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We introduce a novel method to screen the promoters of a set of genes withshared biological function, against a precompiled library of motifs, and findthose motifs which are statistically over-represented in the gene set. The genesets were obtained from the functional Gene Ontology (GO) classification; foreach set and motif we optimized the sequence similarity score threshold,independently for every location window (measured with respect to the TSS),taking into account the location dependent nucleotide heterogeneity along thepromoters of the target genes. We performed a high throughput analysis,searching the promoters (from 200bp downstream to 1000bp upstream the TSS), ofmore than 8000 human and 23,000 mouse genes, for 134 functional Gene Ontologyclasses and for 412 known DNA motifs. When combined with binding site andlocation conservation between human and mouse, the method identifies with highprobability functional binding sites that regulate groups of biologicallyrelated genes. We found many location-sensitive functional binding events andshowed that they clustered close to the TSS. Our method and findings were putto several experimental tests. By allowing a "flexible" threshold and combiningour functional class and location specific search method with conservationbetween human and mouse, we are able to identify reliably functional TF bindingsites. This is an essential step towards constructing regulatory networks andelucidating the design principles that govern transcriptional regulation ofexpression. The promoter region proximal to the TSS appears to be of centralimportance for regulation of transcription in human and mouse, just as it is inbacteria and yeast.

Wide-Scale Analysis of Human Functional Transcription Factor Binding Reveals a Strong Bias towards the Transcription Start Site