Massachusetts General Hospital

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Genomic Analysis of Stress and Inflammation

• Massachusetts General Hospital
• Departments of Medicine and Genetics
• Harvard Medical School
• Boston University

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PGA components and projects
Projects
Component Centers

• Genetic dissection of signal transduction
• Host-pathogen interactions Pseudomonas and CF
• Definition of Protein networks
• Macrophage activation by metabolic and pathogen
  stresses

• Microarray and sequencing Brian Seed, PhD
• Education and Training
• Fred Ausubel, PhD
• Proteomics
• Jack Szostak, PhD
• Human Tissue and Animal Models
• Mason Freeman, MD
• Bioinformatics
• Temple Smith, PhD
• George Church, PhD

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Components Microarrays

• Microarray generation
• Human and mouse cDNA arrays
• Bacterial arrays
• Specialty arrays (e.g., inflammatory gene
  subsets)
• Sequencing and cDNA library generation
• Array verification and generation
• Identification of genes isolated by RNA display
• cDNA library production

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Education and Training

• Genomics training course - hands-on lab
  experience
• Web based genomics training
• Visiting scientist project advice
• Seminar series in genomics
• Bioinformatics training, undergrad work study

5
Proteomics

• Development of RNA display technology for
  identification of protein-protein interactions
• PDZ domains
• Kinase substrate identification

6
Human Tissue / Animal Models

• Acquisition and process of human tissues for gene
  expression profiling and immunohistochemistry
• Atherosclerotic lesions (carotid, coronary,
  periph.)
• Hearts (idiopathic cardiomyopathy, CAD)
• Lungs (cystic fibrosis, emphysematous)
• Mouse and Cell Models
• CD14 and toll receptor null (endotoxin/bacterial
  signaling)
• CD36 null (lipid uptake)
• SR-A null (lipid uptake and bacterial
  interactions)
• Cystic Fibrosis (with Gerry Pier and Fred
  Ausubel)

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Animal Models

• Conditional KO mice
• Based on homologous recombination in bacteria
• Conditional alleles generated by site-specific
  recombinase action
• Flexible, medium throughput technology
• In vivo imaging of transgenic reporter mice
• Fluorescence imaging of cells in living animals
• Ear, dorsal skin chamber and cranial windows
• Provides information about the activation of
  genes in the organismic environment

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Generation of Mutant Collections

• Pseudomonas Transposon Insertion Collection
• High-quality non-redundant collection of multiple
  insertions in all non-essential genes
• Somatic cell mutant cell lines
• Reporter cell lines that facilitate the rapid
  identification of mammalian somatic cell
  mutations affecting signal transduction
• Mutant progeny derived from those lines

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Bioinformatics

• Database design and management
• Software development (data entry and tracking
  software)
• Web access of data for internal and external
  users
• Data analysis software
• Bioinformatics education

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Genetic dissection of signal transduction

• To create reporter cell lines that allow
  identification of genes promoting activation of
  stress and inflammation pathways.
• To develop and exploit automated sib selection
  strategies to identify new molecules that
  activate stress and inflammation
  pathways.
• To use microarray analysis to understand the
  genotypes of mutant cell lines bearing lesions in
  stress and inflammation signal transduction
  pathways.

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Stages in the enrichment of an NF-kB inducer
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In Vivo Imaging

• Direct visualization of reporter gene output in
  transgenic animals
• Can be performed using the same reporters used in
  high-throughput discovery screens
• Allows responding cell populations to be
  identified in vivo
• Responding cells can be culled and phenotyped

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Host/Pathogen interactions

• The mucoid derivatives of PA14 from Aim 2 will be
  used to infect CF mice. The P. aeruginosa
  microarray (Aim 1) and a murine microarray
  consisting of the currently available UniGene
  clusters will be used to determine gene
  expression profiles for the pathogen and the
  host, respectively, before and during the
  infection process.
• Mucoid derivatives of P. aeruginosa PA14 mutants
  attenuated for pathogenicity in model
  non-vertebrate hosts will be introduced into CF
  mice to identify virulence-related factors
  required for infection of the CF lung.
• P. aeruginosa PA14 mutants identified in Aim 4
  that display defects in CF lung pathogenesis will
  be used to infect CF mice and expression of both
  P. aeruginosa and mouse genes will be analyzed
  using the P. aeruginosa and mouse DNA
  microarrays.

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Bacterial Pathogen
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P. aeruginosa Kills C. elegans and Colonizes the
C. elegans Intestine
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P. aeruginosa
E. coli
80
60
nematodes killed
40
20
0
0
20
40
60
80
Hours of Feeding on P. aeruginosa
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P. aeruginosa Pathogenicity-Related Genes
Identified by Screening in Model Hosts

• 32 Pathogenicity genes identified out of 8,000
  random transposon insertions screened.
• Need to screen 30,000 random insertions to reach
  saturation.
• Obtain full set of virulence-related genes by
  screening in several model invertebrate hosts.
• Because screening for mutant phenotypes is rate
  limiting, construct non-redundant insertion
  library (4800 nonessential genes) to screen the
  rest of the genome.

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Advantages of a Non-Redundant Library

• Simplifies screening in multiple hosts.
• Multiple insertion alleles of certain genes will
  be useful for confirming the phenotypes of
  insertion mutations.
• gt 80 savings in time for each screen in a model
  host.
• The library can be expanded until it is
  saturated.
• Obtain information about genes that are NOT
  required for pathogenesis as well as genes that
  are.
• The PCR products used to construct the library
  can be used to synthesize a micro-array of
  (non-essential) ORFs for PA14 expression studies.

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Raw Data Archive - sequences - trace files
archive raw data
BLAST/FASTA
present?
present?
present?
present?
y
y
y
y
mutant priority definition 1 annotated ORF 2 not
annot. ORF, putative ORF 3 annotated ORF, put.
Promoter 4 not annot. ORF, not put. ORF, put.
Promoter 5 not annot. ORF, not put. ORF, not put.
Promoter
n
n
n
n
contaminant?
y
n
novel PA14 specific
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Building the non-redundant PA14 mutant library
select seq. for inclusion in NR1PA14
PA14 redundant intermediate library
tested in any host?
Sibling present in NR1PA14?
n
n
combine these criteria for mutant priority
definition
y
y
PA14 non-redundant library (NR1PA14)
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Definition of protein networks by RNA display

• To create a cellular protein-RNA fusion library
  from pooled mRNA from normal human and mouse
  tissues
• To use isolated domains from proteins transducing
  stress and inflammation pathway signals to
  identify interaction partners of those
  proteins
• To automate the detection of interactions between
  signal transduction proteins and their target
  proteins.
• To make slick slides for presentations

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RNA-Protein Fusions
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mRNA Display
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Decoding Protein-Protein Interactions
Challengeto identify all binding partners of a
given bait protein
Solution pass a library of cellular mRNA fusions
over the protein identify which fusions bind to
the bait protein
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Deconvoluting mRNA-Protein Fusion Targets with
Microarrays
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Cellular Library Features

• No cloning
• Randomly primed cDNA
• Direct assembly of library in vitro
• Libraries are large enough to contain all
  possible start and end points for every protein
  fragment

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Cellular Libraries Selections
Cellular protein domain library
Target (bait)
Select 1-4 rounds
PCR
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Identification of Kinase Substrates
Library of fusions prepared from cellular RNA
Phosphorylate fusions with kinase in vitro
Immunoprecipitate phosphorylated fusions with
anti-phosphotyrosine Ab
PCR RNA from phosphorylated fusions
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Other Selections in Progress- PDZ domains
binding known targets- coiled-coil partners-
calmodulin binding proteins
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Macrophage Activation

• To perform comparative gene expression studies
  assessing the impact of key proteins in
  inflammatory and stress response pathways, using
  macrophages taken from wild type and knock-out
  mice.
• To explore concordances between murine and human
  macrophage expression, and to establish baseline
  profiles revealing the consequences of various
  sample collection practices.
• To analyze gene expression in aortas taken from
  normal and apo E null mice and from coronary
  arteries of mice following allogeneic heart
  transplantation
• To conduct parallel investigations on the gene
  expression profiles of human carotid
  endarterectomy, coronary endarterectomy, and
  heart transplant specimens, and to establish, if
  possible, the characteristic gene clustering
  features of these conditions.

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LPS signal transduction pathway
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OxLDL
LDL
A.
B.
800
600
400
200
0
wt
cl 3
P388D1
cl 4
cl 5
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RNA display