BackgroundBi-directional gene pairs have received considerable attention for their prevalence in vertebrate genomes. However, their biological relevance and exact regulatory mechanism remain less understood. To study the inner properties of this gene organization and the difference between bi- and uni-directional genes, we conducted a genome-wide investigation in terms of their sequence composition, functional association and regulatory motif discovery.ResultsWe identified 1210 bi-directional gene pairs based on the GRCh37 assembly data, accounting for 11.6% of all the human genes owning RNAs. CpG islands were detected in 98.42% of bi-directional promoters and 61.07% of unidirectional promoters. Functional enrichment analysis in GO and GeneGO both revealed that bi-directional genes tend to be associated with housekeeping functions in metabolism pathways and nuclear processes, and 46.84% of the pair members are involved in the same biological function. By fold-enrichment analysis, we characterized 73 and 43 putative transcription factor binding sites(TFBS) that preferentially occur in bi-directional promoters from TRANSFAC and JASPAR database respectively. By text mining, some of them were verified by individual experiments and several novel binding motifs were also identified.ConclusionsBi-directional promoters feature a significant enrichment of CpG-islands as well as a high GC content. We provided insight into the function constraints of bi-directional genes and found that paired genes are biased toward functional similarities. We hypothesized that the functional association underlies the co-expression of bi-directional genes. Furthermore, we proposed a set of putative regulatory motifs in the bi-directional promoters for further experimental studies to investigate transcriptional regulation of bi-directional genes.