Identification of conserved domains in the promoter regions of nitric oxide synthase 2: implications for the species-specific transcription and evolutionary differences

BMC Genomics Identification of conserved domains in the promoter regions of nitric oxide synthase 2: implications for the species-specific transcription and evolutionary differences Daniel Rico 2 Juan M Vaquerizas 1 Hernn Dopazo 1 Lisardo Bosc 0 0 Instituto de Investigaciones Biomedicas Alberto Sols (CSIC-UAM) , Arturo Duperier 4, 28029 Madrid , Spain 1 Bioinformatics Department, Centro de Investigacion Principe Felipe , Autopista del Saler 16, 46013 Valencia , Spain 2 Centro Nacional de Investigaciones Cardiovasculares , Melchor Fernandez Almagro 3, 28029 Madrid , Spain Background: The majority of the genes involved in the inflammatory response are highly conserved in mammals. These genes are not significantly expressed under normal conditions and are mainly regulated at the transcription and prost-transcriptional level. Transcription from the promoters of these genes is very dependent on NF-B activation, which integrates the response to diverse extracellular stresses. However, in spite of the high conservation of the pattern of promoter regulation in B-regulated genes, there is inter-species diversity in some genes. One example is nitric oxide synthase 2 (NOS-2), which exhibits a species-specific pattern of expression in response to infection or pro-inflammatory challenge. Results: We have conducted a comparative genomic analysis of NOS-2 with different bioinformatic approaches. This analysis shows that in the NOS-2 gene promoter the position and the evolutionary divergence of some conserved regions are different in rodents and non-rodent mammals, and in particular in primates. Two not previously described distal regions in rodents that are similar to the unique upstream region responsible of the NF-B activation of NOS-2 in humans are fragmented and translocated to different locations in the rodent promoters. The rodent sequences moreover lack the functional B sites and IFN- response sites present in the homologous human, rhesus monkey and chimpanzee regions. The absence of B binding in these regions was confirmed by electrophoretic mobility shift assays. Conclusion: The data presented reveal divergence between rodents and other mammals in the location and functionality of conserved regions of the NOS-2 promoter containing NF-B and IFN response elements. - Background The biological activity of most genes involved in adaptive responses is regulated mainly at the level of transcription, and to a lower extent at the post-transcriptional level [1]. A primary example is the highly conserved mammalian inflammatory response, which involves the coordinated transcriptional induction of multiple genes. In this process, an important integrating role is played by the transcription factor NF-B [2,3]. Extensive and detailed research has revealed common, evolutionarily conserved patterns in the regulation of NF-B 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 significantly, with a more restricted inducibility in primate species 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 promoter 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 epithelial 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 NOS2 promoters correlate with differences in NOS-2 expression 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 3fold increase in the activity of promoter regions containing 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-B activation is required for cytokine induction of both human and rodent NOS-2. Mutational analysis of putative NF-B sites in the 7.2 kb promoter region of the human NOS-2 promoter identified four B sites between -5.2 and -6.1 kb, a region termed the distal NF-B enhancer region [13,18]. We have compared the distribution of B and other transcription factor binding sites (TFBSs) in the promoter region of NOS-2 in seven different mammals to evaluate their relative degree of evolutionary conservation and to investigate whether a pattern of changes in their promoter sequences could be established. For this analysis, we downloaded the corresponding 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 compared the evolutionary divergence in the enhancer and proximal region of the NOS-2 promoter to obtain information on the relative selective pressure on these sequences. Taken together, the data obtained are in agreement 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 different 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 agreement was not exact, most Mulan aligned sequences coincided 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 counterparts. 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 mammals and the available fish orthologues (tetraodon, (...truncated)