BMC Genomics 2007, 8:271 http://www.biomedcentral.com/1471-2164/8/271 Table 1: NF-κB binding sites in NOS-2 promoters. Sequences were scanned with Match using TRANSFAC Professional 8.2 and minimizing false positives. Coincidence with experimentally demonstrated κB sites is indicated Position Strand Matrix match Comments Human Mouse Rat -119 -4357 -5213 -5802 -8279 +192 +191 -103 -996 -997 -998 -2796 -5347 -5343 -5342 -1601 -2394 -2399 -2458 -2459 -2460 -8999 -9000 -9655 + - - - + + + - + - + + + - + & - - - + + - + + - - 0.908 0.987 0.935 0.908 0.913 0.894 0.913 0.917 0.989 0.999 0.979 0.918 0.971 0.967 0.997 0.917 0.989 0.993 0.989 0.999 0.979 0.980 0.937 0.936 Controversial (8) c-Rel. Described (13) Described (13) Described (19) Described (11, 31) c-Rel. Described (11, 31) c-Rel. c-Rel. c-Rel. ing tool interconnected with Mulan, a strategy that searches for TFBSs conserved among all the sequences aligned (i.e. multi-conserved sites). We included all mam- mal NOS-2 promoters in the analysis and tried all possi- ble combinations of human vs. other mammal species alignments. A schematic representation of the results is shown (Fig. 3) and detailed information about these con- served TFBSs is available in additional file 1. It is remark- able the conservation of these sites in the distal regulatory domain in primates, dog and cow, but not in rodents. None of the human AP-1, IRF-1 and STAT-1 sites are con- served in the rodents' distal regions. Only the equivalent κB sites -5201 (human) and -8307 (rodents) are detected as conserved between primates and rodents with MultiTF, but we proved that this site is not functional in mouse by EMSAs (see below). A unique proximal conserved κB site was found in human that was present also in the dog and rodent NOS-2 promoters (at -115) and it corresponds to the functional κB binding sites from the promoters of mouse (at -110; -85 in the literature) and rat (at -71) NOS- 2 genes. Sequence conservation of this site in human NOS-2 is interesting since apparently it is not functional. In this connection it might prove significant that the human and chimpanzee NOS-2 promoters contain an (A)n simple repeat downstream of this putative -115 κB site, between -94 and -75 bp. This sequence does not sFDciergitupatirlieodn3afancatloysris of NF-κB, AP-1 and IFN-γ dependent tran- Detailed analysis of NF-κB, AP-1 and IFN-γ depend- ent transcription factors. Schematic representation of the relevant TFBSs in the promoter of NOS-2 from different species (plotted from additional data file 1). All the con- served sites in human and other mammals are shown, and conservation with other species is indicated. occur in dog, mouse or rat (alignments provided in the additional data file 2). This (A)n repeat in primates might be responsible, at least in part, for the lesser importance or loss of function of this human κB site (see Discussion). NF-κB does not bind to mouse motifs homologous to the human NF-κB enhancer region All the previous data suggested that the distal rodent regions homologous to the human enhancer are probably not functional, at least not in response to pro-inflamma- tory stimuli. In contrast, the enhancer region is likely to be important not only in human, but also probably in other primates and dog too, based on their sequence similarity, position from the TSS and pattern of conserved TFBSs. As NOS-2 induction by LPS is totally dependent of NF-κB, we decided to test NF-κB binding to some regions in the mouse enhancer region by EMSA, in macrophages exposed to LPS. EMSA probes were designed based on mouse-human MEME alignments with the human sequences containing functional human κB sites. The functional mouse κB site at -103 (-87) was used as a pos- itive control (Fig. 4). None of the three new putative mouse κB sequences tested was capable of binding NF-κB, and this probably indicates that this region does not oper- ate as an NF-κB enhancer of NOS-2 transcription in mouse. Discussion An immense amount of non-coding genomic sequence is now available from different species, allowing the com- Page 5 of 10 (page number not for citation purposes)