2003 Inferring Connection Maps from AfCS Experimental Data and Legacy Data

2003 COMPONENTS Parts-List INTERACTIONS AND NETWORKS COMPUTATIONAL MODELS Alliance for Cellular Signaling Context-Specific 2003

Our experiments measure genes, proteins and key metabolites. What are the underlying biological relationships amongst these entities? The cell functions as an integrated system involving all these players. How can we analyze our data to reveal this interconnectedness? Data Analysis 2003

Reconstructing Networks Legacy Data Protein Interactions Biochemical Pathways Published Literature AfCS Data Microarray Data Yeast Two- Hybrid Data RNAi Data Protein Data Perturbation Data Microscopy Data Reconstructing Networks

2003 Signal Transduction in a Cell from Downward, Nature, August (2001)

2003 Significance analysis of microarrays * (SAM) (R. Tibshirani, G. Chu 2002) Objective: The replicated expression for each gene is taken for the 4hr time condition (untreated vs ligand) to determine whether the gene is statistically differentially up- or down- regulated. The t-statistics for all the genes are ordered and noted. The labels are then permutated and the t-statistic is calculated again. After many iterations, the cumulative t-statistics is averaged for each gene. Finally, for a given false positive rate, [called “False Discovery Rate” or FDR], the significant genes are selected. For each gene, define the adjusted “t-statistic” as follows:  treated -  untreated  + adjustment factor   mean of replicates   standard deviation for the gene

2003

“mitogenic” ligands FDR = 1% FDR = 35% FDR = 18% FDR = 1%- 3% Two-way dendrogram using significantly expressed genes (4hr) 2670 unique genes

2003 Concordance of significantly up (+) or down (-) regulated genes mitogenic ligands (FDR = 1%) 756 (-) 1082 (+) 337 (-) 135 (-) 553 (-) 147 (-) “down-regulated” matches “up-regulated” matches 3 (-) 446 (-) 887 (+) 96 (-) Mosaic plot 578 (+) 73 (+) 597 (+) 117 (+) 47 (+) 477 (+) 117 (+) 4 (+)6 (+)3 (+) 796 (-) 854 (+) 5 (+)4 (+) 3 (-) 10 (+) 1 (-) 3 (-) 2 (-) 3 (-) 72 (+) 18 (+) 341 (-) 143 (-) 152(-) 80(+) 108 (+) 171 (-) 163 (+) 151 (-) 119 (-) Discordance matrix Example: CD40L had 756 down-regulated and 1082 up-regulated genes. Those which were similarly regulated in AIG: 337 down 578 up. 72 (-)

2003 Beyond Clustering How can we obtain biological information from array data at the level of individual genes and correlations in expression between genes? Can we use the correlations to build a connection network that reflects correlations in expression? Is there biological significance to this?

2003 Two-way hierarchical cluster: mean ratio (vs control) of phosphoprotein levels and ligand Note: the ligands that elicit an ERK response (chemokines + AIG, CD40L) clustered together. A correspondence plot below also showed the grouping.

2003 Similarity measures between genes under different conditions with respect to expression levels for… … groups of genes  clustering methods … pairs of genes  correlation methods Linear correlation  (x – x mean ) (y – y mean ) [  (x – x mean ) 2  (y- y mean ) 2 ] ½ Partial correlation = r 2 xy = r xy.[z] r xy - r x[z] r y[z] [(1- r 2 x[z] ) (1- r 2 y[z] ) ] ½ “marginal” global correlation (for ligand j ) r 2 all xy - r 2 all xy except ligand j Gene network maps AfCS expression data with literature information

2003 Transcription factor encoded by fos is stabilized by ERK and continues to affect other IE genes such as jun from Nature Cell Biology August 2002

2003 Schematic interpretation of ERK signal duration for IE gene product for fos Cross-correlation matrices Transcription response from “non-ERK” ligands Transcription response from “ERK” response ligands

2003 Microarray analysis model using gene expression profiles DNA Gene AGene BGene CGene D Protein mRNA P P Signal transduction is most likely regulated on the protein level, but the downstream signal on the transcriptional level is the resultant output from the upstream (outside the nucleus) signal input. The signal information processing complexity is now increased on the transcription level but some information flows upstream and oscillates in an input/output fashion.

2003 Mitogen-Activated Protein Kinase Pathways Mediated by ERK, JNK, and p38 Protein Kinases G. L. Johnson and R. Lapadat Science 2002 December 6; 298: (in Review)

2003 Transcriptional effects downstream from proteins recruited in MAPK cascades (Hazzalin, et al,Nature Cell Biology (2002)

2003 “marginal correlation” “marginal” global correlation (for ligand j ) difference in correlation = r 2 all xy - r 2 all xy except ligand j Red indicates positive influence on the gene upon removing ligand j Green indicates negative influence on the gene upon removing ligand j

2003 “Marginal” correlation IE genes downstream from MAPK Ligand n=33 Idea: indicates the “leverage” on the global correlation coefficient for a gene for the particulat ligand

2003 Marginal Correlations between Genes Provides a “biologically”-driven approach to discriminating ligand responses at the gene and gene-product level. Serves as a pathway driven hypothesis generation method for QRTPCR. Suggests ideal double ligand experiments to explore major signaling pathways that lead to downstream gene expression changes.

2003 “Marginal” correlation signatures IE genes downstream from MAPK Ligand n=33  Correlation coefficient green = negative red = positive Mitogenic ligand

2003 “Marginal” correlation signatures IE genes downstream from MAPK Ligand n=33  Correlation coefficient green = negative red = positive chemokines No obvious pattern so consider data reduction

2003 mitogenicchemokines

2003 For the case of ligand 2MA… cAMP responsive element modulator

2003 Marginal Correlations averaged over Pathway-Specific Genes

2003 Marginal Correlations averaged over Pathway-Specific Genes

2003 Marginal Correlations averaged over Pathway-Specific Genes

2003 Marginal Correlations averaged over Pathway-Specific Genes

2003 transcription factor binding sites immediately upstream from “immediate- early” genes fos & jun (Hazzalin, et al,Nature Cell Biology (2002) = expression measured indirectly in ligand AfCS experiment

2003 Difference in IE gene cross-correlations from ligands that involve ERK pathway Critical level p = Partial correlations Ligands that stimulate ERK Note: junB expression wasn’t detected

2003 Difference in IE gene cross-correlations from ligands that involve ERK pathway Critical level p = Partial correlations ERK CREM a h k Possible interpretation of a gene regulatory network

2003 J Biol Chem 1998 Nov 20;273(47): The transcription factors ID{-3321=Elk-1} and ID{-11291=Serum Response Factor} are necessary for GH-stimulated transcription of ID{-3796=c-fos} through the Serum Response Element (SRE). Proc Natl Acad Sci U S A 1991 Jun 15;88(12): Furthermore, expression of antisense ID{2352=CREM} enhances ID{-3796=c-fos} basal and cAMP-induced transcription. Neurol Res 2000 Mar;22(2): In the non-trauma patients 36% expressed ID{-3796=c-fos} and 73% expressed ID{-6204=c-jun} mRNA, with all patients studied expressing ID{-3796=c-Fos} and ID{-6204=c- Jun} proteins. Mol Cell Biol 1991 Jan;11(1): We observe that the expression of endogenous ID{-6204=c-jun} and ID{-6205=jun B} genes is induced by E1A, which directly transactivates the promoters of ID{-3796=c-fos}, ID{-6204=c- jun}, and ID{-6205=jun B}. Genes Correlated by Gene Expression from Legacy Data extracted from Pathway Assist (Stratagene Database)

2003 Connections at the Protein Level from Legacy Data extracted using Pathway Assist (Stratagene)

2003

Full view of two-way dendrogram Two-Way Dendrogram from AfCS ligand screen using the probes (genes) relating to the “immediate-early” genes (with additional genes that encode MAPK proteins involved in the cascade). Summary: The transcription profiles of these selected genes distinguished the “mitogenic” ligands (AIG, CPG, CD40L, IL-4, IL10, LPS) from the “non-mitogenic” at the 2hr / 4hr time period. Since the upstream MAPK-ERK pathway is involved in cell proliferation this would be expected under ideal experimental conditions. The fact that a distinct two-way “bicluster” (mitogenic ligands are clustered to the IE genes from MAPK-ERK) as a first-pass result of the microarray experiment is highly encouraging. This “semi-supervised” approach indicates our expression data is biologically informative.

2003 Kohn’s Mammalian Cell Cycle Map (with AfCS genes)

2003 Kohn’s Mammalian Cell Cycle Map (with AfCS genes)

2003 Kohn’s Mammalian Cell Cycle Map (with AfCS genes)

2003

Non-mitogenic ligand response gene correlations Mitogenic ligand response gene correlations

2003 MYC Box and related genes

2003 MYC Connection Map Genetic regulatory module generated by partial correlations critical value = 10 -6

2003 Literature-derived expression-based connection maps for all AfCS proteins AfCS proteins with no known connections

2003

Pathway database, analysis tools and GUI for Cellular Signaling 2003

Creation of an integrated signaling GUI and database system Design of a system for testing legacy pathways against AfCS experimental data Reconstruction of signaling pathways Creation of tools for validation of pathway models Pathway Reconstruction and Analysis 2003

System Architecture 2003