Accessory gene regulator AHL Acyl homoserine lactone AI Autoinducer AIP Autoinducing peptide ChIP Chromatin immunoprecipitation COP Complex or pathway Da Dalton DNA Deoxyribonucleic acid HMM Hidden Markov model M Molar (1 mol/l) mRNA Messenger RNA PCA Principal component analysis QS Quorum sensing QSAR Quantitative structure-activity relationship RNA Ribonucleic acid SAM S-adenosyl methionine SM Small molecule SNP Single nucleotide polymorphism