BACKGROUND
the binding of gene-specific transcription factors  to the tf binding sites  in the promoter region is required for the initiation of gene transcription.
in the last decade, extensive efforts have been made to identify tfbss in saccharomyces cerevisiae using both experimental and computational approaches  <cit> .
these data are useful for studying the spatial distribution of tfbss on yeast promoters.
this distribution may be used to address questions on gene regulation such as whether there exists a "preferred zone" in the promoter for tf binding and how strongly the position of a tfbs affects the transcriptional output of a gene.
in s. cerevisiae, several studies have revealed that tfbss are not uniformly distributed over the promoter region  <cit> .
for example, using computational tools to search for over-represented binding motifs in various groups of genes in s. cerevisiae, hughes et al.
<cit>  found that tfbss are highly enriched in the region approximately between  <dig> and  <dig> bp upstream of the translation start site.
similarly, harbison et al.
<cit>  found from chip-chip data that 74% of the tfbss are located between  <dig> and  <dig> bp upstream of the translation start site.
these studies have provided a rough spatial distribution of tfbss.
however, improvements can be made.
first, since the transcription of a gene into mrna begins at the transcription start site , it is biologically more relevant to study the tfbs distribution relative to the tss than to the translation start codon.
in this study we have used the tss as the reference point, taking advantage of the high-resolution tss data recently generated by rna-seq  <cit> .
second, in previous studies the distribution was inferred using the number of tfbss at each site of the promoter region, but the promoter length varies greatly among genes  <cit> .
for example, there are  <dig> sequences at position - <dig> bp  but only  <dig> sequences at position - <dig> bp .
clearly, the number of tfbss at a site should be normalized by the number of promoter sequences that cover that site.
two factors have been known to affect the distribution of tfbss in eukaryotes: nucleosome positioning and presence of tata-box  <cit> .
as the primary building block of eukaryotic chromatins, a nucleosome is formed by tightly wrapping ~ <dig> bp of dna around a histone octamer  <cit> .
the presence of nucleosomes is a major barrier for direct tf-tfbs interactions.
studies on nucleosome positioning have revealed that many yeast genes contain a nucleosome free region  in their promoters, which exposes tfbss to tfs  <cit> .
tfbss are mainly located in the nfr in nfr-containing promoters but are more evenly distributed in nfr-less promoters, suggesting a significant effect of nucleosome positioning on tfbs distribution  <cit> .
the tata box is usually located ~40- <dig> bp upstream of the tss in many yeast genes and facilitates in directing rna polymerase ii to the downstream tss  <cit> .
in s. cerevisiae, ~20% of the genes contain a tata-box in their promoter regions  <cit> .
the tata box-containing genes and tata box-less genes are known to use different transcription initiation pathways  <cit> .
it has been shown that promoters with a tata box have distinct structure features, such as higher nucleosome occupancy and higher dna bendability near the tss than do tata box-less promoters, which may affect the tfbs distribution  <cit> .
in this study, we re-evaluated the effects of nucleosome positioning and the presence/absence of tata-box on the distribution patterns of tfbs inferred by our improved methods.
in addition, we also studied whether there is a relationship between tfbs distribution and the length of the 5' untranslated region .
although the average length of 5'utrs is rather constant among diverse organisms , the length substantially varies among genes in the same genome  <cit> .
it is not known if this variation in 5'utr length has any relationship with the tfbs distribution and other promoter structural features, although it is well established that 5'utr plays important roles in post-transcriptional regulation, including modulation of the mrna subcellular localization, mrna stability, transport of mrnas out of the nucleus and translation efficiency  <cit> .
finally, we assessed the effects of the tfbs distribution on the output of gene transcription, which can be measured in two ways:  transcription abundance  under a given condition and  transcriptional plasticity, which is defined as the capacity for a gene to change its transcriptional level under different conditions.
RESULTS
a tfbs distribution peak at ~ <dig> bp upstream of the tss
we defined the promoter region of a gene as the intergenic region of the gene from its translation start codon to the coding boundary of the nearest upstream gene.
we used the tss as the reference point to infer the spatial distribution of tfbss by measuring the frequency of tfbss at each position in the promoter.
for comparison, we also obtained a distribution using the translation start codon as the reference point.
since the total number and content of tfbss strongly depend on the criteria for defining tfbss, we analyzed four different sets of tfbs data to ensure the robustness of our conclusions .
with the tss as the reference point, we found tfbss to be highly enriched from ~ <dig> to  <dig> bp upstream of the tss with a sharp peak at - <dig> bp; the four different tfbs datasets yield highly consistent overall patterns .
the frequencies of tfbss at the peak are significantly higher than the average randomized distribution of tfbss, indicating a strong positioning bias of tfbss relative to the tss .
in contrast, with the start codon as the reference point, only a plateau, but no sharp peak, is observed .
similar tfbs distributions were obtained when we excluded bidirectional promoters, each of which is a promoter region shared by two divergently transcribed adjacent genes , highlighting the robustness of our results.
to statistically evaluate if the tfbss have more biased distribution inferred using the tss as the reference point than does the distribution obtained using the translation start codon as the reference point, we calculated the localization bias of the peak which is defined as the ratio of the maximal value of the tfbs frequencies divided by the average value of the tfbs frequencies .
we found that localization bias of the peak inferred by the tss  is significant higher than that inferred by the start codon  supporting the existence of a preferred range of distances from tfbss to the tss.
to ensure the robustness of the above results, we also inferred the distribution of tfbss retrieved from swissregulon  <cit> , which contained  <dig> high confidence binding sites for  <dig> tfs with a posterior probability >  <dig>  .
similar to the above results, the tfbss are highly enriched in the region of 80- <dig> bp upstream of the tss, and the distribution of tfbss is less biased when the start codon is used as the reference .
based on  <dig> bootstraps, the localization bias of the peak of tfbs distribution inferred by the tss  is significant higher than that inferred by the start codon  .
therefore, our analysis appearsto be robust.
interestingly, the frequency of tfbss immediately downstream of the peak, including the 5'utr region, is significantly lower than random expectation and the frequency in any other intergenic regions and random expectation, revealing a tfbs-depleted region from - <dig> bp to the 5'utr .
this region roughly corresponds to the location of the core promoter  <cit> .
the core promoter contains dna elements that directly interact with the general transcription machinery and can extend  <dig> bp upstream or downstream of the tss  <cit> .
this tfbs-depleted region can be explained by the selection against potential spatial competition between tf binding and the general transcription complex.
the depletion of tfbs in the 5'utr region indicates that tfbss strongly prefer the location upstream to the region where the general transcription complex binds.
strong selective constraints on the distances between tss and tfbss
if the distance between a tfbs and the tss is important for the tf function, dna insertion and deletion events in this region should tend to be selectively disadvantageous.
to test this hypothesis, we estimated the frequency of deletion polymorphisms using the genome-wide polymorphism data of  <dig> s. cerevisiae strains from diverse ecological niches and geographic locations  <cit> .
for all promoters with an identified tss location, we computed the average occurrence of deletion events per sequence at each position relative to the tss .
figure 1c shows that the frequency of deletion polymorphisms monotonically decreases as the distance to the tss decreases.
in addition, the frequency of deletion polymorphisms in the region from ~- <dig> bp to tss is significantly lower than the random expectation.
fewer deletion polymorphisms in the tss proximal region suggest a lower level of tolerance of deletion mutations.
our data shows that most tfbss are located within the first  <dig> bp upstream of the tss and that the number of tfbss decreases as the distance to the tss increases.
therefore, the frequency of deletion polymorphisms is negatively correlated with tfbs occurrence, suggesting that the distance between a tfbs and the tss is under purifying selection.
in contrast, no significant variation in snp frequency is detected among different promoter regions , probably because the occurrence of snp does not change the tfbs-tss distance.
effects of nucleosome occupancy and presence/absence of tata-box on the tfbs distribution
we calculated the average nucleosome occupancy in the  <dig> bp region surrounding the tss for all yeast genes using the recently released nucleosome occupancy data by kaplan et al.
<cit>  .
the genome average nucleosome occupancy reveals a nfr in the ~ <dig> bp region immediately upstream of the tss , which is consistent with a previous study based on different nucleosome data  <cit> .
as shown in figure 2a, there is a negative correlation between tfbs density and nucleosome occupancy.
the peak of the tfbs distribution matches the valley of nucleosome occupancy, indicating that tfbss are predominantly enriched in the nfr.
the negative correlation between the tfbs distribution and nucleosome occupancy has been observed on individual tfs, such as abf <dig>  reb <dig>  mbp <dig> and rsc <dig>  <cit> .
the consistency between the genome-wide scale tfbs distribution and the distribution for individual genes suggests that the presence of nfr is an important factor for the uneven spatial distribution of tfbss in s. cerevisiae.
to further investigate how low nucleosome occupancy surrounding the tss affects the distribution of tfbss, we used the k-means clustering algorithm to classify all genes into four clusters based on their nucleosome positioning patterns in the promoter region  <cit>  .
the four clusters  contain  <dig> ,  <dig>   <dig> and  <dig>  promoters, respectively .
type i promoters have the most conspicuous  <dig> bp nfr with well defined boundaries, whereas type iv promoters are mostly occupied by nucleosomes.
similar to the type i promoters, type ii and iii promoters also contain a nucleosome-depleted region upstream of the tss, but their nfrs lack a well positioned upstream nucleosome .
interestingly, these four types of promoters display distinct tfbs distribution patterns .
type i promoters have the most uneven tfbs distribution among all groups, with a very sharp peak at ~ <dig> bp upstream of the tss; whereas type iv promoters show no sharp peak and are broadly distributed between - <dig> and - <dig> bp .
the presence of a nfr close to the tss results in the enrichment of tfbss within the nfr.
although the tfbss of type iv promoters are relatively broadly distributed in the promoter region, the frequencies of tfbss are significantly higher than in other types of promoters at most positions.
on average, more tfbss are present in a type iv promoter than a promoter with a nfr, suggesting a more sophisticated regulation for genes with type iv promoters.
tirosh et al.
<cit>  identified two extreme classes of promoters based on nucleosome occupancy: depleted proximal-nucleosome  and occupied proximal-nucleosome .
they showed that tfbss are strongly enriched in the nfr of dpn promoters, but are more evenly distributed throughout opn promoters.
dpn and opn genes are similar to the type i and iv genes, respectively, regarding the nucleosome occupancy pattern and the tfbs distribution.
however, our normalized distribution of tfbss on the type iv promoters provides extra information: tfbss of type iv promoters are not only more uniformly distributed, but with a significant higher density of tfbss compared to type i promoters.
therefore, our results suggest that although the presence of a nucleosome may reduce the accessibility of tfbss for the tfs, it does not necessarily reduce the occurrence of tfbss.
indeed, more tfbss are found in nucleosome-occupied promoters, suggesting that genes with nucleosome-occupied promoters tend to be regulated by more tfs.
we compared the tfbs distributions between  <dig>  tata box-containing and  <dig>  tata box-less genes.
as shown in figure 2d, the tfbss in tata box-less genes show a sharp peak upstream of the tss, whereas a high plateau instead of a clear peak of the tfbs distribution is observed in tata box-containing genes.
in addition, higher frequencies of tfbss are observed in tata box-containing genes than in tata box-less genes at all positions, indicating that tata box-containing promoters tend to contain more tfbss.
therefore, the tata box-containing genes have a similar tfbs distribution pattern to type iv genes, suggesting that these genes may be under more intricate regulation.
association of different 5'utr lengths with distinct tfbs distributions and nucleosome occupancy patterns
the observation of a higher variation in 5'utr length within species than between species motivated us to investigate its relationship with promoter structures.
to study this question, we divided all s. cerevisiae genes into three groups according to the lengths of 5'utr: short , medium  and long .
distinct tfbs distributions are observed among the three groups.
as shown in figure 2e, the long 5'utr genes have the highest frequency of tfbss and most broadly distributed tfbss in the promoter region.
especially, long 5'utr genes contain a significant higher frequency of tfbss in the 5'utr region than short 5'utr genes .
in contrast, the tfbss of short 5'utr genes are enriched in a short region of 80- <dig> bp upstream of the tss, showing a negative correlation between 5'utr length and uneven distribution of tfbss.
we repeated these analyses by dividing all genes in to four groups of 5'utr length and observed a similar trend.
to obtain further information about the relationship between 5'utr length and the distribution of tfbss, we studied the correlations of 5'utr length with nucleosome positioning and the presence/absence of tata box.
first, we found a significant positive correlation between 5'utr length and the nucleosome occupancy level near the tss .
specifically, the average 5' utr length of type iv genes is nearly as twice long as that of type i genes , while the type ii and iii genes have intermediate 5' utr lengths .
second, long 5'utr genes tend to have much higher nucleosome occupancy near the tss compared with short 5'utr genes .
in addition, we also observed a positive correlation between the 5' utr length and presence of tata-box .
tata box-containing genes are enriched in long 5'utr genes , and tata box-less genes tend to have short 5'utrs  .
therefore, tata box-containing genes have, on average, longer 5'utrs than tata box-less genes  .
we speculate that promoter structural features, such as nucleosome occupancy and presence/absence of tata box, affect the 5'utr length, because they play a more important role in transcription regulation than does the 5'utr length.
however, we cannot rule out the possibility that the reverse is true.
effects of promoter features on gene transcription profiles
to test if the tfbs distribution pattern affects gene transcriptional regulation, we infer the tfbs distribution patterns for genes with different transcriptional plasticity values and transcriptional abundances .
we calculated the transcriptional plasticity value for each gene from over  <dig> profiles of microarray data  and ranked all genes by their transcriptional plasticity values.
the tfbs distribution patterns were computed for  <dig> genes with the highest plasticity and for  <dig> genes with the lowest plasticity, respectively.
as shown in figure 4a, the tfbss of low plasticity genes form a sharp peak around  <dig> bp upstream of the tss, showing a highly uneven tfbs distribution.
in contrast, the tfbss of high plasticity genes are broadly distributed in a  <dig> bp region, from - <dig> to - <dig> bp upstream of the tss.
more importantly, the density of tfbss in this region is even higher for high plasticity genes.
this result suggests that the large number of tfbss in the promoter is an important factor for the plasticity of gene expression.
we also studied the tfbs distribution patterns for genes with different expression levels under normal growth conditions.
nagalakshmi et al.
<cit>  used rna-seq data to divide yeast genes into three expression groups: high, medium and low.
we found that the tfbss of the three groups are not very different with regard to the distribution shape in the promoter region .
however, significant differences can be seen in terms of the frequency of tfbss.
indeed, the overall density of tfbss is positively correlated with the expression abundance, suggesting that genes with more tfbss in their promoter regions tend to have a higher expression level.
discussion
our study reveals that tfbss are highly enriched in a narrow region ~ <dig> bp upstream of the tss, whereas no clear distribution peak is observed when the translation start codon is used as the reference point .
this result differs from the observation of previous studies  <cit> .
the difference arose because different methods were used to infer the distribution of tfbss.
as mentioned above, previous studies did not normalize the number of tfbss observed at a position by the number of promoters that covered that position.
one can easily imagine that the proximal regions of the translation start site have the largest number of sequences, whereas more upstream regions have fewer sequences.
our revised distribution of tfbss suggests that the distance between tfbss and tss is important for transcriptional regulation.
the distribution pattern of tfbss can be valuable for binding motif prediction because candidate tfbss at different locations should be given different weights.
when genome-wide tss and tfbs data becomes available for other closely related species of s. cerevisiae, it will be interesting to see whether the spatial distribution of tfbss have been conserved in evolution.
our observation that both nucleosome positioning and presence/absence of tata box affect the distribution of tfbss is consistent with previous studies  <cit> .
the dna sequence in the nfr is exposed and therefore accessible for tfs, because no chromatin remodelling and no dna unwrapping are required for tf-tfbs interaction, which is an advantageous structure for constantly transcribed genes.
therefore, it is believed that there are more tfbss in the nfr than in a nucleosome occupied region  <cit> .
this is true in general, but the nucleosome occupancy pattern in the promoter region does not strongly affect the total number of tfbss in the promoter.
in fact, the tfbs density for nucleosome-occupied promoters  tends to be even higher than the peak of nfr-containing promoters .
more tfbss are also observed in the tata-box containing genes .
it has been shown that the genes with nucleosome occupied promoters and tata-box containing genes have higher expression variability under different conditions, compared to the other types of genes  <cit> .
our gene ontology analysis reveals that type iv genes are significantly enriched in oxidation reduction and response to various stimuli , similar to the tata box-containing genes  <cit> .
stress-response genes need to be finely regulated, and so their expression level can be dramatically changed under different conditions.
in addition, genes with high expression plasticity also contain more tfbss in the promoter region .
therefore, the presence of more tfbss in the promoters of these genes provides higher flexibility for different tfbs combinations under different conditions.
CONCLUSIONS
our study showed that the spatial distribution of tfbss has a sharp peak at  <dig> bp upstream of the tss, which is inside the nucleosome-depleted region.
in contrast, no clear peak of the tfbs distribution was observed using the translation start codon as the reference point.
the frequency of deletion polymorphisms monotonically decreases as the distance to tss decreases, while no significant variation in nucleotide polymorphism frequency was observed along the promoter region, suggesting that the distance between tfbss and the tss is functionally constrained.
our study further indicated that the tfbs distribution pattern is affected by nucleosome occupancy and presence of tata-box and that the distribution pattern affects the transcription level and transcription plasticity of the gene.

 the binding of gene-specific transcription factors to the tf binding sites in the promoter region is required for the initiation of gene transcription. in the last decade, extensive efforts have been made to identify tfbss in saccharomyces cerevisiae using both experimental and computational approaches. these data are useful for studying the spatial distribution of tfbss on yeast promoters. this distribution may be used to address questions on gene regulation such as whether there exists a "preferred zone" in the promoter for tf binding and how strongly the position of a tfbs affects the transcriptional output of a gene. in s. cerevisiae, several studies have revealed that tfbss are not uniformly distributed over the promoter region. for example, using computational tools to search for over-represented binding motifs in various groups of genes in s. cerevisiae, hughes et al. found that tfbss are highly enriched in the region approximately between and bp upstream of the translation start site. similarly, harbison et al. found from chip-chip data that 74% of the tfbss are located between and bp upstream of the translation start site. these studies have provided a rough spatial distribution of tfbss. however, improvements can be made. first, since the transcription of a gene into mrna begins at the transcription start site, it is biologically more relevant to study the tfbs distribution relative to the tss than to the translation start codon. in this study we have used the tss as the reference point, taking advantage of the high-resolution tss data recently generated by rna-seq. second, in previous studies the distribution was inferred using the number of tfbss at each site of the promoter region, but the promoter length varies greatly among genes. for example, there are sequences at position - bp but only sequences at position - bp. clearly, the number of tfbss at a site should be normalized by the number of promoter sequences that cover that site. two factors have been known to affect the distribution of tfbss in eukaryotes: nucleosome positioning and presence of tata-box. as the primary building block of eukaryotic chromatins, a nucleosome is formed by tightly wrapping ~ bp of dna around a histone octamer. the presence of nucleosomes is a major barrier for direct tf-tfbs interactions. studies on nucleosome positioning have revealed that many yeast genes contain a nucleosome free region in their promoters, which exposes tfbss to tfs. tfbss are mainly located in the nfr in nfr-containing promoters but are more evenly distributed in nfr-less promoters, suggesting a significant effect of nucleosome positioning on tfbs distribution. the tata box is usually located ~40- bp upstream of the tss in many yeast genes and facilitates in directing rna polymerase ii to the downstream tss. in s. cerevisiae, ~20% of the genes contain a tata-box in their promoter regions. the tata box-containing genes and tata box-less genes are known to use different transcription initiation pathways. it has been shown that promoters with a tata box have distinct structure features, such as higher nucleosome occupancy and higher dna bendability near the tss than do tata box-less promoters, which may affect the tfbs distribution. in this study, we re-evaluated the effects of nucleosome positioning and the presence/absence of tata-box on the distribution patterns of tfbs inferred by our improved methods. in addition, we also studied whether there is a relationship between tfbs distribution and the length of the 5' untranslated region. although the average length of 5'utrs is rather constant among diverse organisms, the length substantially varies among genes in the same genome. it is not known if this variation in 5'utr length has any relationship with the tfbs distribution and other promoter structural features, although it is well established that 5'utr plays important roles in post-transcriptional regulation, including modulation of the mrna subcellular localization, mrna stability, transport of mrnas out of the nucleus and translation efficiency. finally, we assessed the effects of the tfbs distribution on the output of gene transcription, which can be measured in two ways: transcription abundance under a given condition and transcriptional plasticity, which is defined as the capacity for a gene to change its transcriptional level under different conditions.  a tfbs distribution peak at ~ bp upstream of the tss we defined the promoter region of a gene as the intergenic region of the gene from its translation start codon to the coding boundary of the nearest upstream gene. we used the tss as the reference point to infer the spatial distribution of tfbss by measuring the frequency of tfbss at each position in the promoter. for comparison, we also obtained a distribution using the translation start codon as the reference point. since the total number and content of tfbss strongly depend on the criteria for defining tfbss, we analyzed four different sets of tfbs data to ensure the robustness of our conclusions. with the tss as the reference point, we found tfbss to be highly enriched from ~ to bp upstream of the tss with a sharp peak at - bp; the four different tfbs datasets yield highly consistent overall patterns. the frequencies of tfbss at the peak are significantly higher than the average randomized distribution of tfbss, indicating a strong positioning bias of tfbss relative to the tss. in contrast, with the start codon as the reference point, only a plateau, but no sharp peak, is observed. similar tfbs distributions were obtained when we excluded bidirectional promoters, each of which is a promoter region shared by two divergently transcribed adjacent genes, highlighting the robustness of our results. to statistically evaluate if the tfbss have more biased distribution inferred using the tss as the reference point than does the distribution obtained using the translation start codon as the reference point, we calculated the localization bias of the peak which is defined as the ratio of the maximal value of the tfbs frequencies divided by the average value of the tfbs frequencies. we found that localization bias of the peak inferred by the tss is significant higher than that inferred by the start codon supporting the existence of a preferred range of distances from tfbss to the tss. to ensure the robustness of the above results, we also inferred the distribution of tfbss retrieved from swissregulon, which contained high confidence binding sites for tfs with a posterior probability >. similar to the above results, the tfbss are highly enriched in the region of 80- bp upstream of the tss, and the distribution of tfbss is less biased when the start codon is used as the reference. based on bootstraps, the localization bias of the peak of tfbs distribution inferred by the tss is significant higher than that inferred by the start codon. therefore, our analysis appearsto be robust. interestingly, the frequency of tfbss immediately downstream of the peak, including the 5'utr region, is significantly lower than random expectation and the frequency in any other intergenic regions and random expectation, revealing a tfbs-depleted region from - bp to the 5'utr. this region roughly corresponds to the location of the core promoter. the core promoter contains dna elements that directly interact with the general transcription machinery and can extend bp upstream or downstream of the tss. this tfbs-depleted region can be explained by the selection against potential spatial competition between tf binding and the general transcription complex. the depletion of tfbs in the 5'utr region indicates that tfbss strongly prefer the location upstream to the region where the general transcription complex binds. strong selective constraints on the distances between tss and tfbss if the distance between a tfbs and the tss is important for the tf function, dna insertion and deletion events in this region should tend to be selectively disadvantageous. to test this hypothesis, we estimated the frequency of deletion polymorphisms using the genome-wide polymorphism data of s. cerevisiae strains from diverse ecological niches and geographic locations. for all promoters with an identified tss location, we computed the average occurrence of deletion events per sequence at each position relative to the tss. figure 1c shows that the frequency of deletion polymorphisms monotonically decreases as the distance to the tss decreases. in addition, the frequency of deletion polymorphisms in the region from ~- bp to tss is significantly lower than the random expectation. fewer deletion polymorphisms in the tss proximal region suggest a lower level of tolerance of deletion mutations. our data shows that most tfbss are located within the first bp upstream of the tss and that the number of tfbss decreases as the distance to the tss increases. therefore, the frequency of deletion polymorphisms is negatively correlated with tfbs occurrence, suggesting that the distance between a tfbs and the tss is under purifying selection. in contrast, no significant variation in snp frequency is detected among different promoter regions, probably because the occurrence of snp does not change the tfbs-tss distance. effects of nucleosome occupancy and presence/absence of tata-box on the tfbs distribution we calculated the average nucleosome occupancy in the bp region surrounding the tss for all yeast genes using the recently released nucleosome occupancy data by kaplan et al.. the genome average nucleosome occupancy reveals a nfr in the ~ bp region immediately upstream of the tss, which is consistent with a previous study based on different nucleosome data. as shown in figure 2a, there is a negative correlation between tfbs density and nucleosome occupancy. the peak of the tfbs distribution matches the valley of nucleosome occupancy, indicating that tfbss are predominantly enriched in the nfr. the negative correlation between the tfbs distribution and nucleosome occupancy has been observed on individual tfs, such as abf reb mbp and rsc. the consistency between the genome-wide scale tfbs distribution and the distribution for individual genes suggests that the presence of nfr is an important factor for the uneven spatial distribution of tfbss in s. cerevisiae. to further investigate how low nucleosome occupancy surrounding the tss affects the distribution of tfbss, we used the k-means clustering algorithm to classify all genes into four clusters based on their nucleosome positioning patterns in the promoter region. the four clusters contain, and promoters, respectively. type i promoters have the most conspicuous bp nfr with well defined boundaries, whereas type iv promoters are mostly occupied by nucleosomes. similar to the type i promoters, type ii and iii promoters also contain a nucleosome-depleted region upstream of the tss, but their nfrs lack a well positioned upstream nucleosome. interestingly, these four types of promoters display distinct tfbs distribution patterns. type i promoters have the most uneven tfbs distribution among all groups, with a very sharp peak at ~ bp upstream of the tss; whereas type iv promoters show no sharp peak and are broadly distributed between - and - bp. the presence of a nfr close to the tss results in the enrichment of tfbss within the nfr. although the tfbss of type iv promoters are relatively broadly distributed in the promoter region, the frequencies of tfbss are significantly higher than in other types of promoters at most positions. on average, more tfbss are present in a type iv promoter than a promoter with a nfr, suggesting a more sophisticated regulation for genes with type iv promoters. tirosh et al. identified two extreme classes of promoters based on nucleosome occupancy: depleted proximal-nucleosome and occupied proximal-nucleosome. they showed that tfbss are strongly enriched in the nfr of dpn promoters, but are more evenly distributed throughout opn promoters. dpn and opn genes are similar to the type i and iv genes, respectively, regarding the nucleosome occupancy pattern and the tfbs distribution. however, our normalized distribution of tfbss on the type iv promoters provides extra information: tfbss of type iv promoters are not only more uniformly distributed, but with a significant higher density of tfbss compared to type i promoters. therefore, our results suggest that although the presence of a nucleosome may reduce the accessibility of tfbss for the tfs, it does not necessarily reduce the occurrence of tfbss. indeed, more tfbss are found in nucleosome-occupied promoters, suggesting that genes with nucleosome-occupied promoters tend to be regulated by more tfs. we compared the tfbs distributions between tata box-containing and tata box-less genes. as shown in figure 2d, the tfbss in tata box-less genes show a sharp peak upstream of the tss, whereas a high plateau instead of a clear peak of the tfbs distribution is observed in tata box-containing genes. in addition, higher frequencies of tfbss are observed in tata box-containing genes than in tata box-less genes at all positions, indicating that tata box-containing promoters tend to contain more tfbss. therefore, the tata box-containing genes have a similar tfbs distribution pattern to type iv genes, suggesting that these genes may be under more intricate regulation. association of different 5'utr lengths with distinct tfbs distributions and nucleosome occupancy patterns the observation of a higher variation in 5'utr length within species than between species motivated us to investigate its relationship with promoter structures. to study this question, we divided all s. cerevisiae genes into three groups according to the lengths of 5'utr: short, medium and long. distinct tfbs distributions are observed among the three groups. as shown in figure 2e, the long 5'utr genes have the highest frequency of tfbss and most broadly distributed tfbss in the promoter region. especially, long 5'utr genes contain a significant higher frequency of tfbss in the 5'utr region than short 5'utr genes. in contrast, the tfbss of short 5'utr genes are enriched in a short region of 80- bp upstream of the tss, showing a negative correlation between 5'utr length and uneven distribution of tfbss. we repeated these analyses by dividing all genes in to four groups of 5'utr length and observed a similar trend. to obtain further information about the relationship between 5'utr length and the distribution of tfbss, we studied the correlations of 5'utr length with nucleosome positioning and the presence/absence of tata box. first, we found a significant positive correlation between 5'utr length and the nucleosome occupancy level near the tss. specifically, the average 5' utr length of type iv genes is nearly as twice long as that of type i genes, while the type ii and iii genes have intermediate 5' utr lengths. second, long 5'utr genes tend to have much higher nucleosome occupancy near the tss compared with short 5'utr genes. in addition, we also observed a positive correlation between the 5' utr length and presence of tata-box. tata box-containing genes are enriched in long 5'utr genes, and tata box-less genes tend to have short 5'utrs. therefore, tata box-containing genes have, on average, longer 5'utrs than tata box-less genes. we speculate that promoter structural features, such as nucleosome occupancy and presence/absence of tata box, affect the 5'utr length, because they play a more important role in transcription regulation than does the 5'utr length. however, we cannot rule out the possibility that the reverse is true. effects of promoter features on gene transcription profiles to test if the tfbs distribution pattern affects gene transcriptional regulation, we infer the tfbs distribution patterns for genes with different transcriptional plasticity values and transcriptional abundances. we calculated the transcriptional plasticity value for each gene from over profiles of microarray data and ranked all genes by their transcriptional plasticity values. the tfbs distribution patterns were computed for genes with the highest plasticity and for genes with the lowest plasticity, respectively. as shown in figure 4a, the tfbss of low plasticity genes form a sharp peak around bp upstream of the tss, showing a highly uneven tfbs distribution. in contrast, the tfbss of high plasticity genes are broadly distributed in a bp region, from - to - bp upstream of the tss. more importantly, the density of tfbss in this region is even higher for high plasticity genes. this result suggests that the large number of tfbss in the promoter is an important factor for the plasticity of gene expression. we also studied the tfbs distribution patterns for genes with different expression levels under normal growth conditions. nagalakshmi et al. used rna-seq data to divide yeast genes into three expression groups: high, medium and low. we found that the tfbss of the three groups are not very different with regard to the distribution shape in the promoter region. however, significant differences can be seen in terms of the frequency of tfbss. indeed, the overall density of tfbss is positively correlated with the expression abundance, suggesting that genes with more tfbss in their promoter regions tend to have a higher expression level. discussion our study reveals that tfbss are highly enriched in a narrow region ~ bp upstream of the tss, whereas no clear distribution peak is observed when the translation start codon is used as the reference point. this result differs from the observation of previous studies. the difference arose because different methods were used to infer the distribution of tfbss. as mentioned above, previous studies did not normalize the number of tfbss observed at a position by the number of promoters that covered that position. one can easily imagine that the proximal regions of the translation start site have the largest number of sequences, whereas more upstream regions have fewer sequences. our revised distribution of tfbss suggests that the distance between tfbss and tss is important for transcriptional regulation. the distribution pattern of tfbss can be valuable for binding motif prediction because candidate tfbss at different locations should be given different weights. when genome-wide tss and tfbs data becomes available for other closely related species of s. cerevisiae, it will be interesting to see whether the spatial distribution of tfbss have been conserved in evolution. our observation that both nucleosome positioning and presence/absence of tata box affect the distribution of tfbss is consistent with previous studies. the dna sequence in the nfr is exposed and therefore accessible for tfs, because no chromatin remodelling and no dna unwrapping are required for tf-tfbs interaction, which is an advantageous structure for constantly transcribed genes. therefore, it is believed that there are more tfbss in the nfr than in a nucleosome occupied region. this is true in general, but the nucleosome occupancy pattern in the promoter region does not strongly affect the total number of tfbss in the promoter. in fact, the tfbs density for nucleosome-occupied promoters tends to be even higher than the peak of nfr-containing promoters. more tfbss are also observed in the tata-box containing genes. it has been shown that the genes with nucleosome occupied promoters and tata-box containing genes have higher expression variability under different conditions, compared to the other types of genes. our gene ontology analysis reveals that type iv genes are significantly enriched in oxidation reduction and response to various stimuli, similar to the tata box-containing genes. stress-response genes need to be finely regulated, and so their expression level can be dramatically changed under different conditions. in addition, genes with high expression plasticity also contain more tfbss in the promoter region. therefore, the presence of more tfbss in the promoters of these genes provides higher flexibility for different tfbs combinations under different conditions.  our study showed that the spatial distribution of tfbss has a sharp peak at bp upstream of the tss, which is inside the nucleosome-depleted region. in contrast, no clear peak of the tfbs distribution was observed using the translation start codon as the reference point. the frequency of deletion polymorphisms monotonically decreases as the distance to tss decreases, while no significant variation in nucleotide polymorphism frequency was observed along the promoter region, suggesting that the distance between tfbss and the tss is functionally constrained. our study further indicated that the tfbs distribution pattern is affected by nucleosome occupancy and presence of tata-box and that the distribution pattern affects the transcription level and transcription plasticity of the gene. 
our analysis of sequence variation data among yeast strains revealed very low deletion polymorphisms in the region between the distribution peak and the tss, suggesting that the distances between tfbss and the tss have been selectively constrained in evolution. as in previous studies, we found that the nucleosome occupancy and the presence/absence of tata-box in the promoter region affect the tfbs distribution pattern. the tfbs distribution pattern is affected by nucleosome occupancy and presence of tata-box and it affects the transcription level and transcription plasticity of the gene. we found a sharp peak for the distribution of tfbss at ~ bp upstream of the tss, but no clear peak when the translation start codon was used as the reference point. previous studies used the translation start codon as the reference point to infer the tfbs distribution. in addition, we found that there exists a correlation between the 5'utr length and the tfbs distribution pattern and we showed that the tfbs distribution pattern affects gene transcription level and plasticity. how the transcription factor binding sites are distributed in the promoter region have implications for gene regulation. in this study, we reexamined the spatial distribution of tfbss, investigated various promoter features that may affect the distribution, and studied the effect of tfbs distribution on transcriptional regulation. the spatial distribution of tfbss obtained using the tss as the reference point shows a much sharper peak than does the distribution obtained using the translation start codon as the reference point. however, it is biologically more relevant to use the transcription start site as the reference point.
 how the transcription factor binding sites are distributed in the promoter region have implications for gene regulation. previous studies used the translation start codon as the reference point to infer the tfbs distribution. however, it is biologically more relevant to use the transcription start site as the reference point. in this study, we reexamined the spatial distribution of tfbss, investigated various promoter features that may affect the distribution, and studied the effect of tfbs distribution on transcriptional regulation.  we found a sharp peak for the distribution of tfbss at ~ bp upstream of the tss, but no clear peak when the translation start codon was used as the reference point. our analysis of sequence variation data among yeast strains revealed very low deletion polymorphisms in the region between the distribution peak and the tss, suggesting that the distances between tfbss and the tss have been selectively constrained in evolution. as in previous studies, we found that the nucleosome occupancy and the presence/absence of tata-box in the promoter region affect the tfbs distribution pattern. in addition, we found that there exists a correlation between the 5'utr length and the tfbs distribution pattern and we showed that the tfbs distribution pattern affects gene transcription level and plasticity.  the spatial distribution of tfbss obtained using the tss as the reference point shows a much sharper peak than does the distribution obtained using the translation start codon as the reference point. the tfbs distribution pattern is affected by nucleosome occupancy and presence of tata-box and it affects the transcription level and transcription plasticity of the gene. 