THE EVOLUTION OF GENOMIC IMPRINTING AND X CHROMOSOME INACTIVATION IN MAMMALS

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1 THE EVOLUTION OF GENOMIC IMPRINTING AND X CHROMOSOME INACTIVATION IN MAMMALS Timothy Alexander Hore A thesis submitted for the degree of Doctor of Philosophy of The Australian National University. August, 2008

2 DECLARATION Except where specific reference is made to other sources, the work presented in this thesis is the work of the author. It has not been submitted, in whole or in part for any other degree. Timothy Alexander Hore

3 ACKNOWLEDGEMENTS Firstly, I would like to sincerely thank Professor Jenny Graves for her extraordinary supervision throughout my time in her lab. I am extremely grateful for her support, patience and guidance over the past four and a half years. My impressions of her work ethic, intellect and unwavering enthusiasm will stay with me always. It has been an immense pleasure to be associated with your science Jenny; thankyou for every minute of it. I would like to express my genuine gratitude to my supervisors Janine Deakin and Edda Koina. Thankyou both for being excellent mentors. Janine, you have provided constant support and immense encouragement since taking me on as a summer student in I really appreciate it! Edda, thankyou for your warm personality, generosity and scientific expertise. To my surrogate supervisors Tariq Ezaz and Paul Waters, thankyou for your tremendous aid at different times during my project. I will always appreciate your friendship, advice and your willingness to help. To Rob Rapkins, who I worked closely with and followed on from; I am grateful for your generosity and for teaching me so much when I first arrived. I would like to express my sincere appreciation to all collaborators and co-authors. In particular, thankyou to Matthew Wakefield for his innovative thinking and bioinformatic assistance. A huge thankyou to Marilyn Renfree and Andrew Pask from the University of Melbourne for their contribution of wallaby tissue and expertise in marsupial biology and genomic imprinting. Thankyou also to my international collaborators, in particular Lance Davidow of Massachusetts General Hospital and the Opossum genome sequencing team from the BROAD institute. In addition, I would like to thank Frank Grutzner for access to the precious platypus tissue series, which has immensely benefited so many in the lab as well as myself. Thankyou to Lauren Marrotte for being so generous with her resources and time, chaperoning me through many wallaby dissections. Further tissue contributions from Tariq Ezaz and Alex Quinn (bearded dragon), Junior Cullen and Scott Hammond

4 (cattle), and Lyn Hinds (wallaby) have also been greatly appreciated. Without your help, I would have no results! I would like to say a massive thankyou to members of the lab not yet mentioned, for their help and friendship:- Shafagh Al-Nadaf, Amber Alsop, Hannah Bender, Julie Chaumeil, Margaret Delbridge, Ruth Doherty, Nisrine El-Mogharbel, Barbara Harriss, Jason Limnios, Daniel McMillan, Pat Miethke, Amir Mohammadi, Liz Murchison, Veronica Murtagh, Denis O'Meally, Hardip Patel, Vidushi Patel, Natasha Sankovic, Carly Smith, Rami Stiglec, Katherine Thompson, Mary Wallis and Ke-Jun Wei. In particular, thanks to Rob, Paul, Tariq, Denis, Julie and Hardip for being good fun and great to bounce ideas off. To my family and friends from New Zealand, and now Australia, thankyou for accepting my commitment to my studies and for giving me the support to complete them. In particular, to my parents Ralph and Brenda for giving me everything I could need and the drive to achieve anything I could imagine. Thankyou to my siblings and their spouses Ben and Vanessa, Cath and Graham, Liz and Willy, Steve and Kim. I really appreciate your encouragement and I value the impact you all have had on my life. Lastly, to my girlfriend Donna. I am extremely grateful for your love and patience, particularly during tough days, late nights and tight deadlines. I could not have got here without you and I am extremely lucky to have such a wonderful person to share my life with.

5 ABSTRACT Genomic imprinting is responsible for monoallelic gene expression that depends on the sex of the parent from which the alleles (one active, one silent) were inherited. X- chromosome inactivation is also a form of monoallelic gene expression. One of the two X chromosomes is transcriptionally silenced in the somatic cells of females, effectively equalising gene dosage with males who have only one X chromosome that is not complemented by a gene poor Y chromosome. X chromosome inactivation is random in eutherian mammals, but imprinted in marsupials, and in the extraembryonic membranes of some placentals. Imprinting and X inactivation have been studied in great detail in placental mammals (particularly humans and mice), and appear to occur also in marsupial mammals. However, both phenomena appear to have evolved specifically in mammals, since there is no evidence of imprinting or X inactivation in non-mammalian vertebrates, which do not show parent of origin effects and possess different sex chromosomes and dosage compensation mechanisms to mammals. In order to understand how imprinting and X inactivation evolved, I have focused on the mammals most distantly related to human and mouse. I compared the sequence, location and expression of genes from major imprinted domains, and genes that regulate genomic imprinting and X-chromosome inactivation in the three extant mammalian groups and other vertebrates. Specifically, I studied the evolution of an autosomal region that is imprinted in humans and mouse, the evolution of the X-linked region thought to control X inactivation, and the evolution of the genes thought to establish and control differential expression of various imprinted loci. This thesis is presented as a collection of research papers that examines each of these topics, and a review and discussion that synthesizes my findings. The first paper reports a study of the imprinted locus responsible for the human Prader- Willi and Angelman syndromes (PWS and AS). A search for kangaroo and platypus orthologues of PWS-AS genes identified only the putative AS gene UBE3A, and showed it was in a completely different genomic context to that of humans and mice. The only PWS gene found in marsupials (SNRPN) was located in tandem with its ancient paralogue SNRPB, on a different chromosome to UBE3A. Monotremes

6 apparently have no orthologue of SNRPN. The several intronless genes of the PWS-AS domain also have no orthologues in marsupials or monotremes or non-mammal vertebrates, but all have close paralogues scattered about the genome from which they evidently retrotransposed. UBE3A in marsupials and monotremes, and SNRPN in marsupials were found to be expressed from both alleles, so are not imprinted. Thus, the PWS-AS imprinted domain was assembled from many non-imprinted components relatively recently, demonstrating that the evolution of imprinting has been an ongoing process during mammalian radiation. In the second paper, I examine the evolution of the X-inactivation centre, the key regulatory region responsible for X-chromosome inactivation in humans and mice, which is imprinted in mouse extraembryonic membranes. By sequencing and aligning flanking regions across the three mammal groups and non-mammal vertebrates, I discovered that the region homologous to the X-inactivation centre, though intact in birds and frogs, was disrupted independently in marsupial and monotreme mammals. I showed that the key regulatory RNA of this locus (X-inactive specific transcript or XIST) is absent, explaining why a decade-long search for marsupial XIST was unsuccessful. Thus, XIST is eutherian-specific and is therefore not a basic requirement for X- chromosome inactivation in all mammals. The broader significance of the findings reported in these two papers is explored with respect to other current work regarding the evolution and construction of imprinted loci in mammals in the form of a review. This comparison enabled me to conclude that like the PWS-AS domain and the X-inactivation centre, many domains show unexpected construction from disparate genomic elements that correlate with their acquisition of imprinting. The fourth and last paper examines the evolution of CCCTC-binding Factor (CTCF) and its parologue Brother Of Regulator of Imprinted Sites (BORIS) which contribute to the establishment and interpretation of genomic imprinting at the Insulin-Like Growth Factor 2/H19 locus. In this paper I show that the duplication of CTCF giving rise to BORIS occurred much earlier than previously recognised, and demonstrate that a major change in BORIS expression (restriction to the germline) occurred in concert with the evolution of genomic imprinting. The papers that form the bulk of this thesis show that

7 the evolution of epigenetic traits such as genomic imprinting and X-chromosome inactivation is labile and has apparently responded rapidly to different selective pressures during the independent evolution of the three mammal groups. I have introduced these papers, and discussed them generally in terms of current theories of how and why these forms of monoallelic expression have evolved in mammals.

9 TABLE OF CONTENTS CHAPTER 1: INTRODUCTION GENETICS, GENOMICS AND EPIGENETICS Chromatin Other modulators of epigenetic gene control GENOMIC IMPRINTING Definition Phylogenetic distribution of genomic imprinting Significance Imprinted gene ontogeny Genomic distribution of imprinted genes Imprinted gene regulation X CHROMOSOME INACTIVATION Therian sex chromosomes and X inactivation Regulation of therian X inactivation Monotreme sex chromosomes and X inactivation X inactivation and genomic imprinting AIMS OF THE PRESENT STUDY Core aim Methodology and chapter summary CHAPTER 2: RECENT ASSEMBLY OF AN IMPRINTED DOMAIN FROM NON-IMPRINTED COMPONENTS Contribution to publication Publication (Rapkins et al., 2006) CHAPTER 3: THE REGION HOMOLOGOUS TO THE X-CHROMOSOME INACTIVATION CENTRE HAS BEEN DISRUPTED IN MARSUPIAL AND MONOTREME MAMMALS Contribution to publication Publication (Hore et al., 2007a)... 57

10 CHAPTER 4: CONSTRUCTION AND EVOLUTION OF IMPRINTED LOCI IN MAMMALS...79 Contribution to publication...79 Publication (Hore et al., 2007b)...80 CHAPTER 5: THE EVOLUTION OF EPIGENETIC REGULATORS CTCF AND BORIS/CTCFL IN AMNIOTES...89 Contribution to publication...89 Publication (Hore et al., 2008)...90 CHAPTER 6: DISCUSSION AND CONCLUSION THE EVOLUTION OF IMPRINTED LOCI Evolution of the PWS-AS locus Evolution of the XIC Evolution of other imprinted loci Kinship and the evolution of imprinting THE EVOLUTION OF REGULATORS OF IMPRINTING AND X INACTIVATION The evolution of CTCF, BORIS and imprinting BORIS expression in ovary and oocytes CTCF, BORIS, cancer and reprogramming THE EVOLUTION OF X INACTIVATION Dosage compensation in monotremes Marsupial X inactivation CONCLUSION REFERENCES...133

11 APPENDIX 1: TABLE OF IMPRINTED GENES APPENDIX 2: THE EVOLTION OF IMPRINTING: CHROMOSOMAL MAPPING OF ORTHOLOGUES OF MAMMALIAN IMPRINTED DOMAINS IN MONOTREME AND MARSUPIAL MAMMALS Contribution to publication Publication (Edwards et al., 2007) APPENDIX 3. THE STATUS OF DOSAGE COMPENSATION IN THE MULTIPLE X CHROMOSOMES OF THE PLATYPUS Contribution to publication Publication (Deakin et al., 2008) APPENDIX 4: GENOME OF THE MARSUPIAL MONODELPHIS DOMESTICA REVEALS INNOVATION IN NON-CODING SEQUENCES Contribution to publication Publication (Mikkelsen et al., 2007)

Weird animal genomes and sex chromosome evolution

Weird animal genomes and sex chromosome evolution Genomes of distantly related mammals, other vertebrates Sex chromosome origins, evolution, fate Evolution of X and Y genes Jenny Graves La Trobe University,