Development and Application of a High- Throughput Rnai Screen to Reveal Novel Components of the DNA Sensing Pathway
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Development and Application of a High- Throughput RNAi Screen to Reveal Novel Components of the DNA Sensing Pathway The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Roy, Matthew Stephen. 2013. Development and Application of a High-Throughput RNAi Screen to Reveal Novel Components of the DNA Sensing Pathway. Doctoral dissertation, Harvard University. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:11124826 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA 2013 – Matthew Stephen Roy All rights reserved. Dissertation Advisor: Nir Hacohen Matthew Stephen Roy Development and application of a high-throughput RNAi screen to reveal novel components of the DNA sensing pathway Abstract The mammalian immune system has evolved a complex and diverse set of mechanisms to detect and respond to pathogens by recognizing conserved molecular structures and inducing protective immune responses. While many of these mechanisms are capable of sensing diverse molecular structures, a large fraction of pathogen sensors recognize nucleic acids. Pathogen-derived nucleic acids trigger nucleic acid sensors that typically induce anti-viral or anti-microbial immunity, however host-derived nucleic acids may also activate these sensors and lead to increased risk of inflammatory or autoimmune disease. Animal models and humans lacking key DNA nucleases, such as Trex1/Dnase3, accumulate intracellular DNA and develop progressive autoimmunity marked by increased Type-I Interferon (IFN) expression and inflammatory signatures. Double-stranded DNA (dsDNA) is a potent inducer of the Type-I IFN response. Many of the sensors and signaling components that drive the IFN signature following simulation with transfected dsDNA (also called ʻInterferon Stimulatory DNAʼ or ʻISDʼ) remain unknown. We set out to identify novel components of the ISD pathway by developing a large-scale loss-of-function genetic perturbation screen of 1003 candidate genes. We interrogated multiple human and murine primary and immortalized cells, tested several Type-I IFN reporters, and considered multiple loss-of-function strategies before proceeding with an RNAi screen whereby mouse embryonic fibroblasts were stimulated with ISD and Type-IFN pathway activation was assessed by measuring Cxcl10 protein by ELISA. iii Candidate genes for testing in the RNAi screen were curated from quantitative proteomic screens, IFN-beta and ISD stimulated mRNA expression profiles, and a selection of domain-based proteins including helicases, cytoplasmically located DNA- binding proteins and a set of potential negative regulators including phosphatases, deubiquitinases and known signaling proteins. We identified a number of novel ISD pathway components including Abcf1, Ptpn1 and Hells. We validated hits through siRNA-resistant cDNA rescue, chemical inhibition or targeted knockout. Additionally, we evaluated protein-protein interactions of our strongest validated hits to develop a network model of the ISD pathway. In addition to the identification of novel ISD pathway components, our enriched screening data set may provide a useful resource of candidate genes involved in the response to cytosolic DNA. iv Table of Contents Abstract ............................................................................................................................. iii List of Figures and Tables ................................................................................................ vii List of Abbreviations ......................................................................................................... xi Acknowledgements ......................................................................................................... xiii Attributions ...................................................................................................................... xv Chapter 1: Introduction: The Role of Nucleic Acids In Eliciting an Immune Response 1.1 – Introduction ................................................................................................. 2 1.2 – Nucleic acid recognition by Toll-like receptors ............................................ 3 1.3 – Detection of RNA by cytosolic sensors ....................................................... 6 1.4 – Detection of DNA by cytosolic sensors ....................................................... 8 1.5 – Inflammasome activation by cytosolic DNA ................................................ 9 1.6 – Interferon activation by cytosolic DNA sensors ......................................... 10 1.7 – Endogenous DNA ligands and autoimmunity ............................................ 17 1.8 – Conclusion ................................................................................................ 21 Chapter 2: Development of a High-Throughput Screening Method to Detect Nucleic Acid Responses 2.1 – Introduction: Identification of a cellular based system to detect nucleic acid responses ........................................................................................................... 24 2.2 – Ligands and transfection reagents ............................................................ 25 2.3 – Identification of cells for use in genetic and biochemical screen ............... 33 2.4 – Development of a quantitative assay to detect type-I interferon responses of nucleic acids ....................................................................................................... 46 2.5 – Genetic perturbation using RNA interference ............................................ 64 2.6 – Screening strategy .................................................................................... 69 2.7 – Conclusion ................................................................................................ 82 Chapter 3: Generation of a candidate gene set by curation and quantitative proteomics 3.1 – Introduction: candidate gene selection ...................................................... 84 3.2 – Candidate gene selection: interferon-regulated, DNA-stimulated gene from published arrays ................................................................................................. 84 3.3 – Candidate gene selection: DNA SILAC ..................................................... 90 3.4 – Candidate gene selection: helicases ......................................................... 93 3.5 – Candidate gene selection: cytoplasmic DNA-binding proteins .................. 98 3.6 – Candidate gene selection: putative negative regulators and signaling molecules ........................................................................................................... 99 3.7 – Conclusion .............................................................................................. 104 v Chapter 4: A High-throughput Loss-of-Function RNAi Screen for of the ISD-Sending Pathway Reveals Identifies Known Components and Novel Regulators 4.1 – Introduction ............................................................................................. 110 4.2 – Pilot screen: phosphatases and deubiquitinases .................................... 110 4.3 – High-throughput loss-of-function RNAi screen ........................................ 115 4.4 – Database development ........................................................................... 119 4.5 – Secondary screening .............................................................................. 133 4.6 – Conclusion .............................................................................................. 143 Chapter 5: Validation and Characterization of Novel Regulators of the DNA Sensing Pathway 5.1 – Introduction ............................................................................................. 145 5.2 – Validation of putative DNA-sensors ........................................................ 145 5.3 – Abcf1 ........................................................................................................ 149 5.4 – Ifit1 ........................................................................................................... 157 5.5 – Reep4 ....................................................................................................... 167 5.6 – Putative ISD-sensing pathway signaling molecules ................................ 170 5.7 – Putative ISD-sending pathway candidates with no known ISD-interaction partners ............................................................................................................ 171 5.8 – Conclusion .............................................................................................. 179 Chapter 6: Concluding Remarks and Network Analysis 6.1 – Overview: Screening development, analysis and outcome .................... 182 6.2 – Protein-protein interaction network analysis ........................................... 184 6.3 – Predictions and concluding remarks ....................................................... 191 Chapter 7: Material and Methods ....................................................................................... 198 References ................................................................................................................... 204 vi List of Figures and Tables Figure 1.1 Nucleic acid sensors