Genetic analysis of p38 mitogen-activated protein kinase signaling in innate immunity and stress physiology of Caenorhabditis elegans by Daniel J. Pagano B.S. Biology Drexel University, 2007 SUBMITTED TO THE DEPARTMENT OF BIOLOGY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY AT THE MASSACHUSETTS INSTITUTE OF TECHNOLOGY FEBRUARY 2014 TECHN)LOGY 0 2014 Daniel J. Pagano. All rights reserved. The author hereby grants to MIT permission to reproduce and to distribute publicly paper and electronic "ARIES copies of this thesis document in whole or in part in any medium now known or hereafter created. /_1 Signature of Author: Department of Biology November 4, 2013 Certified by: Dennis H. Kim Associate Professor of Biology Thesis Supervisor Accepted by: t Amy E. Keating Associate Professor of Biology Co-Chairman of the Graduate Committee Department of Biology i ii Genetic analysis of p38 mitogen-activated protein kinase signaling in innate immunity and stress physiology of Caenorhabditis elegans by Daniel J. Pagano Submitted to the Department of Biology on January 13, 2014 in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ABSTRACT Host-microbe interactions play an important role in the physiology and evolution of animals. Interactions with microbes can generally be considered beneficial or pathogenic to the host. The ability of an organism to mount an immune response to infection by pathogenic microbes is critical to its survival, and basic mechanisms of innate immunity are conserved in evolutionarily diverse species. A pivotal signaling pathway in the evolutionarily conserved innate immune responses of multicellular organisms is the stress-activated p38 mitogen-activated protein kinase (MAPK) pathway. This thesis focuses on the physiological role of p38 MAPK signaling in the host defense of Caenorhabditiselegans. In Chapter Two, I report the identification and characterization of the conserved ATF/CREB bZIP protein ATF-7 as the key transcriptional regulator of the PMK- 1 p38 MAPK-dependent immune response to pathogens. These data suggest a model in which the PMK- 1 p3 8 MAPK phosphorylates ATF-7 and switches it from a transcriptional repressor to a transcriptional activator of effector genes of the innate immune response of C. elegans. In Chapter Three, I characterize the roles of PMK- 1 and a second p38 MAPK ortholog, PMK-2, which are encoded in an operon, in tissue-specific signaling mechanisms involved in host defense. I show that PMK-2 functions redundantly with PMK- 1 in the nervous system to mediate neurobehavioral responses to pathogens. Furthermore, I demonstrate a role for the miR-58/80-82 family of microRNAs in regulating the tissue expression of pmk-2, which suggests a role for microRNAs in the establishment of tissue-specific expression of co-operonic genes. The work described in this thesis establishes the ancient evolutionary origins of the p38 MAPK- CREB/ATF pathway in innate immunity, and establishes a role for microRNAs in defining the tissue-expression pattern of co-operonic p38 MAPK genes. New directions for further understanding the ancient evolutionary mechanisms of p38 MAPK signaling and their tissue- specific regulation are discussed in Chapter Four. Thesis Supervisor: Dennis H. Kim Title: Associate Professor of Biology iii iv Acknowledgments There are many people that I would like to acknowledge and thank for help in my graduate studies. First and foremost, I would like to thank my advisor, Dennis Kim, for his constant support, encouragement, and enthusiasm. Dennis created an environment in which I could pursue my interests independently but was always present when I needed help or guidance. I would like to thank the past and present members of the Kim lab that I worked with for discussions and creating a friendly and fun environment to work in: Annie Inman, Chi-Fong Wang, Claire Richardson, Doug Cattie, Elena Kingston, Howard Chang, Jen Paek, Josh Meisel, Janelle Whitney, Julianna LeMieux, Kirthi Reddy, Matt Meisner, Matt Youngman, Odile Kamanzi, Pete Kulalert, Rob Shivers, Sonika Reddy, Stephanie Kinkel, Tristan Kooistra, Zoe Hilbert, and Zo6 Rogers. I am especially indebted to Rob Shivers and Matt Youngman for teaching me the basics of working with C. elegans. I would like to thank my neighbors in the Horvitz lab for sharing protocols, reagents, and strains. I especially thank Na An for strains, and Nick Paquin and Nikhil Bhatla for discussions. I would like to thank the members of my thesis committee, Rick Young and Jeroen Saeij, for their advice and guidance during my graduate studies. I would also like to thank Victor Ambros for joining my committee for my thesis defense. I would like to acknowledge a number of teachers and previous advisors, Stanley Krantz, Aleister Saunders, Douglas Auld, Benjamin Doranz, and Lee Goldstein, who played an instrumental role in my decision to pursue my graduate degree. I would like to thank Kaitlin for the time and adventures we have shared together and for her support while I finished my thesis work presented here. Finally, I would like to thank my family for their love and support. v vi Table of Contents Title Page ......................................................................................................................................... i Abstract .......................................................................................................................................... iii Acknowledgm ents ........................................................................................................................... v Table of Contents .......................................................................................................................... vii Chapter One: Introduction .......................................................................................................... I Introduction ..................................................................................................................................... 3 Host defense .................................................................................................................................... 3 C elegans-pathogeninteractions .................................................................................................... 6 Stress- activated M APKs ................................................................................................................. 8 Stress-activated M APKs in C. elegans ......................................................................................... 10 Figure 1. p38 M APK signaling in C. elegans ......................................................................... I I Figure 2. JNK M APK signaling in C. elegans........................................................................ I I In developm ent of neural asymmetry ..................................................................................... I I In stress responses and host defense ....................................................................................... 12 Innate im m unity ................................................................................................................ 13 Aversive learning ............................................................................................................. 14 Summ ary ....................................................................................................................................... 15 References ..................................................................................................................................... 17 vii Chapter Two: Phosphorylation of the Conserved Transcription Factor ATF-7 by PMK-1 p38 MAPK Regulates Innate Immunity in Caenorhabditiselegans.................................... 21 S um m ary ....................................................................................................................................... 23 Introduction ................................................................................................................................... 24 R esu lts ........................................................................................................................................... 2 6 Isolation and characterization of mutant alleles of atf-7...................................................... 26 Table 1. List of isolates from a screen for mutants with diminished GFP expression from the agIs219 transgene and enhanced susceptibility to killing by P. aeruginosa P A 14 . ................................................................................................................................ 2 7 Figure 1. Characterization of atf-7 mutants that affect signaling downstream of PM K-I p38 M A PK ....................................................................................................... 31 Figure 2. Mutant alleles of C. elegans atf-7, an ortholog of the mammalian ATF2/ATF7/CREB5 family of bZIP transcription factors............................................ 33 atf-7 loss-of-function alleles suppress the immunodeficiency phenotype of pmk-I............ 34 Figure 3. The loss-of-function atf-7(qd22 qdi30) mutation suppresses the immunodeficient phenotype caused by deficient signaling in the PMK- 1 pathway ........ 37 Regulation of PMK- 1-regulated genes by ATF-7 .............................................................. 35 Figure 4. ATF-7 regulation of PMK- 1-regulated genes .............................................