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 'Interferon Stimulatory DNA' or 'ISD') 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.
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.
|Advisor:||Hacohen, Nir, Turley, Shannon|
|Commitee:||Benoist, Christophe, Kagan, Jonathan, Kramnik, Igor|
|Department:||Biology: Medical Sciences, Division of|
|School Location:||United States -- Massachusetts|
|Source:||DAI-B 74/10(E), Dissertation Abstracts International|
|Keywords:||Cytosol, DNA sensing, Innate immunity, RNAi|
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