oPOSSUM: integrated tools for analysis of regulatory motif over-representation

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/

Figure 1.

Determination of one-to-one orthologs for human and mouse genes. An initial set of homologs was downloaded from EnsEMBL v41 (30). All homologs annotated as ‘one2one’ are extracted. To select the closest putative ortholog pairs from homologs with ‘one2many’ or ‘many2many’ relationships, we check for upstream conservation using the whole-genome human–mouse alignments (6). We re-annotate unambiguously aligned homologs as putative one-to-one orthologs, adding 195 gene pairs to our set and bringing the total number of orthologs to 15 162.

Figure 2.

Identification of transcription start regions (TSRs) using a combination of EnsEMBL annotations and CAGE data. To improve our alignments, we determine putative alternative TSSs for the human and mouse genes. For each gene, the entire repertoire of transcripts from both EnsEMBL core genes and EST genes are retrieved. The TSSs for all transcripts are recorded, followed by a clustering step such that TSSs within 500 bp of one another are merged to form a transcriptional start region (TSR). For each TSR containing a transcript annotated as ‘known’ or ‘novel’, we accept the TSR as is. For TSRs based solely on EST gene transcripts, we require a minimum of 5 CAGE tags as evidence for transcription initiation.

Figure 3.

(A) A screenshot of the output of the oPOSSUM Human SSA analysis, with TFBS profiles ranked by Z-score. The arrows allow the user to sort and re-order the results by Fisher score, TF name, TF class, TF supergroup or TF profile information content (IC). Each TF name links to a pop-up window displaying the TFBS profile information. (B) Pop-up window displaying genes that contain a particular TFBS (in this case, MEF2A; partial list shown), as well as the promoter coordinates associated with each gene, and the motif locations and scores. Sites in overlapping alternative promoters are highlighted for emphasis. Such sites are only counted once in the statistical analysis.