A GENOME-WIDE SIRNA SCREEN REVEALS DIVERSE CELLULAR PROCESSES AND PATHWAYS THAT MEDIATE GENOMIC STABILITY A DISSERTATION SUBMITTED TO THE DEPARTMENT OF CHEMICAL AND SYSTEMS BIOLOGY AND THE COMMITTEE ON GRADUATE STUDIES OF STANFORD UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Renee Darlene Paulsen May 2010 © 2010 by Renee Darlene Paulsen. All Rights Reserved. Re-distributed by Stanford University under license with the author. This work is licensed under a Creative Commons Attribution- Noncommercial 3.0 United States License. http://creativecommons.org/licenses/by-nc/3.0/us/ This dissertation is online at: http://purl.stanford.edu/zx436yn9136 Includes supplemental files: 1. Table S1. H2AX Signal and Cell Cycle Distribution for Genome (NT dataset). The following list shows the percentage ... (Paulsen_ST1.xlsx) 2. Table S2. List of Genes with Significant H2AX Values (NT dataset). The following list shows genes that had a H2AX s... (Paulsen_ST2.xlsx) 3. Table S3. Genes that Caused Extensive Cell Death. The following is a list of genes that when knocked down lead to w... (Paulsen_ST3.xlsx) 4. Table S4. Categories of Enrichment Determined by DAVID Bioinformatic Database and Ingenuity Pathway Analysis. The g... (Paulsen_ST4.xlsx) 5. Table S5. List of Deconvoluted Genes. The following is a list of genes for which we individually tested four differ... (Paulsen_ST5.xlsx) 6. Table S6. Individual Components of Gene Modules and Networks Enriched Amongst Screen Hits. The individual modules i... (Paulsen_ST6.xlsx) 7. Table S7: 53BP1 Staining Results and Table S8: Phospho-H3 Recovery Assay Results. (Paulsen_ST7_ST8.xlsx) 8. Table S9. Table of Genes Identified in This Screen and Other Screens. Hits from complimentary siRNA screens were co... (Paulsen_ST9.xlsx) 9. Table S10: H2AX Values From Retesting mRNA Processing Genes and the Effect of RNAseH Treatment, Table S11: mRNA Pro... (Paulsen_ST10_ST11_ST12.xlsx) ii I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Karlene Cimprich, Primary Adviser I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Tobias Meyer I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Clifford Wang I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Joanna Wysocka Approved for the Stanford University Committee on Graduate Studies. Patricia J. Gumport, Vice Provost Graduate Education This signature page was generated electronically upon submission of this dissertation in electronic format. An original signed hard copy of the signature page is on file in University Archives. iii ABSTRACT Genome instability has long been known to be a hallmark of cancerous cells, but the cellular causes and consequences of such instability are still not fully understood. Mutations, translocations, DNA rearrangements, as well as chromosomal loss can all result in the loss of genomic integrity. To prevent the disruption of cellular homeostasis due to DNA damage accumulation, cells contain pathways to sense and respond to DNA damage including cell cycle checkpoints and numerous DNA repair processes, collectively known as the DNA damage response (DDR). Mutations in many of the genes involved in the DDR are linked to several diseases, including premature aging, neurodegeneration and cancer. These signaling pathways are especially critical during DNA replication when the DNA is unwound and vulnerable to processing. Here, the cell relies on the S-phase checkpoint to sense DNA damage at the sites of replication forks and to facilitate a number of downstream pathways to maintain genomic stability. These processes include blocking further origin firing, facilitating DNA repair, preventing cell cycle progression, and stabilizing stalled replication forks. Here, two genome-wide siRNA screens were employed to identify additional genes involved in genome stabilization by monitoring phosphorylation of the histone variant H2AX, an early mark of DNA damage. The first screen looked at H2AX phosphorylation that occurred simply by individual protein depletion, and the second screen used a low level of a replication inhibitor, aphidicolin, to specifically identify genes that were needed to prevent DNA damage during S-phase, potentially due to the loss of replication fork stabilization mechanisms. While the results from the second screen are still undergoing further characterization, we did discover hundreds of genes whose down-regulation led to elevated levels of H2AX phosphorylation (γH2AX) in the absence of any external stress. From this gene set, we identified many gene networks that were significantly enriched amongst our screening hits as well as several intriguing individual genes that were chosen for follow up study. iv From our screen, we found a widespread role for mRNA processing factors, predominantly mRNA splicing proteins, in preventing γH2AX, with the loss of nearly ninety mRNA processing genes capable of inducing DNA damage. Additionally, we discovered that in some cases, loss of proper mRNA processing caused damage due to aberrant RNA-DNA structure formation, along with defects in the replication progression. Furthermore, we connected increased γH2AX levels to the neurological disorder, Charcot-Marie-Tooth (CMT) syndrome, and we found a role for several CMT proteins in the DNA damage response with loss causing DNA damage sensitivities and DNA repair deficiencies. Finally, the genome-wide siRNA screen also lead to the discovery of the histone methyltransferase PR-Set7/Set8 as a novel mediator of genomic integrity. We demonstrated that Set8 has multi-faceted roles to facilitate replication progression and prevent DNA damage formation. Cumulatively, this thesis highlights that preservation of genome stability is mediated by a larger network of biological processes than previously appreciated, and understanding all mechanisms in place to prevent DNA damage should help elucidate how cells prevent mutations, translocations, and ultimately, oncogenic transformation. v ACKNOWLEDGMENTS I am deeply grateful for all of the faculty, family, and friends who have helped during the course of my PhD. First and foremost, thank you to my advisor, Karlene Cimprich for guiding me throughout the process, and serving as a good mentor and role model both in science and life. Thank you also to my committee members, Tobias Meyer, Joanna Wysocka, Cliff Wang, and Teresa Wang for their helpful suggestions and useful criticism along the way. Many thanks also go to the Cimprich lab members both past and present. Thanks to Sharon Barr and Ryan Bombargden for getting me up and running in the lab. Thanks to Jia-Ren Lin, Michelle Zeman, Claudia Choi, and Anna Guan for being excellent grad student lab mates along the way. Also thanks to Deena Soni for all of the help with the siRNA screen and Robert Driscoll, Anja Duursma, Julie Sollier, Thomas Wechsler, and Andrew Kile for all of the good ideas and fresh enthusiasm you have brought to the lab. A special thanks also to M.C. Yee who is simply an inspiration in so many ways. Thank you for all of your help scientifically as well as mentoring and family support. I would not have made it through graduate school without you, and I greatly appreciate all you have done for me and my family. A special thanks also to Chris Van, Debbie Chang, and Angie Hahn for all of the advice, commiseration sessions, support, food, and friendship. Graduate school wouldn’t have been nearly as much fun without you all of you. Outside of the Cimprich lab thank you to Roy Wollman, Annette Salmeen, Phil Vitorino, Lin Gan, David Solow-Cordero, and Jason Wu for all of your help with the genome wide screen, analysis, and interpretation. You made it all possible and thankfully make sense. Also a special thanks to Blueshift biotechnologies, now part of MDS analytical technologies for letting us invade with an idea, and leave with beautiful scientific results. Jayne Hesley, Steve Miller, and Evan Cromwell, we would still be screening the genome if it were not for you and your brilliant instrumentation. Thank you also to my friends in the CSB department, Emily Eggler, Andy Poon, Paul Rack, Mark Sellmyer, and the many others who have come and gone along the way. vi Thank you also to my family for supporting me throughout this process. Thanks to my parents Stanley and Nancy Baack for instilling in me the values to succeed in life. You have always been a constant source of support. Thank you to my sister, Jennifer Baack, who has patiently proof read countless manuscripts, and papers. Thank you also to my extended family, Wes and Pat Paulsen. I am so blessed to have two sets of supporting parents in my life, and I am very grateful to Pat for all of her help during this process. Finally, thank you to my husband, my best friend, and the love of my life TJ Paulsen. You have stood by my side, listened to my complaints, been a constant source of reason, stolen me from the bench when appropriate, and made me a better person by knowing you. You and Meagan have been the joy of my life and I cannot tell you how thankful I am for your love, comfort, and support