Epistasis Analysis Using Microarrays Chris Workman

Experiments with Microarrays Cool technology, but how do we use it? How is it useful? Identify “marker genes” in disease tissues Classification, diagnostics Toxicology, stress response Drug candidate screens, basic science Genetic factors Measuring interactions (chIP-on-chip)

Overview Expression profiling in single-deletions Epistasis analysis using single- and double- deletions Epistasis analysis, genetic and environmental factors Reconstructing pathways that explain the genetic relationships between genes

Expression Profiling in 276 Yeast Single-Gene Deletion Strains (“The Rosetta Compendium”) Only 19 % of yeast genes are essential in rich media Giaever, et. al. Nature (2002)

Clustered Rosetta Compendium Data

Gene Deletion Profiles Identify Gene Function and Pathways

Principle of Epistasis Analysis

Experimental Design Compare single-gene deletions to wild type Compare to the double knockout to wild type

Experimental Design: Single vs Double-Gene Deletions

Classical Epistasis Analysis Using Microarrays to Determine the Molecular Phenotypes Time series expression (0-24hrs) every 2hrs

Mixing Genetic and Environmental Factors

Expression in Single-Gene Deletions (yeast mec1 and dun1 deletion strains)

Chen-Hsiang Yeang, PhD Craig Mak MIT UCSD UC Santa Cruz Yeang, Jaakkola, Ideker. J Comp Bio (2004) Yeang, Mak, et. al. Genome Res (2005)

Measurements Networks “Systems level” understanding Treat disease Synthetic biology In silico cells

Measurements Networks “Systems level” understanding Treat disease Synthetic biology In silico cells Test & Refine

Displaying deletion effects Published work: “Epistasis analysis using expression profiling” (2005)

Relevant Interactions Subset of Rosetta compendium used 28 deletions were TF (red circles) 355 diff. exp. genes (white boxes)  P < TF-deletion effects (grey squiggles)

Network Measurements Yeast under normal growth conditions Promoter binding ChIP-chip / location analysis Lee, et. al. Science(2002) Protein-protein interaction Yeast 2-hybrid Database of Interaction Proteins (DIP) Deane, et. al. Mol Cell Proteomics (2002)

ChIP Measurement of Protein-DNA Interactions (Chromatin Immunoprecipitation)

Step 1: Network connectivity (chIP-chip analysis) ~ 5k genes (white boxes) ~ 20k interactions (green lines)

Step 2: Network annotation (gene expression analysis) What parts are wired together How and why the parts are wired together the way they are Measure variables that are a function of the network (gene expression). Monitor these effects after perturbing the network (TF knockouts).

Inferring regulatory paths = = Direct Indirect

Annotate: inducer or repressor OR

Annotate: inducer or repressor

Computational methods Problem Statement: Find regulatory paths consisting of physical interactions that “explain” functional relationship Method: A probabilistic inference approach  Yeang, Ideker et. al. J Comp Bio (2004) To assign annotations Formalize problem using a factor graph Solve using max product algorithm  Kschischang. IEEE Trans. Information Theory (2001)  Mathematically similar to Bayesian inference, Markov random fields, belief propagation

Inferred Network Annotations A network with ambiguous annotation

Test & Refine

Which deletion experiments should we do first? A mutual information based score For each candidate experiment (gene  ) Variability of predicted expression profiles  Predict profile for each possible set of annotations  More variable = more information from experiment Reuse network inference algorithm to compute effect of deletion!

Ranking candidate experiments

We target experiments to one region of network Expression for: SOK2 , HAP4 , MSN4 , YAP6 

Expression of Msn4 targets Average signed z-score

Expression of Hap4 targets

Yap6 targets are unaffected

Refined Network Model Caveats Assumes target genes are correct Only models linear paths Combinatorial effects missed Measurements are for rich media growth

Using this method of choosing the next experiment Is it better than other methods? How many experiments? Run simulations vs: Random Hubs

Simulation results # simulated deletions profiles used to learn a “true” network

Current Work Treat disease “Systems level” understanding Test & Refine Networks Transcriptional response to DNA damage Measurements

Acknowledgments Trey Ideker Craig MakChen-Hsiang Yeang Tommi Jaakkola Scott McCuine Maya Agarwal Mike Daly Ideker lab members Funding grants from NIGMS, NSF, and NIH Tom Begley Leona Samson