BMC Genomics 2007, 8:271 http://www.biomedcentral.com/1471-2164/8/271 scription factor NF-κB [2,3]. Extensive and detailed research has revealed common, evolutionarily conserved patterns in the regulation of NF-κB target genes [4-8]. However, the NOS-2 gene presents an exception. The NOS-2 coding region is highly conserved in all vertebrates [9,10], but its transcriptional regulation differs signifi- cantly, with a more restricted inducibility in primate spe- cies than that seen in rodents and other mammals. We have analyzed whether these different responses could be explained, at least in part, by divergent evolution of the NOS-2 promoter sequence. Extensive studies of the mouse NOS-2 promoter have shown that only the proximal 1 kb sequence of the 5'- flanking region is necessary for complete inducibility by LPS and cytokine treatment [11-13]. To confer full pro- moter activity in the rat, 2 kb of additional 5' flanking region are required [14]. In contrast, the proximal region of the human NOS-2 promoter shows no inducibility: the proximal 3.7 kb sequence does not respond to LPS or cytokines in DLD-1 colon cells [15] or A549 lung epithe- lial cells [16]; and although the 4.7 kb upstream region has basal promoter activity in liver (AKN-1) and A549 cells, it does not show any cytokine-inducible activity [17]. These differences between human and rodent NOS- 2 promoters correlate with differences in NOS-2 expres- sion and NO synthesis, which is markedly less inducible in human cells. Vera et al. (1996) [17] cloned 16 kb of the human NOS-2 5'-upstream flanking region and generated deletional NOS-2 promoter sequences ranging in size from 1.3 to 16 kb. Compared to the 1.3 kb sequence, they observed a 3- fold increase in the activity of promoter regions contain- ing the -5.8 kb sequence, a 4-fold increase with the -7.2 kb sequence, and a 9-fold increase with the -16 kb sequence. Moreover, deletion of the region between -2.1 and -4.7 kb showed that this sequence lacks cytokine responsiveness. NF-κB activation is required for cytokine induction of both human and rodent NOS-2. Mutational analysis of putative NF-κB sites in the 7.2 kb promoter region of the human NOS-2 promoter identified four κB sites between -5.2 and -6.1 kb, a region termed the distal NF-κB enhancer region [13,18]. We have compared the distribu- tion of κB and other transcription factor binding sites (TFBSs) in the promoter region of NOS-2 in seven differ- ent mammals to evaluate their relative degree of evolu- tionary conservation and to investigate whether a pattern of changes in their promoter sequences could be estab- lished. For this analysis, we downloaded the correspond- ing promoter sequences from EnsEMBL. An 11 kb sequence spanning from -10 kb to +1 kb was first obtained from the Human Genome, and the available homologues in other species (orthologues) were then directly selected and downloaded. Using this strategy, we identified multiple conserved TFBSs that can be related to the activity of these promoters, at the time that we com- pared the evolutionary divergence in the enhancer and proximal region of the NOS-2 promoter to obtain infor- mation on the relative selective pressure on these sequences. Taken together, the data obtained are in agree- ment with the different inducibility of NOS-2 observed in mammals. Results Analysis of the promoter region of NOS-2 reveals different degrees of sequence conservation among mammals The -10 kb to +1 kb sequence of NOS-2 genes from differ- ent species were aligned by four independent methods to identify conserved regulatory sequences (see Methods). Mulan's graphical alignment is presented in Fig. 1A, with the human sequences as the reference. Although agree- ment was not exact, most Mulan aligned sequences coin- cided with the alignments generated by AVID and BlastZ (zPICTURE), and with MEME detected motifs. Because chimp is phylogenetically very close to human, most of the conserved regions were identical to their human coun- terparts. Consequently, analysis of these sequences was not very informative, and the alignments of chimpanzee sequences are not presented. In contrast, although human and murine gene promoters were generally similar, there were some significant differences. No similarities were found between any of the analyzed promoters in mam- mals and the available fish orthologues (tetraodon, fugu and zebrafish). The proximal region of the NOS-2 pro- moter was well conserved in all species analyzed, and two domains could be defined: one from -700 bp to +500 bp, the other from -1.3 kb to -1.0 kb. We also noted that whereas some regions were conserved only between dog and human (and therefore chimp), there was a region, located between -3 kb and -4 kb, that was conserved between rodents and primates but absent in dog. This analysis also showed that domains similar to the NF-κB enhancer region located between -5.2 and -6.1 kb in the human NOS-2 promoter [13,19] are also found in chimp, dog, cow, macaque, mouse and rat orthologues (Fig. 1A). The human NOS-2 distal NF-κB enhancer has two disrupted similar motifs in rodents The MEME output revealed that the distal NF-κB enhancer region in the human NOS-2 promoter and the similar sequences in dog, mouse and rat contain two non-over- lapping and very conserved motifs. Sequence analysis showed that this region is split in the rodent promoters (Fig. 1B) with one of the component motifs translocated further upstream (-7618 in mouse and -7388 in rat, com- pared to -5114 in human, -5552 in macaque, -6733 in cow and -4781 in dog). The position of the other motif was maintained positioning all the mammalian species Page 2 of 10 (page number not for citation purposes)