Bernard et al. BMC Genomics 2010, 11:166 http://www.biomedcentral.com/1471-2164/11/166 same might not be true for TC[-39,-26]-PLM. As a conse- quence, large pyrimidine-rich regions could have been preferentially maintained during evolution in promoters with a TA-PLM whereas they may have been counter- selected, at least upstream of the TSS, in promoters with a functional TC[-39,-26]-PLM. It is important to note that only TC[-39,-26]-PLMs exhibited the TATA-box topological constraints in the TA-less promoter set. Altogether, our results (i) confirmed the importance of TC-microsatellites (TC{n} or TTC{n} for instance - Figure 3.A) in plant promoters and (ii) predicted the existence of a novel class of functional elements, the TC[-39,-26]- PLMs characterized by a sharp peak in the [-39, -26] region and observed in almost 18% of A. thaliana promoters. We investigated the putative presence of TC[-39,-26]- PLMs in other eukaryotic genomes. We analyzed 15802 Homo sapiens promoters and 15833 Mus musculus pro- moters with an experimentally supported TSS [39]. We observed neither TC[-39,-26]-PLMs nor any other PLMs at the TATA-box expected region. Thus, both the TC- microsatellites and the TC[-39,-26]-PLMs observed in plants are absent in both human and mouse. These observations suggest an evolutionary link between the presence of the TC[-39,-26]-PLMs and the TC- microsatellites. TC[-39,-26]-elements derived from three seed motifs As for all identified TATAΔ-PLMs, any 6-base-long TC [-39,-26]-PLM may be a functional PLM, be a part of a larger functional PLM or contain a smaller functional PLM. Any of the TC[-39,-26]-PLMs can potentially over- lap by 5 consecutive bases with at least one other TC [-39,-26]-PLM. Among the promoters studied, we found only 3 overlapping TC[-39,-26]-PLMs: CTTCTT, TTCTTC and TCTTCT. The trinucleotide repetition made up of 2 T bases and one C base is characteristic of self-overlapping motifs involved in DNA recognition by transcription factors [40]. Furthermore, we analyzed the effect of extending each of the 29 TC[-39,-26]-PLMs on SMS, i.e. the distribution score. Extended PLMs with higher SMS scores than those of the initial 6-base-long PLM were considered to be functional candidates. CTTCTT and TTCTTC could be extended up to the 9- base-long TCTTCTTCT PLM that exhibited the highest SMS (Additional file 1). Together, these results provide evidence in favour of the TCTTCTTCT motif being a functional TC[-39,-26]-PLM. Extension of most of the other TC[-39,-26]-PLMs did not generate PLMs with higher SMS than the SMS of the initial 6-base-long PLM. Note that the TC-microsatellites observed in the gene 5’ UnTranslated Regions (UTRs) could be extended up to a 13-base-long PLM (data not shown). The short- est core-PLMs were CTC, TCT and CTT, found within Page 5 of 15 27 TC[-39,-26]-PLMs, suggesting that these trinucleotides could be the core of the functional TC[-39,-26]-PLMs. Both the trinucleotide repetitions and the different T and C environments might therefore have a role in TC [-39,-26]-PLM function. Three observations are in favour of a putative role of most TC[-39,-26]-PLMs independently of the presence of other PLMs in the same region. First, TC[-39,-26]-PLMs were often observed without any of the other PLMs sharing the same preferential position in [-39, -26]. Sec- ond, TC[-39,-26]-PLMs are not extensions of either a TATA-box or its variants. Thus, among the [-39,-26]- PLM-containing promoters, 80% contained PLM-motifs belonging to only one PLM class, either a TATA-box, a TATAΔ- or a TC[-39,-26]-PLM. Third, it was possible to construct oriented graphs displaying the motif diver- gence for both TA- and TC-PLMs (Figure 2 and 4). Concerning the TA-PLMs, the graph root or seed is the TATAWA-PLM. For a given TA-PLM, the number of promoters containing it depends on both the distance from the seed, i.e. the number of substitutions between the given PLM and the seed-PLM and the existence or not of a more divergent sequence. We applied the same approach to the conserved TC[-39,-26]-PLMs and found that they could be organized into a unique, closed and oriented graph (see Methods section). Three seeds were suggested: TCTTCT, TTTCTT and TTCTTC (Figure 4). Similar to that observed for TA-PLMs, these three TC [-39,-26]-PLMs were those most frequently observed in promoters with the number of promoters containing the other TC[-39,-26]-PLMs depending on both the dis- tance from these seeds and the existence or not of a more divergent sequence. In conclusion, all [-39,-26]- PLMs can be detected independently in different pro- moters and may be organized into different groups with apparent evolutionary links between the given PLM and the related seed. TC[-39,-26]-element-containing genes are preferentially involved in protein metabolism While most promoters contain only one class of [-39,-26]- PLM, some contain more than one (Figure 5). To investigate the functional significance of the different [-39,-26]-PLM classes we decided to construct four pro- moter sets each characterized by the exclusive presence of one of the classes of PLM in the [-39, -26] region. We distinguished (i) the 1496 promoters containing only a TATA-box, (ii) the 1919 containing only a TC [-39,-26]-PLM, (iii) the 2773 containing only a TATAΔ- PLM and (iv) as a negative reference the 7194 promo- ters without any [-39,-26]-PLM, called hereafter the [-39,- 26]-PLM-less set. Gene Ontology (GO) annotations [41] have been used previously in the prediction of the functional role of