BMC Genomics 2006, 7:133 http://www.biomedcentral.com/1471-2164/7/133 SoFTcrSigatShtu,toertlheroagpt5olobuetslohfnungmutaconleBog1etind(eiens)dmeonusisteiegseine5s)Kabnpd utposAtrleua(minof the Scatter plot of nucleotide densities in 5 Kbp upstream of the TSS, that belong to B1 (in mouse genes) and to Alu (in orthologous human genes). For both species the values pre- sented by a point are obtained by averaging over 200 genes that belong to a "gene set". There are 27 gene sets, that were obtained by sorting the 5400 human promoters (that have mouse othologues) according to their Alu content, and divid- ing them into 27 bins (of 200 genes in each). Each mouse gene set contains the orthologous genes of the correspond- ing human gene-set. since Alu and the mouse SINE elements B1 and B2 spread in the rodent and human genomes after the divergence of rodents and primates. We checked whether this trend is present also in the regulatory regions of genes, of size 5 kbp, and we found a positive correlation between B1 and ALU densities even in this relatively short region. In more than 5400 pairs of orthologous genes, that had available upstream sequences in both human and mouse, we found positive correlation (see methods) of 0.495 (pval<1e-05). In addition we ordered the genes by increasing Alu con- tent in the 5000 upstream bp region, and divided the genes on this basis into 27 sets, with each set containing 200 genes. We then computed the average Alu and B1 associated nucleotide densities for each of the 27 sets, and plotted the two average Alu/B1 contents (Fig 5). Each point in Fig 5 represents a group of 200 genes, with the x (y) coordinates representing the Alu (B1) densities in human (mouse). The monotonic behaviour evident in Fig 5 indicates that the Alu and B1 contents of orthologous genes are largely similar in human and mouse. This simi- larity is far from being obvious, since spreading of Alu and B1 across the genome is believed to have happened after the divergence of primates and rodents. Conclusion Summary This research suggests that evolution used transposable elements to insert modules of transcription factor binding motifs into promoters and, by means of their presence, assemble higher level regulatory networks. In order to explore this question we focused on Alu elements, which are good potential candidates to be part of the building blocks of regulatory networks for two reasons. First, Alu elements are abundant in the upstream region of the TSS of genes, and second, Alu elements contain dozens of putative BSs for TFs. Some of these BSs were found before and their association with Alu was also reported, whereas in some cases although the BSs were found, the fact that they reside on Alu went unnoticed. Finally, we list here also BSs on Alu that were not identified previously. Our findings imply that the biological pathway on which Alu- mediated regulation appears to have the most significant impact is the development process. Many of the TFs that have binding motifs on Alu are associated with develop- ment; moreover, some of these BSs were previously dem- onstrated to be functional in vivo and essential to regulation of some target genes. We have also shown that a few subsequences on Alu, that were known to be highly conserved, provide many of the binding sites for the transcription factors. We studied the Alu distribution in the upstream region of genes and found that while Alu are "repelled" from the 500 bp long region immediately preceding the transcription starts site, the 4500 bp long sequence further upstream is enriched (compared to the entire genome). The Alu content in the promoters varies between functional families of genes. Comparison of Alu in human and B1 in mice shows a sig- nificant correlation between their distributions (with respect to orthologs), and high similarity between the binding motifs that reside on the two mobile elements. Several particular cases (TFs and genes) were discussed in detail. Future directions Most importantly, more experiments are needed to verify that the BMs that were found by us (see Table 1 and Addi- tional data file 1) indeed bind their corresponding TFs and, furthermore, to verify their regulatory roles. Of par- ticular interest is the role of Alu in developmental proc- esses. We found that promoters of genes involved in biosynthesis and metabolism are enriched (via Alu) in BMs of several developmental TFs (Table 4). It is our belief that since development and cell proliferation (of which biosynthesis is an essential component) are opposing processes [50], these BMs play an inhibitory role, sup- pressing proliferation during differentiation. This hypoth- Page 11 of 15 (page number not for citation purposes)