pairs of TFBS proﬁles include combinations of Mef-2, SRF and Sp-1. The inclusion of alternative promoters provides notable improvements in the Human SSA and Human CSA analyses. The same datasets were used to validate our previous and current human oPOSSUM analyses systems. Demarcation of additional promoter boundaries increases the signal in the discovery process, improving the signal for both over-represented single TFBSs and combinations of TFBSs in the gene sets analyzed. Worm SSA. Worm SSA was tested on a set of well- characterized nematode muscle genes (Table S10) (26). Analysis of 1000 bp of upstream sequence, using the top 10% of conserved regions (minimum of 60% sequence identity), a matrix match threshold of 80% and the worm proﬁles, identiﬁed the putative muscle1 motif with a Z-score of 20.6 and a Fisher score 50.01 (Table 4). This is, however, somewhat circular, given that 19 of the 41 input genes were used to generate the putative muscle-speciﬁc worm proﬁles. Analysis using the JASPAR CORE proﬁles ranked SP1 and Su(H) within the top 10 scoring proﬁles (Table S10B). Studies in Xenopus and Drosophila provide evidence that MyoD triggers Notch signaling through Su(H) for muscle determination (27,28). Although SP1 has been implicated in muscle CRMs, it is a general TF involved in the expression of many diﬀerent genes and binds to GC-rich motifs. Yeast SSA. The yeast CLB2 gene cluster is comprised of 32 genes whose pattern of expression peaks at late G2/early M phase of the cell cycle (Table S11). Transcription of these genes is regulated by two TFs: FKH, which is a component of the TF SFF, and MCM1, a member of the early cell cycle box (ECB) binding complex. Analysis of 500 bp of upstream sequence using a matrix match threshold of 85% ranked ECB, MCM1 and FKH1 in the top ﬁve scoring TFBS proﬁles (Table 5), which is consistent with the literature (29). Nucleic Acids Research, 2007, Vol. 35, Web Server issue W249 Web server The four oPOSSUM systems, Human SSA, Human CSA, Worm SSA and Yeast SSA, have been integrated into a use- friendly website at: http://www.cisreg.ca/oPOSSUM/. We recommend that users of the system begin with the SSA to quickly identify TFBSs that may be relevant to their input data sets. For sets of human and mouse genes, this can be followed with the CSA, which takes longer to process, but which can provide insights into TFBSs that may be acting in concert to regulate the set of genes. The web implementation allows for analysis in default and custom modes. Default mode processing is faster as TFBS counts have been pre-calculated and stored for pre-deﬁned conservation levels, matrix match thresholds and promoter lengths. In either mode, the user is required to select a species and to enter a list of gene identiﬁers (EnsEMBL, RefSeq, HGNC and Entrez Gene are sup- ported for human). A number of options are available to specify the TFBS proﬁle set to be used in the analysis. Finally, the conservation level, matrix match threshold and the promoter length can be varied. In the custom mode, users may deﬁne their own background set, which provides users with more control, but results in more variable processing speeds depending on the size of the background set and the parameters selected. Upon submission, oPOSSUM SSA generates a sum- mary of the input parameters, and produces a single table that ranks the over-represented TFBSs by descending Z-score. The table may be sorted by TF name, TF class, supergroup, information content (IC), Z-score and Fisher score (Figure 3A). Pop-up windows linked to each TFBS foreground count display the genes in which the putative site is located, the promoter region(s) for each gene, as well as the TFBS’s co-ordinates and score (Figure 3B). TFBSs that occur in overlapping promoter regions are marked by an asterisk and highlighted in yellow. The TF names are linked to the JASPAR database for easy access to information regarding the binding Table 4. oPOSSUM results for worm skeletal muscle genes using worm proﬁles Worm Muscle1 Muscle2 LIN-14 Muscle3 YEAST ECB MCM1 FKH1 CCA LYS14 Status Putative Putative Putative Putative TF Class Unclassiﬁed MADS Forkhead Unclassiﬁed C6_Zinc ﬁnger IC 11.34 11.97 9.13 16.67 Target gene hits 6 4 9 4 IC 16.65 9.15 13.28 16.93 17.02 Target gene hits 13 10 30 3 6 Table 5. oPOSSUM results for the yeast CLB2 gene cluster Background TFBS rate 0.0025 0.0022 0.0143 0.0029 Background TFBS rate 0.0019 0.0073 0.0305 0.0017 0.0030 Target TFBS rate 0.0156 0.0089 0.0280 0.0064 Target TFBS rate 0.0131 0.0165 0.0473 0.0040 0.0053 Z-score 20.56 11.19 9.12 5.02 Z-score 32.87 13.71 12.26 7.08 5.20 Fisher score 4.24E–04 1.39E 02 1.17E 01 6.96E 02 Fisher score 8.68E 09 1.08E 02 4.05E 02 2.02E 01 9.41E 02