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66 Gonye et al.4.7. Interpreting the PAINT ResultsHypothesized gene-TRE network from the enrichment analysis (step 20)indicates those TREs that are significantly overrepresented in the promoters ofthe entire input gene list as compared with the promoters in the reference (allpromoters in PAINT promoter database). The significantly above-randomnature of occurrence of certain TREs makes these ideal candidates for furtherexperimental validation. Additional association of subsets of the master list ofgenes, after the first cutoff of “differentially expressed” is applied, is often usedto identify subsets of genes sharing some type of more detailed behavior, typicallyeither by coexpression grouping by any of a plurality of clustering algorithmsor by functional grouping, for example, in conjunction with the geneontology annotation. When using PAINT for TRNA, these subgroupings can beused to determine if any of the binding sites found on promoters of differentiallyexpressed genes are diagnostic for any specific gene behavior (coexpressioncluster, functional subgroup, and so on). Therefore, the desired resultwould be identification of a TRE determined to be statistically enriched in oneor a few of the subgroups, but not all. When a cluster membership file is provided,PAINT will generate visualizations with the genes reordered into theirrespective groups based on the list order. Thus, group-enriched TREs willappear on the Feasnet image as a vertical collection of red boxes, which mirrorthe limits of the gene list for the group. The enrichment can be more easily visualizedby graphing the –log(p-value) for each TRE of interest for each subgroupas shown in Fig. 4. The enrichment p-values of TREs in each subgroup can beobtained from the Significance of TRE occurrence (in clusters compared witha reference) section of the PAINT output, following the linkOverrepresentation in either raw or FDR-adjusted p-values. In the exampledepicted in Fig. 4, the TRE V$AML1_Q6 is significantly enriched in onlygroup 8, whereas the TRE V$GATA3_03 is enriched in both group 5 and group7. The resultant biological inference is that these specific TREs, and their cognateTF, are specifically involved in the regulation of that subgroup of genes.AcknowledgmentsThe authors acknowledge the financial support for this work from DARPABioCOMP initiative under the project “Multi-Timescale Complex Adaptation,”no. F30602-01-2-0578, PI: James Schwaber.References1. Liang, P. and Pardee, A. B. (1992) Differential display of eukaryotic messengerRNA by means of the polymerase chain reaction. Science 257, 967–971.2. Velculescu, V. E., Zhang, L., Vogelstein, B., and Kinzler, K. W. (1995) Serialanalysis of gene expression. Science 270, 484–487.