BMC Genomics 2006, 7:133 http://www.biomedcentral.com/1471-2164/7/133 BMs tend to cluster together on specific parts of Alu consensus sequences, that also tend to be more conserved during evolution We checked the BM's locations along the consensus sequences on both strands of the eight Alu subfamilies. In general, most of the putative BSs were found on the sense strand. It is interesting to note that Alu elements show a 55% to 45% bias towards antisense orientation [34] rela- tive to nearby genes. It would be of considerable interest to check experimentally whether this orientational differ- ence is reflected in functional roles of the TFs. The target sites that passed T5 cover, on average, 50% of the consen- sus sequence of each subfamily. On the other hand, the total length of all the binding motifs found on one Alu consensus sequence exceeds, on the average, 420 nucle- otides (150% of the Alu's length). This redundancy in putative target sites is caused by the fact that several PSSMs have very similar structure, giving top scores to the same DNA sequence. The short sequences GGTAATCCC on the 5' to 3' strand and its complementary sequence GGGATTACC serve as a putative BS of five TFs, four of which are homeodomain proteins (Table 1 and Additional data file 1, TFs and loca- tions marked by *). This sequence is part of the consensus of all eight Alu subfamilies, and in the five middle AluS subfamilies the BS appears on both the left and right mon- omers. Moreover, this is precisely the sequence identified by Britten [23] as one of the most conserved subsequences on Alu, which was seen as a surprise since it did not have any role in retrotransportation or for any other function. Later on, it was found that in the germ line the Specific Alu Binding Protein (SABP) can bind to this sequence and prevent methylation [35], but since the importance of this function of is still not understood, and hence the high level of conservation is still a mystery. Another region is a putative harbour for several BMs is the middle poly A- stretch (that bridges the two Alu monomers). We found this sequence to be a putative target site for many binding motifs such as all MEF2 family members, HNF1.03, OC.2 and PAX4 (marked by ** in Table 1 and Additional data file 1). These putative target sites were also found to accu- mulate less mutations than the expected rate [23]. More recently both the central poly A stretch and the subse- quence mentioned above [36-38] were found to be asso- ciated with A-to-I editing. Existing evidence for TF binding to Alu (on DNA in solution) Having identified BSs of various TFs on Alu, we searched for existing experimental evidence for binding and regula- tion associated with these motifs. Indeed, several TFs that are known to bind to motifs that passed our thresholds have already been shown to bind to DNA, and the fact that the binding sequences reside on Alu has been noticed (denoted by * on Table 2). We show in Table 2 four addi- tional BMs that we found and report here for the first time; their corresponding TFs were shown to bind to the target sites identified by us, but the fact that they reside on Alu has not been previously noted. These TFs (bold in Table 2) are PITX2, Nkx2.5 Gfi-1 and LUN1. The most striking example was LUN-1; Chu et al. [39] observed (by EMSA) that it binds to 55 DNA sequences, 27 of which are palindromic; we discovered that all of these 27 reside on Table 2: TFs for which the binding to the Alu-associated BM has been experimentally verified. Most passed T1 and FDR of 20%. The last two (marked by **) did not pass our promoter regions search, but were reported in the literature as bound to Alu. Four TFs (bold) are new: they were found by us but their being bound to Alu went unnoticed. The first column is the PSSM symbol from MatInspector [30] or TRANSCFAC [31] the second and third columns are the gene symbol and description of the biological process. The last two columns list those target genes of each TF that have on-Alu BSs, and the appropriate references. Binding Motif name TF Gene Symbol Page 4 of 15 (page number not for citation purposes) V$PITX2_Q2 V$NKX25.01 V$LYF1.01* V$GFI1.01 V$LXRE.01* V$ER.01–02 V$RAR.01* V$GC.01* V$LUN1 V$PAX6.02** V$YY1.01** PITX2 Nkx2.5 ZNFN1A1 (LYF1) Gfi-1 LXREα ER RARA SP1 LUN1 PAX6** YY1** Biological process involvement Eye, heart, tooth and abdominal organs Heart formation T-cell differentiation Hematopoietic differentiation Retonic acid receptors family. Embryonic morphogenesis Nuclear factor. Tumer suppressor Lung development Pancreas development Nuclear factor Target genes with Alu target sites PLOD1 PLOD1 CD8 AZU, P21, JAK3, ACT, AAT BRCA1, ERF-3 Keratin K18 [40,42] Ref [42] [27] [41] [56] [18] [22] [21] [39] [45] [20]