BACKGROUND
in recent years large numbers of novel cis-regulatory sequences have been described that are conserved in stress-response genes, but the role of these elements and their binding transcription factors  remains unknown  <cit> .
the identification of the binding tfs is therefore a major challenge for bioinformaticians and experimentalists.
using database-assisted analysis of cis-sequences it is possible to generate hypotheses on the nature of the binding tf  <cit> , but the experimental validation of these predictions is often missing.
the current work investigates how efficient a database-assisted approach leads to the prediction of the correct tf or tf-families that bind to conserved cis-sequences in co-regulated genes.
the identification of cis-regulatory sequences has been facilitated using bioinformatic and web-queryable resources  <cit> .
one approach is the detection of cis-elements annotated in existing resources by performing a database-assisted analysis  <cit> .
another approach is the de novo discovery of conserved sequence patterns in sets of co-regulated genes without knowing if these sequences have been associated with a function before.
this involves detection of over-represented sequences in the upstream region of co-regulated genes by using pattern mining programs such as meme, alignace, consensus, co-bind, bioprospector, mitra, or integrative frameworks such as best  <cit> .
to discover conserved sequence motifs in promoters of up-regulated a. thaliana genes, best was applied in the work presented here.
co-regulated genes were identified using drought responsive microarray expression data annotated to the pathoplant database  <cit> .
the identified motifs were classified with stamp  <cit>  and compared to known cis-regulatory sequences annotated to the athamap, place and agris databases  <cit> .
furthermore, the newly developed footprintdb repository, built on top of 3d-footprint, was employed to predict interacting tfs  <cit> .
to confirm bioinformatic predictions,  <dig> cis-sequences were used for the isolation of interacting tfs in a yeast one-hybrid screening.
the specificity of each tf-dna interaction was further validated in yeast.
furthermore, two tfs were used for the generation of synthetic factors that activate reporter gene expression under the control of synthetic promoters harbouring corresponding cis-sequences in a p. patens expression system.
RESULTS
identification of seven motif families in drought-responsive a. thaliana genes
the goal of this work was the identification of transcription factors binding to cis-regulatory sequences conserved in promoters of drought-responsive genes.
thus, the first part aimed to identify sequence motifs harbouring such cis-sequences.
figure  <dig> gives an overview of the workflow.
in a first step, microarray experiments annotated to the pathoplant database were employed to identify gene sets two- to tenfold up-regulated by drought in roots and shoots.
a total of  <dig> queries were performed.
additional file  <dig> lists the parameters for these queries and also the number of induced genes obtained in each of the  <dig> queries.
the  <dig> queries yielded  <dig> up-regulated gene groups containing up to  <dig> co-regulated genes.
in a second step, conserved sequence motifs within the upstream region of the  <dig> co-regulated gene groups were identified with the software package best  <cit> .
for motif detection,  <dig>  bp upstream of all genes in a co-induced gene group were screened with this software.
this analysis resulted in  <dig> sequence motifs,  <dig> from roots,  <dig> from shoots, and  <dig> from combinations of timepoints or tissues.
in a third step, all sequences from the  <dig> motifs were subjected to an in silico expression analysis  <cit> .
with this tool, motifs specific for drought responsive genes were identified.
a total of  <dig> motifs containing cis-sequences that pass this analysis remained,  <dig> from roots,  <dig> from shoots and  <dig> from combinations of timepoints or tissues.
these sequences and the alignments generating the  <dig> sequence motifs are shown in additional file  <dig>  single sequences that pass the in silico expression analysis criteria are shown in bold in this file.
these  <dig> sequence motifs were further analysed using stamp .
stamp determines the relationship between the motifs and also calculates similarities to known cis-regulatory sequences  <cit> .
to illustrate the similarities among the  <dig> sequence motifs, a tree was generated and illustrated by the program mega  <cit> , which is shown in figure  <dig>  based on the relationship between the motifs,  <dig> motif groups were designated.
stamp was also employed for the identification of motif similarities by comparing the  <dig> motifs with known cis-elements from plant databases athamap, agris and place  <cit> .
additional file  <dig> summarises these results.
several motifs from group i, that harbours  <dig> motifs, show significant similarities  to a g-box motif and to abscisic acid-responsive elements  known as bzip binding sites.
for example with the place database eight motifs show similarities with the gboxlerbcs sequence  <cit>  and nine motifs show similarities to four different abres from place .
these abres are associated with abiotic stress response  <cit> .
twenty motifs cluster within the remaining groups  and display less significant similarities  to a wide array of cis-elements, with many of them so far not being associated with abiotic stress response .
bioinformatic prediction of tfs interacting with conserved cis-sequences
the collection of sequence motifs detected with best were further analysed with footprintdb, which currently contains over  <dig>  unique tfs and their experimentally-associated dna motifs  <cit> .
the flowchart in figure  <dig> illustrates this approach.
this strategy retrieved a. thaliana candidates for all motif groups but group v. these predictions further confirm that  <dig> group i motifs display obvious similarities to g-box, abre-binding motifs recognised by tfs of the abf family , and in general reproduce the stamp analysis explained earlier.
the remaining groups do not have a clear resemblance to any documented dna binding specificity, and thus proteins from a variety of families were retrieved.
in total,  <dig> unique a. thaliana protein sequences were retrieved for the  <dig> drought-related motifs .
cis
-elements/sequence motifs.
isolation of tfs interacting with conserved cis-sequences
sequences from the identified dna motifs, which are conserved in the promoter of genes simultaneously induced by drought were cloned  into the yeast phisi vector with the aim to carry out yeast one-hybrid  screens, in order to identify tfs that are able to activate the transcription from these putative cis-regulatory sequences.
out of these  <dig> sequence motifs,  <dig> individual cis-sequences shown in table  <dig> and representing all seven motifs groups, were used for y1h.
additional file  <dig> shows the result of the y1h screen.
a total of  <dig> different tfs belonging to  <dig> different families were identified.
amongst the identified factors,  <dig> were already described as playing a role in various stress responses.
five were found to be associated with abiotic stresses , three with biotic stresses , and three with both types of stress .
moreover, this analysis led in most of the cases to the identification of specific associations between a motif group and a tf family.
table  <dig> and figure 4a summarise these findings.
table  <dig> shows all selected tfs that were able to activate the screened sequence but unable to activate the mutated sequence in yeast .
sequence  <dig> from motif group i and sequence  <dig> from motif group v preferably select bhlh tfs .
instead, both sequences from motif group ii preferably select nac factors.
interestingly, sequences from motif group iii and vi, which are closely related , preferably select myb tfs.
for the other sequences, no preferred tf family members were selected .
cis
-sequences selected for further experimental and bioinformatics analysis
cis
proveniences of selected motif sequences.
query refers to figure  <dig>  the genes and positions relative to transcription start refer to the genomic context of the cloned cis-elements.
cis
-sequences
seq.
cis
n.b.
: myb refers to the r2r3-myb class of myb tf.
cis
bold characters: mutated nucleotides; x: interactions tested in transient assays using physcomitrella patens protoplasts.
within motif group i, two different sequences were assayed.
for the first one, aacgtggg, no specific class of tf was found to preferentially activate transcription in yeast.
in this assay, five different tfs, belonging to five different families , were identified .
conversely, seven bhlh tfs  were able to activate the transcription from the second sequence, gcacgtggag, revealing some transcriptional specificity .
interestingly, both elements contain the acgt core sequence, which once mutated  prevented the identified tfs to activate transcription in yeast, indicating its preponderant role in these interactions.
the y1h analysis of group v motifs, which belong to the cluster showing the highest level of sequence similarity with group i, led to the identification of four bhlh tfs.
in fact, these bhlh factors were found to activate transcription in yeast from the atgtgatgc sequence, but not from the gcatcaccc sequence, for which tfs from two other families  were identified.
the careful analysis of these group v motif sequences and their corresponding mutated versions atgcaatgc and gcatagccc) exposed that when the atgtgatgc sequence is concatenated, an e-box  is created, whereas this is not the case with the gcatcaccc sequence.
the fact that sequences from group i and v display strong similarities with either the well described g  or e boxes probably explains why bhlh tfs were found to be associated with these two motif groups.
group ii is closely related, at the sequence level, to group i and v. however, unlike groups i and v, only two nac tfs were found to interact with the two sequences tested , with one  being identical for both sequences .
from group iii, four sequences were analysed, namely cacctaac, accacaacc , accaaacat and tcggaccaa.
from these ac-rich sequences only r2r3-myb tfs were identified as able to activate transcription in yeast , with the exception of tcggaccaa, for which no interacting tf was identified .
the analysis of the ctctctcac element from group iv concluded with the identification of three factors: one nac and two bzip  tfs .
finally, three different sequences belonging to group vii were also analysed, namely tctcctccac, ctttcccc and cctccttct, from which no clear pattern of binding emerged.
the screening of the tctcctccac sequence led to the identification of three different classes of tfs .
three additional factors belonging to three further classes  where identified with the ctttcccc element.
finally, no binding tfs for the cctccttct element were retrieved from regia in yeast .
validation of transcription factor predictions with yeast-one-hybrid experimental data
in order to benchmark the accuracy of bioinformatically predicted candidate tfs, they were compared to those experimentally identified by y1h screening.
key search parameters were tuned as explained in methods and in figure  <dig>  and a trade-off between precision and sensitivity could be achieved.
in summary, we were able to lower the blastp e-value threshold to 1e- <dig> without a significant sensitivity loss, but fixed the stamp cutoff to 1e- <dig> to avoid a major sensitivity drop while ensuring reliable short cis-element alignments  <cit> .
we also found that filtering out candidate tfs with poorly conserved interface residues, those directly contacting dna nitrogen bases, was helpful in order to discard false predictions.
a cutoff of 50% interface similarity was observed to be adequate, increasing specificity to  <dig> , as shown in additional file  <dig> .
tested cis-elements were classified as predictable if the footprintdb repository contained at least one annotated tf with a significantly similar motif , which was homologous to the experimental y1h-inferred factors.
thirteen of the  <dig> cis-elements tested in the y1h experiment were accordingly called ‘predictable', but only  <dig> of them  yielded true positives among footprintdb results.
the rest had significantly divergent dna motifs annotated in the database, with e-values over the cutoff, and therefore could not be identified .
it is important to note that in all predictable cases the correct binding tfs were identified by homology to annotated plant regulatory proteins, including a. thaliana, petunia hybrida, oryza sativa or phaseolus vulgaris.
on the other hand, group ii , iii  and vii  elements could not be successfully matched as the idef <dig> and gcn4-like motifs  were not part of footprintdb when the predictions were made.
overall, after conducting the benchmark experiment using as input the  <dig> cis-sequences in table  <dig>  we conclude that we correctly predicted  <dig> out of  <dig>  of the tfs isolated in the y1h assays, finding on average  <dig> validated tf every  <dig> predictions.
if only predictable sites are considered, these numbers improve to  <dig> out of  <dig>  and  <dig> confirmed tf every  <dig> predictions, respectively.
finally, when the complete analysis was repeated with input dna motifs  instead of individual cis-sequences, the fraction of successfully recovered tfs dropped to  <dig> out of  <dig> , yielding  <dig> confirmed tf every  <dig> predictions.
these results and the complete benchmark reports can be found in additional file  <dig> .
functional analysis of tfs interacting with conserved cis-sequences
in this study, some factors were found to be able to activate transcription in yeast from various cis-sequences conserved in the promoters of genes that are simultaneously induced by drought.
amongst these, two were selected for more detailed analysis, nac protein at3g <dig> and myb <dig> at1g <dig>  these selected tfs were able to induce transcription in y1h experiments from three  and two  different motif groups, respectively .
the ability of these two factors to interact with their dna target in vivo was analysed in transient expression assays, using physcomitrella patens protoplasts.
in this experiment, the dna binding domain  of the nac protein and myb <dig> was fused to the vp <dig> activation domain, resulting in the formation of two synthetic tfs.
these chimeric proteins were then assayed against different conserved cis-sequences, namely ctctctcac and gctgccggaga , and cacctaac and accacaacc, for the nac and myb <dig> dbd, respectively .
this analysis confirms the results obtained in y1h experiments.
interestingly, the transcriptional activity supported by the nac dbd was a lot stronger with the gctgccggaga sequence than with ctctctcac, strengthening the idea that the gctgccggaga element is most probably a new nac target sequence .
conversely, similar activity was observed when myb <dig> dbd was assayed against either, cacctaac or accacaacc, suggesting that myb <dig> could activate the transcription from a wide variety of ac-rich cis-sequences .
the cis-sequences and transcription factors identified in the present study may play a role in drought-responsive gene expression in a. thaliana.
in the context of this assumption, several cis-sequences were used to generate synthetic promoters, which were tested in transgenic a. thaliana plants.
however, no drought-responsive reporter gene expression has been observed in these transgenic lines so far .
it will be interesting to see if overexpression or knock-down mutations of the identified transcription factors will have any effect on drought-responsiveness of the plants.
discussion
the application of bioinformatic resources has led to the identification of large numbers of novel cis-regulatory sequences conserved in stress response genes  <cit> .
often the role of these elements and their binding transcription factors has remained unknown.
the primary goal of the present work was the identification of tfs interacting with cis-sequences conserved in co-regulated genes.
a total of  <dig> sequence motifs classified into  <dig> motif families were identified from microarray data on drought responsive gene expression.
these motifs were bioinformatically predicted to interact with tfs belonging to specific tf families.
these predictions were then tested experimentally using yeast one-hybrid screenings leading to the identification of novel tf-dna interactions.
initially, the two bioinformatic approaches employed, preferably predicted tfs interacting with putative binding sites for motif group i that harbours  <dig> of a total of  <dig> motifs.
using the stamp web server as well as footprintdb, these show significant similarities  to g-box and abre elements.
however, no consistency in tf predictions was obtained with these database-assisted analyses using the other motifs.
this first set of results exposed the natural limitations of this approach, mainly associated with the data content of the underlying scanned databases.
for example, predictions using the stamp webserver depend on previously annotated data derived from databases such as transfac, jaspar, place, agris, and athamap  <cit> .
the content of these repositories is primarily derived from functionally known regulatory sequences, which are expected to be only a tiny fraction of all cis-elements.
similarly, footprintdb  contains tfs and their experimentally-associated dna motifs from these and other databases.
a main difference to the stamp webserver is that footprintdb motifs are associated to their cognate binding tfs, and their dna-binding interfaces are annotated using 3d-footprint structural data  <cit> .
however, stamp as well as footprintdb detect and predict tf-dna interactions based on known data.
therefore, tf-dna interactions not readily predicted with stamp and footprintdb may represent novel or to date unknown tf-dna interactions.
this was substantiated in the present study by using sequences enriched in promoters of drought-responsive genes, but not readily associated with interacting tfs for experimental analysis using y1h-screenings.
towards these ends two different sequences from motif group i were assayed.
for the first one, aacgtggg, no specific class of tf was found to preferentially induce transcription in yeast.
conversely, seven bhlh tfs were able to activate the transcription from the second sequence, gcacgtggag.
both elements contain the acgt core sequence, which once mutated  prevented transcription to be activated.
these observations suggest that sequences aacgtggg and gcacgtggag are bona fide binding sites for bhlh <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig>  motif group v showed the highest levels of sequence similarity with group i and drove the identification of four bhlh tfs.
in fact, the bhlh tfs were found to prompt transcription in yeast from cis-element atgtgatgc, but not from gcatcaccc.
when sequence atgtgatgc is concatenated, an e-box  is created , whereas this is not the case with gcatcaccc.
the fact that sequences from group i and v contain or display strong similarities with either the well described g-box  or e-box probably explains why bhlh tfs were found to be associated with these two motif groups.
in fact, numerous studies have shown that bhlh tfs regulate the activity of their target gene promoters through these types of cis-regulatory sequences  <cit> .
however, no bzip tfs were identified as interacting with the motif that contains the acgt core, suggesting that the interactions with the bhlh tfs are quite specific and that the full-length sequence of the motifs may have a role in this specificity  <cit> .
group ii is closely related, at the sequence level, to group i and v. however, unlike group i and v, only two nac tfs were found to interact with the two sequences tested , with one  being identical for both sequences .
a wide variety of nac binding sites have been identified so far in various plant species, revealing the large diversity of the sequences that can be recognised by this group of tfs  <cit> .
the gccacgtcagc sequence displays similarities with the core sequence ca  g    that is recognised by the rice  and barley  idef <dig> nac proteins, suggesting that the identified nac could activate the transcription in yeast from it  <cit> .
this assertion was supported by the lack of transcriptional activity associated with the mutagenesis of this element .
interestingly, the gctgccggaga sequence appears to be quite divergent when compared to the other described nac binding sites even though both nac tfs were able to activate the transcription in yeast from it, suggesting that the gctgccggaga dna sequence could be a new cis-regulatory element that could be targeted by nac proteins .
from group iii, the ac-rich sequences cacctaac, accacaacc , accaaacat and tcggaccaa identified only r2r3-myb tfs to be able to activate transcription in yeast .
this result is in agreement with previous studies that have demonstrated that numerous r2r3-myb proteins from different plant species are able to bind to and induce transcription from ac-rich sequences  <cit> .
one of the studied sequences  contains the well-described ac-i cis-regulatory element  to which the a. thaliana myb <dig> tf  was shown to bind to in vitro and activate transcription from it in yeast  <cit> .
this result was confirmed in our screen .
similarly, we found that two other r2r3-myb subgroup  <dig> members , namely myb <dig> and myb <dig>  were also able to activate transcription in yeast from the same element  <cit> .
the overlap between the r2r3-myb proteins that interact with the cacctaac  and the accacaacc  sequences was quite high, as  <dig> out of the  <dig> interacting r2r3-mybs  were common to both elements .
interestingly, only four r2r3-mybs prompted transcription in yeast from the accaaacat element, from which three belong to the same r2r3-myb subgroup, namely subgroup  <dig>   <cit> .
group vi, such as goup iii, is composed of ac-rich motifs.
from this motif group only one putative cis-regulatory sequence was assayed, namely ccaactaa, from which only r2r3-myb tfs  were also identified .
eleven  and  <dig>  of the identified r2r3-mybs were identical with the r2r3-mybs found to interact with the cacctaac and accacaacc sequences, respectively.
when concatenated, this motif partially contains the r2r3-myb targeted ac-ii element  from which the identified tfs are likely to induce transcription in yeast, based on the absence of activation with the mutated version  of the element  <cit> .
surprisingly, as no acgt core sequence  is present in this motif, the analysis of the ctctctcac element from group iv leads to the identification of two bzip  factors .
nevertheless, a detailed analysis of this sequence showed that a bzip-like dna target  similar to two gcn4-like  motifs found in the promoter of the wheat storage protein genes was present, suggesting that this sequence could be mainly recognised by this class of bzip factors  <cit> .
while these y1h results on their own already provide valuable biological insight, they also served in this work as a reference dataset.
indeed they were also used to benchmark the predictive value of the footprintdb approach for recognizing putative binding tfs with different parameter settings.
two stamp e-value cutoffs were tested.
while a stringent 1e- <dig> threshold yielded the most specific predictions, it was at the cost of a significantly reduced sensitivity .
on the contrary, a relaxed 1e- <dig> cut-off increased the sensitivity  by compromising specificity.
however, this loss of specificity could be corrected by lowering the blastp e-value cut-off to 1e- <dig>  in addition, we observed a further gain in specificity by applying an interface similarity filter for candidate tfs, requiring them to have at least 50% similar interface residues to be considered.
altogether, these settings yield a 69% sensitivity and a 92% specificity.
another important observation regarding the performance was that individual input cis-elements worked much better than sequence motifs, which presumably increase the chance of obtaining significant but irrelevant alignments of dna sequences, which are the cornerstone of this approach.
in addition to the y1h screenings, the in vivo interaction of two tfs with their dna target was analysed in transient expression assays using physcomitrella patens protoplasts.
in this experiment, two synthetic tfs were generated by fusing the dna binding domain  of the nac protein  and myb <dig>  to the vp <dig> activation domain.
these chimeric proteins were then assayed against the conserved cis-sequences, ctctctcac and gctgccggaga, and cacctaac and accacaacc, for the nac and myb <dig> dbd, respectively .
interestingly, the transcriptional activity supported by the nac dbd was a lot stronger with the gctgccggaga sequence than with ctctctcac one, strengthening the idea that the gctgccggaga element is most probably a new nac target sequence .
to date the nac protein encoded by the arabidopsis gene at3g <dig> had not been investigated experimentally.
comparing the proposed novel nac binding site gctgccggaga with known arabidopsis nac binding sites reveals a large variability of binding sites recognised by nac proteins.
for example, the nam  and nap  proteins bind to the cis-sequences aagggatga and cacgtaagt, respectively  <cit> .
the high variability of nac binding sites is also illustrated by the a-rich binding site of ataf <dig>  <cit> .
nac transcription factors are involved in a wide array of abiotic stress responses  <cit> .
interestingly, the nac transcription factors anac <dig> , anac <dig> , and anac <dig>  bind to the sequence annnnntcnnnnnnnacacgcatgt, a drought responsive cis-sequence  <cit> .
although this sequence is part of a drought responsive promoter , overexpression of anac <dig>  anac <dig>  and anac <dig> in transgenic plants did not up-regulate erd <dig> expression indicating that other interacting factors may be necessary for the induction of the erd <dig> gene  <cit> .
in this context it is interesting to note that none of the cis-sequences investigated here, although enriched in drought responsive genes, individually confer drought responsive gene expression .
among these sequences was also the novel nac binding site gctgccggaga.
similarly it may be suggested that the investigated cis-sequences are each part of a combinatorial element that requires a second binding site for an interacting transcription factor in close vicinity for its functionality  <cit> .
when the myb <dig> dbd was assayed against cacctaac and accacaacc in physcomitrella patens protoplasts using a synthetic tf, similar activity was observed with both cis-sequences, suggesting that myb <dig> could induce transcription from a wide variety of ac-rich cis-sequences .
myb <dig> belongs to subgroup  <dig> of r2r3myb tfs  <cit> .
the gene is known to be up-regulated by chitin but no further functional analysis has been carried out to date  <cit> .
there are  <dig> r2r3-myb tfs in the genome of arabidopsis <cit>  and random binding site selection experiments with myb <dig>   <dig>   <dig>  and  <dig> revealed that these factors always bind to cis-sequences with one or two conserved acc and/or aac core sequences  <cit> .
consequently, the adjacent nucleotides can vary significantly and arabidopsis myb factors have a relatively degenerate binding site recognition.
consistent with known myb binding sites, the synthetic tf containing the myb <dig> dbd activates gene expression from two synthetic promoters harbouring the acc and aac motif .
although the myb <dig> binding site cacctaac does not confer drought responsive reporter gene expression in transgenic arabidopsis , this motif has been found in arabidopsis in the promoter of three myb <dig> target genes that are involved in vasculature development, one of the main components of the transpiration stream, which indirectly may participate in plant adaptation to drought stress  <cit> .
such observations reinforce the postulate that a combination of regulatory elements is required to integrate the environmental signals on a specific gene promoter  <cit> .
CONCLUSIONS
the work presented demonstrates the successful integration of several bioinformatic resources to predict and validate tfs interacting with conserved sequence motifs in co-regulated genes.
predictions were confirmed by using a yeast-one-hybrid approach to identify interacting tfs belonging to the predicted tf families.
tf-dna interactions were further experimentally validated in yeast and with a physcomitrella patens transient expression system, leading to the discovery of several novel tf-dna interactions.
our work establishes a novel approach to identify tfs interacting with conserved cis-sequences.
this approach may facilitate the experimental identification of tfs because a candidate tf-family can be predicted bioinformatically using the footprintdb database.
although the limitation of this approach is the content of the databases used, footprintdb is particularly valuable because it contains data for tf-dna interactions from many different species.
therefore, this database may help to identify a dna binding protein domain for any submitted sequence.
afterwards one may check plant tf databases for the predicted dna binding domain to identify novel sequence-specific tf-dna interactions.

 in recent years large numbers of novel cis-regulatory sequences have been described that are conserved in stress-response genes, but the role of these elements and their binding transcription factors remains unknown. the identification of the binding tfs is therefore a major challenge for bioinformaticians and experimentalists. using database-assisted analysis of cis-sequences it is possible to generate hypotheses on the nature of the binding tf, but the experimental validation of these predictions is often missing. the current work investigates how efficient a database-assisted approach leads to the prediction of the correct tf or tf-families that bind to conserved cis-sequences in co-regulated genes. the identification of cis-regulatory sequences has been facilitated using bioinformatic and web-queryable resources. one approach is the detection of cis-elements annotated in existing resources by performing a database-assisted analysis. another approach is the de novo discovery of conserved sequence patterns in sets of co-regulated genes without knowing if these sequences have been associated with a function before. this involves detection of over-represented sequences in the upstream region of co-regulated genes by using pattern mining programs such as meme, alignace, consensus, co-bind, bioprospector, mitra, or integrative frameworks such as best. to discover conserved sequence motifs in promoters of up-regulated a. thaliana genes, best was applied in the work presented here. co-regulated genes were identified using drought responsive microarray expression data annotated to the pathoplant database. the identified motifs were classified with stamp and compared to known cis-regulatory sequences annotated to the athamap, place and agris databases. furthermore, the newly developed footprintdb repository, built on top of 3d-footprint, was employed to predict interacting tfs. to confirm bioinformatic predictions, cis-sequences were used for the isolation of interacting tfs in a yeast one-hybrid screening. the specificity of each tf-dna interaction was further validated in yeast. furthermore, two tfs were used for the generation of synthetic factors that activate reporter gene expression under the control of synthetic promoters harbouring corresponding cis-sequences in a p. patens expression system.  identification of seven motif families in drought-responsive a. thaliana genes the goal of this work was the identification of transcription factors binding to cis-regulatory sequences conserved in promoters of drought-responsive genes. thus, the first part aimed to identify sequence motifs harbouring such cis-sequences. figure  gives an overview of the workflow. in a first step, microarray experiments annotated to the pathoplant database were employed to identify gene sets two- to tenfold up-regulated by drought in roots and shoots. a total of queries were performed. additional file lists the parameters for these queries and also the number of induced genes obtained in each of the queries. the queries yielded up-regulated gene groups containing up to co-regulated genes. in a second step, conserved sequence motifs within the upstream region of the co-regulated gene groups were identified with the software package best. for motif detection,  bp upstream of all genes in a co-induced gene group were screened with this software. this analysis resulted in sequence motifs, from roots, from shoots, and from combinations of timepoints or tissues. in a third step, all sequences from the motifs were subjected to an in silico expression analysis. with this tool, motifs specific for drought responsive genes were identified. a total of motifs containing cis-sequences that pass this analysis remained, from roots, from shoots and from combinations of timepoints or tissues. these sequences and the alignments generating the sequence motifs are shown in additional file single sequences that pass the in silico expression analysis criteria are shown in bold in this file. these sequence motifs were further analysed using stamp. stamp determines the relationship between the motifs and also calculates similarities to known cis-regulatory sequences. to illustrate the similarities among the sequence motifs, a tree was generated and illustrated by the program mega, which is shown in figure  based on the relationship between the motifs, motif groups were designated. stamp was also employed for the identification of motif similarities by comparing the motifs with known cis-elements from plant databases athamap, agris and place. additional file summarises these results. several motifs from group i, that harbours motifs, show significant similarities to a g-box motif and to abscisic acid-responsive elements known as bzip binding sites. for example with the place database eight motifs show similarities with the gboxlerbcs sequence and nine motifs show similarities to four different abres from place. these abres are associated with abiotic stress response. twenty motifs cluster within the remaining groups and display less significant similarities to a wide array of cis-elements, with many of them so far not being associated with abiotic stress response. bioinformatic prediction of tfs interacting with conserved cis-sequences the collection of sequence motifs detected with best were further analysed with footprintdb, which currently contains over unique tfs and their experimentally-associated dna motifs. the flowchart in figure  illustrates this approach. this strategy retrieved a. thaliana candidates for all motif groups but group v. these predictions further confirm that group i motifs display obvious similarities to g-box, abre-binding motifs recognised by tfs of the abf family, and in general reproduce the stamp analysis explained earlier. the remaining groups do not have a clear resemblance to any documented dna binding specificity, and thus proteins from a variety of families were retrieved. in total, unique a. thaliana protein sequences were retrieved for the drought-related motifs. cis -elements/sequence motifs. isolation of tfs interacting with conserved cis-sequences sequences from the identified dna motifs, which are conserved in the promoter of genes simultaneously induced by drought were cloned into the yeast phisi vector with the aim to carry out yeast one-hybrid screens, in order to identify tfs that are able to activate the transcription from these putative cis-regulatory sequences. out of these sequence motifs, individual cis-sequences shown in table  and representing all seven motifs groups, were used for y1h. additional file shows the result of the y1h screen. a total of different tfs belonging to different families were identified. amongst the identified factors, were already described as playing a role in various stress responses. five were found to be associated with abiotic stresses, three with biotic stresses, and three with both types of stress. moreover, this analysis led in most of the cases to the identification of specific associations between a motif group and a tf family. table  and figure 4a summarise these findings. table  shows all selected tfs that were able to activate the screened sequence but unable to activate the mutated sequence in yeast. sequence from motif group i and sequence from motif group v preferably select bhlh tfs. instead, both sequences from motif group ii preferably select nac factors. interestingly, sequences from motif group iii and vi, which are closely related, preferably select myb tfs. for the other sequences, no preferred tf family members were selected. cis -sequences selected for further experimental and bioinformatics analysis cis proveniences of selected motif sequences. query refers to figure  the genes and positions relative to transcription start refer to the genomic context of the cloned cis-elements. cis -sequences seq. cis n.b.: myb refers to the r2r3-myb class of myb tf. cis bold characters: mutated nucleotides; x: interactions tested in transient assays using physcomitrella patens protoplasts. within motif group i, two different sequences were assayed. for the first one, aacgtggg, no specific class of tf was found to preferentially activate transcription in yeast. in this assay, five different tfs, belonging to five different families, were identified. conversely, seven bhlh tfs were able to activate the transcription from the second sequence, gcacgtggag, revealing some transcriptional specificity. interestingly, both elements contain the acgt core sequence, which once mutated prevented the identified tfs to activate transcription in yeast, indicating its preponderant role in these interactions. the y1h analysis of group v motifs, which belong to the cluster showing the highest level of sequence similarity with group i, led to the identification of four bhlh tfs. in fact, these bhlh factors were found to activate transcription in yeast from the atgtgatgc sequence, but not from the gcatcaccc sequence, for which tfs from two other families were identified. the careful analysis of these group v motif sequences and their corresponding mutated versions atgcaatgc and gcatagccc) exposed that when the atgtgatgc sequence is concatenated, an e-box is created, whereas this is not the case with the gcatcaccc sequence. the fact that sequences from group i and v display strong similarities with either the well described g or e boxes probably explains why bhlh tfs were found to be associated with these two motif groups. group ii is closely related, at the sequence level, to group i and v. however, unlike groups i and v, only two nac tfs were found to interact with the two sequences tested, with one being identical for both sequences. from group iii, four sequences were analysed, namely cacctaac, accacaacc, accaaacat and tcggaccaa. from these ac-rich sequences only r2r3-myb tfs were identified as able to activate transcription in yeast, with the exception of tcggaccaa, for which no interacting tf was identified. the analysis of the ctctctcac element from group iv concluded with the identification of three factors: one nac and two bzip tfs. finally, three different sequences belonging to group vii were also analysed, namely tctcctccac, ctttcccc and cctccttct, from which no clear pattern of binding emerged. the screening of the tctcctccac sequence led to the identification of three different classes of tfs. three additional factors belonging to three further classes where identified with the ctttcccc element. finally, no binding tfs for the cctccttct element were retrieved from regia in yeast. validation of transcription factor predictions with yeast-one-hybrid experimental data in order to benchmark the accuracy of bioinformatically predicted candidate tfs, they were compared to those experimentally identified by y1h screening. key search parameters were tuned as explained in methods and in figure  and a trade-off between precision and sensitivity could be achieved. in summary, we were able to lower the blastp e-value threshold to 1e- without a significant sensitivity loss, but fixed the stamp cutoff to 1e- to avoid a major sensitivity drop while ensuring reliable short cis-element alignments. we also found that filtering out candidate tfs with poorly conserved interface residues, those directly contacting dna nitrogen bases, was helpful in order to discard false predictions. a cutoff of 50% interface similarity was observed to be adequate, increasing specificity to, as shown in additional file. tested cis-elements were classified as predictable if the footprintdb repository contained at least one annotated tf with a significantly similar motif, which was homologous to the experimental y1h-inferred factors. thirteen of the cis-elements tested in the y1h experiment were accordingly called ‘predictable', but only of them yielded true positives among footprintdb results. the rest had significantly divergent dna motifs annotated in the database, with e-values over the cutoff, and therefore could not be identified. it is important to note that in all predictable cases the correct binding tfs were identified by homology to annotated plant regulatory proteins, including a. thaliana, petunia hybrida, oryza sativa or phaseolus vulgaris. on the other hand, group ii, iii and vii elements could not be successfully matched as the idef and gcn4-like motifs were not part of footprintdb when the predictions were made. overall, after conducting the benchmark experiment using as input the cis-sequences in table  we conclude that we correctly predicted out of of the tfs isolated in the y1h assays, finding on average validated tf every predictions. if only predictable sites are considered, these numbers improve to out of and confirmed tf every predictions, respectively. finally, when the complete analysis was repeated with input dna motifs instead of individual cis-sequences, the fraction of successfully recovered tfs dropped to out of, yielding confirmed tf every predictions. these results and the complete benchmark reports can be found in additional file. functional analysis of tfs interacting with conserved cis-sequences in this study, some factors were found to be able to activate transcription in yeast from various cis-sequences conserved in the promoters of genes that are simultaneously induced by drought. amongst these, two were selected for more detailed analysis, nac protein at3g and myb at1g these selected tfs were able to induce transcription in y1h experiments from three and two different motif groups, respectively. the ability of these two factors to interact with their dna target in vivo was analysed in transient expression assays, using physcomitrella patens protoplasts. in this experiment, the dna binding domain of the nac protein and myb was fused to the vp activation domain, resulting in the formation of two synthetic tfs. these chimeric proteins were then assayed against different conserved cis-sequences, namely ctctctcac and gctgccggaga, and cacctaac and accacaacc, for the nac and myb dbd, respectively. this analysis confirms the results obtained in y1h experiments. interestingly, the transcriptional activity supported by the nac dbd was a lot stronger with the gctgccggaga sequence than with ctctctcac, strengthening the idea that the gctgccggaga element is most probably a new nac target sequence. conversely, similar activity was observed when myb dbd was assayed against either, cacctaac or accacaacc, suggesting that myb could activate the transcription from a wide variety of ac-rich cis-sequences. the cis-sequences and transcription factors identified in the present study may play a role in drought-responsive gene expression in a. thaliana. in the context of this assumption, several cis-sequences were used to generate synthetic promoters, which were tested in transgenic a. thaliana plants. however, no drought-responsive reporter gene expression has been observed in these transgenic lines so far. it will be interesting to see if overexpression or knock-down mutations of the identified transcription factors will have any effect on drought-responsiveness of the plants. discussion the application of bioinformatic resources has led to the identification of large numbers of novel cis-regulatory sequences conserved in stress response genes. often the role of these elements and their binding transcription factors has remained unknown. the primary goal of the present work was the identification of tfs interacting with cis-sequences conserved in co-regulated genes. a total of sequence motifs classified into motif families were identified from microarray data on drought responsive gene expression. these motifs were bioinformatically predicted to interact with tfs belonging to specific tf families. these predictions were then tested experimentally using yeast one-hybrid screenings leading to the identification of novel tf-dna interactions. initially, the two bioinformatic approaches employed, preferably predicted tfs interacting with putative binding sites for motif group i that harbours of a total of motifs. using the stamp web server as well as footprintdb, these show significant similarities to g-box and abre elements. however, no consistency in tf predictions was obtained with these database-assisted analyses using the other motifs. this first set of results exposed the natural limitations of this approach, mainly associated with the data content of the underlying scanned databases. for example, predictions using the stamp webserver depend on previously annotated data derived from databases such as transfac, jaspar, place, agris, and athamap. the content of these repositories is primarily derived from functionally known regulatory sequences, which are expected to be only a tiny fraction of all cis-elements. similarly, footprintdb contains tfs and their experimentally-associated dna motifs from these and other databases. a main difference to the stamp webserver is that footprintdb motifs are associated to their cognate binding tfs, and their dna-binding interfaces are annotated using 3d-footprint structural data. however, stamp as well as footprintdb detect and predict tf-dna interactions based on known data. therefore, tf-dna interactions not readily predicted with stamp and footprintdb may represent novel or to date unknown tf-dna interactions. this was substantiated in the present study by using sequences enriched in promoters of drought-responsive genes, but not readily associated with interacting tfs for experimental analysis using y1h-screenings. towards these ends two different sequences from motif group i were assayed. for the first one, aacgtggg, no specific class of tf was found to preferentially induce transcription in yeast. conversely, seven bhlh tfs were able to activate the transcription from the second sequence, gcacgtggag. both elements contain the acgt core sequence, which once mutated prevented transcription to be activated. these observations suggest that sequences aacgtggg and gcacgtggag are bona fide binding sites for bhlh and motif group v showed the highest levels of sequence similarity with group i and drove the identification of four bhlh tfs. in fact, the bhlh tfs were found to prompt transcription in yeast from cis-element atgtgatgc, but not from gcatcaccc. when sequence atgtgatgc is concatenated, an e-box is created, whereas this is not the case with gcatcaccc. the fact that sequences from group i and v contain or display strong similarities with either the well described g-box or e-box probably explains why bhlh tfs were found to be associated with these two motif groups. in fact, numerous studies have shown that bhlh tfs regulate the activity of their target gene promoters through these types of cis-regulatory sequences. however, no bzip tfs were identified as interacting with the motif that contains the acgt core, suggesting that the interactions with the bhlh tfs are quite specific and that the full-length sequence of the motifs may have a role in this specificity. group ii is closely related, at the sequence level, to group i and v. however, unlike group i and v, only two nac tfs were found to interact with the two sequences tested, with one being identical for both sequences. a wide variety of nac binding sites have been identified so far in various plant species, revealing the large diversity of the sequences that can be recognised by this group of tfs. the gccacgtcagc sequence displays similarities with the core sequence ca g that is recognised by the rice and barley idef nac proteins, suggesting that the identified nac could activate the transcription in yeast from it. this assertion was supported by the lack of transcriptional activity associated with the mutagenesis of this element. interestingly, the gctgccggaga sequence appears to be quite divergent when compared to the other described nac binding sites even though both nac tfs were able to activate the transcription in yeast from it, suggesting that the gctgccggaga dna sequence could be a new cis-regulatory element that could be targeted by nac proteins. from group iii, the ac-rich sequences cacctaac, accacaacc, accaaacat and tcggaccaa identified only r2r3-myb tfs to be able to activate transcription in yeast. this result is in agreement with previous studies that have demonstrated that numerous r2r3-myb proteins from different plant species are able to bind to and induce transcription from ac-rich sequences. one of the studied sequences contains the well-described ac-i cis-regulatory element to which the a. thaliana myb tf was shown to bind to in vitro and activate transcription from it in yeast. this result was confirmed in our screen. similarly, we found that two other r2r3-myb subgroup members, namely myb and myb were also able to activate transcription in yeast from the same element. the overlap between the r2r3-myb proteins that interact with the cacctaac and the accacaacc sequences was quite high, as out of the interacting r2r3-mybs were common to both elements. interestingly, only four r2r3-mybs prompted transcription in yeast from the accaaacat element, from which three belong to the same r2r3-myb subgroup, namely subgroup. group vi, such as goup iii, is composed of ac-rich motifs. from this motif group only one putative cis-regulatory sequence was assayed, namely ccaactaa, from which only r2r3-myb tfs were also identified. eleven and of the identified r2r3-mybs were identical with the r2r3-mybs found to interact with the cacctaac and accacaacc sequences, respectively. when concatenated, this motif partially contains the r2r3-myb targeted ac-ii element from which the identified tfs are likely to induce transcription in yeast, based on the absence of activation with the mutated version of the element. surprisingly, as no acgt core sequence is present in this motif, the analysis of the ctctctcac element from group iv leads to the identification of two bzip factors. nevertheless, a detailed analysis of this sequence showed that a bzip-like dna target similar to two gcn4-like motifs found in the promoter of the wheat storage protein genes was present, suggesting that this sequence could be mainly recognised by this class of bzip factors. while these y1h results on their own already provide valuable biological insight, they also served in this work as a reference dataset. indeed they were also used to benchmark the predictive value of the footprintdb approach for recognizing putative binding tfs with different parameter settings. two stamp e-value cutoffs were tested. while a stringent 1e- threshold yielded the most specific predictions, it was at the cost of a significantly reduced sensitivity. on the contrary, a relaxed 1e- cut-off increased the sensitivity by compromising specificity. however, this loss of specificity could be corrected by lowering the blastp e-value cut-off to 1e- in addition, we observed a further gain in specificity by applying an interface similarity filter for candidate tfs, requiring them to have at least 50% similar interface residues to be considered. altogether, these settings yield a 69% sensitivity and a 92% specificity. another important observation regarding the performance was that individual input cis-elements worked much better than sequence motifs, which presumably increase the chance of obtaining significant but irrelevant alignments of dna sequences, which are the cornerstone of this approach. in addition to the y1h screenings, the in vivo interaction of two tfs with their dna target was analysed in transient expression assays using physcomitrella patens protoplasts. in this experiment, two synthetic tfs were generated by fusing the dna binding domain of the nac protein and myb to the vp activation domain. these chimeric proteins were then assayed against the conserved cis-sequences, ctctctcac and gctgccggaga, and cacctaac and accacaacc, for the nac and myb dbd, respectively. interestingly, the transcriptional activity supported by the nac dbd was a lot stronger with the gctgccggaga sequence than with ctctctcac one, strengthening the idea that the gctgccggaga element is most probably a new nac target sequence. to date the nac protein encoded by the arabidopsis gene at3g had not been investigated experimentally. comparing the proposed novel nac binding site gctgccggaga with known arabidopsis nac binding sites reveals a large variability of binding sites recognised by nac proteins. for example, the nam and nap proteins bind to the cis-sequences aagggatga and cacgtaagt, respectively. the high variability of nac binding sites is also illustrated by the a-rich binding site of ataf. nac transcription factors are involved in a wide array of abiotic stress responses. interestingly, the nac transcription factors anac, anac, and anac bind to the sequence annnnntcnnnnnnnacacgcatgt, a drought responsive cis-sequence. although this sequence is part of a drought responsive promoter, overexpression of anac anac and anac in transgenic plants did not up-regulate erd expression indicating that other interacting factors may be necessary for the induction of the erd gene. in this context it is interesting to note that none of the cis-sequences investigated here, although enriched in drought responsive genes, individually confer drought responsive gene expression. among these sequences was also the novel nac binding site gctgccggaga. similarly it may be suggested that the investigated cis-sequences are each part of a combinatorial element that requires a second binding site for an interacting transcription factor in close vicinity for its functionality. when the myb dbd was assayed against cacctaac and accacaacc in physcomitrella patens protoplasts using a synthetic tf, similar activity was observed with both cis-sequences, suggesting that myb could induce transcription from a wide variety of ac-rich cis-sequences. myb belongs to subgroup of r2r3myb tfs. the gene is known to be up-regulated by chitin but no further functional analysis has been carried out to date. there are r2r3-myb tfs in the genome of arabidopsis and random binding site selection experiments with myb and revealed that these factors always bind to cis-sequences with one or two conserved acc and/or aac core sequences. consequently, the adjacent nucleotides can vary significantly and arabidopsis myb factors have a relatively degenerate binding site recognition. consistent with known myb binding sites, the synthetic tf containing the myb dbd activates gene expression from two synthetic promoters harbouring the acc and aac motif. although the myb binding site cacctaac does not confer drought responsive reporter gene expression in transgenic arabidopsis, this motif has been found in arabidopsis in the promoter of three myb target genes that are involved in vasculature development, one of the main components of the transpiration stream, which indirectly may participate in plant adaptation to drought stress. such observations reinforce the postulate that a combination of regulatory elements is required to integrate the environmental signals on a specific gene promoter.  the work presented demonstrates the successful integration of several bioinformatic resources to predict and validate tfs interacting with conserved sequence motifs in co-regulated genes. predictions were confirmed by using a yeast-one-hybrid approach to identify interacting tfs belonging to the predicted tf families. tf-dna interactions were further experimentally validated in yeast and with a physcomitrella patens transient expression system, leading to the discovery of several novel tf-dna interactions. our work establishes a novel approach to identify tfs interacting with conserved cis-sequences. this approach may facilitate the experimental identification of tfs because a candidate tf-family can be predicted bioinformatically using the footprintdb database. although the limitation of this approach is the content of the databases used, footprintdb is particularly valuable because it contains data for tf-dna interactions from many different species. therefore, this database may help to identify a dna binding protein domain for any submitted sequence. afterwards one may check plant tf databases for the predicted dna binding domain to identify novel sequence-specific tf-dna interactions. 
tf-dna interactions were further experimentally validated in yeast and with a physcomitrella patens transient expression system, leading to the discovery of several novel tf-dna interactions. using the pathoplant database, up-regulated gene groups were identified with microarray data for drought-responsive gene expression from arabidopsis thaliana. application of the binding site estimation suite of tools discovered conserved sequence motifs within the corresponding promoters. using the stamp web-server, sequence motifs were classified into motif families for which similarities with known cis-regulatory sequences were identified. the present work demonstrates the successful integration of several bioinformatic resources with experimental approaches to predict and validate tfs interacting with conserved sequence motifs in co-regulated genes. this starts with the selection of co-regulated gene sets and leads first to the prediction and then to the experimental validation of tfs interacting with cis-sequences conserved in the promoters of these co-regulated genes. predictions were confirmed by using a yeast-one-hybrid approach to select interacting tfs belonging to the predicted tf families. in contrast, the identification of the transcription factors interacting with these cis-sequences is much more elaborate. all motifs were subjected to a footprintdb analysis to predict interacting dna binding domains from plant tf families.
 using motif detection programs it is fairly straightforward to identify conserved cis-sequences in promoters of co-regulated genes. in contrast, the identification of the transcription factors interacting with these cis-sequences is much more elaborate. to facilitate this, we explore the possibility of using several bioinformatic and experimental approaches for tf identification. this starts with the selection of co-regulated gene sets and leads first to the prediction and then to the experimental validation of tfs interacting with cis-sequences conserved in the promoters of these co-regulated genes.  using the pathoplant database, up-regulated gene groups were identified with microarray data for drought-responsive gene expression from arabidopsis thaliana. application of the binding site estimation suite of tools discovered conserved sequence motifs within the corresponding promoters. using the stamp web-server, sequence motifs were classified into motif families for which similarities with known cis-regulatory sequences were identified. all motifs were subjected to a footprintdb analysis to predict interacting dna binding domains from plant tf families. predictions were confirmed by using a yeast-one-hybrid approach to select interacting tfs belonging to the predicted tf families. tf-dna interactions were further experimentally validated in yeast and with a physcomitrella patens transient expression system, leading to the discovery of several novel tf-dna interactions.  the present work demonstrates the successful integration of several bioinformatic resources with experimental approaches to predict and validate tfs interacting with conserved sequence motifs in co-regulated genes. databasesarabidopsis thalianaphyscomitrella patensyeast one-hybridmicroarraytranscription factorcis-element 