1. Selection of genes: Choose threshold on fit score to include known EWS/FLI1-induced genes. Model-based approach for analysis of transcriptome perturbation reveals Ewing oncogene interaction Network G. Stoll 1, A. Zinovyev 1, F. Tirode 2, K. Laud-Duval 2, O. Delattre 2, E. Barillot 1 1. Service Bioinformatique, Institut Curie, Paris, France 2. Unité 830 INSERM, Institut Curie, Paris, France Method: from non-linear fit of transcriptome time series to network reconstruction Application: tumor phenotype induced by EWS/FLI1 oncogene in Ewing tumor cells Pulse 5 parameters Switch 4 parameters Amplitude Score=(weight)*(Amplitude) /(Distance to curve) 2 2 Non-linear fit of simple transition (“switch”) and double transition (“pulse”). Gene selection according to fit score. Selection refinement from genes involved in cell-cycle and apoptosis: (from GO terms and BROAD database). Construction of a list of links (influence) between genes based on review papers. Construction of network explaining behavior on cell- cycle and apoptosis. shRNA of EWS/FLI1 Inductible promotor by DOXYCYCLN +DOX: Inhibition of EWS/FLI1 -DOX Experimental data: Inhibition, inhibition + reactivation of EWS/FLI1 produce transcriptome time series. Non-linear fit on every probeset. Construction of an annotated database of links based on review papers. Review ref, Experimental ref, Link, Type of link, Delay, Confidence, Tissue, Comment. Construction of an annotated network of EWS/FLI1 effect on cell-cycle and apoptosis. Contradictory path analysis: suggestion of experimental design. Block contradictory pathsBlock non-contradictory paths Experimental design: block experimentally all non-contradictory paths to APAF1. If expression of APAF1 does not change => need external connection (maybe direct or indirect target of EWS/FLI1) Example: IGFBP3 Example: E2F2 Inhibition of EWS/FLI1 Inhibition & reactivation of EWS/FLI1 Score of fit: 3.2 Time of transition: 6.0 (days) Length of transition: 4.7 (days) Score of fit: 0.8 Time of transition: 1.3 (days) Length of transition: -1.4 (days) Score of fit: 11.4 Time of pulse: 10.1 (days) Length of pulse: 9.4 (days) Score of fit: 4.0 Time of pulse: 9.0 (days) Length of pulse: -16.0 (days) Blue: transcriptome data. Red: non-linear fitting curve Contradictory paths: conflict between annotated genes (red or green) and influences. Example: network analysis around APAF1 PMID (rev.) PMID (exp.) LinkType Delay Confidence Tissue Comment Partially supported by ANR STICON project (http://bioinfo-out.curie.fr/projects/sitcon/) Selected genes are correlated or anti-correlated with EWS/FLI1. Example of selected genes: caspase 3(-), TNFaIP(-), c-mycBP(+), p53(+), cyclin D1(+), cyclin D2(+), catepsin B(-), E2F2(+), E2F5(+), IGFBP3(-), TGFb2(-) Non-proliferating cells Proliferating cells Correlated genes (with EWS/FLI1) annotated in green Anti-correlated genes (with EWS/FLI1) annotated in red Positive influence: green edge Negative influence: red edge -DOX Reactivation df EWS/FLI1