Nucleic Acids Research, 2007, Vol. 35, Web Server issue W245–W252 doi:10.1093/nar/gkm427 oPOSSUM: integrated tools for analysis of regulatory motif over-representation Shannan J. Ho Sui1,2, Debra L. Fulton1,2, David J. Arenillas1,3, Andrew T. Kwon1,2 and Wyeth W. Wasserman1,3,* 1Centre for Molecular Medicine and Therapeutics, Child and Family Research Institute, 2Genetics Graduate Program and 3Department of Medical Genetics, University of British Columbia, Vancouver BC, Canada Received January 31, 2007; Revised April 13, 2007; Accepted May 10, 2007 ABSTRACT The identification of over-represented transcription factor binding sites from sets of co-expressed genes provides insights into the mechanisms of regulation for diverse biological contexts. oPOSSUM, an internet-based system for such studies of regulation, has been improved and expanded in this new release. New features include a worm-specific version for investigating binding sites conserved between Caenorhabditis elegans and C. briggsae, as well as a yeast-specific version for the analysis of co-expressed sets of Saccharomyces cerevisiae genes. The human and mouse applications feature improvements in ortholog mapping, sequence alignments and the delineation of multiple alternative promoters. oPOSSUM2, introduced for the analysis of over- represented combinations of motifs in human and mouse genes, has been integrated with the original oPOSSUM system. Analysis using user-defined background gene sets is now supported. The transcription factor binding site models have been updated to include new profiles from the JASPAR database. oPOSSUM is available at http://www. cisreg.ca/oPOSSUM/ INTRODUCTION Functional genomics research often generates lists of genes with observed common properties, such as coordi- nated expression. For many studies, a key challenge is the generation of relevant and testable hypotheses about the regulatory networks and pathways that underlie observed co-expression. Our strategy for elucidating regulatory mechanisms identiﬁes over-represented sequence motifs that are present in the upstream regulatory regions of genes. The motifs may represent transcription factor binding sites (TFBSs) that have a role in regulating expression. oPOSSUM (1) and oPOSSUM2 (2) were developed to identify over-represented, predicted TFBSs and combina- tions of predicted TFBSs, respectively, in sets of human and mouse genes. The user inputs a list of related genes, selects the TFBS proﬁle set to be included in the analysis, and the algorithm determines which, if any, predicted TFBSs occur in the promoters of the set of input genes more often than would be expected by chance. Both analytic approaches rely on a database of aligned, orthologous human and mouse sequences, and the delineation of conserved regions within which TFBS predictions are analyzed. While the approach does not explicitly address uncharacterized transcription factors (TFs), the eﬀective coverage is broadened by the fact that members within certain structural families of TFs can exhibit similarities in binding speciﬁcity. While intra-class similarity is not always the case, as exempliﬁed by the zinc-ﬁnger family of TFs (3), the observation holds true for many TF families (4,5). Here we describe the new release of the oPOSSUM system, which integrates the two previously developed applications, and has been expanded to accommodate new species (yeast and worms). It also includes new methods for orthology assignment, transcription start site (TSS) determination and sequence alignment. MATERIALS AND METHODS Over-representation analysis oPOSSUM single site analysis (SSA). The oPOSSUM system for identifying over-represented TFBSs in sets of co-expressed genes ﬁrst focused on SSA (1). Two scores were developed to assess over-representation, one at the TFBS occurrence level and the other at the gene level. The Z-score, based on the normal approximation to the binomial distribution, indicates how far and in what direction the number of TFBS occurrences deviates from *To whom correspondence should be addressed. Tel: +1 604 875 3812; Fax: +1 604 875 3819; Email: wyeth@cmmt.ubc.ca 2007 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.