Mannaert et al. BMC Genomics 2010, 11:658 http://www.biomedcentral.com/1471-2164/11/658 used strategy to discover potential regulatory elements. Such elements are expected to be present in the vicinity of transcription factors or developmental genes, such as Hox genes [42]. We identified 33 evolutionarily con- served non-protein coding sequences, or so-called phy- logenetic footprints, with the software tracker [45] (Additional file 1). To distinguish putatively transcribed footprints, we blast searched all footprints against the NCBI database of ESTs, which resulted in the identifica- tion of eight footprints between 29 and 876 bp long, not located within untranslated regions (UTRs), with one or more EST matches (Additional file 2). Because EST data are absent for many organisms, including coelacanth and caecilians, we did not find ESTs corresponding to these footprints for every organism. However, given the high degree of sequence conservation and the finding of ESTs of each footprint in at least two different organ- isms, we believe it is possible that transcription and per- haps the function of these elements are conserved. Therefore, we consider these footprints to be putative ncRNA genes, and the other, non-transcribed footprints as potential cis-regulatory elements (Figure 1). Non-coding RNAs are functional molecules that are not translated into a protein. Instead, they are involved in post-transcriptional modification or DNA replication or have a regulatory function, and can be found in inter- genic regions, introns and even in the UTR of genes or overlapping with protein coding genes [46]. The gene regulatory RNAs are usually small and act in trans by post-transcriptional silencing of target genes through the binding of complementary sites. Next to the large number of small ncRNAs, an increasing number of long ncRNAs is being described, which are at least 200 to over 10,000 nucleotides long (reviewed by [47]). Long ncRNAs can act independently of a target sequence, in cis, by interfering with the transcription of a neighboring gene, or in trans, by recruiting proteins that alter the chromatin state [47]. One example of a regulatory ncRNA in the HoxC cluster is HOTAIR, which epigen- etically represses transcription of 40 kb across the HoxD locus [48]. Screening of all the expressed footprints against the Functional RNA Database [49] did not result in the identification of any classified ncRNA. However, four of them produced significant matches with putative RNAs predicted by Evofold, which is a method to iden- tify functional RNA structures in vertebrates by using a combined probabilistic model of RNA structure and sequence evolution [50]. In addition, in footprint fp1, also known as CR3 [51], a significant RNA secondary structure was predicted (p = 0.92). Seven of the expressed footprints lie in the intergenic region between Hoxd-11 and Hoxd-10 (Figure 1). What tracker consid- ers to be multiple footprints may correspond to a single potential ncRNA gene, as indicated by alignment of the Page 5 of 9 region between Hoxd-11 and Hoxd-10 with the corre- sponding ESTs (not shown). Two of the expressed foot- prints were previously identified as regulatory regions RVIII/RIX (fp26) and RX (fp20) ([52-55]. The fact that these two regulatory regions appear to be transcribed may shed new light on how they function. Cis-regulatory elements can be anticipated in inter- genic regions, in introns and in the 5’ and 3’ UTR of protein-coding genes, and may act as a promoter, enhancer, repressor or insulator [42]. In total, 24 foot- prints (fps) are putative cis-regulatory elements. Nine of these were found in the intergenic regions, including the area downstream of Evx-2; four footprints are located in an intron, five located in the 5’ UTR and six located in the 3’ UTR of the Hox and Evx-2 genes. Three of the intergenic footprints were identified in previous studies and are known as RXI (fp18) and RXII (fp9 and fp10) [23,56]. Two other intergenic footprints (fp 2 and fp3) are extremely conserved in all species used in this study and are located downstream Evx-2. The reason for this high degree of conservation however remains unclear [51]. One footprint (fp23) that was found in the intron of Hoxd-11 contains a HB1 element, which consists of homeodomain binding sites. This element is previously described from the intron of Hoxa7, the Drosophila homolog Ubx, the introns of Hox4 genes and the intron of Hoxa-11 [57-60]. Of the five footprints in the 5’UTR, two also include the promoter region. The 5’ and 3’ UTRs of mRNAs play an important role in the post- transcriptional regulation of gene expression through the presence of cis-acting elements and through interac- tion with micro-RNAs (miRNAs) [61,62]. Conserved sequences in 3’ UTRs may contain potential target sites for miRNAs that are involved in post-transcriptional gene silencing. Micro-RNAs are short, single-stranded RNA molecules of ~22 nucleotides that show at least partial complementarities to their target mRNA. If the miRNA is only partially complementary, a perfect match between the seed (nucleotides 2 to 7) of the miRNA and the target mRNA is necessary for inhibition of transla- tion or for promotion of deadenylation [63,64]. There- fore all six footprints located in the 3’UTR of the genes were screened for the presence of hypothetical target sites for miRNAs. One footprint (fp15), located in the 3’UTR of Hoxd-13, contains a short motif of seven nucleotides, with perfect match to the seed of the miRNA miR-26. Reconstruction of conserved element evolution Gains and losses of putative ncRNAs and cis-regulatory elements in vertebrate posterior HoxD clusters were mapped on a vertebrate timescale under the Dollo parsi- mony criterion, i.e. assuming a single origin (Figure 3). Two putative cis-regulatory elements have been lost in