Defining a Role for SARM

1
Defining a Role for SARM in Pathogen
Resistance and PA14 Unigene Library
Construction Nicole T. Liberati Group Meeting
6/10/02
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Requirement for a p38 MAP kinase signaling
pathway in C. elegans immunity
PATHOGEN
???
ESP-8 (MAPKKK)
ESP-2 (MAPKK)
PMK-1 (p38 MAPK)
???
??? IMMUNE RESPONSE
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PA14 Slow Killing Assay
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Longevity Assay
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Slow Killing Assay at 20C
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Genomic Structure of F13B10.1
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SARM Long
SARM Short
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No C. elegans or D. melanogaster mutants available
SARM Expression patterns
C. elegans expressed at all ages - highest in
oocytes and embryos D. Melanogaster (Ect4) a)
repressed by Dorsal b) expressed in
the dorsal ectoderm H. sapiens Multiple Tissue
Northern
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Mink et al Genomics(2001) 74234-44
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hSARM Experimental Overview

• Mammalian Studies
• 1) siRNA in immune cells
• 2) hSARM overexpression in TLR/NFKB/IRF3
  reporter assays
• 3) hSARM targeted deletion in mice
• 4) hSARM homozygous null Embryonic Stem cells
• C. elegans Studies
• 1) phospho-p38 Western
• 2) F13B10.1 deletion screen
• 3) GFP expression study
• 4) F13B10.1 overexpression analysis
• WT or TIR mutant rescue esp phenotype in
  RNAi-treated worms
• DKs sek-1 reporter strains/p38 western
• 5) Interactome

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hSARM Experimental Overview

• Mammalian Studies
• 1) siRNA in immune cells
• 2) hSARM overexpression in TLR/NFKB/IRF3
  reporter assays
• 3) hSARM targeted deletion in mice
• 4) hSARM homozygous null Embryonic Stem cells
• C. elegans Studies
• 1) phospho-p38 Western
• 2) F13B10.1 deletion screen
• 3) GFP expression study
• 4) F13B10.1 overexpression analysis
• WT or TIR mutant rescue esp phenotype in
  RNAi-treated worms
• DKs sek-1 reporter strains/p38 western
• 5) Interactome

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Mouse adapter protein northern blots
BMDM DC RAW264.7
BMDM DC RAW264.7
___ ___ ___ - - - LPS
___ ___ ___ - - - LPS
TRIF SARM b-actin
MyD88 TRAM b-actin
MAL gives very faint hardly detectable bands
H. Byorkbaka and M. Freeman
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hSARM Experimental Overview

• Mammalian Studies
• 1) siRNA in immune cells
• 2) hSARM overexpression in TLR/NFKB/IRF3
  reporter assays
• 3) hSARM targeted deletion in mice
• 4) hSARM homozygous null Embryonic Stem cells
• C. elegans Studies
• 1) phospho-p38 Western
• 2) F13B10.1 deletion screen
• 3) GFP expression study
• 4) F13B10.1 overexpression analysis
• WT or TIR mutant rescue esp phenotype in
  RNAi-treated worms
• DKs sek-1 reporter strains/p38 western
• 5) Interactome

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TLR2/1 or 2/6 TLR3
TLR4
TLR5/7/8/9
MD2
?
MyD88 MyD88? TRIF/ Mal
MyD88 ? MyD88 TICAM1
Mal
?

NFkB IRF3
NFkB
NFkB
NFkB IRF3
TNFa IFNb IL1 RANTES IL6 IL8
TNFa IL1 IL6 IL8
TNFa IFNb IL1
RANTES IL6 IL8
TNFa IL1 IL6 IL8
ONeill and Fitzgerald
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Cloning the human SARM cDNA
IMAGE clone
EagI
EagI
Corrected 240 bp PCR product Cloned into pBSKII
to generate hSARM 240/pBSKII
EagI
A partial EagI/NheI IMAGE clone Fragment cloned
into hSARM240/pBSKII to generate hSARM/pBSKII
EagI
NheI
PCR products of full length cDNA subcloned into
pUNI system and FLAG fusion vector.
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TIR domain Crystal Structure
TLR2
TIR consensus
TLR5
hSARM
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hSARM Experimental Overview

• Mammalian Studies
• 1) siRNA in immune cells
• 2) hSARM overexpression in TLR/NFKB/IRF3
  reporter assays
• 3) hSARM targeted deletion in mice
• 4) hSARM homozygous null Embryonic Stem cells
• C. elegans Studies
• 1) phospho-p38 Western
• 2) F13B10.1 deletion screen
• 3) GFP expression study
• 4) F13B10.1 overexpression analysis
• WT or TIR mutant rescue esp phenotype in
  RNAi-treated worms
• DKs sek-1 reporter strains/p38 western
• 5) Interactome

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Targeted Deletion Strategy
5 arm
3 arm
neo
TK
Transfect C129 ES cells with targeting construct
Positive selection (neo resistance) and Negative
selection (thymidine kinase)
Southern screen for integrated selection marker
Inject mutant ES cells into Black6 blastocyst
generating chimeric embryos that are introduced
into foster mothers
Search for germ line transmission among offspring
of chimeric founders
Breed heterzygous offspring to generate
homozygous null progeny-PCR screening
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SARM Mouse Targeted Deletion Mutant Strategy
15kb
WT
E
E
1
3
2
5
4
6
8
9
7
MT
E
E
1
neo
8 kb
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200 pg control
200 pg control
20 pg control
20 pg control
Ladder
HindIII
Ladder
BamHI
HindIII
BamHI
EcoRI
EcoRI
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hSARM Experimental Overview

• Mammalian Studies
• 1) siRNA in immune cells
• 2) hSARM overexpression in TLR/NFKB/IRF3
  reporter assays
• 3) hSARM targeted deletion in mice
• 4) hSARM homozygous null Embryonic Stem cells
• C. elegans Studies
• 1) phospho-p38 Western
• 2) F13B10.1 deletion screen
• 3) GFP expression study
• 4) F13B10.1 overexpression analysis
• WT or TIR mutant rescue esp phenotype in
  RNAi-treated worms
• DKs sek-1 reporter strains/p38 western
• 5) Interactome

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RETROVIRAL INSERTIONAL MUTAGENESIS
in diploid loci
paternal maternal
mutagenic virus
no phenotype
heterozygous at viral insertion site
spontaneous LOH (loss of heterozygosity)
induction of MR/ non-disjunction selection for
homozygosity at integration site
homozygous at viral insertion site
phenotype ..
selection for phenotype
locate retroviral insertion site to identify
disrupted gene responsible for phenotype
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Independent Recombination Between LoxP Variants
loxP
loxP
loxP
loxP
R1
R2
TK
IRES
LOH
loxP
loxP
loxP
loxP
R1

R2
TK
IRES
R1
R2
TK
IRES
Tat - cre
R1
R1
R2
R2
R1
R1
R2
R2
R1 , R2 , TK -
R1
R2
R1
R2
selection for phenotype
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hSARM Experimental Overview

• Mammalian Studies
• 1) siRNA in immune cells
• 2) hSARM overexpression in TLR/NFKB/IRF3
  reporter assays
• 3) hSARM targeted deletion in mice
• 4) hSARM homozygous null Embryonic Stem cells
• C. elegans Studies
• 1) phospho-p38 Western
• 2) F13B10.1 deletion screen
• 3) GFP expression study
• 4) F13B10.1 overexpression analysis
• WT or TIR mutant rescue esp phenotype in
  RNAi-treated worms
• DKs sek-1 reporter strains/p38 western
• 5) Interactome

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Phospho-p38 Western of RNAi-treated worm lysates
RNAi Vector Control
RNAi Vector Control
F13B10.1 RNAi
Control lysate
pmk RNAi
pmk RNAi
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hSARM Experimental Overview

• Mammalian Studies
• 1) siRNA in immune cells
• 2) hSARM overexpression in TLR/NFKB/IRF3
  reporter assays
• 3) hSARM targeted deletion in mice
• 4) hSARM homozygous null Embryonic Stem cells
• C. elegans Studies
• 1) phospho-p38 Western
• 2) F13B10.1 deletion screen
• 3) GFP expression study
• 4) F13B10.1 overexpression analysis
• WT or TIR mutant rescue esp phenotype in
  RNAi-treated worms
• DKs sek-1 reporter strains/p38 western
• 5) Interactome

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C. Elegans Deletion Screen Overview
A/B PCR Screen
PspGI
C/D PCR Screen
PspGI
PspGI
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Deletion Screen Overview
- -
PspG1
A/B
C/D
A/B
C/D
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- - - PspGI
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Sequencing a) correct part of genome
b) quality poor
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hSARM Experimental Overview

• Mammalian Studies
• 1) siRNA in immune cells
• 2) hSARM overexpression in TLR/NFKB/IRF3
  reporter assays
• 3) hSARM targeted deletion in mice
• 4) hSARM homozygous null Embryonic Stem cells
• C. elegans Studies
• 1) phospho-p38 Western
• 2) F13B10.1 deletion screen
• 3) GFP expression study
• 4) F13B10.1 overexpression analysis
• WT or TIR mutant rescue esp phenotype in
  RNAi-treated worms
• DKs sek-1 reporter strains/p38 western
• 5) Interactome

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F13B10.1 GFP Expression Constructs
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hSARM Experimental Overview

• Mammalian Studies
• 1) siRNA in immune cells
• 2) hSARM overexpression in TLR/NFKB/IRF3
  reporter assays
• 3) hSARM targeted deletion in mice
• 4) hSARM homozygous null Embryonic Stem cells
• C. elegans Studies
• 1) phospho-p38 Western
• 2) F13B10.1 deletion screen
• 3) GFP expression study
• 4) F13B10.1 overexpression analysis
• WT or TIR mutant rescue esp phenotype in
  RNAi-treated worms
• DKs sek-1 reporter strains/p38 western
• 5) Interactome

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SARM Interactors
Mkk4 interactor
Personal communication, Pierrot-Vidalain and M.
Vidal
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Acknowledgements
Ausubel Lab Dennis Kim Rhonda Feinbaum Dan
Lee Jas Villanueva Sachiko Miyata Jonathan
Urbach Tao Wei Rubin Lab Chris
Sassetti Mekalanos Lab Gene Targeting Facility
Freeman Lab Scott Kim Harry Byörkbacka Francois G
olenbock Lab Kate Fitzgerald Vidal Lab Pierrot
Olivier Ruvkun Lab Sylvia Lee Ho Yi Wei
Ching Seed Lab Naifang Lu Yi Yang
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TraSH Methodology Probe Production
5) Reverse Transcription with fluorophore to
produce labeled cDNA 6) Hybridize differentially
labeled probe pools to microarray
Sassetti, C. M., Boyd, D. H., and Rubin, E. J.,
(2001). PNAS 9812712-12717
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TraSH Methodology Detection of the Presence or
Absence of Tn Mutants in a Pool of Mutants
Sassetti, C. M., Boyd, D. H., and Rubin, E. J.,
(2001). PNAS 9812712-12717
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pMFLGM.GB- 2xT7 (pMAR 2XT7)
lacZ
Mariner Transposase
?pir-dependent ori
29 bp IR
T7 Promoter
pMAR 2xT7
Gm resistance
T7 Promoter
Tra genes
29 bp IR
Amp resistance
Inserted into the PA14 genome
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TraSH PCR products
1 primer
2 primers
Mar1xT7 DNA
Mar1xT7 DNA
Mutant 1
Mutant 1
Mutant 2
Mutant 3
Mutant 4
Mutant 5
Mutant 2
Mutant 3
Mutant 4
Mutant 5
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Mar1xT7 DNA
Mar1xT7 RNA
Mar1xT7 PCR
Mutant 1
control
Mutant 2
Mutant 3
Mutant 4
Mutant 5
2 primer
Ladder
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1 primer control
1 primer control
Mutant 3
Mutant 1
Mutant 2
Mutant 4
Mutant 5
control
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Gentimycin Titration
Irgasan Titration