SEQUENCING AND CHARACTERIZATION OF HOX GENE CLUSTERS IN JAPANESE LAMPREY (LETHENTERON JAPONICUM) TARANG KUMAR MEHTA BSc. Biomedical Science (Honours) Nottingham Trent University, 2004 MRes. Molecular and Cellular Biology Nottingham Trent University, 2007 A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY INSTITUTE OF MOLECULAR AND CELL BIOLOGY & DEPARTMENT OF PAEDIATRICS, YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2013 DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. ……………………………………….. Tarang Kumar Mehta 23rd August 2013 (Revised 14th February 2014) Acknowledgements There are several people I would like to acknowledge: First and foremost I would like to thank my supervisor, Professor Byrappa Venkatesh for his patience, superb mentorship, and the amount of time he devoted into improving my scientific writing and presentation skills; I would also like to thank my thesis-advisory committee (TAC) members, Dr Paul Robson, Dr Samuel Chong, and Dr Sydney Brenner for their guidance and suggestions throughout the course of my PhD; Dr Alice Tay for her advice on presentations and at conferences. Additionally, I would also like to thank the DNA Sequencing Facility, Chew Ah-Keng and other lab members, who were excellent in handling and processing my samples both efficiently and effectively; Dr Ravi Vydianathan for his all-round mentoring throughout my PhD and work towards my first manuscript. He showed me the ropes in all scientific disciplines and without which, I would not have been able to progress as a scientist; Tay Boon-Hui for her constant guidance and support in scientific techniques, and her immaculate lab-managing skills allowing for a safe lab working environment; Sumanty Tohari for maintaining a healthy balance between social and work life activities and making the lab an all round enjoyable place to not only grow as a scientist, but as a person too; Dr Alison Lee for her valuable comments and work on the manuscript, and with Michelle Lian, the both of them have done a fantastic job in assembling the Japanese lamprey genome and have helped me with various aspects of i bioinformatics. In the same breath I must also thank the collaborative effort between Dr. Sydney Brenner’s lab at Okinawa Institute of Science and Technology (OIST, Japan), and our lab in IMCB, for generating the sequencing data for the Japanese lamprey genome; Lim Zhi-Wei for teaching me zebrafish transgenics and being an all-round approachable individual in the lab; The Zebrafish facility for doing an excellent job in maintaining my fish according to all standard animal regulations; All present and former members of the Comparative and Medical Genomics Laboratory and DNA Sequencing Facility in IMCB, Singapore for making the laboratory a pleasant and beneficial place to work and grow as a scientist; The staff of A*Star, Singapore International Graduate Award (SINGA) and Yong Loo Lin School of Medicine, NUS for handing me this fantastic opportunity to work in a great environment, among great scientists. I would also like to thank them both the efficient handling of administrative affairs; All of the interesting and diverse group of friends that I have made here in Singapore who have made my experiences both here and in South-East Asia both exciting and enjoyable; Finally, I would like to thank my late father and mother, and my sister who without their efforts and support, I would not have progressed so far in life. ii Table of contents Acknowledgements ............................................................................................. i List of tables ..................................................................................................... vii List of figures ................................................................................................. viii List of abbreviations .......................................................................................... x Chapter 1: Introduction ...................................................................................... 1 1.1 Hox proteins ........................................................................................ 1 1.2 Hox gene clusters ................................................................................ 2 1.3 Function of Hox proteins in metazoans............................................... 6 1.3.1 Cnidaria ........................................................................................ 6 1.3.2 Protostomes .................................................................................. 7 1.3.3 Deuterostomes – Ambulacraria .................................................... 8 1.3.4 Deuterostomes – Vertebrates ....................................................... 8 1.4 Expression and regulation of Hox genes ........................................... 11 1.5 Cis-regulatory elements ..................................................................... 19 1.5.1 Methods to predict cis-regulatory elements .................................. 20 1.5.2 Comparative genomics approach to predict cis-regulatory elements .. .................................................................................................... 24 1.5.3 Testing the function of predicted cis-regulatory elements ............. 24 1.6 Genome duplication in the stem vertebrate lineage .......................... 27 1.7 Jawless vertebrates (cyclostomes) ..................................................... 29 1.8 Hox genes in jawless vertebrates (cyclostomes) ............................... 32 1.9 Objectives of my work ...................................................................... 35 Chapter 2: Materials and methods ................................................................... 38 2.1 Japanese lamprey BAC libraries ....................................................... 38 2.2 Screening of BAC libraries ............................................................... 38 2.3 Screening potential positive clones ................................................... 39 2.4 Sequencing ends of BAC inserts ....................................................... 40 2.5 Shotgun-sequencing of BAC clones ................................................. 40 2.5.1 Large scale isolation of plasmid DNA ....................................... 41 2.5.2 Sequencing plasmid DNA .......................................................... 41 2.6 Assembling BAC shotgun reads ....................................................... 42 iii 2.7 Japanese lamprey whole genome sequence ...................................... 43 2.8 Annotation and analysis of genes ...................................................... 43 2.9 Predicting conserved noncoding elements (CNEs) ........................... 45 2.10 Functional assay of CNEs in transgenic zebrafish ............................ 46 Chapter 3: Results – Hox clusters in the Japanese lamprey ............................. 49 3.1 Screening of lamprey BAC libraries ................................................. 49 3.2 Sequencing and assembly of BAC clones ......................................... 50 3.3 Mining the Japanese lamprey genome assembly for Hox genes ....... 53 3.4 Determining orthology of lamprey Hox gene clusters ...................... 62 3.4.1 Phylogenetic analysis ................................................................. 62 3.4.2 Analysis of gene synteny ........................................................... 71 3.5 Sizes of Japanese lamprey Hox clusters ............................................ 73 3.6 Absence of Hox12 gene in lamprey .................................................. 78 3.7 microRNA in Japanese lamprey Hox clusters ................................... 79 Chapter 4: Results – Conserved noncoding elements (CNEs) in the Hox gene clusters ............................................................................................................. 82 4.1 Introduction ....................................................................................... 82 4.2 CNEs in lamprey and representative gnathostome Hox loci ............. 82 4.3 Functional assay of CNEs ................................................................. 97 4.3.1 Functional assay of αCNE2 ....................................................... 98 4.3.2 Functional assay of αCNE5 ..................................................... 101 4.3.3 Functional assay of βCNE2 and βCNE3 .................................. 103 4.3.4 Summary of expression patterns driven by selected lamprey CNEs .................................................................................................. 105 Chapter 5: Results – Genome duplications in the lamprey lineage ............... 107 5.1 Japanese lamprey Hox clusters and their duplication ..................... 107 5.2 Comparative analysis of non-Hox genes in lamprey and representative gnathostomes ...................................................................... 108 5.3 Lamprey and gnathostome genome duplication history ................. 110 Chapter 6: Discussion .................................................................................... 117 Bibliography .................................................................................................. 128 Appendix .......................................................................................................