W248 Nucleic Acids Research, 2007, Vol. 35, Web Server issue regulated by FoxM1, a member of the forkhead family of TFs, using BT-20 cells that had been transfected with FoxM1 siRNA. They identiﬁed a set of 27 genes that were speciﬁcally regulated in cells transfected with FoxM1 siRNA (Table S4). The 27 Aﬀymetrix UG144A identiﬁers were mapped to 27 EnsEMBL gene identiﬁers and submitted to Human SSA with default parameters. Of these, 22 genes had a unique mouse ortholog and were used in the oPOSSUM analysis. While a speciﬁc proﬁle for FoxM1 is not present in JASPAR CORE, other members of the forkhead family were ranked in the top 10 highest scoring TFBS proﬁles (Table 1). There is also a known association between HNF4, the highest scoring TFBS proﬁle, and the forkhead TF, FOXO1 in the regulation of gluconeogenic gene expression in hepato- cytes (20), which may explain the over-representation of the HNF4 proﬁle. We previously identiﬁed over-represented Fos binding sites in a set of genes induced after transformation by c-Fos in rat ﬁbroblast cells (1,21). We analyzed 160 orthologous genes from the original list of 252 induced genes (Table S7). This is a notable improvement over the previous version where only 98 genes were included in the oPOSSUM analysis. The Fos TFBS proﬁle ranked Table 1. oPOSSUM results for human FoxM1-regulated gene cluster JASPAR CORE TF Class second in the list of over-represented TFBSs (Table s7). Inspection of the results using the JASPAR PhyloFACTS proﬁles with default parameters illustrates how inclusion of this new set of proﬁles provides additional, meaningful information (Table 2). The highest ranked PhyloFACTS motif (TGANTCA) is noted by JASPAR as being most similar to the binding proﬁle for AP-1, and the third highest scoring motif (TGASTMAGC) is most similar to the bZIP TF NF-E2. AP-1 complexes are comprised of Fos and Jun proteins, and the structurally related NF-E2 and AP-1 TFs bind similar sequence motifs (22). Human CSA. The CSA was validated on a set of mouse skeletal muscle genes comprised of the union of the results of the microarray studies of Moran et al. (23) and Tomczak et al. (24) (Table S9). To avoid circularity, we removed muscle-speciﬁc genes used to generate the JASPAR binding site proﬁles for Mef-2, Myf, Sp-1, SRF and Tef. These factors occur in clusters in CRMs that contribute to skeletal muscle-speciﬁc expression (25). Table 3 lists the top ﬁve over-represented pairwise TFBS combinations for this set of genes, along with the JASPAR class each TF proﬁle clustered to, and the Fisher score obtained for each pair. The ﬁve most over-represented HNF4 Fos Pbx FOXI1 RORA1 TAL1-TCF3 Staf Foxa2 Foxd3 TEAD TGANTCA GGGYGTGNY TGASTMAGC GGARNTKYCCA GGGAGGRR TF name (Class ID) MEF2A (class 4) MEF2A (class 4) Myf (class 22) SP1 (class (31) Agamous (class 1) Target gene hits Background TFBS rate Target TFBS rate Z-score Fisher score 13 15 5 16 4 12 3 13 13 6 Target gene hits 46 82 43 44 111 0.0054 0.0111 0.0019 0.0153 0.0020 0.0052 0.0014 0.0152 0.0172 0.0028 Background TFBS rate 0.0011 0.0059 0.0013 0.0016 0.0171 0.0085 0.0146 0.0033 0.0186 0.0029 0.0066 0.0021 0.0174 0.0194 0.0037 Target TFBS rate 0.0023 0.0083 0.0024 0.0026 0.0202 TF class name MADS MADS bHLH Zn-ﬁnger, C2H2 MADS TF name (Class ID) Myf (class 22) ZNF42_1–4 (class 25) SRF (class 1) SRF (class 1) MEF2A (class 4) TF class name bHLH Zn-ﬁnger, C2H2 MADS MADS MADS 7.19 5.72 5.57 4.49 3.54 3.30 3.16 3.04 2.93 2.85 18.05 15.64 15.64 12.54 11.98 Z-score Fisher score 2.64E 02 4.29E 01 3.10E 01 9.05E 02 5.04E 01 5.88E 02 3.03E 01 4.83E 01 5.27E 01 4.70E 01 1.40E 04 4.98E 02 1.19E 03 1.11E 03 3.16E 01 Score 1.65E 06 4.24E 06 2.52E 05 2.68E 05 7.63E 05 Nuclear bZIP Homeo Forkhead Nuclear Receptor bHLH Zn-Finger, C2H2 Forkhead Forkhead TEA AP-1 – NF-E2 – MAZ IC 9.62 10.67 14.64 13.18 17.42 14.07 17.54 12.43 12.94 15.67 IC 12.06 14.18 16.60 17.13 14.00 Table 2. oPOSSUM results for c-Fos-regulated gene cluster JASPAR PhyloFACTS Similar to Table 3. oPOSSUM results for skeletal muscle genes identiﬁed by Moran et al. and Tomczak et al