Transcriptome of 3 Ricin Resistant ES cell lines: RR814, RR10 and RR17

1. Transcriptome of 3 Ricin Resistant ES cell lines: RR814, RR10 and RR17 AIM: Identification of genes differentially expressed associated with the different mutants.

2. Summary of the analysis • The experiments & the data - 5 “conditions” to analyze • Differentially expressed transcripts • Pathways ORA with all differentially expressed genes • Gene lists • Gene lists in each condition: • Pathways overrepresentation analysis • Gene ontology overrepresentation analysis • Interactions • Gene lists obtained by Venn diagrams • Genes that change profile of expression between conditions-by mutant • Genes that change profile of expression between condition-by parental cell • Genes with high values of fold changes • Genes expressed in all mutants with different expression profile • Others (interactive) • Candidate genes to identify the mutants • Annotations of the probes

3. DIFFERENTIALLY EXPRESSED (D.E.) GENES Differential gene expression of 3 ricin resistant ES cell mutant, RR10, RR17 and RR814 was investigated using 2 colors microarrays. Cell lines AB2.2 and NN5 were used as a reference. Data was normalized and analyzed using Rosetta Resolver 6 error model (data provided by Dr Chris Lindsay). D.E. genes were defined as having a fold change of gene expression > 1.5 (or 2) and a corrected p-value of <0.01. The number of DE transcripts for each comparisons is shown in Fig.1.

4. THE EXPERIMENTS (called CONDITIONS • for the analysis purpose) Differential gene expression of 3 ricin resistant ES cell mutant, RR10, RR17 and RR814 was investigated using 2 colors microarrays. Cell lines AB2.2 and NN5 were used as a reference. This gives 5 “conditions” (name given by the software) to analyze: RR10 vs AB2.2 RR10 vs NN5 RR17vs AB2.2 RR17 vsNN5 RR814 vs NN5

5. Fig. 1 Differentially expressed probes in the ricin resistant ES cell lines, compared with 2 parental ES cell lines.

6. 2. OVER REPRESENTED PATHWAYS • The list of D.E genes represented in each experiment was uploaded to InnateDB (www.innated.ca) to investigate over represented pathways in each mutant respect to a parental cell line. • Pathway over representation analysis (ORA) was performed in InnateDB using Benjamini and Hochberg corrections for multiple testing and a Hypergeometric algorithm. • For visual simplification, the gene list associated to each pathway is not always included in the table and are available in the excel files associated along pathways ID for further information.

7. PATHWAYS ANALYSIS IN D.E. GENES First approach to know pathways altered in the mutants with reference to a given parental cell: 33 pathways were associated with all D.E genes (corrected p-value < 0.05) : OVER REPRESENTED PATHWAYS IN ALL THE D.E. GENES (FC>1.5; P<0.01) OVER REPRESENTED PATHWAYS (Hypergeometric algorithm, Benjamin Hochberg correction) OF D.E. GENES ( FC > 1.5, P < 0.01) IN EACH CONDITION.

8. Condition 2 Condition 1 If you are interested in a particular pathway(s), it is possible to visualize it with certain localization of all the genes products involved along colours indicating up/down regulation, like the pictures in this slide (e-mail me the pathways list and I will e-mail you back the figure) ECM-receptor interaction Pathway as an example Condition 3 Condition 4

9. PATHWAYS ANALYSIS IN D.E. GENES Integrin cell surface interactions: This pathways presents several genes clearly differentiated for RR17. Most of the genes are up reg. Col11a1, Cdh2, Col4a1, Col4a2 and Fbn1 are up reg exclusively for RR17. For RR10 all genes involved in this pathway are down regulated. RR814 did not present DE genes in this pathway.

10. PATHWAYS ANALYSIS IN D.E. GENES Mammalian Wnt signaling pathway: This pathways presents also a clear alteration differentiated for RR17 with several genes up regulated: Dkk2, Dkk3, Dkk1, Wnt2, Wnt3, Cd44 and Sfrp2. Only few genes are DE in the other conditions: - Only Dkk1 is DE, down reg, in RR814. - Only Wnt4, Wnt2 and Wnt10b are DE, down reg, in RR10.

11. PATHWAYS ANALYSIS IN D.E. GENES Focal adhesion: This pathways presents as well a differential pattern between RR10 and RR17. Particularly, Col3a1 presented a large value of Fold change for RR17 (up reg). Collagen adhesion via alpha 2 beta 1 glycoprotein: Differentiation between RR10 and RR17. Col11a1 and Col4a2 up for RR17. Col1a1 and Cola2 down for RR10. Nothing for RR814.

12. PATHWAYS ANALYSIS IN D.E. GENES Complement and coagulation cascades: 2 genes up reg for RR8814: Hc and A2m Again, RR10 and RR17 have genes with a differential pattern: Up reg only in RR17: serpine1, C2, Thbd, Plat. ----- For this data may be useful to screen and visualise the graphs and the localization of the genes.

13. 2.1 OVER REPRESENTED PATHWAYS IN RR10 vsAB2.22.1.1 Pathways analysis in D.E. genes (at least 1.5 fold, P < 0.01)Pathways 2.1.2 Pathways analysis of up regulated genes (at least 1.5 fold, P < 0.01) 2.1.3 Pathways analysis of down regulated genes (at least 1.5 fold, P < 0.01) 2.2 OVER REPRESENTED PATHWAYS IN RR10 vs NN5 2.1.1 Pathways analysis in D.E. genes (at least 1.5 fold, P < 0.01)Pathways 2.1.2 Pathways analysis of up regulated genes (at least 1.5 fold, P < 0.01) Pathways2.1.3 Pathways analysis of down regulated genes (at least 1.5 fold, P < 0.01) Pathways

14. 2.3 OVER REPRESENTED PATHWAYS IN RR17 vsAB2.22.1.1 Pathways analysis in D.E. genes (at least 1.5 fold, P < 0.01)Pathways 2.1.2 Pathways analysis of up regulated genes (at least 1.5 fold, P < 0.01) Pathways2.1.3 Pathways analysis of down regulated genes (at least 1.5 fold, P < 0.01) Pathways2.4 OVER REPRESENTED PATHWAYS IN RR17 vs NN5 2.1.1 Pathways analysis in D.E. genes (at least 1.5 fold, P < 0.01)Pathways 2.1.2 Pathways analysis of up regulated genes (at least 1.5 fold, P < 0.01) Pathways2.1.3 Pathways analysis of down regulated genes (at least 1.5 fold, P < 0.01) Pathways

15. 2.5 OVER REPRESENTED PATHWAYS IN RR814 vs AB2.22.5.1 Pathways analysis in D.E. genes (at least 1.5 fold, P < 0.01)link to RR814 vs NN5 Pathways ORA in all DE genes 2.5.2 Pathways analysis of up regulated genes (at least 1.5 fold, P < 0.01) link to RR814 vs NN5 Pathways ORA in up reg genes2.5.3 Pathways analysis of down regulated genes (at least 1.5 fold, P < 0.01) link to RR814 vs NN5 Pathways ORA in down reg genes

16. 3. OVER REPRESENTED GENE ONTOLOGY TERMS • The list of D.E genes represented in each experiment was uploaded to InnateDB (www.innated.ca) to identify gene ontology terms significantly associated with DE genes in each experiment. • Gene ontology over representation analysis was performed in InnateDB using Benjamini and Hochberg corrections for multiple testing and a Hypergeometric algorithm.

17. Example:GENE ONTOLOGY TERMS OVER REPRESENTATION ANALYSIS (GO ORA) - RR814 vsNN5GO ORA in D.E. genes (at least 1.5 fold, P < 0.01) GO term NameGO ORA of up regulated genes (at least 1.5 fold, P < 0.01)link to RR814 vs NN5 GO ORA of up reg genesGO ORA of down regulated genes (at least 1.5 fold, P < 0.01)link to RR814 vs NN5 GO ORA of down reg genes

18. The following slides show the Venn diagrams allowing analysis of D.E. genes by parameters (cell line, mutant, up/down regulation). As a result, we have here 60 different list of genes (i.e. different compartments in the diagrams plus up/down regulation). Let me know what list(s) are more relevant for the biological question(s), i.e. down regulated genes expressed only in a given mutant, and we can go further in any region(s) of the Venn diagrams. To start, I centred the attention in the five D.E. genes present in ALL condition because they show certain pattern related with the mutants.

19. VENN DIAGRAMS AN EXAMPLE: AB2.2-Differential Gene expression between RR10 and RR17 Fold change>=2, p-value<0.01 RR10 861 744 1605 RR17 1394 1044 2438 ?! 597 298 895 1118 610 1728 710

20. AN EXAMPLE: AB2.2-Differential Gene expression between RR10 and RR17 Fold change>=2, p-value<0.01 Up or down in both “crossed” +27 -27 +17 -17 +10 -10 (7)

21. AB2.2: Differential Gene expression between RR10 and RR17 Fold change>=2, p-value<0.01 RR10 861 744 1605 RR17 1394 1044 2438 597 298 895 262 432 1118 610 1728 16 710

22. NN5: Differential Gene expression between RR10, RR17 and RR814 Fold change>=2, p-value<0.01 RR10 594 496 1063 RR17 674 415 1089 50 118 25 193 528 311 839 580 290 870 9 10 1 6 17 12 4 6 22 RR814 153 38 191 119 24 143

23. RR10: Differential Gene expression between NN5 and AB2.2 Fold change>=2, p-value<0.01 AB2.2 861 744 1605 NN5 594 469 1063 554 507 1061 306 237 1 544 288 231 519

24. RR17: Differential Gene expression between NN5 and AB2.2 Fold change>=2, p-value<0.01 AB2.2 1394 1044 2438 NN5 674 415 1089 831 720 1551 561 322 4 887 111 91 202

25. 5 genes in the “core difference”: They are expressed in all experiments but with different expression profile Fold change>=2, p-value<0.01

26. What do these 5 genes do? Lectin-gene information X-linked lymphocyte-regulated 4B-gene information link to OMIM (human diseases) results or Lgals7 Suggestion: pubgenegives networks of literature

27. Network based on co-occurrence in article for the "Xlr4b" Gene and Proteins in Musmusculus: Pubgene search for Xlr4b

28. Network based on co-occurrence in article for the “Lgals7" Gene and Proteins in Musmusculus: Pubgene search for Lgals7

29. Candidate Genes for RT-PCRSelection based in D.E. genes in only one mutant, reference cell line or condition, with high fold change. Gene info Gene infoOMIM

30. Candidate Genes for RT-PCRSelection based in D.E. genes in only one mutant, reference cell line or condition, with high fold change.

31. GENERAL FILES:1. INTERACTIONSThese files give you the interactions described to date for the D.E. genes detected.Interactions of the D.E. genes with all databases2. COMPLETE LIST of D.E. GENES ALONG THEIR CHARACTERIZATION (GO TERMS) AND EXPRESSION PROFILE IN EACH CONDITIONclick here