BACKGROUND
interferon-gamma  is a potent pro-inflammatory cytokine that regulates a spectrum of biological processes, and is produced primarily in response to infection  <cit> .
ifn-γ signal transduction begins at the cell surface with the formation of a heteromeric protein complex that includes ifn-γ, ifn-γ receptor- <dig>  and ifn-γ receptor- <dig>  <cit> .
associated with the ifn-γ receptors are members of the janus kinase  family of tyrosine kinases, which become activated upon formation of the ifn-γ receptor complex, and in turn phosphorylate the signal transducer and activator of transcription- <dig>  transcription factor  <cit> .
upon its phosphorylation, stat <dig> homo-dimerizes, and is transported into the nucleus where it binds to the gamma activated sequence  to activate the expression of ifn-γ-responsive genes  <cit> .
one example of a stat1-responsive gene is the interferon regulatory factor- <dig>  gene, an important ifn-γ-responsive transcription factor which contains a functional gas  <dig> bp upstream of its first exon  <cit> .
in addition, stat <dig> functions as a component of the ifn stimulated gene factor  <dig>  transcription factor complex, which also includes stat <dig> and interferon regulatory factor- <dig>   <cit> .
the isgf <dig> complex is formed in response to signaling by ifn family members, including ifn-α, that associate with ifn-α receptor- <dig> and ifn-α receptor- <dig>  upon its transportation into the nucleus, isgf <dig> binds to ifn-stimulated response elements  to activate the expression of ifn-α-responsive genes.
how gene expression is regulated by the association of transcription factors to their target sequences is a central question in mammalian biology.
compared with lower organisms such as s. cerevisiae and d. melanogaster, the genomic regions responsible for regulating the expression of mammalian genes are much more difficult to predict, and may be located far away from a gene's transcriptional start site .
furthermore, the debate over what constitutes a gene was further intensified with the development of tiling arrays for the human genome, and the discovery that much of the human transcriptome is coded for by regions of the genome that lie outside of exons as they have been classically defined  <cit> .
chromatin immunoprecipitation microarray  technology provided additional insight into the regulation of the human transcriptome when it was employed to examine the transcription factor binding sites  of sp <dig>  cmyc, and p <dig> on chromosomes  <dig> and  <dig>  <cit> .
interestingly, only 22% of these tfbss were located in the upstream regions of genes, the regions that have classically been defined as "promoter" regions.
the genomic relationship between sites of transcription factor binding, and sites of transcription, was further elucidated in an elegant study of estrogen receptor  binding across chromosomes  <dig> and  <dig>  <cit> .
remarkably, the rna polymerase ii  complex was found to associate in an estrogen-dependent manner with the majority of tested tfbss, even those located far from the nearest tss.
using a chromatin capture assay, an er binding site located over  <dig> kbp from the nrip- <dig> gene was shown to function as an enhancer of nrip- <dig> transcription.
these results showed that, when bound to its tfbs, the er can act as an enhancer to regulate the expression of target genes by associating, often across large chromosomal distances, with rnapolii.
recently, the response to ifn-γ has been the subject of microarray expression analyses as well as chip-chip analyses for stat <dig>  <cit> .
hartman et al.
compared the locations of stat <dig> binding sites on chromosome  <dig>  with the expression level of the nearest gene, and noted that only 21% of stat <dig> binding sites were within  <dig> kbp of the start of the nearest gene.
hartman et al.
also suggested that a novel mechanism may alter the specificity of stat <dig> binding, depending on whether it is induced by ifn-α or ifn-γ.
elucidating the rationale with which transcription factor binding sites are deployed across the genome will likely be of great benefit to our understanding of the complexities of transcriptional regulation in mammals.
we have examined the data associated with several recent tiling array experiments, and show here that stat binding and mrna transcription in response to ifn are much more closely associated than previous reports would suggest.
our results indicate that, in contrast to other mammalian transcription factors such as the er, stat <dig> binds closely to the tsss of its target genes.
our findings also suggest that stat <dig> may play an important role in recruiting the rnapolii complex to the promoters of ifn-γ-inducible genes.
RESULTS
stat <dig> binding and mrna transcription in response to ifn-γ are correlated at the probe level for chromosome  <dig> tiling arrays
to examine the relationship between stat <dig> binding and mrna transcription in response to ifn-γ, we obtained the data associated with hartman et al.
's tiling array analysis of gene transcription and stat <dig> binding for chromosome  <dig> following treatment with ifn-γ and ifn-α  <cit> .
we were particularly interested in determining whether there was any evidence for localized coupling of differential stat <dig> binding and differential mrna expression.
to our surprise, there was a pronounced probe level correlation between differential stat <dig> binding in response to ifn-γ, and differential mrna expression in response to ifn-γ .
a similar probe level relationship between stat <dig> binding, stat <dig> binding, and differential expression, was also apparent for the responses of cells to ifn-α .
furthermore, probes that detected differential stat <dig> binding in response to ifn-α also detected substantial differential expression in response to ifn-γ , and consistent with this, there was a high probe level correlation between the binding of stat <dig> or stat <dig> in response to ifn-α, and the binding of stat <dig> in response to ifn-γ .
consistent with hartman et al.
's conclusion that the binding preference of stat <dig> may differ in response to ifn-α versus ifn-γ  <cit> , some differences in the binding sites of stat <dig> were apparent for these stimuli , although this was primarily true only of some of the relatively weak stat <dig> binding sites.
for relatively strong stat <dig> binding sites, the binding preferences in response to ifn-γ versus ifn-α showed substantial correlation .
the findings outlined above strongly suggest that there is a high degree of overlap between the positions where stat <dig> and stat <dig> bind in response to either ifn-γ or ifn-α, which is perhaps not surprising, since stat <dig> binds dna as part of the isgf <dig> transcription factor complex, of which stat <dig> is also a component  <cit> .
consistent with this, correlation was also apparent between the transcriptional response of cells to ifn-γ and the transcriptional response of cells to ifn-α .
of greater interest, however, is the clear probe level correlation between the binding of stat <dig> or stat <dig>  and the differential expression in response to ifn-γ or ifn-α.
given that probes for the chromosome  <dig> arrays are  <dig> –  <dig> bp long  <cit> , our observations indicate that the locations at which stat <dig> or stat <dig> associate with chromosome  <dig> are in the majority of cases very close to regions of chromosome  <dig> that are transcriptionally regulated by ifn-γ or ifn-α.
detection of stat <dig> binding sites within the encode region
to examine stat <dig> binding in response to ifn-γ at a higher resolution than is permitted by cdna microarray technology, we obtained data associated with a recent chip-chip analysis of stat <dig> binding in response to ifn-γ  <cit>  across the  <dig> mb encyclopedia of dna elements  region  <cit> .
a moderately conservative differential intensity cut-off of  <dig>   was selected by inspection of a quantile-quantile plot of the differential intensities .
in total,  <dig> probes satisfied this cut-off.
these probes all clustered to  <dig> distinct genomic sites,  <dig>  of which were within  <dig> kbp of the nearest 1st exon of a gene .
this method appears to identify fewer stat <dig> binding sites than are identified on the ucsc genome browser  <cit> , presumably because our significance cut-off is higher.
when the alternative transcripts of three genes, atp11a, mcf2l, and rab11-fip <dig>  were accounted for,  <dig> of the  <dig>  of the stat <dig> binding sites we identified were within  <dig> kbp of the nearest 1st exon of a gene .
of the  <dig> genes identified in this manner, three  are known ifn-γ-responsive genes  <cit> .
in the case of irf- <dig>  a stat <dig> binding site immediately upstream of the first exon has been previously identified by in vivo footprinting  <cit> .
the majority of stat <dig> binding sites exhibit localized suppression of rnapolii binding in non-stimulated cells
our finding that ifn-γ-induced stat <dig> binding to chromosome  <dig> is closely associated with increased mrna transcription suggested that stat <dig> may bind proximally to rnapolii within the promoters of ifn-γ-responsive genes.
we were therefore interested in examining the binding of rnapolii to regions near stat <dig> binding sites.
to this end, we obtained data associated with bieda et al.
's recent analysis of rnapolii binding within the encode region for non-stimulated helas <dig> cells  <cit> .
remarkably, the binding of rnapolii in non-stimulated cells was negatively correlated  with the binding of stat <dig> in response to ifn-γ for  <dig> of the  <dig> binding sites within the encode region .
this is indicative of a diminished level of rnapolii binding, relative to the average level of rnapolii binding for the entire encode region.
localized suppression of rnapolii binding is likely to reflect the locations at which modifications in the chromatin state, or possibly other factors, prevent the association of rnapolii .
rnapolii binds to stat <dig> binding sites near the irf <dig> and ifnar <dig> genes in non-stimulated cells
of the five stat <dig> binding sites that did not exhibit suppressed rnapolii binding in non-stimulated cells , three were located within  <dig> kbp upstream of the irf <dig> gene , and one was located near the start of the ifnar <dig> gene .
in each case, stat <dig> binding in response to ifn-γ was positively correlated with rnapolii binding, relative to the average level of rnapolii binding across the encode region.
binding of rnapolii and localized mrna transcription are associated with stat <dig> binding sites near the irf <dig> and ifnar <dig> genes in non-stimulated cells
our finding that ifn-γ-induced stat <dig> binding to chromosome  <dig> is closely associated with increased mrna transcription indicated that stat <dig> binds closely to transcriptionally-active regions of the genome.
our subsequent identification of stat <dig> binding sites that are bound by rnapolii near the start positions of the irf <dig> and ifnar <dig> genes in non-stimulated cells suggested that basal levels of mrna transcription might be detectable at these sites.
to investigate this possibility, we obtained data associated with a recent analysis of basal mrna transcription in non-stimulated hela cells for  <dig> human chromosomes  <cit> .
pleasingly, most exons of the irf <dig> and ifnar <dig> genes exhibited clear peaks of basal mrna detection .
basal mrna transcription was apparent not only within exons, but also introns and extragenic regions .
all peaks of stat <dig> binding near the irf <dig> and ifnar <dig> genes overlapped with peaks of basal mrna transcription as well as peaks of basal rnapolii binding , thus reinforcing our finding using hartman et al.
's chromosome  <dig> data  <cit>  that stat <dig> binding to chromosome  <dig> is closely associated with increased mrna transcription in response to ifn-γ.
stat <dig> binds proximally to the start positions of genes with key roles in the cellular response to ifn-γ
when conducting microarray analyses of cellular responses to stimuli, it is often difficult to distinguish genes that are obligatory components of the cell's response from genes that are induced only in particular cell types or under certain conditions, and may therefore be less critical to the outcome of the response.
it is interesting to note that within the encode region, stat <dig> bound most closely to start positions of two genes with key roles in ifn signal transduction, irf <dig> and ifnar <dig> .
in fact, these are the only genes for which proximal stat <dig> binding in the encode region was observed that have known roles in ifn signal transduction.
we wondered therefore whether stat <dig> binding sites are also closely associated with the start positions of other "key" ifn-γ-inducible genes.
to this end, we identified  <dig> genes for which the transcriptional response to ifn-γ is conserved between human microglial cells and mouse livers, and are thus likely to be essential components of the responses of most cells to ifn-γ .
remarkably, the expression levels of seven of these genes are known to be regulated by stat <dig> binding sites located within just  <dig> bp of the start of each gene .
this close proximity with which stat <dig> regulates the expression of key ifn-γ-responsive genes is in stark contrast to the relatively large distances between most stat <dig> binding sites within the encode region, and the start positions of the nearest genes .
this suggests that genes with stat <dig> binding sites that are not located close to the start of the coding region could be more susceptible to tissue-specific mechanisms of regulation, for example by chromatin modification of the promoter.
discussion
whilst it is clear that mammalian promoter regions are often enriched for tfbss, much remains to be learnt about how mammalian tfbss are distributed throughout the genome, and what determines the positioning of tfbss.
we analyzed data from hartman et al.
's study of ifn-induced stat binding and mrna transcription for chromosome  <dig>  <cit> , and to our surprise, we observed a marked probe level correlation between stat <dig> binding and differential expression in response to ifn-γ.
although all chip-chip analyses of transcription factor binding have the potential to detect some rnapolii binding sites due to interactions between rnapolii and the transcription factor of interest, our results suggest that the vast majority of stat <dig> binding sites, if not all stat <dig> binding sites, are proximal to transcriptionally active regions.
the extent to which we observed this phenomenon indicates that stat <dig> binding sites are located proximally to regions that are bound by the transcriptional apparatus.
in addition to the discovery of a probe level correlation between mrna transcription and stat <dig> binding in response to ifn-γ, our analysis of hartman et al.
's chromosome  <dig> tiling array data for stat binding and mrna transcription in response to ifn  <cit>  revealed probe level correlations for various combinations of stat <dig> binding, stat <dig> binding, and mrna transcription in response to treatment with either ifn-γ or ifn-α.
together, these observations suggest that differences in the binding specificities between stat <dig> and stat <dig> are predominantly quantitative rather than qualitative.
consistent with this idea, ifn-α and ifn-γ were recently shown to induce the transcription of similar sets of genes  <cit> .
the substantial correlation we observed between the binding sites of stat <dig> in response to ifn-α vs ifn-γ perhaps casts some doubt over the significance of hartman et al.
's observation that the binding preference of stat <dig> is differentially regulated in response to ifn-α as compared to ifn-γ  <cit> .
consistent with our observation of a probe level correlation between stat <dig> binding and mrna expression in response to ifn-γ for chromosome  <dig>  we found that stat <dig> binds specifically to sites within the encode region that exhibit either diminished or enhanced binding of rnapolii in non-stimulated cells.
collectively, these findings suggest that stat <dig> binds proximally to regions of the genome that are involved in the initiation of rna transcription, since this would explain both the correlation with mrna transcription that we identified using the chromosome  <dig> tiling array data, and the correlation with differential rna polymerase binding that we identified using the encode tiling array data.
the primary mechanism by which stat <dig> activates gene transcription may therefore be to recruit rna polymerase ii to the promoters of ifn-γ-responsive genes.
there have been several reports implicating stat <dig> in the recruitment of components of the transcriptional apparatus to the promoters of particular ifn-responsive genes.
in  <dig>  stat <dig> was shown to synergize with nf-κb at the promoter of the cxcl <dig> gene to bind the transcriptional co-activator, creb-binding protein, which may in turn recruit the rnapolii complex  <cit> .
in  <dig>  it was shown that irf <dig>  may recruit the rnapolii complex to the promoter of interferon-stimulated gene  <dig> in a histone deacetylase-dependent manner  <cit> .
in  <dig>  stat <dig> was shown to recruit the dna helicase protein mini-chromosome maintenance- <dig> to the promoters of the genes encoding irf <dig>  tap <dig>  guanylate-binding protein- <dig>  and class ii transactivator in response to ifn-γ  <cit> .
furthermore, transcription of the aforementioned genes in response to ifn-γ was dependent on the activity of the mini-chromosome maintenance- <dig> protein, and the binding of the mini-chromosome maintenance- <dig> protein to the irf <dig> gene was consistent with the binding of rnapolii to the irf <dig> gene  <cit> .
our findings that stat <dig> binds to positions on chromosome  <dig> that exhibit ifn-γ-inducible mrna transcription, and to positions within the encode region that exhibit either enhanced or diminished rnapolii binding , are in strong agreement with the above reports.
furthermore, our results generalize the findings of the previous reports, since they suggest that recruitment of the rnapolii complex to the promoters of ifn-γ-responsive genes is the dominant mechanism by which stat <dig> activates gene transcription.
at stat <dig> binding sites that are normally inaccessible to rnapolii, this could involve chromatin modification to make the site more accessible to rnapolii.
at stat <dig> binding sites that exhibit basal levels of rnapolii binding, stat <dig> may simply enhance rnapolii binding.
such a mechanism of transcriptional regulation contrasts with that of the er, which can regulate the transcription of genes from distant er tfbss by acting as an enhancer for rnapolii activity  <cit> .
interestingly, two stat <dig> binding sites upstream of two well characterized mediators of ifn signal transduction, ifnar <dig> and irf <dig>  were the closest two stat <dig> binding sites to the start of any gene in the encode region .
this could simply be a coincidence, however our identification of ten genes for which transcription in response to ifn-γ is conserved between different tissues of humans and mice provides an interesting alternative explanation.
whilst many genes are probably induced by ifn-γ only in certain cell types or under certain conditions, these ubiquitously-responsive genes are likely to play key roles in the response to ifn-γ of numerous cell types .
consistent with this hypothesis, genes with important functions in cytokine signaling were identified, including socs- <dig> and - <dig>  <cit> , irf <dig>  <cit> , and the chemokines cxcl <dig> and cxcl <dig>  <cit> .
remarkably, seven of the ten ubiquitously-ifn-γ-inducible genes that we identified are known to be regulated by stat <dig> binding sites that are located within  <dig> bp from the start of each gene.
of the remaining three, little is known about the roles of gadd45γ and id <dig> in response to ifn-γ, and socs- <dig> may be regulated primarily by irf <dig>  and not stat <dig>  <cit> .
the close proximity with which stat <dig> regulates the expression of "core" ifn-γ-inducible genes suggests that many of the stat <dig> binding sites identified by chip-chip may be involved in the regulation of genes, alternative gene transcripts, or non-coding rnas that are induced by ifn-γ only in certain cell types or only under certain conditions.
as illustrated in figure  <dig>  increased regulatory opportunities may be available for genes that are regulated by stat <dig> from a distance, compared with genes that are regulated by stat <dig> close to the start of the coding region.
this model contrasts with a recent report showing that different combinations of transcription factor binding sites in the proximal promoter region are important for determining tissue-specific gene expression  <cit> .
however, given that biological systems are inherently variable, it seems unlikely that the two models are necessarily mutually exclusive.
CONCLUSIONS
our analysis of stat <dig> binding and mrna transcription reveals that the binding of stat <dig> is closely associated with ifn-γ-inducible transcriptional activity.
furthermore, our findings implicate stat <dig> in the regulation of rnapolii binding, and suggest that recruitment of the rnapolii complex to the promoters of ifn-γ-responsive genes could be a general mechanism by which stat <dig> activates transcription.
finally, we propose that stat <dig> may strategically associate with the start positions of key ifn-γ-responsive genes to ensure that they are ubiquitously, rather than conditionally, induced in response to ifn-γ.

 interferon-gamma is a potent pro-inflammatory cytokine that regulates a spectrum of biological processes, and is produced primarily in response to infection. ifn-γ signal transduction begins at the cell surface with the formation of a heteromeric protein complex that includes ifn-γ, ifn-γ receptor- and ifn-γ receptor-. associated with the ifn-γ receptors are members of the janus kinase family of tyrosine kinases, which become activated upon formation of the ifn-γ receptor complex, and in turn phosphorylate the signal transducer and activator of transcription- transcription factor. upon its phosphorylation, stat homo-dimerizes, and is transported into the nucleus where it binds to the gamma activated sequence to activate the expression of ifn-γ-responsive genes. one example of a stat1-responsive gene is the interferon regulatory factor- gene, an important ifn-γ-responsive transcription factor which contains a functional gas bp upstream of its first exon. in addition, stat functions as a component of the ifn stimulated gene factor transcription factor complex, which also includes stat and interferon regulatory factor-. the isgf complex is formed in response to signaling by ifn family members, including ifn-α, that associate with ifn-α receptor- and ifn-α receptor- upon its transportation into the nucleus, isgf binds to ifn-stimulated response elements to activate the expression of ifn-α-responsive genes. how gene expression is regulated by the association of transcription factors to their target sequences is a central question in mammalian biology. compared with lower organisms such as s. cerevisiae and d. melanogaster, the genomic regions responsible for regulating the expression of mammalian genes are much more difficult to predict, and may be located far away from a gene's transcriptional start site. furthermore, the debate over what constitutes a gene was further intensified with the development of tiling arrays for the human genome, and the discovery that much of the human transcriptome is coded for by regions of the genome that lie outside of exons as they have been classically defined. chromatin immunoprecipitation microarray technology provided additional insight into the regulation of the human transcriptome when it was employed to examine the transcription factor binding sites of sp cmyc, and p on chromosomes and. interestingly, only 22% of these tfbss were located in the upstream regions of genes, the regions that have classically been defined as "promoter" regions. the genomic relationship between sites of transcription factor binding, and sites of transcription, was further elucidated in an elegant study of estrogen receptor binding across chromosomes and. remarkably, the rna polymerase ii complex was found to associate in an estrogen-dependent manner with the majority of tested tfbss, even those located far from the nearest tss. using a chromatin capture assay, an er binding site located over kbp from the nrip- gene was shown to function as an enhancer of nrip- transcription. these results showed that, when bound to its tfbs, the er can act as an enhancer to regulate the expression of target genes by associating, often across large chromosomal distances, with rnapolii. recently, the response to ifn-γ has been the subject of microarray expression analyses as well as chip-chip analyses for stat. hartman et al. compared the locations of stat binding sites on chromosome with the expression level of the nearest gene, and noted that only 21% of stat binding sites were within kbp of the start of the nearest gene. hartman et al. also suggested that a novel mechanism may alter the specificity of stat binding, depending on whether it is induced by ifn-α or ifn-γ. elucidating the rationale with which transcription factor binding sites are deployed across the genome will likely be of great benefit to our understanding of the complexities of transcriptional regulation in mammals. we have examined the data associated with several recent tiling array experiments, and show here that stat binding and mrna transcription in response to ifn are much more closely associated than previous reports would suggest. our results indicate that, in contrast to other mammalian transcription factors such as the er, stat binds closely to the tsss of its target genes. our findings also suggest that stat may play an important role in recruiting the rnapolii complex to the promoters of ifn-γ-inducible genes.  stat binding and mrna transcription in response to ifn-γ are correlated at the probe level for chromosome tiling arrays to examine the relationship between stat binding and mrna transcription in response to ifn-γ, we obtained the data associated with hartman et al.'s tiling array analysis of gene transcription and stat binding for chromosome following treatment with ifn-γ and ifn-α. we were particularly interested in determining whether there was any evidence for localized coupling of differential stat binding and differential mrna expression. to our surprise, there was a pronounced probe level correlation between differential stat binding in response to ifn-γ, and differential mrna expression in response to ifn-γ. a similar probe level relationship between stat binding, stat binding, and differential expression, was also apparent for the responses of cells to ifn-α. furthermore, probes that detected differential stat binding in response to ifn-α also detected substantial differential expression in response to ifn-γ, and consistent with this, there was a high probe level correlation between the binding of stat or stat in response to ifn-α, and the binding of stat in response to ifn-γ. consistent with hartman et al.'s conclusion that the binding preference of stat may differ in response to ifn-α versus ifn-γ, some differences in the binding sites of stat were apparent for these stimuli, although this was primarily true only of some of the relatively weak stat binding sites. for relatively strong stat binding sites, the binding preferences in response to ifn-γ versus ifn-α showed substantial correlation. the findings outlined above strongly suggest that there is a high degree of overlap between the positions where stat and stat bind in response to either ifn-γ or ifn-α, which is perhaps not surprising, since stat binds dna as part of the isgf transcription factor complex, of which stat is also a component. consistent with this, correlation was also apparent between the transcriptional response of cells to ifn-γ and the transcriptional response of cells to ifn-α. of greater interest, however, is the clear probe level correlation between the binding of stat or stat and the differential expression in response to ifn-γ or ifn-α. given that probes for the chromosome arrays are – bp long, our observations indicate that the locations at which stat or stat associate with chromosome are in the majority of cases very close to regions of chromosome that are transcriptionally regulated by ifn-γ or ifn-α. detection of stat binding sites within the encode region to examine stat binding in response to ifn-γ at a higher resolution than is permitted by cdna microarray technology, we obtained data associated with a recent chip-chip analysis of stat binding in response to ifn-γ across the mb encyclopedia of dna elements region. a moderately conservative differential intensity cut-off of was selected by inspection of a quantile-quantile plot of the differential intensities. in total, probes satisfied this cut-off. these probes all clustered to distinct genomic sites, of which were within kbp of the nearest 1st exon of a gene. this method appears to identify fewer stat binding sites than are identified on the ucsc genome browser, presumably because our significance cut-off is higher. when the alternative transcripts of three genes, atp11a, mcf2l, and rab11-fip were accounted for, of the of the stat binding sites we identified were within kbp of the nearest 1st exon of a gene. of the genes identified in this manner, three are known ifn-γ-responsive genes. in the case of irf- a stat binding site immediately upstream of the first exon has been previously identified by in vivo footprinting. the majority of stat binding sites exhibit localized suppression of rnapolii binding in non-stimulated cells our finding that ifn-γ-induced stat binding to chromosome is closely associated with increased mrna transcription suggested that stat may bind proximally to rnapolii within the promoters of ifn-γ-responsive genes. we were therefore interested in examining the binding of rnapolii to regions near stat binding sites. to this end, we obtained data associated with bieda et al.'s recent analysis of rnapolii binding within the encode region for non-stimulated helas cells. remarkably, the binding of rnapolii in non-stimulated cells was negatively correlated with the binding of stat in response to ifn-γ for of the binding sites within the encode region. this is indicative of a diminished level of rnapolii binding, relative to the average level of rnapolii binding for the entire encode region. localized suppression of rnapolii binding is likely to reflect the locations at which modifications in the chromatin state, or possibly other factors, prevent the association of rnapolii. rnapolii binds to stat binding sites near the irf and ifnar genes in non-stimulated cells of the five stat binding sites that did not exhibit suppressed rnapolii binding in non-stimulated cells, three were located within kbp upstream of the irf gene, and one was located near the start of the ifnar gene. in each case, stat binding in response to ifn-γ was positively correlated with rnapolii binding, relative to the average level of rnapolii binding across the encode region. binding of rnapolii and localized mrna transcription are associated with stat binding sites near the irf and ifnar genes in non-stimulated cells our finding that ifn-γ-induced stat binding to chromosome is closely associated with increased mrna transcription indicated that stat binds closely to transcriptionally-active regions of the genome. our subsequent identification of stat binding sites that are bound by rnapolii near the start positions of the irf and ifnar genes in non-stimulated cells suggested that basal levels of mrna transcription might be detectable at these sites. to investigate this possibility, we obtained data associated with a recent analysis of basal mrna transcription in non-stimulated hela cells for human chromosomes. pleasingly, most exons of the irf and ifnar genes exhibited clear peaks of basal mrna detection. basal mrna transcription was apparent not only within exons, but also introns and extragenic regions. all peaks of stat binding near the irf and ifnar genes overlapped with peaks of basal mrna transcription as well as peaks of basal rnapolii binding, thus reinforcing our finding using hartman et al.'s chromosome data that stat binding to chromosome is closely associated with increased mrna transcription in response to ifn-γ. stat binds proximally to the start positions of genes with key roles in the cellular response to ifn-γ when conducting microarray analyses of cellular responses to stimuli, it is often difficult to distinguish genes that are obligatory components of the cell's response from genes that are induced only in particular cell types or under certain conditions, and may therefore be less critical to the outcome of the response. it is interesting to note that within the encode region, stat bound most closely to start positions of two genes with key roles in ifn signal transduction, irf and ifnar. in fact, these are the only genes for which proximal stat binding in the encode region was observed that have known roles in ifn signal transduction. we wondered therefore whether stat binding sites are also closely associated with the start positions of other "key" ifn-γ-inducible genes. to this end, we identified genes for which the transcriptional response to ifn-γ is conserved between human microglial cells and mouse livers, and are thus likely to be essential components of the responses of most cells to ifn-γ. remarkably, the expression levels of seven of these genes are known to be regulated by stat binding sites located within just bp of the start of each gene. this close proximity with which stat regulates the expression of key ifn-γ-responsive genes is in stark contrast to the relatively large distances between most stat binding sites within the encode region, and the start positions of the nearest genes. this suggests that genes with stat binding sites that are not located close to the start of the coding region could be more susceptible to tissue-specific mechanisms of regulation, for example by chromatin modification of the promoter. discussion whilst it is clear that mammalian promoter regions are often enriched for tfbss, much remains to be learnt about how mammalian tfbss are distributed throughout the genome, and what determines the positioning of tfbss. we analyzed data from hartman et al.'s study of ifn-induced stat binding and mrna transcription for chromosome, and to our surprise, we observed a marked probe level correlation between stat binding and differential expression in response to ifn-γ. although all chip-chip analyses of transcription factor binding have the potential to detect some rnapolii binding sites due to interactions between rnapolii and the transcription factor of interest, our results suggest that the vast majority of stat binding sites, if not all stat binding sites, are proximal to transcriptionally active regions. the extent to which we observed this phenomenon indicates that stat binding sites are located proximally to regions that are bound by the transcriptional apparatus. in addition to the discovery of a probe level correlation between mrna transcription and stat binding in response to ifn-γ, our analysis of hartman et al.'s chromosome tiling array data for stat binding and mrna transcription in response to ifn revealed probe level correlations for various combinations of stat binding, stat binding, and mrna transcription in response to treatment with either ifn-γ or ifn-α. together, these observations suggest that differences in the binding specificities between stat and stat are predominantly quantitative rather than qualitative. consistent with this idea, ifn-α and ifn-γ were recently shown to induce the transcription of similar sets of genes. the substantial correlation we observed between the binding sites of stat in response to ifn-α vs ifn-γ perhaps casts some doubt over the significance of hartman et al.'s observation that the binding preference of stat is differentially regulated in response to ifn-α as compared to ifn-γ. consistent with our observation of a probe level correlation between stat binding and mrna expression in response to ifn-γ for chromosome we found that stat binds specifically to sites within the encode region that exhibit either diminished or enhanced binding of rnapolii in non-stimulated cells. collectively, these findings suggest that stat binds proximally to regions of the genome that are involved in the initiation of rna transcription, since this would explain both the correlation with mrna transcription that we identified using the chromosome tiling array data, and the correlation with differential rna polymerase binding that we identified using the encode tiling array data. the primary mechanism by which stat activates gene transcription may therefore be to recruit rna polymerase ii to the promoters of ifn-γ-responsive genes. there have been several reports implicating stat in the recruitment of components of the transcriptional apparatus to the promoters of particular ifn-responsive genes. in stat was shown to synergize with nf-κb at the promoter of the cxcl gene to bind the transcriptional co-activator, creb-binding protein, which may in turn recruit the rnapolii complex. in it was shown that irf may recruit the rnapolii complex to the promoter of interferon-stimulated gene in a histone deacetylase-dependent manner. in stat was shown to recruit the dna helicase protein mini-chromosome maintenance- to the promoters of the genes encoding irf tap guanylate-binding protein- and class ii transactivator in response to ifn-γ. furthermore, transcription of the aforementioned genes in response to ifn-γ was dependent on the activity of the mini-chromosome maintenance- protein, and the binding of the mini-chromosome maintenance- protein to the irf gene was consistent with the binding of rnapolii to the irf gene. our findings that stat binds to positions on chromosome that exhibit ifn-γ-inducible mrna transcription, and to positions within the encode region that exhibit either enhanced or diminished rnapolii binding, are in strong agreement with the above reports. furthermore, our results generalize the findings of the previous reports, since they suggest that recruitment of the rnapolii complex to the promoters of ifn-γ-responsive genes is the dominant mechanism by which stat activates gene transcription. at stat binding sites that are normally inaccessible to rnapolii, this could involve chromatin modification to make the site more accessible to rnapolii. at stat binding sites that exhibit basal levels of rnapolii binding, stat may simply enhance rnapolii binding. such a mechanism of transcriptional regulation contrasts with that of the er, which can regulate the transcription of genes from distant er tfbss by acting as an enhancer for rnapolii activity. interestingly, two stat binding sites upstream of two well characterized mediators of ifn signal transduction, ifnar and irf were the closest two stat binding sites to the start of any gene in the encode region. this could simply be a coincidence, however our identification of ten genes for which transcription in response to ifn-γ is conserved between different tissues of humans and mice provides an interesting alternative explanation. whilst many genes are probably induced by ifn-γ only in certain cell types or under certain conditions, these ubiquitously-responsive genes are likely to play key roles in the response to ifn-γ of numerous cell types. consistent with this hypothesis, genes with important functions in cytokine signaling were identified, including socs- and -, irf, and the chemokines cxcl and cxcl. remarkably, seven of the ten ubiquitously-ifn-γ-inducible genes that we identified are known to be regulated by stat binding sites that are located within bp from the start of each gene. of the remaining three, little is known about the roles of gadd45γ and id in response to ifn-γ, and socs- may be regulated primarily by irf and not stat. the close proximity with which stat regulates the expression of "core" ifn-γ-inducible genes suggests that many of the stat binding sites identified by chip-chip may be involved in the regulation of genes, alternative gene transcripts, or non-coding rnas that are induced by ifn-γ only in certain cell types or only under certain conditions. as illustrated in figure increased regulatory opportunities may be available for genes that are regulated by stat from a distance, compared with genes that are regulated by stat close to the start of the coding region. this model contrasts with a recent report showing that different combinations of transcription factor binding sites in the proximal promoter region are important for determining tissue-specific gene expression. however, given that biological systems are inherently variable, it seems unlikely that the two models are necessarily mutually exclusive.  our analysis of stat binding and mrna transcription reveals that the binding of stat is closely associated with ifn-γ-inducible transcriptional activity. furthermore, our findings implicate stat in the regulation of rnapolii binding, and suggest that recruitment of the rnapolii complex to the promoters of ifn-γ-responsive genes could be a general mechanism by which stat activates transcription. finally, we propose that stat may strategically associate with the start positions of key ifn-γ-responsive genes to ensure that they are ubiquitously, rather than conditionally, induced in response to ifn-γ. 
this finding establishes a generalized basis for the positioning of stat binding sites within the genome, and supports a role for stat in the direct recruitment of the rna polymerase ii complex to the promoters of ifn-γ-responsive genes. this finding was consistent between different tiling microarray platforms, and between different measures of transcriptional activity, including differential binding of rna polymerase ii, and differential mrna transcription. re-analysis of tiling microarray data from a recent study of ifn-γ-induced stat chip-chip and mrna expression revealed that stat binding is tightly associated with localized mrna transcription in response to ifn-γ. close relationships were also apparent between stat binding, stat binding, and mrna transcription in response to ifn-α. signal transducer and activator of transcription proteins are key regulators of gene expression in response to the interferon family of anti-viral and anti-microbial cytokines. together, our results indicate that stat binds proximally to regions of the genome that exhibit regulated transcriptional activity. we have examined the genomic relationship between stat binding and regulated transcription using multiple tiling microarray and chromatin immunoprecipitation microarray experiments from public repositories. furthermore, we found that sites of stat binding within the encyclopedia of dna elements region are precisely correlated with sites of either enhanced or diminished binding by the rna polymerase ii complex.
 signal transducer and activator of transcription proteins are key regulators of gene expression in response to the interferon family of anti-viral and anti-microbial cytokines. we have examined the genomic relationship between stat binding and regulated transcription using multiple tiling microarray and chromatin immunoprecipitation microarray experiments from public repositories.  in response to ifn-γ, stat bound proximally to regions of the genome that exhibit regulated transcriptional activity. this finding was consistent between different tiling microarray platforms, and between different measures of transcriptional activity, including differential binding of rna polymerase ii, and differential mrna transcription. re-analysis of tiling microarray data from a recent study of ifn-γ-induced stat chip-chip and mrna expression revealed that stat binding is tightly associated with localized mrna transcription in response to ifn-γ. close relationships were also apparent between stat binding, stat binding, and mrna transcription in response to ifn-α. furthermore, we found that sites of stat binding within the encyclopedia of dna elements region are precisely correlated with sites of either enhanced or diminished binding by the rna polymerase ii complex.  together, our results indicate that stat binds proximally to regions of the genome that exhibit regulated transcriptional activity. this finding establishes a generalized basis for the positioning of stat binding sites within the genome, and supports a role for stat in the direct recruitment of the rna polymerase ii complex to the promoters of ifn-γ-responsive genes. 