The BB promoter contains cis-elements in the 5' UTR and 5' non-transcribed region
A 3.5 kb genomic fragment of the BB
locus consisting of 1.3 kb of 5' non-transcribed promoter sequence, 669 bp of 5' UTR containing two introns, the coding sequence (CDS) and 199 bp of 3' UTR contains all regulatory elements sufficient for rescuing levels of BB
]. In order to isolate cis
-elements required for the regulation of BB
expression, we fused 1035 bp upstream of the BB
start codon to a BB
cDNA. This initial construct, termed minimal promoter (pBBmin), consists of 366 bp non-transcribed sequence and the entire 5' UTR and is sufficient to rescue the petal overgrowth phenotype of bb-1
mutant plants, indicating that it contains all necessary regulatory elements for BB
expression regulation (Figure ).
Figure 1 Promoter deletion analysis. The initial construct of pBBmin is shown on top. The gray arrow indicates BB cDNA; while 5' UTR and introns are marked with blue and red lines, respectively. Lines below indicate the promoter constructs for the sequential deletion (more ...)
To further delimit regulatory elements within this stretch of sequence we removed successive 50-bp fragments from the 5' end of the non-transcribed sequence in four steps, until only 141 bp upstream of the transcription initiation site remained. In a parallel experiment only the 366 bp of non-transcribed sequence were fused to an alternative 5' UTR (omega sequence, [10
]) or to 90 bp or 170 bp of the endogenous 5'UTR sequence. The resulting seven promoter deletion constructs (illustrated in Figure ) were all fused to the BB
cDNA and transformed into bb-1
mutant plants. We generated lines homozygous for a single transgene insertion derived from three independent primary transformants and measured petal size of 3-5 plants as a read-out for transcriptional activity of the promoters to be tested. The results from the individual lines for each construct are shown in Additional file 1
: Figure S1. This assay allowed us to determine not only the qualitative functionality of the promoter sequences, i.e. the correct spatial expression pattern in petals, but also quantitatively assess the levels of expression. This is because petal size strictly depends on BB
dosage, with a mere two-fold reduction in expression in heterozygous plants leading to a significant increase in petal size [6
]. We note that this approach might miss specific regulatory sequences that are only active in organs other than petals. For better visualisation we normalised the average petal sizes to the Landsberg errecta
) wild-type value. To test for significant changes we performed a pair-wise t
-test on all plants derived from one construct compared to wild-type and to account for multiple testing we used the Benjamini-Hochberg correction [11
]. While removing the first 50 bp from the 5’ non-transcribed sequence did not affect the rescuing activity of the construct, eliminating a further 50 bp resulted in one transgenic line in smaller than wild-type petals, suggesting the existence of a potential negative regulatory element within this second 50-bp fragment. However, as two of the three lines measured did not show any significant changes to wild type, this result needs to be confirmed independently. Removal of 150 bp from the 5' non-transcribed promoter sequence resulted in an almost 40% increase in petal size in the transgenic plants (compared to an 80% increase vs. L.er
in non-transgenic bb-1
mutants), indicating that the third 50-bp fragment contains important positively acting promoter elements. An effect of similar strength was observed, when parts or all of the 5' UTR were removed (constructs "-5'UTR" and "+170", Figure ), indicating that the 5' UTR also contains sequences important for normal BB
expression and that the non-transcribed sequence on its own is not sufficient to condition rescuing levels of BB
expression. As removal of sequences from the 5' UTR or from the non-transcribed region only caused a loss of rescuing activity to what is seen in bb1/+
heterozygous plants, there appear to be two independent regulatory inputs, acting on either of these regions, with only their combined action leading to wild-type BB
expression levels. Also, positive and negative inputs seem to be integrated by the BB
As cis-regulatory elements within the 5' UTR could affect BB transcription as well as its translation, which we cannot monitor at present, we decided to focus our further analysis on the non-transcribed sequences.
The BB CDS is strongly conserved within Brassicaceae
Putative E3 ubiqutin ligases related to BB can be found in several plant species [6
], suggesting that BB
function is conserved among plants. This would open up the possibility to use phylogenetic footprinting (see below) to identify cis-
regulatory promoter elements by virtue of their conservation. As a step towards isolating the non-transcribed promoter sequences, we first isolated the BB
genomic coding sequence (CDS) from seven species in six different Brassicaceae genera: Arabidopsis thaliana, Arabidopsis lyrata, Arabis alpina, Cardamine hirsuta, Iberis amara, Sisymbrium officenale
and Thalspi perfoliata
. Together these genera represent examples for almost the entire Brassicaceae family [12
The genomic BB
sequences for A. thaliana
and A. lyrata
were isolated from public databases (see Materials and Methods). For the other species, we used primers designed against the BB
CDS from A. thaliana
and amplified the CDS sequences by PCR. All sequences were compared to the A. thaliana
reference using the mVISTA tool [14
]. The alignment of the isolated BB
CDS showed a very high degree of conservation within all isolated genera (Figure ). The most divergent sequence stretches were found in regions corresponding to introns within A. thaliana
(marked in white on the VISTA plot in Figure ), suggesting that the intron-exon structure of BB
is well conserved. Interestingly, not all introns show such a low conservation. In the VISTA plot using a 100-bp sliding window analysis with a minimum conservation width of 100 bp, introns in the middle part of the BB CDS show levels of conservation above 70% throughout the seven species (marked in red on the VISTA plot in Figure ), suggesting that these introns contain functional elements. To confirm that the isolated sequences are really the CDS of the orthologues of BB
and not of the homologous BIG BROTHER-RELATED
, At3g19910) gene, we performed a phylogenetic analysis using the CDS of the A. thaliana
and A. lyrata BBR
as outgroup. In this analysis all BB
CDS clearly clustered together, indicating that we isolated the orthologues of BB
and not of BBR
Figure 2 VISTA plot of BB CDSs. a) VISTA plot of pair-wise comparisons of different Brassicacea species to A. thaliana. Intron and exon annotation of A. thaliana is shown on top (exons are represented by thick lines, introns by thin lines) and filled portions (more ...)
For four of the seven species (A. thaliana, A. lyrata, C. hirsuta and S. officinale) we were able to isolate larger genomic fragments containing also 5’ non-transcribed sequences. The VISTA alignment against A. thaliana using these larger fragments showed a high level of conservation also within the 5' UTR and the approximately 300 bp of non-transcribed sequence (Figure ). Taken together, these results show that the BB gene is well conserved within Brassicaceae. The conservation is not only limited to the transcribed exon sequence of BB, but also non-transcribed and intronic regions show relatively high levels of sequence similarity.
Phylogenetic footprinting reveals strong conservation of the 5' non-transcribed sequence
In addition to the protein coding sequence, regulatory elements important for proper qualitative and quantitative expression also tend to be conserved to maintain the function of a given genomic locus. The high sequence conservation of BB
CDSs and non-transcribed sequences within Brassicaceae encouraged us to use phylogenetic footprinting, i.e. to systematically compare the non-transcribed regions of the BB
loci to further delimit cis
-regulatory elements based on their conservation across taxa [7
]. To this end, we used thermal asymmetric interlaced-PCR (TAIL PCR) to amplify genomic sequences extending 5' from the highly conserved BB
]. We were able to isolate approximately 1 kb of non-transcribed sequence from T. perfoliatum, A. alpina, S. officinale, I. amara
and C. hirsuta
. For A. lyrata
and Brassica oleracea
we found aligning sequences in available databases (see Materials and Methods). All isolated DNA sequences contained the entire minimal promoter which we determined in previous experiments (Figure ).
The alignment of all BB promoter sequences showed a very high degree of conservation within the non-transcribed sequence. 174 bp of the 366 bp of the non-transcribed sequence (47.5%) of A. thaliana showed conservation of up to 100% within all Brassicaceae promoters analysed. In comparison only 24 bp of the 669 bp of 5' UTR (3.5%) showed conservation to such high levels (Figure ). This indicates that high levels of sequence diversity can be found between the BB loci in the selected species, and that conversely the high levels of conservation within the non-transcribed sequence are not only due to the close phylogenetic relationship of the taxa in question.
Figure 3 Phyogenetic footprinting of BB promoter. a) VISTA plot of pair-wise comparisons of different Brassicaceae and A. thaliana. Graph illustrates a 20 bp sliding window comparison and conservation of more than 90% with a minimum width of 20 bp is marked colour (more ...)
Pair-wise alignments of the individual sequences with the A. thaliana
sequence using mVISTA [14
] showed that the highly conserved regions fall into five blocks of 24-53 bp according to the A. thaliana
promoter sequence. For better visualisation of the high similarity we used a 20 bp sliding window analysis in which blocks with 90% similarity or more are marked in color (solid red or blue graphs in Figure ). The first four blocks were located within the non-transcribed region and only one block of 24 bp was located within the 5' UTR right next to the annotated transcriptional start site (see boxes in Figure and underlined stretches in Figure ). Three of the four conserved blocks within the non-transcribed sequence were located in the proximal 165 bp upstream from the transcription start site (Figure ), in the sequence that was still present in the "-200" construct tested in Figure .
To identify putative regulatory motifs within this alignment, we scanned for putative cis
-regulatory elements using the FootPrinter algorithm [18
]. This algorithm uses a motif discovery approach on a comparison of a set of homologous sequences. The program searches for defined k-
mers (motives), one from each sequence, within the set of sequences allowing for nucleotide substitutions within the k-
mers depending on the phylogenetic relation, i.e. more closely related species are expected to share more similarity. In this way the algorithm allows the identification of regulatory cis
-elements, even if promoter sequences are too divergent to be accurately aligned [18
] like the 5' UTR of the BB
minimal promoter sequence. In our alignment the FootPrinter software predicted four putative binding sites within the non-transcribed sequence and one at the start of the 5' UTR (see red dashed boxes in Figure ), all of which were within the highly conserved sequences. However, the software did not predict a binding site within all conserved sequences. Two of the putative binding sites were located within one block of conserved sequence whereas one other block did not contain any predicted binding site (see Figure red block and red underlined sequence in Figure ).
Taken together the phylogenetic footprinting of BB promoters from eight Brassicaceae species showed that the non-transcribed sequences and the area around the transcriptional start site are highly conserved, suggesting the presence of cis-regulatory elements. The cis-elements predicted to be present in the 5' UTR based on our previous results (Figure ) were not found, suggesting that the input pathways controlling BB expression via cis-elements within this region may have diverged. As one of the highly conserved blocks was located right in front of the annotated transcriptional start site and did not contain any predicted cis-elements according to the FootPrinter results, this region may contain mainly sequence elements important for basic transcription initiation. Therefore we focused our further characterization on the four other highly conserved blocks, which we termed conserved block A, B, C and D (CA to CD, Figure black boxes).
Putative auxin binding sites are not functional
To test whether the conserved domains of the pBBmin promoter contain any known binding sites for trans
-acting transcription factors we searched for binding sites within the 5 conserved blocks using the PLACE database [19
]. PLACE predicted several putative binding sites for transcription factors. However, most of the binding sites showed matches only in 4 nucleotides and were therefore ignored. Interestingly, block CB and CD contained full matches of the AUXIN REPONSE FACTOR (ARF) binding site (TGTCTC, marked as blue boxes in Figure ). Another putative binding site for a MYB transcription factor was predicted within the conserved block upstream of the transcriptional initiation site.
In order to test whether the predicted ARF binding sites are functional we performed an auxin induction assay in seedlings on a pBBmin construct fused to the LUCIFERASE
) reporter gene. LUC activity was measured after 1 h and 3 h induction with different concentrations of the auxin transport inhibitor NPA or the auxin analogon NAA and measurements normalized against the mock control. For the treatments with NPA we were not able to find significant changes and NAA only lead to a modest induction (Figure ). For comparison, Pufky et al. [21
] showed that genome wide a large number of genes containing the ARF binding site are upregulated more than two-fold after 60min of induction. Also, Ulmasov et al. [22
] showed an 8-fold induction by auxin of GUS fused to a promoter containing the TGTCTC motif.
These results are in line with previous published literature showing that the genomic protein fusion of BB to GUS could not be induced by auxin. Also, the bb-1
mutant was shown to have no altered sensitivity to any tested phytohormones, suggesting that BB acts independently of the major phytohormones [6
]. Therefore we concluded that the predicted ARF binding sites are most likely not functional.
Brassicaceae promoters are functional in A. Thaliana
One important assumption in the phylogenetic footprinting analysis is that conservation of sequence also represents functional conservation. However, as the sequences around the highly conserved elements in the different Brassicaceae species show a high level of diversity, it remains to be tested whether the mere conservation of small sequence stretches results in functional conservation of the promoters. To address this, we tested the functionality of some of the isolated BB promoters from other species in A. thaliana. To this end, the BB promoter sequences of A. alpina, I. amara, S. officinale and T. perfoliatum were fused to the BB cDNA from A. thaliana and transformed into bb-1 mutant A. thaliana plants. All chosen promoter sequences showed high levels of conservation within the non-transcribed sequence; the 5' UTR, however, was very variable (Figure ).
For all constructs, we again established homozygous lines derived from three independent primary transformants each and measured their petal sizes. All tested lines showed a significant decrease in petal size, suggesting that the respective promoters are largely functional in A. thaliana (Figure ). The level of phenotypic rescue did not depend on the similarity within the 5' UTR, as also the promoters of I. amara and T. perfoliatum with their divergent 5' UTR sequences relative to A. thaliana were able to rescue the bb-1 phenotype to a similar extent as the other promoters (Figure ). These results suggest that the sequence conservation in the 5' non-transcribed region is indeed indicative of functional conservation. Whether in the I. amara and T. perfoliatum promoters other sequences fulfill the same function as the 5' UTR sequences in A. thaliana will need to be resolved by further experimentation.
Conserved cis-elements are important for regulation of BB expression
To determine the function of the predicted conserved cis-elements in A. thaliana, we implemented a more refined promoter deletion analysis. In an initial experiment we removed all conserved elements together (delCA-CB-CC-CD in Figure ). As BB expression in A. thaliana is also influenced by cis-elements within the 5' UTR we predicted that such a construct would not completely abolish expression from this promoter, but should significantly decrease the rescuing activity, if the deleted elements play an important role. Similarly, we also addressed the function of each conserved element individually. To this end we removed each conserved block independently from the minimal promoter. To control for the influence of transcription initiation site we also included a single deletion of the 65 bp neighbouring the transcription start site. All constructs were fused to the BB cDNA from A. thaliana and transformed into bb-1 mutants. As before, we assayed the activity of the promoter constructs by analysing homozygous transgenic lines derived from three individual primary transformants.
Figure 4 Promoter deletion assay for conserved elements. Top line indicates the non-transcribed sequence of pBBmin with highly conserved blocks shown as blocks (dashed boxes contain predicted binding motifs from FootPrinter, dotted box does not contain a predicted (more ...)
The results of this deletion series are summarized in Figure . As before, the average petal size is normalized to the L.er wild-type value. The deletion of all four conserved elements led to a significant decrease of the rescuing activity, resulting in a 40% increase of petal size relative to wild-type and control transgenic plants with the unmodified minimal promoter construct. Thus, the removal of the four highly conserved blocks of promoter sequence strongly impairs functionality (Figure ). By contrast, removal of none of the elements by themselves caused a decrease of the rescuing activity. Rather, weak repressive effects of CB and CD are suggested by the slightly smaller than wild-type petals in the corresponding transgenic lines. This suggests that more complex interactions between the CA, CB, CC and CD regions may be involved in BB expression regulation.
To address such potential interactions between different binding sites and thus potentially different inputs, we generated deletions of pairs of the four conserved elements and measured their functionality as before. The analysis of the different double deletions showed a complex picture. Of particular interest was the result of deleting both CB and CD. While both single deletions seemed to enhance the activity of the promoter derivatives (see above), combined removal of CB and CD interfered with the rescuing activity of the construct to a similar level as removing all four conserved elements did.
As a next step, we deleted all possible combinations of three of the four conserved elements, resulting in constructs with only one of the elements remaining. To our surprise, measurements of petal sizes in the resulting transgenic lines indicated that all of the deleted constructs were still able to rescue the bb-1 phenotype to essentially wild-type levels. Thus, with respect to the combination of CB and CD, the additional removal of either CA or CC restored the activity of the promoter. This suggests that in the absence of the CB and CD sequences, repressive factors bind to the modified promoter, and that this binding is abolished when either CA or CC are removed in addition.
Taken together, the functional analysis of the conserved elements within the non-transcribed sequence suggests that these elements harbor binding sites for important trans-acting factors. However, none of the analyzed elements seems to be essential by itself (i.e. they can all be individually deleted without a loss of promoter activity), suggesting that multiple, potentially interacting pathways promote BB expression.