Cbio pathway analysis, modeling, data analysis

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TGF-beta signaling pathway analysis
Massague lab(TGF-beta)
Cbiopathway analysis, modeling, data analysis
Urs RutishauserHakim Djaballah(siRNA / HTS
facility)
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TGF-beta pathwayTGFb
TGF?1
TGF? RII Type II receptor
Alk5 Type I receptor
Joan Massagué
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TGF-beta pathwayBMP
Activin RII Type II receptor
Alk2 Type I receptor
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TGF-beta pathway
42 different ligands (incl. antagonists)
2 I-Smads
5 different Type II receptors
7 different Type I receptors
1 Co-Smad
6 R-Smads
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Complexity at the ligand/receptor level
Gene expression program 1
Gene expression program 2
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Questions

• What other components are involved?
• Identify new pathway members
• Understand signaling mechanisms Crosstalk
  between different receptors
• Explanation for role-reversal of TGF-beta from
  growth-suppressor to activator?

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Biological assay

• Activation and perturbation (siRNA)
• Test different combinations of ligands and
  overexpressed / repressed receptors
• Live-cell assays (time course)
• High-throughput assays

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Assay system

• Activation of the pathway
• Phosphorylation / localization of Smad1/2 (in
  parallel)
• Signal transduction to the nucleus
• Transcription of Smad1/2-responsive targets genes

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Probing the TGF-beta pathwayStimulation with
different combinations and concentrations of
ligands // RNAi knockdowns
42 different ligands
cytoplasm
5 different Type II receptors
Smad1 (BMP)Smad2 (TGF-beta)
7 different Type I receptors
nucleus
activated Smad complex
co-activator or co-repressor
cell-specific DNA-binding cofactors
Smad1/2-responsive genes
Smad4
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Probing the TGF-beta pathwayStimulation with
different combinations and concentrations of
ligands // RNAi knockdowns
42 different ligands
cytoplasm
5 different Type II receptors
Smad1 (BMP)Smad2 (TGF-beta)
7 different Type I receptors
nucleus
1
activated Smad complex
co-activator or co-repressor
cell-specific DNA-binding cofactors
Smad1/2-responsive genes
Smad4
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Pathway activationnuclear translocation of
Smad-FP fusions Option 1 (red/green) Option 2
(blue/yellow) mRFP1-Smad1 Cerulean-Smad1
EGFP-Smad2 Venus-Smad2
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Probing the TGF-beta pathwayStimulation with
different combinations and concentrations of
ligands // RNAi knockdowns
42 different ligands
cytoplasm
5 different Type II receptors
Smad1 (BMP)Smad2 (TGF-beta)
7 different Type I receptors
nucleus
1
activated Smad complex
co-activator or co-repressor
2
cell-specific DNA-binding cofactors
Smad1/2-responsive genes
Smad4
1
2
Pathway activationnuclear translocation of
Smad-FP fusions Option 1 (red/green) Option 2
(blue/yellow) mRFP1-Smad1 Cerulean-Smad1
EGFP-Smad2 Venus-Smad2
Transcriptiondetect Smad-responsive genes mRFP1
(red) coupled to Smad1-specific promoterVenus
(yellow) coupled to Smad2-specific
promoteralternatively assay Smad-responsive
genes by qPCR
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Assay details
Smad localization assay Cerulean-Smad1
(cyan)Venus-Smad2 (yellow) AlternativesmRFP-Sm
ad1 (red)EGFP-Smad2 (green)
Transcriptional assay
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Assay details
Smad localization assay Cerulean-Smad1
(cyan)Venus-Smad2 (yellow) AlternativesmRFP-Sm
ad1 (red)EGFP-Smad2 (green)
Transcriptional assay Low temporal
resolutionPAI-1-mRFP1 (red)Vent2-Venus
(yellow) Higher temporal resolutionPAI-1-nitrore
ductase (red)Vent2-beta-lactamase (green to blue)
U87MG glioblastoma cell line assay has to
be expanded to multiple cell lines
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Progress

• Lab work
• - make DNA constructs / plasmids
• - test constructs in cells
• make cell lines
• test siRNA
• adapt to HTS

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Cytoscape
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HaCaT-EGFP-Smad2, fixed
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EGFP-Smad2 in HaCaTlive cell imaging,
unstimulated
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EGFP-Smad2 in HaCaT keratinocyteslive cell
imaging, stimulated (TGF-beta 1 h)
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EGFP-Smad1 in HaCaTlive cell imaging,
unstimulated
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EGFP-Smad1 in HaCaTlive cell imaging, stimulated
with BMP 1h
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U87MG Smad2 stimulated
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mRFP1-Smad2
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In Cell analyzer
High-throughput automated individual-cell image
acquisition and analysis Multiple fluorescent
reporters
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Signaling pathway analysisCombination of
computational and wet-lab biology
Genomic data Expression data Computational
models Literature mining
High-throughput experiments Test models
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• Lab work
• - make DNA constructs / plasmids
• - test constructs in cells
• make cell lines
• test siRNA
• adapt to HTS

• Literature / computational
• define known pathway

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Gene Atlas
Gene expression data from 79 human tissues, 60
mouse Idea to analyze expression of ligands,
receptors, Smads across all tissues and look for
preferences / biases Compare these biases to
expression levels of known Smad1/2-responsive
genes ? comparison of MSKCC tumor samples
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Target genes for siRNA experiments
Identify potential pathway members based on
similarities to known pathway members Gene /
Protein expression patterns Protein sequence
and domain composition Genomic proximity
Shared promoter elements