BMC Genomics 2007, 8:271 http://www.biomedcentral.com/1471-2164/8/271 EsFeMiqgSuuAerneocf4espuotfathivee mκBoussiteeNsfOroSm-2tphreo"mhuomtearn enhancer like" EMSA of putative κB sites from the "human enhancer like" sequences of the mouse NOS-2 pro- moter. The mouse -103 κB sequence was compared with the oligonucleotide sequences containing putative κB sites (identified as 'κB-like sites') in EMSAs with nuclear protein extracts from mouse peritoneal macrophages. Equal amounts of labeled oligonucleotide were used for each assay. C: nuclear extracts from control macrophages; LPS: nuclear extracts from macrophages treated with 200 ng/ml of LPS for 1 h. Ex: Extracts from LPS-treated cells in the presence of a 50-fold excess of unlabelled oligonucleotide. The bands cor- responding to the p50/p65 and p50/p50 complexes are indi- cated. n.s. corresponds to the front and non specific binding in the EMSA. parison of large 5' upstream regions from orthologous genes. Functional promoter analysis is normally per- formed by cloning part of the 5' upstream region of the gene into an appropriate reporter vector, such as used for luciferase assays. The regions cloned are normally no longer than 2 or 3 kb, for two reasons. The first is that most regulatory elements are located close to the tran- scription start site. The second reason is technical: cloning of long inserts and cell transfection with large plasmids is complex. The minimal promoter regions of many NF-κB regulated genes, as COX-2, IκBα or IκBβ, in mouse and human are located within the proximal 1 or 2 kb 5' upstream region, and the same applies to the mouse NOS-2 promoter. Thus for these genes, all the important regulatory regions are contained within this proximal 1 or 2 kb sequence. How- ever, eukaryotic promoters frequently contain very distant enhancer elements that are important, or indeed some- times essential, for the regulation of gene transcription [22]. This is the case with the human NOS-2 promoter, where maximal induction in luciferase assays requires a 16 kb promoter [17]. The effort of cloning this insert was necessary because human NOS-2 is poorly induced, or not induced at all, with shorter promoters. This is such a long sequence that the regulatory elements located between -9 kb and -16 kb still remain to be identified. Nevertheless, the important distal NF-κB enhancer region between -5.2 kb and -6 kb in the NOS-2 promoter, con- taining four κB sites, was described by Taylor and co- workers [13], and an additional κB site at -8293 bp was identified by Moss' group [19]. We were interested in exploring larger upstream regions of the NOS-2 promoter in mouse and other vertebrate spe- cies other than human, to see if we could find sequence similarities among them. Promoter length in eukaryotes is very variable; Nardone and co-workers [23] proposed that bioinformatic promoter analysis should extend, ideally, for 50–100 kb in either direction from the gene coding sequence, and should include introns and neighboring genes. Moreover, Dieterich and co-workers [24] estimated that 10 kb upstream from the transcription start site is suf- ficient in most cases. We selected 10 kb upstream plus 1 kb downstream from the TSS for a total of 11 kb per sequence, because this provides a good starting point with enough computational burdens. The 11 kb sequence used in this work contained all the described regulatory regions in human NOS-2 promoter, the longest known of all the promoters studied here. MEME (at the settings used) allowed us to detect 10 conserved motifs ranging from 6 to 120 base pairs, but did not give us information about the overall similarity between sequences; mVISTA, zPIC- TURE and Mulan provided this information. We preferred Mulan for graphical alignments and calculation of ECR lengths and similarities because it can detect sequence rearrangements (AVID/mVISTA cannot) and for its dynamic high power and flexibility. The Mulan graphical alignments are shown in Fig. 1A. MEME was very useful for highlighting the two motifs in rodents that were simi- lar to the human distal NF-κB enhancer region, but which are separated, with one motif located further upstream, in the rodent sequences. This feature was also detected with Mulan and zPICTURE, and we present a schematic model in Fig. 1B to show the position of these regions in the human and mouse promoters. The human NF-κB enhancer region is entirely conserved in dog, cow and macaque; but unlike the case in rodents, there is one unique conserved region. The rodents are phylogeneti- cally closer to human than to dog and cow [25], so we hypothesize that a translocation of part of this ancestral region has occurred in the rodent linage after its specia- tion from primates. In addition to these strategies of analysis, we performed an evolutionary analysis in order to evaluate the selective pressures in the proximal promoter and the enhancer, to gain insight into functional differences among the species studied. The data obtained suggested a similar conserva- tion of the enhancer and the proximal promoter in non- rodent species, while mouse and rat showed a compara- tive higher rate of evolution (in relation to intron Page 6 of 10 (page number not for citation purposes)