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A GENOME-WIDE siRNA SCREEN IDENTIFIES POSITIVE AND NEGATIVE
REGULATORS OF NOD2 SIGNALING
Gabriel NuñezMarch 7th, 2013
Department of PathologyUniversity 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 Introduction
NOD2 and Crohn’s Disease
SNP8R702W
SNP12G908R SNP13
1007fs
Genome-wide siRNA Screening Strategy
0
50
100
150
200
250
300
350
400
450
Assay Baseline knockdown ofPOSITIVEregulator
knockdown ofNEGATIVEregulator
Luciferase Reporter activity
(+ MDP)
(+ MDP)
(+ MDP)
siRNA Screen Schematic
Fluorescence basedCell Viability Assay (n=3)
Luminescence basedNF-κB Assay (n=3)
IL-8 ELISAAssay (n=2)
siRNA Library (20nM, 48 hours)
Total - 68 plates - 18,110 genes
NF-κB Luciferase inhibition and Viability Data
Normal (Bell) Distribution
Normal (Bell) Distribution
(RIPK2)
(Non-targeting)
(Non-targeting)
(Lysed)
PosRegs
NegRegs
siRNA screen identifies known components of the NOD2 signaling pathway (Literature)
Ref: Warner et al., Sci. Signal. 6, rs3 (2013)
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
Comparison of Luciferase and IL-8 assay hits
Positive Regulators (906)
Negative Regulators (928)
Luciferase IL-8
Luciferase IL-8
Overlap94 genes
Overlap77 genes
Top 500Positive Regulators
Top 500Negative Regulators
Luc IL-8 Luc IL-8
Legend
Increased Signal
Not significantly changed
Decreased Signal
Gen
es
non Targe
ting contro
lRIPK2
RELA
UBE2D3
CCL13
DONSON
IL2RA
TNFS
F18
LIME1
DDX6
GRIN3BCAD
DAP3CIRBP
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
13 Validated LUC hits are present within loci associated with CD/IBD by GWAS
siRNA treatment
Nor
mal
ized
LUC
indu
ction
upo
n M
DP sti
mul
ation
NegRegs
CD/IBD loci taken from Jostins et 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)
PosRegs
47
17
7
NOD2 genotyping
NOD2 +/+NOD2 +/-NOD2 -/-
Are any of these putative NOD2 regulators involved in Crohn’s Disease?
Strategy: select CD patients whose monocytes are impaired in their response to MDP despite WT NOD2
Patient stratification based on MDP response and NOD2 genotypeGroup 1 – WT NOD2 that responded normally to MDP Group 2 – homozygous for CD associated NOD2 mutations – MDP unresponsiveGroup 3* – WT NOD2 but unresponsive to MDP (8 patients – Exome sequencing)
* HYPOTHESIS: some other component(s) of the NOD2 signaling pathway may be disrupted
n = 71 U of M CD patientsMichigan IBD patient databank(Dr. Ellen Zimmermann)
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)
non Targe
ting contro
l
IL27RA
NMUR2
CDC25CRGL3
DONSON
ACRV1
loc51252
flj23049
ADAMTS8
GRIN3B
ZNF2
95
Genome A
ve0
0.20.40.60.8
11.21.41.61.8
2
23 Luciferase hits
Rela
tive
Nor
m L
UC
indu
ction
(M
DP)
non targe
ting contro
l
DONSON
GABRA6
ACRV1M
C3RGPT
CDC25C
IL27RA
C1QTNF8
ADAMTS
8
PPAP2CPHF3
RGS12
ZNF2
95
GRIN3B
SUSD
2
ARRDC1
loc5
1252
DYX1C1
MSL
N
tralp
ush
0
10
20
30
40
50
60
70
40 IL-8 hits
Rela
tive
Nor
m IL
-8
indu
ction
(MDP
)
Overall Conclusions from the siRNA screen
- we identified and validated 211 genes that either positively or negatively regulate 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 GWASconsistent with an important role for the NOD2 pathway inthe pathogenesis of Crohn’s disease.
- SNPs from 43 genes validated as NOD2 regulators are beinggenotyped in a cohort of nearly 6000 Crohn’s disease patients and healthy controls