Bernard et al. BMC Genomics 2010, 11:166 http://www.biomedcentral.com/1471-2164/11/166 to previous TFBS predictors [14,15] are based on the positional densities of oligonucleotides rather than on their frequency of occurrence. These tools have been used to characterize core-promoter elements in several model genomes including plants [16-22]. All together, the core-promoter elements listed above seem unable to account for the transcription of all the RNA-polymerase-II transcribed genes. Less conserved core-promoter elements present in small gene sets have been described in previous studies at the gene level. For instance, in the human cytosolic phospholipase A2- alpha gene, an AAGGAG motif in the [-35, -30] area binds TBP and is critical for basal transcriptional activity [23]. In other studies, a TBP has been shown to bind to a TAAGAGA element in the [-23, -17] region of the hepatitis B virus S gene [24]. These experimental obser- vations suggest that core-promoter elements specific to small sets of genes remain to be disclosed. A study of large-scale structural properties of DNA in promoters indicated that the instability of DNA around -30 relative to the TSS necessary for transcription may be due to as yet unidentified motifs other than the TATA-box [25]. It is therefore clear from and despite this growing amount of data that the code embedded within core- promoter sequences has not yet been fully deciphered. In this work, we used an in silico hypothesis-driven approach to predict novel elements potentially recog- nized by the transcriptional complex. We explored the bioinformatics-based evidence that sequences other than the TATA-box and TATA-variants but located in the same region relative to the TSS may be functional core- promoter elements. We therefore searched for short sequences exhibiting similar positional constraints to those of the TATA-box and identified pyrimidine-rich elements distinct from the pyrimidine tract [21,26] as candidate elements for about 18% of the plant genes. To determine their potential functional role, we investigated any association between such identified TC-elements and specific features of the genes containing them, as has been previously shown for the TATA-box. Results Less than 39% of A. thaliana promoters contain a TATA- box or a TATA-variant Our approach was based on three steps. First, we searched for all 6 base long motifs with a statistically significant preferential position within the 300 nucleo- tides upstream of the TSS and called these motifs the Preferentially Located Motifs (PLMs). This method was first described by FitzGerald et al. [27] for the analysis of human promoters and was then applied by others to plant promoters [19,21]. Our results correlated well with previous studies in terms of the spatial representation of the PLMs within the plant promoters. Second, for each Page 2 of 15 PLM, we precisely defined: (i) the preferential position relative to the TSS, i.e. the top of the peak, (ii) the func- tional window, i.e. the peak width, derived from the peak boundaries, (iii) the Score of Maximal Square rela- tive to the base line (SMS, see Methods section) ranking the PLMs in terms of their topological constraint and (iv) the list of genes containing the studied PLM within the functional window. Third, to increase the chance of identifying functional PLMs, we searched for their con- servation in 14927 A. thaliana genes and 18012 O. sativa genes with experimentally supported TSSs. A well-annotated genomic sequence is available for both of these species that diverged approximately 150 million years ago [28,29]. As previously described within the 50 nucleotides upstream of the TSS of both A. thaliana [19,21] and O. sativa [26] we found PLMs made up exclusively or almost exclusively of: (i) T and A nucleo- tides and exhibiting strong topological constraints, i.e. a sharp peak, and (ii) T and C nucleotides and exhibiting low topological constraints, i.e. a wide peak. Among the T and A rich motifs, we found the canonical TATA-box defined by the TATAWA consensus (W for A or T) and TATA-box variants [8]. We wondered whether by analysing the whole promoter set, other motifs with the same strong topological constraints could be missed. It is reasonable to predict that the presence of a PLM characterized by a wide functional window overlapping the TATA-box expected area might hide the strong topological constraints of a PLM specific to a small pro- moter set. To address this issue, we built up promoter sets by successively subtracting from the whole set of promoters, those characterized by different classes of PLMs, as described below. We firstly considered the conservation of PLMs at the genome level and then their conservation at the orthologues level. TATAWA is a particularly well-conserved PLM since it is found in the same promoter region in both plants and animals [30]. Confirming previous results, we found this PLM in both A. thaliana and O. sativa genomes in a preferential position 32 bases upstream of the TSS and strictly located within the [-39, -26] region (Figure 1). A total of 2606 (17.5%) and 2601 (14.4%) promoters in A. thaliana and O. sativa respectively contained a TATAWA within the [-39, -26] functional window. In contrast to the motif counting method highly prone to false positives, our method may slightly underestimate the number of functional motifs. Indeed, as our aim was to characterize and compare different sets of genes each containing defined PLMs (TATA-box or other) within their promoters, we chose only “clean” sets, i.e. those containing only one regulatory element to the detriment of completeness. Several T and A rich motifs partially matching the TATAWA sequence have been shown at the TATA-box