Bernard et al. BMC Genomics 2010, 11:166 http://www.biomedcentral.com/1471-2164/11/166 The number of genes containing the TA-PLMs decreased from the TATA-box to the TATAΔ2-PLMs (Figure 2). Interestingly, even though most of the TATAΔ-PLMs were found to be T and A rich as expected, 3 out of the 4 TATAΔ2-PLMs (CTTAAA, TACTTA and TCTTAA) contained a base C. This sug- gested that some T and C rich PLMs, hereafter referred to as TC-PLMs, could have been missed in the analysis of the whole promoter set. Indeed, it has been shown that, in plants, the region overlapping the TSS is char- acteristically rich in T and C nucleotides described as CpT microsatellites or TC-microsatellites [35,36]. We observed these microsatellites during the analyses of the whole promoter set within a number of different 6- base-long PLMs. They are characterized by a wide func- tional window centred downstream of the TSS often extending several hundred bases. We sometimes observed a small secondary peak located around 30 bases upstream of the TSS which disturbed the broad distribution of some of the TC-microsatellites (Figure 3. A). These TC-microsatellites may hide TC[-39,-26]-PLMs, i.e. TC-PLMs with high topological constraints similar to those of TA-PLMs present within specific sub-sets of genes. Figure 3 Distributions of TTCTTC in A. thaliana promoters aligned relative to the TSS. TTCTTC is one of the 29 conserved TC [-39,-26]-PLMs. Distributions are shown for the whole promoter set containing 14927 promoters (A) and for the 1745 TA-less promoters (B). The y-axis percentage depends on the promoter number in each data set. TTCTTC is preferentially positioned at +29 (SMS 10) and -33 (SMS 8) in the whole promoter set and in the TA-less promoter set respectively. In both cases, the size of the window used to scan the promoters was 2 bases. Page 4 of 15 Conserved TC[-39,-26]-elements are observed in almost 18% of A. thaliana promoters We hypothesised that TC[-39,-26]-PLMs with narrow functional windows could be functional alternatives to TA-PLMs. This being the case we expected to be able to detect these PLMs more easily in promoter sets within which they predominate or within promoters not containing TA-PLMs. We therefore selected a TA-less promoter set by excluding the TA-PLM-containing pro- moters from the whole promoter dataset. Out of the 16 possible dinucleotides, only 3 were found to be con- served PLMs and to shared the TATA-box topological constraints: TA, AT and AA. A total of 1745 A. thali- ana promoters (11.7%) and 5089 O. sativa promoters (28.2%) contained no TATA-box or conserved TATAΔ- PLM or the 3 conserved dinucleotide-PLMs. We examined the possibility of these TA-less promo- ter sets representing sets of poorly annotated promoters due to an incorrect prediction of the TSS position. In both TA-less promoter sets, three arguments supported the validity of the TSS position prediction. First, the observed TC[-39,-26]-PLMs were conserved between A. thaliana and O. sativa and in both plants they exhibited the same strict topological constraints (see below). Sec- ond, the distributions of several motifs known as TFBSs supported by experimental analyses [37,38], such as the Inr or the GGCCC element, shared the same topological constraints in both the whole and the TA-less promoter sets (data not shown). Third, as expected, the GC-com- positional strand bias or GC-skew expected in plant promoters was observed at the TSS location in both promoter sets (data not shown). We therefore searched in the TA-less promoter sets for the presence of 6-base-long conserved PLMs. Out of the 4096 possible motifs, we found 29 conserved PLMs exhibiting a strict functional window in the [-39, -26] area relative to the TSS. These PLMs were present in 2645 A. thaliana (17.7%) and in 2331 O. sativa (12.9%) promoters. In agreement with our hypothesis, the 29 PLMs were TC[-39,-26]-PLMs, i.e. were comprised of T and C bases only. Figure 3 illustrates how the TC[-39,- 26]-PLMs can be clearly distinguished in the TA-less promoter set while they were missed in the whole pro- moter set. Indeed, using the whole promoter set, it is only possible to find the TTCTTC-PLM, which is char- acterized by a wide functional window and by a prefer- ential position 29 bases downstream of the TSS (Figure 3.A). On the contrary, using the TA-less promoter set, we observed both the wide functional window PLM and a distinct TTCTTC-PLM characterized by a sharp peak centred 33 bases upstream of the TSS, i.e. sharing the TA-motif topological constraints (Figure 3.B). We pro- pose that TA-PLMs may be recognized and thus be functional in a T and C rich environment whereas the