The Sea Urchin Regulome in Development Thesis by Meredith Ashby In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy California Institute of Technology Pasadena, California 2007 (Defended October 5, 2006) ii © 2007 Meredith Ashby All Rights Reserved iii Acknowledgements So many people have contributed to the success of this effort. I would foremost like to thank my advisor, Eric Davidson, for creating an intellectual atmosphere where I could indulge my passion for science. Thank you for all the discussions about both the big picture and the fine details, probing questions, encouragement, and especially all the delicious cookies. I would also like to acknowledge Stefan Materna, Titus Brown, Andy Cameron, Rachel Gray, and Lili Chen for their work on the research presented here, and the whole Davidson Group, for making the lab such a fun place to work. Many of you I owe particular thanks to: Cathy Yuh and Titus Brown, who sacrificed hours of potentially productive time helping me out when I first joined the lab; Gabriele Amore, for being the best labmate ever, silly walk and opera singing included; the whole crazy crew who made it fun to get up at 4 am to filter gallons of urchin embryos; and Deanna Thomas and Jane Rigg, for making everything happen smoothly, from the magical appearance of supplies, to travel arrangements without bankruptcy, to publications with beautiful figures and accurate references. Finally, I would like to thank all my friends in Pasadena, who have become family to me ─ Jen Ma, Lara, Raffi, Natasha, Maro, Sevan, and Nancy ─ I never would have made it without all of you; Stefan again, for being the best roommate ever; Mom and Dad, for undying patience and encouragement; and Jen, to whom I still owe three weeks of vacation. And most of all, Ashby, thank you for bringing new magic into my life, and putting up with all the crazy travel while I pursued my passion. iv Abstract During development an organism undergoes many rounds of pattern formation, generating ever greater complexity with each ensuing round of cell division and specification. The instructions for executing this process are encoded in the DNA, in cis- regulatory modules that direct the expression of developmental transcription factors and signaling molecules. Each transcription factor binding site within a cis-regulatory module contributes information about when, where or how much a gene is turned on, and by dissecting the modules driving a given gene, all the inputs governing expression of the gene can be accurately identified. Furthermore, by mapping the output of each gene to the inputs of other genes, it is possible to reverse engineer developmental circuits and even whole networks, revealing common bilaterian strategies for specifying progenitor fields, locking down regulatory states, and driving development forward. The S. purpuratus endomesodermal gene network is one of the best-characterized developmental networks, with interactions between over 40 regulatory genes mapped by perturbation experiments. With the sequencing of the sea urchin genome, it is possible to move towards the definitive completion of this network. By identifying all the transcription factors in the genome and determining their expression patterns, any previously unrecognized players can be incorporated into the network. In addition, such a comprehensive examination of transcription factor usage in maximally indirect development has not been done and will itself yield interesting conclusions. Keywords: cis-regulatory module; gene regulatory network; repression; feedback loop v Table of Contents Acknowledgements .......................................................................................................... iii Abstract............................................................................................................................. iv Table of Contents ...............................................................................................................v Introduction........................................................................................................................1 Chapter 1 Indentification and Characterization of Homeobox Transcription Factor Genes in Strongylocentrotus purpuratus, and Their Expression in Embryonic Development.....31 Chapter 2 Gene Families Encoding Transcription Factors Expressed in Early Development of Strongylocentrotus purpuratus ...............................................................68 Chapter 3 High Regulatory Gene Use in Sea Urchin Embryogenesis: Implications for Bilaterian Development and Evolution............................................................................109 Appendix 1: Supplementary Material for Chapter 1 ......................................................129 Appendix 2: Supplementary Material for Chapter 2 ......................................................138 Appendix 3: Rake Database Accession Numbers..........................................................153 1 Introduction Understanding Development through Gene Regulatory Networks Molecular biology has illuminated how DNA encodes amino acid sequences, and how the cell is able to translate those blueprints into proteins. Understanding in similar detail how DNA also encodes where, when and how much each protein will be expressed has yet to be achieved. Written into genomic DNA is a self-executing set of instructions which precisely directs developmental pattern formation and cell division, ultimately producing the complex body plan of the adult organism. Decrypting that information is one of the most interesting problems in biology. Only recently, with the availability of large amounts of genomic DNA sequence and the advent of high throughput cis- regulatory analysis, has it become possible to peer into the black box, and begin to understand at the molecular level how cis-regulatory information is processed to generate complexity during development, both at the individual gene level and at the gene network level. In essence, cis-regulatory elements are information processing devices hardwired into the genomic DNA sequence, the function of which is to regulate gene expression (Davidson, 2006b). Most commonly, cis-regulatory elements or modules are several hundred base pairs long and are located within a few kilobases of the exons or within the introns of the gene they control, though there are many examples of modules which exert their influence over distances as great as 100 kb. A cis-regulatory module is comprised of multiple binding sites for transcription factors, plus some inter-site sequence, with each 2 specific binding interaction having a functional meaning. A cis-regulatory module typically includes many sites for ubiquitous DNA binding proteins, some of which are involved in DNA looping or required for interaction with the basal transcription apparatus. On average, a module will have binding sites for four to eight different transcription factors (Arnone and Davidson, 1997), and several sites may be present for some factors. To a rough approximation, more sites for a given factor afford the module greater sensitivity to a given regulator. Frequently two or more different transcription factors must be bound to a module in order for a gene to be activated (AND logic). Alternately, any one of several different transcription factors may be sufficient to generate an output, and the strength of the module’s regulatory activity depends additively on the number of relevant interactions (OR logic). Repressor binding sites (NOT logic) are often used to delineate expression boundaries. A gene receives information about when and where it is in the course of development by way of these transcription factor binding interactions. For example, when a signal is received from a neighboring cell at a receptor, it typically causes a cascade of protein-protein interactions, and the information conveyed by the signal ultimately arrives at the nucleus in the form of a DNA binding transcription factor. If the transcription factor is present at sufficient concentration, it will occupy target sites in an array of target cis-regulatory modules, and thus communicate important spatial data to the regulatory apparatus of the cell. Information about the current developmental state of the cell itself is expressed via other transcription factors, which may be turned on or off as a result of previous regulatory events. In this way, cis-regulatory elements read cellular conditions. They function by resolving the multiple developmental inputs they receive 3 into a single directive to the basal transcription apparatus, thereby specifying the appropriate outputs. The recent wealth of genomic data has confirmed that bilaterians as simple as nematodes and as complex as humans use the same basic tool kit of transcription factors and signaling molecules to process spatial and temporal information during development (Erwin and Davidson, 2002). The qualitative complexity of the developmental regulatory tool kit is thus not correlated with genome or proteome size. Rather than relying upon a vastly larger tool kit, complexity is increased with remarkable economy by reusing transcription factors in additional unique ways in the course of later rounds of pattern formation. Every regulatory gene has not just one but many cis-regulatory modules which control the expression of the transcription factor it encodes in different spatial domains at different times in development. One module may activate a gene in one