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A GENOME-WIDE siRNA SCREEN IDENTIFIES POSITIVE AND NEGATIVE REGULATORS OF NOD2 SIGNALING. Gabriel Nuñez March 7 th , 2013 Department of Pathology University of Michigan. Outline. Introduction : NOD2 and Crohn’s Disease
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A GENOME-WIDE siRNA SCREEN IDENTIFIES POSITIVE AND NEGATIVE REGULATORS OF NOD2 SIGNALING Gabriel Nuñez March 7th, 2013 Department of Pathology University of Michigan
Outline • Introduction : NOD2 and Crohn’s Disease • Year 1 – Genome-wide siRNA Screen to identify regulators of NOD2 signaling • Year 2 - Exome sequencing of a small cohort of Crohn’s Disease patients in an attempt to identify clinically relevant components of the NOD2 signaling pathway
NOD2 and Crohn’s Disease SNP8 R702W SNP12 G908R SNP13 1007fs
(+ MDP) (+ MDP) (+ MDP) Genome-wide siRNA Screening Strategy
siRNA Screen Schematic siRNA Library (20nM, 48 hours) Total - 68 plates - 18,110 genes IL-8 ELISA Assay (n=2) Fluorescence based Cell Viability Assay (n=3) Luminescence based NF-κB Assay (n=3)
NF-κB Luciferase inhibition and Viability Data Normal (Bell) Distribution (RIPK2) Pos Regs Neg Regs (Non-targeting) Normal (Bell) Distribution (Non-targeting) (Lysed)
siRNA screen identifies known components of the NOD2 signaling pathway (Literature)
Known Components of the NF-kB signaling pathways such as the Proteasome and Nuclear Pore were hits in the screen Ref: Warner et al., Sci. Signal. 6, rs3 (2013)
Genes that interact with core components of the NOD2 signaling pathway were hits in the screen Ref: Warner et al., Sci. Signal. 6, rs3 (2013)
Comparison of Luciferase and IL-8 assay hits Top 500 Negative Regulators Top 500 Positive Regulators Positive Regulators (906) Luc IL-8 Luc IL-8 Luciferase IL-8 Overlap 94 genes Legend Increased Signal Not significantly changed Decreased Signal Genes Negative Regulators (928) Luciferase IL-8 Overlap 77 genes
Pos Regs CD/IBD loci taken from Jostinset al., (2012). Nature. 491:119. 9 of these genes were also IL-8 hits, 9 more IL-8 specific hits are present in CD/IBD risk loci (not shown)
Are any of these putative NOD2 regulators involved in Crohn’s Disease? n = 71 U of M CD patients Michigan IBD patient databank (Dr. Ellen Zimmermann) Strategy: select CD patients whose monocytes are impaired in their response to MDP despite WT NOD2 Patient stratification based on MDP response and NOD2 genotype Group 1 – WT NOD2 that responded normally to MDP Group 2 – homozygous for CD associated NOD2 mutations – MDP unresponsive Group 3* – WT NOD2 but unresponsive to MDP (8 patients – Exome sequencing) * HYPOTHESIS: some other component(s) of the NOD2 signaling pathway may be disrupted
Ongoing follow up of Crohn’s disease SNPs identified as validated NOD2 regulators • 1021 coding SNPs were identified in genes that hits in our siRNA screen • 135 coding SNPs were identified in genes that were validated hit from our secondary siRNA screen • mass array genotyping of 72 prioritized SNPs from 43 genes is being carried out in a larger cohort of • nearly 6000 CD patients and healthy controls to identify risk alleles (Judy Cho, NIDDK IBD consortium)
Overall Conclusions from the siRNA screen • - we identified and validated 211 genes that either positively or • negativelyregulate NOD2 signaling • - 82 of the validated hits affected both NF-κB induction • and IL-8 secretion • - 22 genes identified as NOD2 regulators in our screen are • found in loci known to be associated with CD risk by GWAS • consistent with an important role for the NOD2 pathway in • the pathogenesis of Crohn’s disease. • - SNPs from 43 genes validated as NOD2 regulators are being • genotyped in a cohort of nearly 6000 Crohn’s disease patients • and healthy controls