BACKGROUND
over the past genomics has markedly changed our view on core-promoter organization  <cit> .
for instance, the tata-box can no longer be regarded as a general feature of polymerase ii core-promoters  <cit> .
indeed, only a small fraction of eukaryotic genes actually harbour a tata-box: less than 20% of genes in both human  <cit>  and yeast  <cit> .
while tata-box-containing promoters support the direct binding of tata-box-binding proteins  and thereby also the formation of the pre-initiation complex, tata-less promoters are recognized by multiple tbp-related proteins and other tbp-associated transcription factors  involved in the recruitment of tbp  <cit> .
indeed some tata-variants and other alternative elements allow the initiation of transcription and participate towards defining distinct patterns of expression  <cit> .
several core-promoter elements or general transcription factor binding sites  have been previously identified in eukaryotes.
they are characterized by a strong positional preference relative to the transcriptional start site  as for instance the tata-box in the  area  <cit> , the initiator element, , around the tss  <cit> , the downstream promoter element in the  area  <cit> , or the iib recognition element immediately upstream of certain tata-boxes  <cit> .
the position of binding sites of proteins belonging to the transcription complex is important for the functioning of promoters since it determines both the tss location  <cit>  and the transcription direction  <cit> .
thus, a strong positional conservation of a novel regulatory element would strongly indicate a functional role.
this concept has led to a generation of tools that in contrast to previous tfbs predictors  <cit>  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  <cit> .
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  area binds tbp and is critical for basal transcriptional activity  <cit> .
in other studies, a tbp has been shown to bind to a taagaga element in the  region of the hepatitis b virus s gene  <cit> .
these experimental observations 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 - <dig> relative to the tss necessary for transcription may be due to as yet unidentified motifs other than the tata-box  <cit> .
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 recognized 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  <cit>  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  <dig> base long motifs with a statistically significant preferential position within the  <dig> nucleotides upstream of the tss and called these motifs the preferentially located motifs .
this method was first described by fitzgerald et al.
<cit>  for the analysis of human promoters and was then applied by others to plant promoters  <cit> .
our results correlated well with previous studies in terms of the spatial representation of the plms within the plant promoters.
second, for each plm, we precisely defined:  the preferential position relative to the tss, i.e.
the top of the peak,  the functional window, i.e.
the peak width, derived from the peak boundaries,  the score of maximal square relative to the base line  ranking the plms in terms of their topological constraint and  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 conservation in  <dig> a. thaliana genes and  <dig> o. sativa genes with experimentally supported tsss.
a well-annotated genomic sequence is available for both of these species that diverged approximately  <dig> million years ago  <cit> .
as previously described within the  <dig> nucleotides upstream of the tss of both a. thaliana  <cit>  and o. sativa  <cit>  we found plms made up exclusively or almost exclusively of:  t and a nucleotides and exhibiting strong topological constraints, i.e.
a sharp peak, and  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  and tata-box variants  <cit> .
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 promoter 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  <cit> .
confirming previous results, we found this plm in both a. thaliana and o. sativa genomes in a preferential position  <dig> bases upstream of the tss and strictly located within the  region .
a total of  <dig>  and  <dig>  promoters in a. thaliana and o. sativa respectively contained a tatawa within the  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  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 expected position  <cit> .
we observed plms with the same positional constraints as tatawa, i.e.
showing a sharp peak in the  region.
these plms may be  functional motifs hereafter named as tataΔ-plms where Δ stands for variant; or  motifs being plms due to their overlap with the tatawa sequence.
this overlap represents an inherent drawback of the method since motifs shorter than  <dig> nucleotides  as well as  <dig> base long motifs  may appear as plms.
tata-variant and tata-box are distinct regulatory elements that need differentiating  <cit> .
for this reason, we searched for tataΔ-plms in sets of promoters not containing the canonical tata-box in the  region.
in this way, we were able to ascertain that the plms found within the promoter sets were bona fide elements under topological constraint.
in a. thaliana and o. sativa respectively  <dig>  and  <dig>  promoters were found without a tatawa in the  region.
relative to the tatawa sequence,  <dig> possible motifs diverged at one position.
out of these  <dig> motifs, only  <dig> were found to represent plms  conserved in both species .
consistent with in vivo experimental results  <cit> , t to a or a to t substitutions were the main differences observed among the tataΔ1-plms.
motifs with a base c or g substitution at the central positions were not considered as plms and were counter-selected.
motifs with a c or g substitution at the t <dig> or a <dig> positions were, however, considered as tataΔ1-plms.
there remained  <dig>  and  <dig>  of a. thaliana and o. sativa genes respectively containing neither a tatawa nor a tataΔ1-plm.
we identified only  <dig> conserved tataΔ2-plms amongst the  <dig> possible motifs  and no conserved tataΔ3-plm.
finally, at the tata-box expected position,  <dig> a. thaliana  and  <dig> o. sativa promoters  contained a tataΔ-plm.
hereafter, tataΔ- and tatawa-plms are collectively referred to as ta-plms.
the number of genes containing the ta-plms decreased from the tata-box to the tataΔ2-plms .
interestingly, even though most of the tataΔ-plms were found to be t and a rich as expected,  <dig> out of the  <dig> tataΔ2-plms  contained a base c. this suggested 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 characteristically rich in t and c nucleotides described as cpt microsatellites or tc-microsatellites  <cit> .
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 functional window centred downstream of the tss often extending several hundred bases.
we sometimes observed a small secondary peak located around  <dig> bases upstream of the tss which disturbed the broad distribution of some of the tc-microsatellites .
these tc-microsatellites may hide tc-plms, i.e.
tc-plms with high topological constraints similar to those of ta-plms present within specific sub-sets of genes.
conserved tc-elements are observed in almost 18% of a. thaliana promoters
we hypothesised that tc-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 promoters from the whole promoter dataset.
out of the  <dig> possible dinucleotides, only  <dig> were found to be conserved plms and to shared the tata-box topological constraints: ta, at and aa.
a total of  <dig> a. thaliana promoters  and  <dig> o. sativa promoters  contained no tata-box or conserved tataΔ-plm or the  <dig> conserved dinucleotide-plms.
we examined the possibility of these ta-less promoter 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-plms were conserved between a. thaliana and o. sativa and in both plants they exhibited the same strict topological constraints .
second, the distributions of several motifs known as tfbss supported by experimental analyses  <cit> , such as the inr or the ggccc element, shared the same topological constraints in both the whole and the ta-less promoter sets .
third, as expected, the gc-compositional strand bias or gc-skew expected in plant promoters was observed at the tss location in both promoter sets .
we therefore searched in the ta-less promoter sets for the presence of 6-base-long conserved plms.
out of the  <dig> possible motifs, we found  <dig> conserved plms exhibiting a strict functional window in the  area relative to the tss.
these plms were present in  <dig> a. thaliana  and in  <dig> o. sativa  promoters.
in agreement with our hypothesis, the  <dig> plms were tc-plms, i.e.
were comprised of t and c bases only.
figure  <dig> illustrates how the tc-plms can be clearly distinguished in the ta-less promoter set while they were missed in the whole promoter set.
indeed, using the whole promoter set, it is only possible to find the ttcttc-plm, which is characterized by a wide functional window and by a preferential position  <dig> bases downstream of the tss .
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  <dig> bases upstream of the tss, i.e.
sharing the ta-motif topological constraints .
we propose that ta-plms may be recognized and thus be functional in a t and c rich environment whereas the same might not be true for tc-plm.
as a consequence, 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-plm.
it is important to note that only tc-plms exhibited the tata-box topological constraints in the ta-less promoter set.
altogether, our results  confirmed the importance of tc-microsatellites  in plant promoters and  predicted the existence of a novel class of functional elements, the tc-plms characterized by a sharp peak in the  region and observed in almost 18% of a. thaliana promoters.
we investigated the putative presence of tc-plms in other eukaryotic genomes.
we analyzed  <dig> homo sapiens promoters and  <dig> mus musculus promoters with an experimentally supported tss  <cit> .
we observed neither tc-plms nor any other plms at the tata-box expected region.
thus, both the tc-microsatellites and the tc-plms observed in plants are absent in both human and mouse.
these observations suggest an evolutionary link between the presence of the tc-plms and the tc-microsatellites.
tc-elements derived from three seed motifs
as for all identified tataΔ-plms, any 6-base-long tc-plm may be a functional plm, be a part of a larger functional plm or contain a smaller functional plm.
any of the tc-plms can potentially overlap by  <dig> consecutive bases with at least one other tc-plm.
among the promoters studied, we found only  <dig> overlapping tc-plms: cttctt, ttcttc and tcttct.
the trinucleotide repetition made up of  <dig> t bases and one c base is characteristic of self-overlapping motifs involved in dna recognition by transcription factors  <cit> .
furthermore, we analyzed the effect of extending each of the  <dig> tc-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 .
together, these results provide evidence in favour of the tcttcttct motif being a functional tc-plm.
extension of most of the other tc-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  could be extended up to a 13-base-long plm .
the shortest core-plms were ctc, tct and ctt, found within  <dig> tc-plms, suggesting that these trinucleotides could be the core of the functional tc-plms.
both the trinucleotide repetitions and the different t and c environments might therefore have a role in tc-plm function.
three observations are in favour of a putative role of most tc-plms independently of the presence of other plms in the same region.
first, tc-plms were often observed without any of the other plms sharing the same preferential position in .
second, tc-plms are not extensions of either a tata-box or its variants.
thus, among the -plm-containing promoters, 80% contained plm-motifs belonging to only one plm class, either a tata-box, a tataΔ- or a tc-plm.
third, it was possible to construct oriented graphs displaying the motif divergence for both ta- and tc-plms .
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-plms and found that they could be organized into a unique, closed and oriented graph .
three seeds were suggested: tcttct, tttctt and ttcttc .
similar to that observed for ta-plms, these three tc-plms were those most frequently observed in promoters with the number of promoters containing the other tc-plms depending on both the distance from these seeds and the existence or not of a more divergent sequence.
in conclusion, all -plms can be detected independently in different promoters and may be organized into different groups with apparent evolutionary links between the given plm and the related seed.
tc-element-containing genes are preferentially involved in protein metabolism
while most promoters contain only one class of -plm, some contain more than one .
to investigate the functional significance of the different -plm classes we decided to construct four promoter sets each characterized by the exclusive presence of one of the classes of plm in the  region.
we distinguished  the  <dig> promoters containing only a tata-box,  the  <dig> containing only a tc-plm,  the  <dig> containing only a tataΔ-plm and  as a negative reference the  <dig> promoters without any -plm, called hereafter the -plm-less set.
gene ontology  annotations  <cit>  have been used previously in the prediction of the functional role of tfbs  <cit> .
here we analysed the go annotations of the four classes of genes defined above, according to the four promoter sets.
as far as biological processes are concerned, the most conspicuous observation was a preferred association between genes containing a tata-box and responses to different stimuli .
this result was expected since in human and in yeast the tata-box-containing genes are more frequently involved in specific biological processes  <cit> .
interestingly, we also found that tc-plm-containing genes are more frequently involved in protein metabolism than any other gene class , i.e.
a basic biological process.
neither the tataΔ-containing genes nor the -plm-less genes showed any significant bias for any biological process categories.
we also analysed the partitioning of genes between the different go cellular components.
again, the tata-box-containing genes presented the most biased partitioning.
the products of these genes are more often constituents of the cell wall, the cytosol and the ribosomes and less frequently constituents of the chloroplasts, the plastids, the golgi apparatus and the mitochondria compared to the products of all the other genes .
products of the tc-plm-containing genes exhibited little biased partitioning between the go cellular component categories.
indeed, only the cell wall component was significantly less represented.
it seems biologically sound to have found a positive correlation between the response to different stimuli and the cell wall go categories in the tata-box-containing genes and a negative correlation between the protein metabolism and the cell wall categories for tc-plm-containing genes.
these observations suggest that tc-plms might have a functional role in transcriptional control that could frequently differ from and perhaps even oppose the tata-box functional role.
it is of particular interest that our observations support the hypothesis that small variations in the tata-box sequence are linked to large changes in gene expression  <cit> .
unfortunately, due to the relatively small number of promoters containing a unique tc-plm, it was deemed impossible to perform the same comparisons between the tc-plm seeds alone and their predicted variants as that performed for the ta-motifs.
it should be noted that by analysing all the genes containing a tc-plm alone we decreased the power of our statistical tests.
we were however able to demonstrate a significant difference between the tata-box and the tc-plm sets concerning protein metabolism and cell wall categories .
gene functional categories have been retrieved from the go categories  <cit>  provided by tair  <cit> .
in the table, each number not in parenthesis is the percentage of genes annotated in a given go category.
we performed the two one-sided fisher exact tests allowing the identification of enrichment  or impoverishment  of go categories in a given set of genes when compared with all the other genes, i.e.
genes within the whole gene set minus genes within the considered gene set.
ns indicates a non-significant difference between percentages.
p-values in parenthesis are less than 5% with the bonferroni correction.
results are only shown for go categories exhibiting at least one bias.
only the canonical tata-box is spatially linked to the initiator element
recent progress in mammalian genomics has defined a more functional image of core-promoters.
two functional categories of core-promoters have been described  <cit> .
the single peak promoters have a tightly defined tss position within a few base pairs while in broad peak promoters several tsss may be found within small clusters spanning tens of base pairs.
a tata-box is more likely to be found within single peak promoters than in broad peak promoters  <cit> .
in genome-wide studies, only the most upstream tsss are often considered and are defined by the available transcript sequences.
there are three main categories of core-promoters described in human  <cit> , i.e.
tata-box-containing promoters with an inr, tata-box-containing promoters without inr and tata-less promoters.
we observed the same relative representation of these three categories in arabidopsis.
consistent with mammalian studies  <cit> , recent evidence suggests that a. thaliana genes containing a tata-box tend to have a sharper dominant peak of tss compared to that of other genes  <cit> .
the distance between tata-box and inr is important for accurate transcription initiation  <cit> .
we considered the distance between the inr  <cit> , called here yr-tss, and the conserved plms characterized using our approach.
as expected, the yr-tss is a conserved plm whose functional window is one base upstream of the tss in both plants .
we first analysed the  <dig> a. thaliana promoters containing a tata-box and we represented the distances between each tatawa in the  region and each dinucleotide ca, ta, tg and cg up to  <dig> bases downstream of the tatawa .
the dinucleotide ca, but not ta, tg or cg showed a strong preferred distance of  <dig> to  <dig> bases from tatawa.
this distance is consistent with the preferential position of both plms and with mammalian results  <cit> .
furthermore, none of the yr motifs showed a preferential distance either from tataΔ- or from tc-plms .
finally, our results provide evidence of a link between the canonical tata-box and the ca-tss:  <dig> to  <dig> bases preferentially separate the ca from the first t of tatawa.
genes containing the tc-element are generally long
in human, the presence of a tata-box in promoters is often associated with a compact gene structure  <cit> .
in a. thaliana, we observed the same bias towards a more compact structure of tata-box-containing genes .
in contrast, the tc-plm-containing genes and the -plm-less genes were overall relatively longer while the gene structure of tataΔ-plm-containing genes showed no bias.
when present, the bias in gene size was mainly explained by the 5'utr and by the length of the coding sequence.
however, to a lesser extent, the enrichment in compact genes amongst those containing a tata-box may be explained by the shorter cumulative length of introns, and by the higher percentage of intronless genes .
structural gene features have been assigned by querying the flagdb++ database  <cit> .
for median length data, we performed two one-sided wilcoxon tests allowing the identification of enrichment in wide  or in compact gene structures  in a set of genes compared with all the other genes, i.e.
genes within the whole gene set minus genes within the considered gene set.
for intron-less gene percentages, we performed two one-sided fisher exact tests allowing the identification of higher  or lower  percentages in a gene set in comparison with all the other genes.
ns indicates a non-significant difference.
p-values in parenthesis are less than 5% with the bonferroni correction.
both the first intron and 3'utr lengths are never biased .
based on the counting of expressed sequence tags   <cit>  or on microarray data  <cit> , gene size and gene expression levels have been shown previously to be inversely correlated in both human and yeast.
this being the case, our observations could be due to the fact that, in general, tata-box genes have higher expression levels than other a. thaliana genes  <cit> .
the differences in gene sizes we observed are consistent with a lower mean expression of tataΔ-plm- than of tata-box-containing genes.
both tc-plm-containing and -plm-less genes could be expressed at lower levels.
tata-box-containing genes have been shown to generally display relatively high specificity of expression compared to housekeeping genes that frequently show high expression levels .
therefore, the question remains as to understand whether the presence of regulatory elements in the  region of promoters is linked directly to gene function per se or to the transcription level that in turn is associated with gene function.
to resolve this we analysed a large set of transcriptome data, distinguishing the two different components of transcription: specificity and level.
specific role played by each class of regulatory element found in the  region on different components of transcription
in eukaryotic genomes, various tfbss have been linked to specific gene expression patterns  <cit> .
we searched for such a link with plms in the  region using a. thaliana transcriptome data obtained with the complete arabidopsis transcriptome microarray   <cit>  and available through the complete arabidopsis transcriptome database: catdb  <cit> .
the dataset included  <dig> hybridizations based on  <dig> different samples covering numerous developmental stages, biotic and abiotic stresses and mutants.
we used normalized data on which positive hybridizations have previously been determined .
for any one gene, the relative number of positive hybridizations was considered as a measure of the range of expression, i.e.
of the specificity.
to measure the global level of expression we computed the median of the signal intensities.
the relationship between the median signal intensity and the percentage of hybridizations was clearly not linear and suggested the existence of different classes of genes with respect to transcriptional regulation.
we thus clustered the transcriptome data into four classes .
first, genes were separated in two classes depending on their expression level above or below the distribution model line, respectively he for those with high levels of expression and le for those with low levels of expression.
second, two classes were defined relating to the specificity of hybridizations and delimited by the two inflections in the cloud of points.
the first class clustered genes positively hybridized in less than 15% of microarray hybridizations  and the second class clustered those with more than 85% of hybridizations .
interestingly, each of these four transcriptional clusters was predominantly made up of genes containing one of a specific class of -plms.
thus,  genes within the transcriptional sr group, i.e.
those that hybridized in specific conditions predominantly contained tc-plm;  genes within the transcriptional wr group, i.e.
housekeeping genes predominantly contained a tataΔ-plm;  the transcriptional he group was enriched with the tata-box-containing genes and  the transcriptional le group was enriched with the -plm-less genes.
these results clearly indicate a link between the different motifs within the core-promoter and transcriptional features of the gene and are in favour of a functionally independent role of the tata-box, the tataΔ- and the tc-plms.
our observations provide further evidence for the involvement of the tata-box in the regulation of transcription level.
in addition, our results suggest that both tataΔ- and tc-plms might be involved in defining the specificity of transcription.
the role of the tataΔ-plms might be to promote a relatively wide range of expression while on the other hand tc-plms may impose narrower limits.
the adverse effect of tatawa and -plm-less within genes on the extreme level of transcription  was particularly evident in the sr group .
the most highly expressed genes  were predominantly those containing tatawa whereas the most lowly expressed genes  were mainly -plm-less genes.
we propose that in the presence of a tata-box, the recognition of the tss depends directly on a small number of tbps and/or of tbp-related protein.
the relaxed recognition by tbps in tataΔ-containing promoters might be responsible for a bias towards the large range of expression.
in the absence of tata-box or tataΔ, the recruitment of the tbp might be mediated by different tbp-associated tfs depending on the gene.
interaction of the different tbp-associated tfs with other specific factors might therefore explain the relatively higher specificity of transcription observed for the tc-plm gene set.
the  <dig> expression categories are described in the figure  <dig>  he, high expression; le, low expression; sr small range of expression; wr, wide range of expression; he+ highest expressions; le+, lowest expressions.
the whole gene set contains the  <dig> genes for which we have both a known tss and catma-expression data.
we performed two one-sided fisher exact tests allowing the identification of higher  or lower  percentages in a gene set in comparison with all other genes, i.e.
genes within the whole gene set minus genes within the considered gene set.
ns indicates non-significant difference.
p-values in parenthesis are less than 5% with the bonferroni correction.
the class of regulatory element in the  region is not conserved in higher plant orthologous gene pairs
with the increasing availability of transcriptome data, the search for conserved tfbss within promoters of genes exhibiting similar transcriptional patterns has gathered much attention.
surprisingly, the conservation of core-promoter elements, widely considered as being those most conserved, has not received such large interest.
one recent study addressing this question did cast doubt on this widely accepted notion  <cit> .
concordantly, in several studies on orthologous relationships between large gene families, we observed no significant conservation of the presence of a tata-box between plant orthologues .
even single copy genes present as orthologues in both a. thaliana and o. sativa are no more conserved than random pairs of genes  <cit> .
the co-occurrence of different elements in the  promoter region could, at least in part, explain an apparent non-conservation at the gene level despite the observed conservation at the genomic one.
this prompted us to reexamine the question comparing core-promoter elements in the  region in a. thaliana and o. sativa.
we analysed the conservation of the three classes of -plms in  <dig> pairs of orthologous genes in a. thaliana and o. sativa characterized by an experimentally supported tss.
while the conservation of tatawa appeared relatively low , it was significantly higher than that expected by chance  .
this was not the case for tataΔ- or tc-plms, which exhibited no conservation between orthologous genes .
our results showing that the tata-box is more involved at the transcriptional level and that both tataΔ- and tc-plms are more involved in specificity are in line with finding.
all together our observations are in accordance with the fact that gene transcription levels are positively correlated between orthologous genes in a. thaliana and o. sativa  <cit>  and that the correlation between transcriptional range and intensity is rather weak  <cit> .
nevertheless, tataΔ- and tc-plms are conserved, both at the sequence level per se and at the occurrence level within the whole genome.
in the  <dig> pairs of orthologous genes between a. thaliana and o. sativa, we searched for the number of genes containing a given -plm  in a. thaliana but not in their respective o. sativa ortholog,  in o. sativa but not in their respective a. thaliana ortholog and  in both orthologous genes.
the c/ ratio indicates the level of observed conservation for each plm class in o. sativa in regard to a. thaliana.
then we compared the ratio to the by chance value, i.e.
the ratio b/: the presence of a given plm in o. sativa orthologous genes from the plm-less a. thaliana genes.
we performed two one-sided fisher exact tests allowing the identification of significantly different percentages.
the underlined data indicate that the observed percentage is higher than expected.
ns indicates non-significant difference.
p-values in parenthesis are less than 5% with the bonferroni correction.
discussion and
CONCLUSIONS
in a. thaliana and o. sativa, we identified a novel class of putative tfbs involving tc-elements that are preferentially located within the same core-promoter region as tata-boxes.
we showed that these tc-elements are structurally distinct from the previously described tc-microsatellites observed in the 5'utr of plant genes.
nevertheless, the presence of both tc-elements and tc-microsatellites in higher plants but not in vertebrates suggests an evolutionary link between these two promoter elements.
previous reports have described the presence of a pyrimidine rich element, named the y-patch, in plant core-promoters  <cit> , and its tendency to be associated with both the tata-box and the inr motif  <cit> .
y-patch motifs and tc-microsatellites are two names used to globally describe t and c rich sequences widely surrounding the tss.
in promoters, these two elements are characterized by two non identical but overlapping groups of motifs.
our results show that the tc-plm exhibits specific characteristics distinguishing it from the other y-patch motifs.
y-patch motifs show frequent occurrence in plant promoters, are present in a wide area around the tss  and may be extended from a 6- to a 10-base-long element without decreasing the score associated to their local overrepresentation .
indeed the tc-plms were only observed in a sub-set of 18% of a. thaliana promoters at the tata-box expected position, were associated with a sharp functional window, were 6-base-long and could not be extended without decreasing their sms.
specific promoter features were frequently observed when genes were classified into groups, i.e.
the tc-element-containing gene group, the tata-box-containing gene group or the tataΔ-plms-containing gene group.
first, our data indicate that the tata-box preferentially associates with a ca-tss.
neither the tataΔ- nor the tc-elements exhibited this apparent functional association.
in addition, no association between the tata-box and any of the three other yr-tss was observed.
second, tc-element-containing genes were predominately large and involved in protein metabolism while tata-box-containing genes were predominantly compact and involved in response to stress and stimulus.
indeed, gene function, specificity and level of expression are linked features.
using an original approach we have been able to distinguish the possible involvement of different -elements in the control of either the level or the specificity of expression.
a global analysis of catma-transcriptome data indicated preferential links between the presence of:  a tata-box and high gene expression;  tc-elements and high specificity of gene expression; and  tataΔ-plms and broad expression patterns, as in housekeeping genes.
all together our observations suggest that tc-elements might be considered as a novel class of plant promoter elements linked directly or indirectly to the regulation of gene expression.
the presence of all three elements, i.e.
tata-boxes, tc- and tataΔ-elements, have been observed in two plants that diverged about  <dig> millions of year ago  <cit> .
we observed a global conservation, i.e.
conservation in the relative number of genes containing one of the three motifs in the two genomes.
nevertheless, only the tata-box showed a low level of conservation between orthologous gene pairs while conservation of either tc- or tataΔ-elements between orthologous gene pairs was no higher than that found between any gene pair.
on the one hand, the low level of conservation at the orthologous gene level is in line with the rapid evolution of core regulatory motifs after gene duplication in a. thaliana  <cit>  and with the notion that a. thaliana and o. sativa may have independently evolved novel tsss  <cit> .
on the other hand, the conservation of the relative number of genes with either a tata-box, a tc- or a tataΔ-element suggests a global conservation of the interspecies variability, noise level and evolvability of gene expression; three processes involving tata sequences  <cit> .
the three classes of core-promoter elements observed in the  region are each present in about 20% of the promoters.
in this study, we used "clean" classes of promoters, i.e.
those containing only one of the three classes of elements.
however in several other promoters, more than one element from a given class may be present.
therefore many functional combinations are expected.
experimental data have shown that the tata-box is involved in the direct binding of tbp  <cit>  and at least some tc- and tataΔ-elements might be involved in the recognition by other tfs recruiting tbp to form the transcription initiation complex at the right position.
consistent with this notion, both tc- and tataΔ-elements are linked to the specificity of expression that could be mediated through different tfs under the control of various signals.
an alternative would be that tata-boxes, tataΔ-elements and tc-elements are all responsible for dna instability around - <dig> relative to the tss but at different levels  <cit>  promoting a continuous range of control on gene transcription.
either way, we believe that our observations call for further biological analysis and that our predictions will be useful for future assessment of the relationships between promoter architecture and gene expression.
in this respect, future studies in model plants should be based on a better description of the different organization of core-promoters and include a more precise definition of the location of alternative tsss based on new sequencing technologies.

 over the past genomics has markedly changed our view on core-promoter organization. for instance, the tata-box can no longer be regarded as a general feature of polymerase ii core-promoters. indeed, only a small fraction of eukaryotic genes actually harbour a tata-box: less than 20% of genes in both human and yeast. while tata-box-containing promoters support the direct binding of tata-box-binding proteins and thereby also the formation of the pre-initiation complex, tata-less promoters are recognized by multiple tbp-related proteins and other tbp-associated transcription factors involved in the recruitment of tbp. indeed some tata-variants and other alternative elements allow the initiation of transcription and participate towards defining distinct patterns of expression. several core-promoter elements or general transcription factor binding sites have been previously identified in eukaryotes. they are characterized by a strong positional preference relative to the transcriptional start site as for instance the tata-box in the area, the initiator element,, around the tss, the downstream promoter element in the area, or the iib recognition element immediately upstream of certain tata-boxes. the position of binding sites of proteins belonging to the transcription complex is important for the functioning of promoters since it determines both the tss location and the transcription direction. thus, a strong positional conservation of a novel regulatory element would strongly indicate a functional role. this concept has led to a generation of tools that in contrast to previous tfbs predictors 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. 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 area binds tbp and is critical for basal transcriptional activity. in other studies, a tbp has been shown to bind to a taagaga element in the region of the hepatitis b virus s gene. these experimental observations 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 - relative to the tss necessary for transcription may be due to as yet unidentified motifs other than the tata-box. 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 recognized 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 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.  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 base long motifs with a statistically significant preferential position within the nucleotides upstream of the tss and called these motifs the preferentially located motifs. this method was first described by fitzgerald et al. for the analysis of human promoters and was then applied by others to plant promoters. our results correlated well with previous studies in terms of the spatial representation of the plms within the plant promoters. second, for each plm, we precisely defined: the preferential position relative to the tss, i.e. the top of the peak, the functional window, i.e. the peak width, derived from the peak boundaries, the score of maximal square relative to the base line ranking the plms in terms of their topological constraint and 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 conservation in a. thaliana genes and o. sativa genes with experimentally supported tsss. a well-annotated genomic sequence is available for both of these species that diverged approximately million years ago. as previously described within the nucleotides upstream of the tss of both a. thaliana and o. sativa we found plms made up exclusively or almost exclusively of: t and a nucleotides and exhibiting strong topological constraints, i.e. a sharp peak, and 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 and tata-box variants. 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 promoter 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. confirming previous results, we found this plm in both a. thaliana and o. sativa genomes in a preferential position bases upstream of the tss and strictly located within the region. a total of and promoters in a. thaliana and o. sativa respectively contained a tatawa within the 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 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 expected position. we observed plms with the same positional constraints as tatawa, i.e. showing a sharp peak in the region. these plms may be functional motifs hereafter named as tataΔ-plms where Δ stands for variant; or motifs being plms due to their overlap with the tatawa sequence. this overlap represents an inherent drawback of the method since motifs shorter than nucleotides as well as base long motifs may appear as plms. tata-variant and tata-box are distinct regulatory elements that need differentiating. for this reason, we searched for tataΔ-plms in sets of promoters not containing the canonical tata-box in the region. in this way, we were able to ascertain that the plms found within the promoter sets were bona fide elements under topological constraint. in a. thaliana and o. sativa respectively and promoters were found without a tatawa in the region. relative to the tatawa sequence, possible motifs diverged at one position. out of these motifs, only were found to represent plms conserved in both species. consistent with in vivo experimental results, t to a or a to t substitutions were the main differences observed among the tataΔ1-plms. motifs with a base c or g substitution at the central positions were not considered as plms and were counter-selected. motifs with a c or g substitution at the t or a positions were, however, considered as tataΔ1-plms. there remained and of a. thaliana and o. sativa genes respectively containing neither a tatawa nor a tataΔ1-plm. we identified only conserved tataΔ2-plms amongst the possible motifs and no conserved tataΔ3-plm. finally, at the tata-box expected position, a. thaliana and o. sativa promoters contained a tataΔ-plm. hereafter, tataΔ- and tatawa-plms are collectively referred to as ta-plms. the number of genes containing the ta-plms decreased from the tata-box to the tataΔ2-plms. interestingly, even though most of the tataΔ-plms were found to be t and a rich as expected, out of the tataΔ2-plms contained a base c. this suggested 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 characteristically rich in t and c nucleotides described as cpt microsatellites or tc-microsatellites. 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 functional window centred downstream of the tss often extending several hundred bases. we sometimes observed a small secondary peak located around bases upstream of the tss which disturbed the broad distribution of some of the tc-microsatellites. these tc-microsatellites may hide tc-plms, i.e. tc-plms with high topological constraints similar to those of ta-plms present within specific sub-sets of genes. conserved tc-elements are observed in almost 18% of a. thaliana promoters we hypothesised that tc-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 promoters from the whole promoter dataset. out of the possible dinucleotides, only were found to be conserved plms and to shared the tata-box topological constraints: ta, at and aa. a total of a. thaliana promoters and o. sativa promoters contained no tata-box or conserved tataΔ-plm or the conserved dinucleotide-plms. we examined the possibility of these ta-less promoter 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-plms were conserved between a. thaliana and o. sativa and in both plants they exhibited the same strict topological constraints. second, the distributions of several motifs known as tfbss supported by experimental analyses, such as the inr or the ggccc element, shared the same topological constraints in both the whole and the ta-less promoter sets. third, as expected, the gc-compositional strand bias or gc-skew expected in plant promoters was observed at the tss location in both promoter sets. we therefore searched in the ta-less promoter sets for the presence of 6-base-long conserved plms. out of the possible motifs, we found conserved plms exhibiting a strict functional window in the area relative to the tss. these plms were present in a. thaliana and in o. sativa promoters. in agreement with our hypothesis, the plms were tc-plms, i.e. were comprised of t and c bases only. figure illustrates how the tc-plms can be clearly distinguished in the ta-less promoter set while they were missed in the whole promoter set. indeed, using the whole promoter set, it is only possible to find the ttcttc-plm, which is characterized by a wide functional window and by a preferential position bases downstream of the tss. 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 bases upstream of the tss, i.e. sharing the ta-motif topological constraints. we propose that ta-plms may be recognized and thus be functional in a t and c rich environment whereas the same might not be true for tc-plm. as a consequence, 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-plm. it is important to note that only tc-plms exhibited the tata-box topological constraints in the ta-less promoter set. altogether, our results confirmed the importance of tc-microsatellites in plant promoters and predicted the existence of a novel class of functional elements, the tc-plms characterized by a sharp peak in the region and observed in almost 18% of a. thaliana promoters. we investigated the putative presence of tc-plms in other eukaryotic genomes. we analyzed homo sapiens promoters and mus musculus promoters with an experimentally supported tss. we observed neither tc-plms nor any other plms at the tata-box expected region. thus, both the tc-microsatellites and the tc-plms observed in plants are absent in both human and mouse. these observations suggest an evolutionary link between the presence of the tc-plms and the tc-microsatellites. tc-elements derived from three seed motifs as for all identified tataΔ-plms, any 6-base-long tc-plm may be a functional plm, be a part of a larger functional plm or contain a smaller functional plm. any of the tc-plms can potentially overlap by consecutive bases with at least one other tc-plm. among the promoters studied, we found only overlapping tc-plms: cttctt, ttcttc and tcttct. the trinucleotide repetition made up of t bases and one c base is characteristic of self-overlapping motifs involved in dna recognition by transcription factors. furthermore, we analyzed the effect of extending each of the tc-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. together, these results provide evidence in favour of the tcttcttct motif being a functional tc-plm. extension of most of the other tc-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 could be extended up to a 13-base-long plm. the shortest core-plms were ctc, tct and ctt, found within tc-plms, suggesting that these trinucleotides could be the core of the functional tc-plms. both the trinucleotide repetitions and the different t and c environments might therefore have a role in tc-plm function. three observations are in favour of a putative role of most tc-plms independently of the presence of other plms in the same region. first, tc-plms were often observed without any of the other plms sharing the same preferential position in. second, tc-plms are not extensions of either a tata-box or its variants. thus, among the -plm-containing promoters, 80% contained plm-motifs belonging to only one plm class, either a tata-box, a tataΔ- or a tc-plm. third, it was possible to construct oriented graphs displaying the motif divergence for both ta- and tc-plms. 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-plms and found that they could be organized into a unique, closed and oriented graph. three seeds were suggested: tcttct, tttctt and ttcttc. similar to that observed for ta-plms, these three tc-plms were those most frequently observed in promoters with the number of promoters containing the other tc-plms depending on both the distance from these seeds and the existence or not of a more divergent sequence. in conclusion, all -plms can be detected independently in different promoters and may be organized into different groups with apparent evolutionary links between the given plm and the related seed. tc-element-containing genes are preferentially involved in protein metabolism while most promoters contain only one class of -plm, some contain more than one. to investigate the functional significance of the different -plm classes we decided to construct four promoter sets each characterized by the exclusive presence of one of the classes of plm in the region. we distinguished the promoters containing only a tata-box, the containing only a tc-plm, the containing only a tataΔ-plm and as a negative reference the promoters without any -plm, called hereafter the -plm-less set. gene ontology annotations have been used previously in the prediction of the functional role of tfbs. here we analysed the go annotations of the four classes of genes defined above, according to the four promoter sets. as far as biological processes are concerned, the most conspicuous observation was a preferred association between genes containing a tata-box and responses to different stimuli. this result was expected since in human and in yeast the tata-box-containing genes are more frequently involved in specific biological processes. interestingly, we also found that tc-plm-containing genes are more frequently involved in protein metabolism than any other gene class, i.e. a basic biological process. neither the tataΔ-containing genes nor the -plm-less genes showed any significant bias for any biological process categories. we also analysed the partitioning of genes between the different go cellular components. again, the tata-box-containing genes presented the most biased partitioning. the products of these genes are more often constituents of the cell wall, the cytosol and the ribosomes and less frequently constituents of the chloroplasts, the plastids, the golgi apparatus and the mitochondria compared to the products of all the other genes. products of the tc-plm-containing genes exhibited little biased partitioning between the go cellular component categories. indeed, only the cell wall component was significantly less represented. it seems biologically sound to have found a positive correlation between the response to different stimuli and the cell wall go categories in the tata-box-containing genes and a negative correlation between the protein metabolism and the cell wall categories for tc-plm-containing genes. these observations suggest that tc-plms might have a functional role in transcriptional control that could frequently differ from and perhaps even oppose the tata-box functional role. it is of particular interest that our observations support the hypothesis that small variations in the tata-box sequence are linked to large changes in gene expression. unfortunately, due to the relatively small number of promoters containing a unique tc-plm, it was deemed impossible to perform the same comparisons between the tc-plm seeds alone and their predicted variants as that performed for the ta-motifs. it should be noted that by analysing all the genes containing a tc-plm alone we decreased the power of our statistical tests. we were however able to demonstrate a significant difference between the tata-box and the tc-plm sets concerning protein metabolism and cell wall categories. gene functional categories have been retrieved from the go categories provided by tair. in the table, each number not in parenthesis is the percentage of genes annotated in a given go category. we performed the two one-sided fisher exact tests allowing the identification of enrichment or impoverishment of go categories in a given set of genes when compared with all the other genes, i.e. genes within the whole gene set minus genes within the considered gene set. ns indicates a non-significant difference between percentages. p-values in parenthesis are less than 5% with the bonferroni correction. results are only shown for go categories exhibiting at least one bias. only the canonical tata-box is spatially linked to the initiator element recent progress in mammalian genomics has defined a more functional image of core-promoters. two functional categories of core-promoters have been described. the single peak promoters have a tightly defined tss position within a few base pairs while in broad peak promoters several tsss may be found within small clusters spanning tens of base pairs. a tata-box is more likely to be found within single peak promoters than in broad peak promoters. in genome-wide studies, only the most upstream tsss are often considered and are defined by the available transcript sequences. there are three main categories of core-promoters described in human, i.e. tata-box-containing promoters with an inr, tata-box-containing promoters without inr and tata-less promoters. we observed the same relative representation of these three categories in arabidopsis. consistent with mammalian studies, recent evidence suggests that a. thaliana genes containing a tata-box tend to have a sharper dominant peak of tss compared to that of other genes. the distance between tata-box and inr is important for accurate transcription initiation. we considered the distance between the inr, called here yr-tss, and the conserved plms characterized using our approach. as expected, the yr-tss is a conserved plm whose functional window is one base upstream of the tss in both plants. we first analysed the a. thaliana promoters containing a tata-box and we represented the distances between each tatawa in the region and each dinucleotide ca, ta, tg and cg up to bases downstream of the tatawa. the dinucleotide ca, but not ta, tg or cg showed a strong preferred distance of to bases from tatawa. this distance is consistent with the preferential position of both plms and with mammalian results. furthermore, none of the yr motifs showed a preferential distance either from tataΔ- or from tc-plms. finally, our results provide evidence of a link between the canonical tata-box and the ca-tss: to bases preferentially separate the ca from the first t of tatawa. genes containing the tc-element are generally long in human, the presence of a tata-box in promoters is often associated with a compact gene structure. in a. thaliana, we observed the same bias towards a more compact structure of tata-box-containing genes. in contrast, the tc-plm-containing genes and the -plm-less genes were overall relatively longer while the gene structure of tataΔ-plm-containing genes showed no bias. when present, the bias in gene size was mainly explained by the 5'utr and by the length of the coding sequence. however, to a lesser extent, the enrichment in compact genes amongst those containing a tata-box may be explained by the shorter cumulative length of introns, and by the higher percentage of intronless genes. structural gene features have been assigned by querying the flagdb++ database. for median length data, we performed two one-sided wilcoxon tests allowing the identification of enrichment in wide or in compact gene structures in a set of genes compared with all the other genes, i.e. genes within the whole gene set minus genes within the considered gene set. for intron-less gene percentages, we performed two one-sided fisher exact tests allowing the identification of higher or lower percentages in a gene set in comparison with all the other genes. ns indicates a non-significant difference. p-values in parenthesis are less than 5% with the bonferroni correction. both the first intron and 3'utr lengths are never biased. based on the counting of expressed sequence tags or on microarray data, gene size and gene expression levels have been shown previously to be inversely correlated in both human and yeast. this being the case, our observations could be due to the fact that, in general, tata-box genes have higher expression levels than other a. thaliana genes. the differences in gene sizes we observed are consistent with a lower mean expression of tataΔ-plm- than of tata-box-containing genes. both tc-plm-containing and -plm-less genes could be expressed at lower levels. tata-box-containing genes have been shown to generally display relatively high specificity of expression compared to housekeeping genes that frequently show high expression levels. therefore, the question remains as to understand whether the presence of regulatory elements in the region of promoters is linked directly to gene function per se or to the transcription level that in turn is associated with gene function. to resolve this we analysed a large set of transcriptome data, distinguishing the two different components of transcription: specificity and level. specific role played by each class of regulatory element found in the region on different components of transcription in eukaryotic genomes, various tfbss have been linked to specific gene expression patterns. we searched for such a link with plms in the region using a. thaliana transcriptome data obtained with the complete arabidopsis transcriptome microarray and available through the complete arabidopsis transcriptome database: catdb. the dataset included hybridizations based on different samples covering numerous developmental stages, biotic and abiotic stresses and mutants. we used normalized data on which positive hybridizations have previously been determined. for any one gene, the relative number of positive hybridizations was considered as a measure of the range of expression, i.e. of the specificity. to measure the global level of expression we computed the median of the signal intensities. the relationship between the median signal intensity and the percentage of hybridizations was clearly not linear and suggested the existence of different classes of genes with respect to transcriptional regulation. we thus clustered the transcriptome data into four classes. first, genes were separated in two classes depending on their expression level above or below the distribution model line, respectively he for those with high levels of expression and le for those with low levels of expression. second, two classes were defined relating to the specificity of hybridizations and delimited by the two inflections in the cloud of points. the first class clustered genes positively hybridized in less than 15% of microarray hybridizations and the second class clustered those with more than 85% of hybridizations. interestingly, each of these four transcriptional clusters was predominantly made up of genes containing one of a specific class of -plms. thus, genes within the transcriptional sr group, i.e. those that hybridized in specific conditions predominantly contained tc-plm; genes within the transcriptional wr group, i.e. housekeeping genes predominantly contained a tataΔ-plm; the transcriptional he group was enriched with the tata-box-containing genes and the transcriptional le group was enriched with the -plm-less genes. these results clearly indicate a link between the different motifs within the core-promoter and transcriptional features of the gene and are in favour of a functionally independent role of the tata-box, the tataΔ- and the tc-plms. our observations provide further evidence for the involvement of the tata-box in the regulation of transcription level. in addition, our results suggest that both tataΔ- and tc-plms might be involved in defining the specificity of transcription. the role of the tataΔ-plms might be to promote a relatively wide range of expression while on the other hand tc-plms may impose narrower limits. the adverse effect of tatawa and -plm-less within genes on the extreme level of transcription was particularly evident in the sr group. the most highly expressed genes were predominantly those containing tatawa whereas the most lowly expressed genes were mainly -plm-less genes. we propose that in the presence of a tata-box, the recognition of the tss depends directly on a small number of tbps and/or of tbp-related protein. the relaxed recognition by tbps in tataΔ-containing promoters might be responsible for a bias towards the large range of expression. in the absence of tata-box or tataΔ, the recruitment of the tbp might be mediated by different tbp-associated tfs depending on the gene. interaction of the different tbp-associated tfs with other specific factors might therefore explain the relatively higher specificity of transcription observed for the tc-plm gene set. the expression categories are described in the figure he, high expression; le, low expression; sr small range of expression; wr, wide range of expression; he+ highest expressions; le+, lowest expressions. the whole gene set contains the genes for which we have both a known tss and catma-expression data. we performed two one-sided fisher exact tests allowing the identification of higher or lower percentages in a gene set in comparison with all other genes, i.e. genes within the whole gene set minus genes within the considered gene set. ns indicates non-significant difference. p-values in parenthesis are less than 5% with the bonferroni correction. the class of regulatory element in the region is not conserved in higher plant orthologous gene pairs with the increasing availability of transcriptome data, the search for conserved tfbss within promoters of genes exhibiting similar transcriptional patterns has gathered much attention. surprisingly, the conservation of core-promoter elements, widely considered as being those most conserved, has not received such large interest. one recent study addressing this question did cast doubt on this widely accepted notion. concordantly, in several studies on orthologous relationships between large gene families, we observed no significant conservation of the presence of a tata-box between plant orthologues. even single copy genes present as orthologues in both a. thaliana and o. sativa are no more conserved than random pairs of genes. the co-occurrence of different elements in the promoter region could, at least in part, explain an apparent non-conservation at the gene level despite the observed conservation at the genomic one. this prompted us to reexamine the question comparing core-promoter elements in the region in a. thaliana and o. sativa. we analysed the conservation of the three classes of -plms in pairs of orthologous genes in a. thaliana and o. sativa characterized by an experimentally supported tss. while the conservation of tatawa appeared relatively low, it was significantly higher than that expected by chance. this was not the case for tataΔ- or tc-plms, which exhibited no conservation between orthologous genes. our results showing that the tata-box is more involved at the transcriptional level and that both tataΔ- and tc-plms are more involved in specificity are in line with finding. all together our observations are in accordance with the fact that gene transcription levels are positively correlated between orthologous genes in a. thaliana and o. sativa and that the correlation between transcriptional range and intensity is rather weak. nevertheless, tataΔ- and tc-plms are conserved, both at the sequence level per se and at the occurrence level within the whole genome. in the pairs of orthologous genes between a. thaliana and o. sativa, we searched for the number of genes containing a given -plm in a. thaliana but not in their respective o. sativa ortholog, in o. sativa but not in their respective a. thaliana ortholog and in both orthologous genes. the c/ ratio indicates the level of observed conservation for each plm class in o. sativa in regard to a. thaliana. then we compared the ratio to the by chance value, i.e. the ratio b/: the presence of a given plm in o. sativa orthologous genes from the plm-less a. thaliana genes. we performed two one-sided fisher exact tests allowing the identification of significantly different percentages. the underlined data indicate that the observed percentage is higher than expected. ns indicates non-significant difference. p-values in parenthesis are less than 5% with the bonferroni correction. discussion and  in a. thaliana and o. sativa, we identified a novel class of putative tfbs involving tc-elements that are preferentially located within the same core-promoter region as tata-boxes. we showed that these tc-elements are structurally distinct from the previously described tc-microsatellites observed in the 5'utr of plant genes. nevertheless, the presence of both tc-elements and tc-microsatellites in higher plants but not in vertebrates suggests an evolutionary link between these two promoter elements. previous reports have described the presence of a pyrimidine rich element, named the y-patch, in plant core-promoters, and its tendency to be associated with both the tata-box and the inr motif. y-patch motifs and tc-microsatellites are two names used to globally describe t and c rich sequences widely surrounding the tss. in promoters, these two elements are characterized by two non identical but overlapping groups of motifs. our results show that the tc-plm exhibits specific characteristics distinguishing it from the other y-patch motifs. y-patch motifs show frequent occurrence in plant promoters, are present in a wide area around the tss and may be extended from a 6- to a 10-base-long element without decreasing the score associated to their local overrepresentation. indeed the tc-plms were only observed in a sub-set of 18% of a. thaliana promoters at the tata-box expected position, were associated with a sharp functional window, were 6-base-long and could not be extended without decreasing their sms. specific promoter features were frequently observed when genes were classified into groups, i.e. the tc-element-containing gene group, the tata-box-containing gene group or the tataΔ-plms-containing gene group. first, our data indicate that the tata-box preferentially associates with a ca-tss. neither the tataΔ- nor the tc-elements exhibited this apparent functional association. in addition, no association between the tata-box and any of the three other yr-tss was observed. second, tc-element-containing genes were predominately large and involved in protein metabolism while tata-box-containing genes were predominantly compact and involved in response to stress and stimulus. indeed, gene function, specificity and level of expression are linked features. using an original approach we have been able to distinguish the possible involvement of different -elements in the control of either the level or the specificity of expression. a global analysis of catma-transcriptome data indicated preferential links between the presence of: a tata-box and high gene expression; tc-elements and high specificity of gene expression; and tataΔ-plms and broad expression patterns, as in housekeeping genes. all together our observations suggest that tc-elements might be considered as a novel class of plant promoter elements linked directly or indirectly to the regulation of gene expression. the presence of all three elements, i.e. tata-boxes, tc- and tataΔ-elements, have been observed in two plants that diverged about millions of year ago. we observed a global conservation, i.e. conservation in the relative number of genes containing one of the three motifs in the two genomes. nevertheless, only the tata-box showed a low level of conservation between orthologous gene pairs while conservation of either tc- or tataΔ-elements between orthologous gene pairs was no higher than that found between any gene pair. on the one hand, the low level of conservation at the orthologous gene level is in line with the rapid evolution of core regulatory motifs after gene duplication in a. thaliana and with the notion that a. thaliana and o. sativa may have independently evolved novel tsss. on the other hand, the conservation of the relative number of genes with either a tata-box, a tc- or a tataΔ-element suggests a global conservation of the interspecies variability, noise level and evolvability of gene expression; three processes involving tata sequences. the three classes of core-promoter elements observed in the region are each present in about 20% of the promoters. in this study, we used "clean" classes of promoters, i.e. those containing only one of the three classes of elements. however in several other promoters, more than one element from a given class may be present. therefore many functional combinations are expected. experimental data have shown that the tata-box is involved in the direct binding of tbp and at least some tc- and tataΔ-elements might be involved in the recognition by other tfs recruiting tbp to form the transcription initiation complex at the right position. consistent with this notion, both tc- and tataΔ-elements are linked to the specificity of expression that could be mediated through different tfs under the control of various signals. an alternative would be that tata-boxes, tataΔ-elements and tc-elements are all responsible for dna instability around - relative to the tss but at different levels promoting a continuous range of control on gene transcription. either way, we believe that our observations call for further biological analysis and that our predictions will be useful for future assessment of the relationships between promoter architecture and gene expression. in this respect, future studies in model plants should be based on a better description of the different organization of core-promoters and include a more precise definition of the location of alternative tsss based on new sequencing technologies. 
here we assessed the possibility of identifying novel functional motifs in plant genes, sharing the tata-box topological constraints. the tata-box and tata-variants are regulatory elements involved in the formation of a transcription initiation complex. our observations suggest that the tc-elements might constitute a class of novel regulatory elements participating towards the complex modulation of gene expression in plants. arabidopsis thaliana transcriptome analysis revealed that tata-box-containing genes were generally those showing relatively high levels of expression and that tc-element-containing genes were generally those expressed in specific conditions.
 the tata-box and tata-variants are regulatory elements involved in the formation of a transcription initiation complex. both have been conserved throughout evolution in a restricted region close to the transcription start site. however, less than half of the genes in model organisms studied so far have been found to contain either one of these elements. indeed different core-promoter elements are involved in the recruitment of the tata-box-binding protein. here we assessed the possibility of identifying novel functional motifs in plant genes, sharing the tata-box topological constraints.  we developed an ab-initio approach considering the preferential location of motifs relative to the tss. we identified motifs observed at the tata-box expected location and conserved in both arabidopsis thaliana and oryza sativa promoters. we identified tc-elements within non-ta-rich promoters bases upstream of the tss. as with the tata-box and tata-variant sequences, it was possible to construct a unique distance graph with the tc-element sequences. the structural and functional features of tc-element-containing genes were distinct from those of tata-box- or tata-variant-containing genes. arabidopsis thaliana transcriptome analysis revealed that tata-box-containing genes were generally those showing relatively high levels of expression and that tc-element-containing genes were generally those expressed in specific conditions.  our observations suggest that the tc-elements might constitute a class of novel regulatory elements participating towards the complex modulation of gene expression in plants. 