BACKGROUND
sequence specific binding of transcription factors in response to diverse cellular input signals is a major determinant in the regulation of transcription.
binding sequences for many factors have been identified by experiment and/or by a wealth of prediction methods .
consensus binding sites were classically determined by selex experiments and verified by emsa while more recently affinity measurements by methods better suited to moderate to large scale experimentation like microarray binding experiments have been used  <cit> .
experimentally determined affinities or frequencies for each base at every position of a binding site can be represented as position weight matrices or sequence logos, which can be used for prediction of new binding sites  <cit> .
it is well known that not all sequences, which a transcription factor strongly binds to in vitro will also be bound in an in vivo context  <cit> .
global chromatin immunoprecipitation can identify the sequences bound by a transcription factor within the cellular context but does not indicate whether the binding site is functional, i.e.
whether the presence of a given tf at this site affects expression of the target gene.
for this, additional information usually derived from microarray data, sequencing or promoter studies is required  <cit> .
relative binding affinity is a good indicator of transcriptional activation or repression in an artificial system as shown for example by kang et al for the zif <dig> dna binding domain joined to repressor or activator domains  <cit> .
a detailed description of the quantitative relationship between affinity and activation potential in the cell is difficult since in vivo activation depends on the presence of co-factors, additional transcription factors and the epigenetic state of the chromatin.
on the other hand, a single high affinity binding site in combination with a minimal promoter frequently does not produce strong target gene activation and reporter constructs therefore usually contain several repeats of consensus binding sites to enhance reporter activity.
in the analysis of specific promoters attention is usually first focussed on consensus sites though the functionality of variant sites for many transcription factors has been shown in vivo and in reporter gene assays.
the effect of variation in a single site on activation and specificity has extensively been investigated in e coli <cit> .
within specific mammalian promoters the influence of variant sites on transcriptional activation has not been explored systematically.
the three gli transcription factors, mediators of the hedgehog signal, comprise a dna binding domain of five zinc fingers, which are very highly homologous in the three glis.
two of the five fingers are responsible for all but one of the protein-dna base contacts  <cit> .
the glis can function as activators and/or repressors and regulate target genes in a highly context specific way.
the consensus binding sequence gaccaccca was first determined by kinzler et al  <cit>  and many direct gli target genes have been identified.
hallikas et al  <cit>  determined the affinities of all single base substitutions in the gli consensus binding sequence using a fusion of luciferase with the gli-dna binding domain in an in vitro assay.
these data together with information on species conservation were used in the novel eel prediction program to identify gli regulated genes within the mouse and human genome.
these predictions were successful in identifying new target genes though some known target genes were not represented in the original version.
this emphasizes the need to characterise in more detail the relationship between affinity and functionality of gli binding sites in functional assays.
we therefore set out to investigate the activity of all single site variants of the consensus gli binding site in a luciferase assay.
frequently gli transcriptional activity is measured in an artificial construct containing multiple copies of the consensus site.
here we use a construct based on the ptch <dig> promoter, which is functional in many different cell types and should approximate a "normal" control of gene expression.
using relatively low gli concentration to enhance specificity we found that a rather large number of variant gli binding sites was able to activate transcription within the ptch <dig> promoter.
we then proceeded to turn variant binding sites into consensus within two unrelated natural promoters containing essential non-consensus gli binding sites and found that activity was not significantly enhanced.
RESULTS
a ptch <dig> reporter system to measure the functionality of variant gli binding sites
the hedgehog receptor ptch <dig> is a well characterised direct gli target gene and its elevated expression is indicative of hh pathway activation.
ptch <dig> expression is driven from several alternative transcription start sites  <cit> .
the ptch <dig> promoter region upstream of exon 1b  has been shown to contain a gli consensus site   <cit>  essential for activation by gli.
we localised a second gli binding site  with a single substitution compared to consensus upstream of bs <dig> at - <dig>  the presence of bs <dig> only is not sufficient for promoter activation by gli in a luciferase assay, but it enhances transcriptional activation in the presence of bs <dig> .
we chose to use the essential bs <dig> site in the ptch <dig> promoter to investigate the influence of all  <dig> possible single base substitutions in the consensus sequence on transcriptional activation.
to facilitate the exchange of consensus by the variant binding site we replaced the consensus site with a linker sequence permitting the test sequence to be quickly inserted into the ptch <dig> luciferase reporter construct  .
together with the variant sequence, a hindiii site was inserted to allow fast identification of plasmids containing the variant sequence.
the base c in position  <dig> relative to the start of the consensus sequence has previously been shown to positively affect gli binding affinity  <cit>  and is included in the construct as part of the hindiii site.
we then tested the functionality of the luciferase reporter system by comparing the ability of gli2act to activate the reporter constructs containing the linker with the consensus sequence ) to the unmodified ptch <dig> promoter luciferase reporter construct  .
all results presented here were obtained with gli2act, which is a strong activator.
when gli <dig> was used comparable results were obtained though activity was lower .
as shown in figure 1b both wild type  and modified ptch <dig> promoter construct ) were strongly induced in response to gli2act with only slightly lower activation for the modified ptch <dig> promoter .
as expected, the inactive variant 6g7g   in ptch1_wt as well as in ptch1_var resulted in strongly reduced reporter activity.
no activation was observed with ptch1_var, with no inserted sequence.
thus, the modified ptch <dig> reporter system is functional and can be used to systematically measure the effect of variation in gli binding sequence on gli target gene activation.
the effect of gli binding site variants on ptch <dig> promoter activation
to determine gli activity for all single site variants of the  <dig> bp consensus binding sequence we co-transfected each ptch1_var luciferase reporter together with gli2act into hacat cells .
as a negative control we used ptch1_var .
to exploit the dynamic range of the reporter system, all assays were performed under optimal transcriptional activation conditions using moderate gli2act levels.
the boxplot  shows the range of activities measured at each position, statistical significance compared to negative control and to consensus is shown in figure 2b.
at first view it is striking that many sequence variants result in reporter activation similar to the consensus gli binding site.
especially in position  <dig> there is no significant difference in the transcriptional activities between consensus and any non-consensus bases .
in contrast, any substitution in position  <dig> or  <dig> leads to loss of activity, consistent with affinity measurements showing complete loss of gli binding if these critical positions are altered .
there are several positions where the identity of the substituted base shows a pronounced effect on transcriptional activation: in position  <dig> , g and t do not lead to reporter gene transcription while a reproducibly equals or even appears to exceed the level of activation by consensus.
a number of variants results in activities intermediate between consensus and background.
taking into account the variability inherent in biological replicates it is not possible to attach significance to relatively small differences in activity.
to exclude the possibility that the linker sequence, which surrounds the binding site differentially affects the activation of the various reporter constructs, we also tested a small number of binding site variants directly within the unmodified ptch <dig> promoter construct by introducing site-specific mutations .
no major discrepancies were observed, suggesting in summary that many variant gli binding sites are functional and can substitute for the consensus.
we then compared the transcriptional activation  to the affinity profile described by hallikas et al  <cit>  and found that a large number of substitutions, which have quite low affinity significantly activate the luciferase reporter.
this may be due to the fact that the nonlinear normalization applied to the raw data very strongly emphasizes the consensus site  <cit> .
conventional competitive emsa measurements on selected binding sequences with linear normalization showed several single substitutions with kd values within a factor of  <dig> of the consensus , which are compatible with the results of the luciferase reporter activity found.
this is also consistent with the existence of many single and several double substitutions in the gli consensus sequence of promoters with known gli dependent function in vivo , which failed to be retrieved in genome-wide in silico searches for gli target genes  <cit>  e.g.
bcl <dig>  il1r <dig>  fst, tgm <dig> .
though not perfectly representing the context of chromatin, luciferase reporter assays can be used to distinguish between potentially functional gli binding sites and apparent binding sites, which do not activate reporter gene activity within their sequence context.
this can be demonstrated clearly for the tgm <dig> promoter, which contains three potential gli binding sites: one consensus sequence and two variants with a c to a substitution in position  <dig>  .
mutation of the consensus sequence to nonbinding 6g7g does not affect reporter activity nor is the consensus site bound by gli in a chip experiment .
in contrast, the variant sites are bound by gli and mutation of either variant site abolishes transcriptional activation .
non-consensus gli binding sites in gli target gene promoters
to further explore the influence of binding affinity on transcriptional activation in a natural promoter context other than ptch <dig> we chose the jun and gli <dig> promoters, both containing functional non-consensus gli binding sites, for further analysis
the human jun   <cit>  and human proximal gli <dig> promoter   <cit>  both contain only one functional gli binding site thus eliminating possible interactions between nearby gli binding sites .
either binding site variant has been shown to be essential for activation by gli and both have significantly lower affinity than the consensus site  and gli <dig> ,  <dig>  for gli <dig>  , double substitutions as found in the jun promoter were not tested under identical conditions,  <cit> .
to compare the activity of the variants to the consensus, we applied site directed mutagenesis to change the wild type variant sites to the consensus sequence  .
the luciferase reporter constructs  were then tested for the response to gli2act in hacat cells and luciferase activity was compared to the respective wild type promoter constructs .
we detected no significant difference between gli consensus and non consensus wild type sequences in the context of either promoter.
these results indicate that relatively low binding site affinity does not prevent activation by gli in a luciferase assay at optimal gli concentration.
recent observations show that lower affinity binding sites for transcription factors can be identified by global chip  <cit>  and occur quite frequently.
large scale affinity measurements as described in  <cit>  showed that a large selection of transcription factors recognises many variations of the primary motifs and that even secondary motifs exist, which may possibly affect changes in transcriptional specificity.
a visible influence of low affinity sites on gene expression in yeast has been described pointing to their potential relevance for modulating gene expression  <cit> .
vokes et al  <cit>  identified a number of gli promoters/enhancers, which behave in a tissue specific way and are influenced by nuclear gli concentration.
in a more global study, groups of sites with high and lower affinities to rest repressor were shown to cluster into groups responsible for activation of target genes expressed commonly, specifically or uniquely in different cell lines  <cit> .
these data imply an important role for lower affinity sites in the context dependent control of transcription and point to the need for more detailed investigation of their function.
CONCLUSIONS
the results presented here specifically focus on the activation potential of binding sites of the gli transcription factors, the mediators of the hedgehog signal.
we measured activation in a standardised luciferase assay in the context of the ptch <dig> promoter testing all single site mutations of the gli consensus binding sequence.
a rather large number of substitutions was shown to be active, which is consistent with the existence of many known gli target gene promoters containing variant sites with lower binding affinity.
taking into account the contribution of a larger subset of binding sites with significant affinity the results presented in this study are likely to be helpful in the prediction and experimental validation of more direct gli target genes.

 sequence specific binding of transcription factors in response to diverse cellular input signals is a major determinant in the regulation of transcription. binding sequences for many factors have been identified by experiment and/or by a wealth of prediction methods. consensus binding sites were classically determined by selex experiments and verified by emsa while more recently affinity measurements by methods better suited to moderate to large scale experimentation like microarray binding experiments have been used. experimentally determined affinities or frequencies for each base at every position of a binding site can be represented as position weight matrices or sequence logos, which can be used for prediction of new binding sites. it is well known that not all sequences, which a transcription factor strongly binds to in vitro will also be bound in an in vivo context. global chromatin immunoprecipitation can identify the sequences bound by a transcription factor within the cellular context but does not indicate whether the binding site is functional, i.e. whether the presence of a given tf at this site affects expression of the target gene. for this, additional information usually derived from microarray data, sequencing or promoter studies is required. relative binding affinity is a good indicator of transcriptional activation or repression in an artificial system as shown for example by kang et al for the zif dna binding domain joined to repressor or activator domains. a detailed description of the quantitative relationship between affinity and activation potential in the cell is difficult since in vivo activation depends on the presence of co-factors, additional transcription factors and the epigenetic state of the chromatin. on the other hand, a single high affinity binding site in combination with a minimal promoter frequently does not produce strong target gene activation and reporter constructs therefore usually contain several repeats of consensus binding sites to enhance reporter activity. in the analysis of specific promoters attention is usually first focussed on consensus sites though the functionality of variant sites for many transcription factors has been shown in vivo and in reporter gene assays. the effect of variation in a single site on activation and specificity has extensively been investigated in e coli. within specific mammalian promoters the influence of variant sites on transcriptional activation has not been explored systematically. the three gli transcription factors, mediators of the hedgehog signal, comprise a dna binding domain of five zinc fingers, which are very highly homologous in the three glis. two of the five fingers are responsible for all but one of the protein-dna base contacts. the glis can function as activators and/or repressors and regulate target genes in a highly context specific way. the consensus binding sequence gaccaccca was first determined by kinzler et al and many direct gli target genes have been identified. hallikas et al determined the affinities of all single base substitutions in the gli consensus binding sequence using a fusion of luciferase with the gli-dna binding domain in an in vitro assay. these data together with information on species conservation were used in the novel eel prediction program to identify gli regulated genes within the mouse and human genome. these predictions were successful in identifying new target genes though some known target genes were not represented in the original version. this emphasizes the need to characterise in more detail the relationship between affinity and functionality of gli binding sites in functional assays. we therefore set out to investigate the activity of all single site variants of the consensus gli binding site in a luciferase assay. frequently gli transcriptional activity is measured in an artificial construct containing multiple copies of the consensus site. here we use a construct based on the ptch promoter, which is functional in many different cell types and should approximate a "normal" control of gene expression. using relatively low gli concentration to enhance specificity we found that a rather large number of variant gli binding sites was able to activate transcription within the ptch promoter. we then proceeded to turn variant binding sites into consensus within two unrelated natural promoters containing essential non-consensus gli binding sites and found that activity was not significantly enhanced.  a ptch reporter system to measure the functionality of variant gli binding sites the hedgehog receptor ptch is a well characterised direct gli target gene and its elevated expression is indicative of hh pathway activation. ptch expression is driven from several alternative transcription start sites. the ptch promoter region upstream of exon 1b has been shown to contain a gli consensus site essential for activation by gli. we localised a second gli binding site with a single substitution compared to consensus upstream of bs at - the presence of bs only is not sufficient for promoter activation by gli in a luciferase assay, but it enhances transcriptional activation in the presence of bs. we chose to use the essential bs site in the ptch promoter to investigate the influence of all possible single base substitutions in the consensus sequence on transcriptional activation. to facilitate the exchange of consensus by the variant binding site we replaced the consensus site with a linker sequence permitting the test sequence to be quickly inserted into the ptch luciferase reporter construct. together with the variant sequence, a hindiii site was inserted to allow fast identification of plasmids containing the variant sequence. the base c in position relative to the start of the consensus sequence has previously been shown to positively affect gli binding affinity and is included in the construct as part of the hindiii site. we then tested the functionality of the luciferase reporter system by comparing the ability of gli2act to activate the reporter constructs containing the linker with the consensus sequence ) to the unmodified ptch promoter luciferase reporter construct. all results presented here were obtained with gli2act, which is a strong activator. when gli was used comparable results were obtained though activity was lower. as shown in figure 1b both wild type and modified ptch promoter construct ) were strongly induced in response to gli2act with only slightly lower activation for the modified ptch promoter. as expected, the inactive variant 6g7g in ptch1_wt as well as in ptch1_var resulted in strongly reduced reporter activity. no activation was observed with ptch1_var, with no inserted sequence. thus, the modified ptch reporter system is functional and can be used to systematically measure the effect of variation in gli binding sequence on gli target gene activation. the effect of gli binding site variants on ptch promoter activation to determine gli activity for all single site variants of the bp consensus binding sequence we co-transfected each ptch1_var luciferase reporter together with gli2act into hacat cells. as a negative control we used ptch1_var. to exploit the dynamic range of the reporter system, all assays were performed under optimal transcriptional activation conditions using moderate gli2act levels. the boxplot shows the range of activities measured at each position, statistical significance compared to negative control and to consensus is shown in figure 2b. at first view it is striking that many sequence variants result in reporter activation similar to the consensus gli binding site. especially in position there is no significant difference in the transcriptional activities between consensus and any non-consensus bases. in contrast, any substitution in position or leads to loss of activity, consistent with affinity measurements showing complete loss of gli binding if these critical positions are altered. there are several positions where the identity of the substituted base shows a pronounced effect on transcriptional activation: in position, g and t do not lead to reporter gene transcription while a reproducibly equals or even appears to exceed the level of activation by consensus. a number of variants results in activities intermediate between consensus and background. taking into account the variability inherent in biological replicates it is not possible to attach significance to relatively small differences in activity. to exclude the possibility that the linker sequence, which surrounds the binding site differentially affects the activation of the various reporter constructs, we also tested a small number of binding site variants directly within the unmodified ptch promoter construct by introducing site-specific mutations. no major discrepancies were observed, suggesting in summary that many variant gli binding sites are functional and can substitute for the consensus. we then compared the transcriptional activation to the affinity profile described by hallikas et al and found that a large number of substitutions, which have quite low affinity significantly activate the luciferase reporter. this may be due to the fact that the nonlinear normalization applied to the raw data very strongly emphasizes the consensus site. conventional competitive emsa measurements on selected binding sequences with linear normalization showed several single substitutions with kd values within a factor of of the consensus, which are compatible with the results of the luciferase reporter activity found. this is also consistent with the existence of many single and several double substitutions in the gli consensus sequence of promoters with known gli dependent function in vivo, which failed to be retrieved in genome-wide in silico searches for gli target genes e.g. bcl il1r fst, tgm. though not perfectly representing the context of chromatin, luciferase reporter assays can be used to distinguish between potentially functional gli binding sites and apparent binding sites, which do not activate reporter gene activity within their sequence context. this can be demonstrated clearly for the tgm promoter, which contains three potential gli binding sites: one consensus sequence and two variants with a c to a substitution in position. mutation of the consensus sequence to nonbinding 6g7g does not affect reporter activity nor is the consensus site bound by gli in a chip experiment. in contrast, the variant sites are bound by gli and mutation of either variant site abolishes transcriptional activation. non-consensus gli binding sites in gli target gene promoters to further explore the influence of binding affinity on transcriptional activation in a natural promoter context other than ptch we chose the jun and gli promoters, both containing functional non-consensus gli binding sites, for further analysis the human jun and human proximal gli promoter both contain only one functional gli binding site thus eliminating possible interactions between nearby gli binding sites. either binding site variant has been shown to be essential for activation by gli and both have significantly lower affinity than the consensus site and gli, for gli, double substitutions as found in the jun promoter were not tested under identical conditions,. to compare the activity of the variants to the consensus, we applied site directed mutagenesis to change the wild type variant sites to the consensus sequence. the luciferase reporter constructs were then tested for the response to gli2act in hacat cells and luciferase activity was compared to the respective wild type promoter constructs. we detected no significant difference between gli consensus and non consensus wild type sequences in the context of either promoter. these results indicate that relatively low binding site affinity does not prevent activation by gli in a luciferase assay at optimal gli concentration. recent observations show that lower affinity binding sites for transcription factors can be identified by global chip and occur quite frequently. large scale affinity measurements as described in showed that a large selection of transcription factors recognises many variations of the primary motifs and that even secondary motifs exist, which may possibly affect changes in transcriptional specificity. a visible influence of low affinity sites on gene expression in yeast has been described pointing to their potential relevance for modulating gene expression. vokes et al identified a number of gli promoters/enhancers, which behave in a tissue specific way and are influenced by nuclear gli concentration. in a more global study, groups of sites with high and lower affinities to rest repressor were shown to cluster into groups responsible for activation of target genes expressed commonly, specifically or uniquely in different cell lines. these data imply an important role for lower affinity sites in the context dependent control of transcription and point to the need for more detailed investigation of their function.  the results presented here specifically focus on the activation potential of binding sites of the gli transcription factors, the mediators of the hedgehog signal. we measured activation in a standardised luciferase assay in the context of the ptch promoter testing all single site mutations of the gli consensus binding sequence. a rather large number of substitutions was shown to be active, which is consistent with the existence of many known gli target gene promoters containing variant sites with lower binding affinity. taking into account the contribution of a larger subset of binding sites with significant affinity the results presented in this study are likely to be helpful in the prediction and experimental validation of more direct gli target genes. 
variant gli binding sites with relatively low affinity can within natural promoters lead to strong transcriptional activation. we quantified transcriptional activation by gli using ptch promoter based luciferase reporters containing all single substitutions of the gli consensus binding site. the affinity of variant single substitutions in gli binding sites has been measured systematically, but the affinities of the variant binding sites appears low compared to the frequency of occurrence of variant sites in known gli target gene promoters. the gli transcription factors, mediators of the hedgehog signal bind with high affinity to the consensus sequence gaccaccca.
 the gli transcription factors, mediators of the hedgehog signal bind with high affinity to the consensus sequence gaccaccca. the affinity of variant single substitutions in gli binding sites has been measured systematically, but the affinities of the variant binding sites appears low compared to the frequency of occurrence of variant sites in known gli target gene promoters.  we quantified transcriptional activation by gli using ptch promoter based luciferase reporters containing all single substitutions of the gli consensus binding site. as expected variants with very low affinity did not activate the reporter. many lower affinity binding sequences are, however, functional in the presence of moderate gli concentration. using two natural non-consensus gli site promoters we showed that substitution of the variant sequences by consensus leads to comparable activity.  variant gli binding sites with relatively low affinity can within natural promoters lead to strong transcriptional activation. this may facilitate the identification of additional direct gli target genes. 