Regulation of germ-line expression of the Caenorhabditis elegans gene fem-1 by maternal transcripts by Cheryl Lynn Johnson A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Molecular Genetics University of Toronto © Copyright by Cheryl Lynn Johnson 2010 Regulation of germ-line expression of the Caenorhabditis elegans gene fem-1 by maternal transcripts Cheryl Lynn Johnson Doctor of Philosophy Department of Molecular Genetics University of Toronto 2010 Abstract In addition to previously identified roles for RNA, several new ways in which RNA serves as a regulator of gene expression have recently been described. RNA molecules are involved in both transcriptional and post-transcriptional forms of regulation, sometimes heritably affecting gene activity. Whereas most previously characterized regulatory roles of RNA involve downregulation, I describe a role for maternal transcripts of a gene in promoting zygotic activity of that gene, which I term the licensing of genetic activity. This regulation occurs in the germ line, a tissue notable for its abundance of genetic surveillance mechanisms. The maternal-effect regulation described here was identified using alleles of a sex- determining gene in Caenorhabditis elegans called fem-1. Females homozygous for fem-1 deletions produce heterozygous offspring that exhibit germ-line feminization and have reduced fem-1 activity and transcript accumulation. This phenotype can be rescued by injecting fem-1 RNA into the maternal germ line. The reduction in activity of the zygotic fem-1 locus is heritable, suggesting that the gene is becoming epigenetically silenced. Thus the maternal fem-1 RNA licenses the activity of the zygotic locus by preventing its silencing. By restricting germ- ii line activity to genes that were expressed in the germ line of the previous generation, this process may contribute to protecting the identity and integrity of the germ line. I performed an RNAi screen of candidate genes to ask whether they are required for maternal-effect silencing or licensing. Several enhancers and suppressors of germ-line feminization in the descendants of fem-1 deficiency homozygotes were identified. Chromatin regulation may be involved, and small-RNA pathways are important for both the silencing and licensing components of fem-1 regulation. Based on my characterization of this phenomenon, I proposed models of how maternal-effect regulation of fem-1 may be mediated. To test predictions of certain models, I examined whether specific characteristics of fem-1 make it susceptible to this silencing. Results of these experiments limit the possible models of maternal- effect regulation and suggest directions for future investigation. iii Acknowledgments My supervisor, Dr. Andrew Spence, is the person who first noticed the unusual inheritance patterns associated with fem-1 deficiency alleles. I am grateful that he perceived my interest in genetics and matched me with such an interesting project. I thank him for his mentorship in designing and performing experiments, thinking about broader scientific ideas, and learning to present with greater clarity. Our discussions were fun and productive, and he always heartened me when I entered his office discouraged. My committee members Dr. Brigitte Lavoie and Dr. Howard Lipshitz also helped me to develop as a scientist. I thank them for their input at committee meetings and for providing feedback about the manuscripts and my thesis. I was challenged and encouraged by them both throughout my degree. My fellow Spence lab members were one of the great joys of graduate school. They were my travelling companions on the road to my Ph.D., and I am so happy and honoured to have been their friend and colleague. Thanks to Kathleen Dawson, Leslie Magtanong, Holly Sassi, Jonathan Boetto, Dr. Fiona Broackes-Carter, Dr. Ramona Cooperstock, Dr. Mara Schvarzstein, Stephanie Grouios and Michael Schertzberg. They embraced me from the first day of my rotation and saved my worm pick just in case I decided to join the lab. Likewise, the Roy lab has been wonderful scientifically and socially in Toronto and at conferences. I’m thankful for the camping, the karaoke, the book club, many a great meal with excellent companions, and the general merriment. They injected a lot of fun into my grad school years and have continuously buoyed my spirits in hard times. My thanks go to Caroline Fernandes who helped with the RNAi screen and to everyone who contributed expertise and reagents as listed throughout this thesis. My family and friends have been invaluable during this undertaking. Having my Aunt George Perrier and my cousin Michelle Perrier-Martinen nearby has been fantastic for my transition from Alberta. My sister Dana Johnson and my friends Lindsay LeBlanc and Loretta Foley continue to keep me laughing and smiling. I look up to Grandpa and Grandma Wakelin who have been unceasingly loving and generous. My parents Randy and Darlene Johnson always have supreme confidence in me. Their love and support keep me going, and I dedicate this work to them. (I got the paper, Dad!) My husband Andrew Keeping makes everything in my world shine brighter. He cheerfully supports me in everything, and that makes all the difference. iv Table of Contents Abstract ........................................................................................................................................... ii Acknowledgments .......................................................................................................................... iv Table of Contents ............................................................................................................................ v List of Tables ................................................................................................................................. xi List of Figures .............................................................................................................................. xiii List of Abbreviations and Gene Names ....................................................................................... xvi Chapter 1 Introduction .................................................................................................................... 1 1 Introduction ................................................................................................................................ 2 1.1 The germ line and epigenetic changes ................................................................................ 2 1.2 Epigenetic phenomena and RNA-mediated effects ............................................................ 3 1.2.1 Mechanisms of heritable change in genetic activity ............................................... 3 1.2.2 Overview of chromatin structure ............................................................................ 4 1.2.3 Overview of RNA regulatory mechanisms ............................................................. 6 1.2.4 RNA-mediated effects that heritably reduce gene activity ..................................... 7 1.2.5 RNA-mediated gene activation ............................................................................. 13 1.2.6 RNA-mediated regulation and heritable effects in C. elegans ............................. 15 1.2.6.1 MicroRNAs ............................................................................................ 15 1.2.6.2 Small interfering RNAs .......................................................................... 19 1.2.6.3 Piwi-interacting RNAs (21U-RNAs) ..................................................... 22 1.2.6.4 Genomic imprinting ................................................................................ 24 1.3 Sex determination and the genetics of fem-1 .................................................................... 25 1.3.1 Genetics in C. elegans ........................................................................................... 25 1.3.2 Sexual dimorphism in C. elegans ......................................................................... 26 1.3.3 Overview of somatic sex determination ............................................................... 28 v 1.3.4 Roles of the fem genes .......................................................................................... 30 1.3.5 Germ-line sex determination ................................................................................. 33 1.4 Development and regulation of the C. elegans germ line ................................................. 36 1.4.1 Overview of germ-line development in C. elegans .............................................. 36 1.4.2 Factors required for establishment and maintenance of the germ-line ................. 38 1.4.3 Surveillance processes in the C. elegans germ line .............................................. 41 1.5 Thesis objectives ............................................................................................................... 44 Chapter 2 Licensing of fem-1 in the germ line by maternal transcripts ........................................ 45 2 Licensing of fem-1 in the germ line by maternal transcripts .................................................... 46 2.1 Abstract ............................................................................................................................. 46 2.2 Introduction ....................................................................................................................... 46 2.3 Materials