Massachusetts General HospitalMassachusetts General Hospital

Departments of Medicine and Departments of Medicine and GeneticsGenetics

Harvard Medical SchoolHarvard Medical School

Boston UniversityBoston University

Genomic Analysis of Stress and Genomic Analysis of Stress and InflammationInflammation

PGA components and projectsPGA components and projects

Genetic dissection of Genetic dissection of signal transductionsignal transduction

Host-pathogen Host-pathogen interactions: interactions: Pseudomonas and CFPseudomonas and CF

Definition of Protein Definition of Protein networksnetworks

Macrophage activation Macrophage activation by metabolic and by metabolic and pathogen stressespathogen stresses

Microarray and sequencingMicroarray and sequencing Brian Seed, PhDBrian Seed, PhD

Education and TrainingEducation and Training

Fred Ausubel, PhDFred Ausubel, PhD ProteomicsProteomics

Jack Szostak, PhDJack Szostak, PhD Human Tissue and Animal Human Tissue and Animal

ModelsModels

Mason Freeman, MDMason Freeman, MD BioinformaticsBioinformatics

Temple Smith, PhDTemple Smith, PhD

George Church, PhDGeorge Church, PhD

Components : MicroarraysComponents : Microarrays

Microarray generationMicroarray generation– Human and mouse cDNA arraysHuman and mouse cDNA arrays– Bacterial arraysBacterial arrays– Specialty arrays (e.g., inflammatory gene Specialty arrays (e.g., inflammatory gene

subsets)subsets)

Sequencing and cDNA library generationSequencing and cDNA library generation– Array verification and generationArray verification and generation– Identification of genes isolated by RNA displayIdentification of genes isolated by RNA display– cDNA library productioncDNA library production

Education and TrainingEducation and Training

Genomics training course - hands-on lab Genomics training course - hands-on lab experienceexperience

Web based genomics trainingWeb based genomics trainingVisiting scientist project adviceVisiting scientist project adviceSeminar series in genomicsSeminar series in genomicsBioinformatics training, undergrad work Bioinformatics training, undergrad work

studystudy

ProteomicsProteomics

Development of RNA display technology Development of RNA display technology for identification of protein-protein for identification of protein-protein interactionsinteractions– PDZ domains PDZ domains – Kinase substrate identificationKinase substrate identification

Human Tissue / Animal ModelsHuman Tissue / Animal Models Acquisition and process of human tissues for gene Acquisition and process of human tissues for gene

expression profiling and immunohistochemistryexpression profiling and immunohistochemistry– Atherosclerotic lesions (carotid, coronary, periph.)Atherosclerotic lesions (carotid, coronary, periph.)– Hearts (idiopathic cardiomyopathy, CAD)Hearts (idiopathic cardiomyopathy, CAD)– Lungs (cystic fibrosis, emphysematous)Lungs (cystic fibrosis, emphysematous)

Mouse and Cell ModelsMouse and Cell Models– CD14 and toll receptor null (endotoxin/bacterial CD14 and toll receptor null (endotoxin/bacterial

signaling)signaling)– CD36 null (lipid uptake)CD36 null (lipid uptake)– SR-A null (lipid uptake and bacterial interactions)SR-A null (lipid uptake and bacterial interactions)– Cystic Fibrosis (with Gerry Pier and Fred Ausubel)Cystic Fibrosis (with Gerry Pier and Fred Ausubel)

Animal ModelsAnimal Models Conditional KO miceConditional KO mice

– Based on homologous recombination in bacteriaBased on homologous recombination in bacteria– Conditional alleles generated by site-specific Conditional alleles generated by site-specific

recombinase actionrecombinase action– Flexible, medium throughput technologyFlexible, medium throughput technology

In vivo imaging of transgenic reporter miceIn vivo imaging of transgenic reporter mice– Fluorescence imaging of cells in living animalsFluorescence imaging of cells in living animals– Ear, dorsal skin chamber and cranial windowsEar, dorsal skin chamber and cranial windows– Provides information about the activation of genes Provides information about the activation of genes

in the organismic environmentin the organismic environment

Generation of Mutant CollectionsGeneration of Mutant Collections Pseudomonas Transposon Insertion CollectionPseudomonas Transposon Insertion Collection

– High-quality non-redundant collection of multiple High-quality non-redundant collection of multiple insertions in all non-essential genesinsertions in all non-essential genes

Somatic cell mutant cell linesSomatic cell mutant cell lines– Reporter cell lines that facilitate the rapid Reporter cell lines that facilitate the rapid

identification of mammalian somatic cell mutations identification of mammalian somatic cell mutations affecting signal transductionaffecting signal transduction

– Mutant progeny derived from those linesMutant progeny derived from those lines

BioinformaticsBioinformatics

Database design and managementDatabase design and managementSoftware development (data entry and Software development (data entry and

tracking software)tracking software)Web access of data for internal and Web access of data for internal and

external usersexternal usersData analysis softwareData analysis softwareBioinformatics educationBioinformatics education

Genetic dissection of signal transductionGenetic 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.

Stages in the enrichment of an NF-kB inducer

In Vivo ImagingIn Vivo Imaging

Direct visualization of reporter gene output in Direct visualization of reporter gene output in transgenic animalstransgenic animals

Can be performed using the same reporters used in Can be performed using the same reporters used in high-throughput discovery screenshigh-throughput discovery screens

Allows responding cell populations to be Allows responding cell populations to be identified in vivoidentified in vivo

Responding cells can be culled and phenotypedResponding cells can be culled and phenotyped

Host/Pathogen interactions 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.

Multi-Host Pathogenesis System

humans mice

nematodesplants

insects

P. aeruginosaStrain PA14

112399-4

Bacterial Pathogen

Pseudomonas aeruginosa Pathogenicity-RelatedGenesIdentified by Genetic Screening

# Screened

# Genes

Assay

Host

LettuceLeaf Infection

9

2500

9

5

8

9

051300-1

All Screens

8000

32

>18

22

30

>17

Wax MothKilling

5000

11

not tested

5

not tested

11

C. elegans Killing

5500

13

10

13

11

10

Number of genes identified, which when mutated result in decreased virulence on the hosts indicated on the left.

% n

em

ato

de

s k

ille

d

Hours of Feeding onP. aeruginosa

P. aeruginosa Kills C. elegans and Colonizes the C. elegans Intestine

0

20

40

60

80

100

0 20 40 60 80

E. coli

P. aeruginosa

E. coli

P. aeruginosa

P. aeruginosaP. aeruginosa Pathogenicity-Related Pathogenicity-Related Genes Identified by Screening in Genes Identified by Screening in Model HostsModel Hosts 32 Pathogenicity genes identified out of 8,000 32 Pathogenicity genes identified out of 8,000

random transposon insertions screened. random transposon insertions screened.

Need to screen 30,000 random insertions to reach Need to screen 30,000 random insertions to reach saturation.saturation.

Obtain full set of virulence-related genes by Obtain full set of virulence-related genes by screening in several model invertebrate hosts. screening in several model invertebrate hosts.

Because screening for mutant phenotypes is rate Because screening for mutant phenotypes is rate limiting, construct non-redundant insertion limiting, construct non-redundant insertion library (4800 nonessential genes) to screen the library (4800 nonessential genes) to screen the rest of the genome.rest of the genome.

Advantages of a Non-Redundant Advantages of a Non-Redundant

LibraryLibrary Simplifies screening in multiple hosts.

Multiple insertion alleles of certain genes will be useful for confirming the phenotypes of insertion mutations.

> 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.

Raw Data Archive- sequences- trace files

public sequences

patent sequences

PA14 genomeIST sequences

PA14-specific Sequence

PAO1

BLAST/FASTA

trashsequences

novel PA14 specific

mutant priority definition

1 annotated ORF2 not annot. ORF, putative ORF3 annotated ORF, put. Promoter4 not annot. ORF, not put. ORF, put. Promoter5 not annot. ORF, not put. ORF, not put. Promoter

new sequences

processednew sequences

archive raw data - transposon clipping- N-stripping

Processing:

n

ypresent?

y

n

present?y

n

present?y

n

present?

y

n

contaminant?

PA14 genomeIST sequences.

PA14 non-genome project sequences

PA14 redundantintermediate library

Building the non-redundant PA14 mutant library

select seq. for inclusion in NR1PA14

tested inany host?

PA14 non-redundantlibrary (NR1PA14)

Siblingpresent inNR1PA14?

library of NR1PA14 redundancies

y

n n

y

combine these criteria for mutant priority definition:

•homology to pathogenicity factors•homology to regulatory proteins•homology to house keeping genes•assignment to a pathway containing pathogenicity factors•coordinate of the mutation along the 5’-3’ axis compared to other mutants targeted in the same gene•PA14-specific?

•annotated ORF•putative ORF•put. Promoter for annotated ORF•not annot. ORF, put. Promoter•not put. ORF, not put. Promoter

Definition of protein networks by RNA displayDefinition 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

RNA-Protein FusionsRNA-Protein Fusions

P5'mRNAProteinN Term

P

P

P

PP

mRNA Display

Decoding Protein-Protein Interactions

Challenge:to 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

Deconvoluting mRNA-Protein Fusion Deconvoluting mRNA-Protein Fusion Targets with MicroarraysTargets with Microarrays

cDNA LibrarymRNA LibrarymRNA-Protein Fusion LibraryProtein Activity SelectionPCRMicroarray

Cellular Library Features

No cloningRandomly primed cDNADirect assembly of library in vitroLibraries are large enough to contain all

possible start and end points for every protein fragment

Cellular Libraries: Selections

Cellular protein

domain library

Target(bait)

Select: 1-4

rounds

PCR

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

Other Selections in ProgressOther Selections in Progress

- PDZ domains binding known targets- PDZ domains binding known targets- coiled-coil partners- coiled-coil partners- calmodulin binding proteins- calmodulin binding proteins

Macrophage ActivationMacrophage 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.

Fig. 2 LPS signaling pathways

Fig. 4

ES cell colony

Undifferentiated, Pluripotent ES Cells

Differentiation into Embryoid Bodies

Harvesting of Embryoid Bodiesand isolation of macrophages

ES differentiated macrophage

Embryoid Body

Differentiation of Embryonic Stem Cells into Macrophages

Embryonic fibroblast layer

ES cells are cultured on embryonic fibroblast feederlayers in media containing LIF-D to inhibit differentiation.

ES cells are cultured in LIF-D free medium for 1-3 hand then trypsinized to form a single cell suspension.ES cells are cultured in semi-solid methyl cellulosemedia containing cytokines for 10 days to promotethe formation of embryoid bodies rich in myeloid cells.

Embryoid bodies are gently washed from the methylcellulose media and cultured in petri dishes in mediacontaining myeloid growth factors. Macrophages producedfrom the embryoid body are isolated by their ability toadhere to bacteriologic plastic.

+ OxLDL + LDLA.

B.

wt cl 3 P388D10

200

400

600

800

PPAR null

125

I-O

xL

DL

de

gra

da

tio

n

cl 4 cl 5

RNA display RNA display

Synthesis of RNA-Protein Fusions

6 7 85 9

987

654321

9

8

//

6 7 854 9

Messenger RNALinker - Puromycin

10

6 7 854 9//

mRNA-Protein Fusion with stable amide linkage

10

987

654321

In vitrotranslation

P

P

P

//

peptide

tRNA

Puromycin