BACKGROUND
ifnγ is a pleiotropic cytokine that plays essential roles in antiviral and anticancer immune responses .
ifnγ binds to its receptor complex and activates receptor-associated jak kinases, which phosphorylate a substantial fraction of cytoplasmic signal transducer and activator of transcription  <dig> .
phosphorylated stat <dig> forms homodimers that translocate to the nucleus and bind ifnγ activation sites .
stat <dig> recruits histone acetyltransferases  and other transcriptional co-activators to acetylate chromatin and facilitate transcription.
genomic studies showed that stat <dig> binds at promoter proximal and distal sites, suggesting a role in remote gene regulation .
indeed, ifnγ induces long range interactions between stat1-bound enhancers and target promoters .
interferon regulatory factor  <dig>  is a primary target gene of stat <dig>  like stat <dig>  irf <dig> also acts as a transcription factor , binding to irf-e motifs and interferon-stimulated response elements   <cit> .
access of both stat <dig> and irf <dig> to target enhancers requires the swi/snf chromatin remodeling complex to counter prc <dig>  which uses the histone methyl transferase ezh <dig> to deposit h3k27me <dig> and block the induction of many other cytokine and cytokine responsive loci  <cit> .
irf <dig> functions at the transcription initiation level by facilitating rna pol ii recruitment to isgs promoters  <cit> .
irf <dig> also binds to remote enhancers of the ciita locus that loop together to form a 3d interconnected hub with the promoter  <cit> .
indeed, chip-chip and chip-seq studies show that irf <dig> binds many remote enhancers , and analysis of  <dig> transcription factors in k <dig> cells revealed that stat1-irf <dig> co-binding is a recurring pattern in ifnγ treated cells  <cit> .
notably, stat <dig> is essential but not sufficient for gene induction  <cit> , and both stat <dig> and irf <dig> are required for the ifnγ-induced expression of ciita, gbp <dig>  and gp <dig>  <cit> .
in addition, stat <dig> complexes with irf <dig> at the lmp <dig> promoter and maintains its constitutive expression  <cit> .
here, we studied the extent of stat <dig> and irf <dig> cooperation in hela cells within isg-rich chromosomal segments encompassing ~10% of all known isgs.
most of these loci responded to ifnγ in hela cells, leaving ~20% resistant isgs.
irf <dig> binding sites outnumbered stat <dig> sites  <dig> to  <dig>  a large fraction of stat1/irf <dig> binding occurred at remote sites and looping studies confirmed the functional role of putative enhancers at the socs <dig> locus.
most stat <dig> binding occurred at or near to irf <dig> sites , but irf <dig> often bound isolated from stat <dig>  dual stat <dig> and irf <dig> but not isolated irf <dig> or stat <dig> binding was linked to isg responsiveness.
finally, several variants affecting stat1/irf <dig> motifs induce or impair binding.
RESULTS
diverse gene responses to ifnγ
to define patterns of tf binding around isgs, we employed tiling arrays to focus on 16 mb distributed across  <dig> distinct chromosomal segments with a high density of isgs .
nine segments were 1 mb genomic regions on six chromosomes centered on specific ifnγ target genes .
two others included a 2 mb segment centered on ciita, and a 5 mb segment covering the complete classical  <dig>  mb mhc locus and an additional  <dig>  mb 5′ region including much of the so-called extended mhc class i region .
within these regions 25%  of the genes are known isgs, ~5× more than the genome-wide isg frequency  and ~15-fold above the average isgs density per mb.
the total number of refseq genes and ucsc known genes in the 16 mb regions is  <dig> .
of these, 95%  were represented on the illumina- <dig> human wg-6v <dig> array used to assess gene expression .
the frequency of refseq genes across the genome is ~6/mb, but most of the  <dig> chromosomal segments in our study were gene dense , especially at the mhc , psme  and ifitm clusters  .
there are also  <dig> pseudogenes across the 16 mb, with most  located at the mhc cluster .
pseudogenes are not represented on the illumina genome wide array we used to study expression.fig.
<dig> ifnγ target subclasses.
a human chromosome ideograms, drawn using ncbi map viewer, show locations of the  <dig> studied chromosomal segments .
b the  <dig> genes within these regions were classified as “ifnγ stimulated genes” , “ifnγ-repressed genes”  or “other genes” according to our expression array data and on prior studies .
known isgs  were either induced  or resistant  in hela cells.
genes were further subcategorized according to their robustness  and timing  of induction/repression .
other genes were termed “not-linked to ifnγ”, or “potential isgs”  if they were either isg paralogs or exhibited ifnγ-induced tf binding at their promoter.
italics indicate the number of ciita targets in each class.
c to validate array data, mrna was isolated from hela cells left untreated or exposed to ifnγ for 6 h. rt-pcr was performed on  <dig> induced  or resistant  isgs, selected based on array data 6 h after ifnγ treatment.
pie diagram shows the percentage of validated genes
signaling pathway target loci show cell-specific responsiveness, but the exact tf binding patterns that distinguish induction versus resistance in a specific cell type are unclear.
thus, we compared the pattern of stat <dig> and irf <dig> binding at different gene types.
for this we compiled a database of ~ all known isgs using our own and prior transcriptome data .
as summarized in fig.
1b, isgs fell into  <dig> classes depending on whether ifnγ caused induction, no effect , or repression, and whether induction/repression were early  or late , and strong  or weak .
the microarray expression data was validated using reverse transcription and quantitative pcr , which confirmed 83%  of indisgs and 87%  of resisgs .
of all  <dig> known isgs on the array,  <dig>  genes were es-indisgs,  <dig>  were ls-indisgs,  <dig>  were ew-indisgs or lw-indisgs, and  <dig> were resisgs .
es-indisgs were distributed at an average density of  <dig> /mb within the studied regions .
the highest density was observed at the ifit and gbp clusters with an average of  <dig>  es-indisgs/mb.
no genes were repressed  at the early 6 h time point, while  <dig> and  <dig> were strongly or weakly repressed at later times, respectively , suggesting indirect regulation of irgs .
the remaining genes that were not ifnγ-responsive either in this or any prior study were termed “other genes”.
in summary, known isgs fall into induced and resistant subclasses in hela cells, providing a useful system to define stat <dig> and irf <dig> binding patterns linked to responsiveness.
validation of stat <dig> and irf <dig> chip-chip analyses
chip-chip was used to locate stat <dig> and irf <dig> sites at promoter proximal and distal sites of the genes of each category.
stat <dig> and irf <dig> chips were performed on chromatin from hela cells that were either untreated or exposed to ifnγ for 6 h. hybridization intensities were normalized to internal standards and values from quadruplicate spots were averaged.
significantly different intensities between chip dna and input dna samples in three biological replicates  were determined using the wilcoxon rank sum test.
peaks representing the significantly enriched dna regions  where the ratio of chip to input dna was ≥  <dig> -fold were visualized in the ucsc browser and plotted on a log <dig> scale.
only  <dig> stat <dig> and  <dig> irf <dig> peaks were identified in untreated cells, rising to  <dig> and  <dig> post-ifnγ treatment, respectively.
browser views are shown in additional file 2: figure s <dig> and can be visualized at http://research.lunenfeld.ca/ifny.
chip-qpcr validated 91%  and 96%  of stat <dig> and irf <dig> chip-chip peaks, respectively .
we compared stat <dig> binding at 6 h  with ifnγ-induced stat <dig> binding after 30 min  <cit> , also assessed in hela cells.
in the 16 mb of dna assessed here, the latter study detected  <dig> stat <dig> sites, of which  <dig> overlapped with the  <dig> stat <dig> sites we detected.fig.
<dig> stat <dig> and irf <dig> binding patterns.
arbitrarily selected chip-chip stat <dig> , irf <dig>  sites were re-examined by chip-qpcr on chromatin from hela cells with no or 6 h of ifnγ treatment.
91% of peaks were validated in both cases.
the frequency of consensus motifs identified by jasper within stat <dig>  or irf <dig>  peaks is indicated
basal tf binding
unphosphorylated stat <dig> has roles in regulating isgs days after ifn treatment  <cit> , but its role in untreated cells is less clear, although stat <dig> nuclear cytoplasmic shuttling occurs even in untreated cells .
basal stat <dig> binding is linked to the nuclear localization of unphosphorylated stat <dig> and contributes to the constitutive expression of some targets  <cit> .
irf <dig> is also expressed to low levels in unstimulated hela cells  <cit>  and it cooperates with stat <dig> to maintain low basal expression levels of lmp <dig>  <cit> .
in addition, there is also some stat <dig> phosphorylation  in untreated cells that contributes to basal activity, as shown elegantly by knockin studies in mice  <cit> .
we detected  <dig> stat <dig> and  <dig> irf <dig> binding sites in untreated cells, accounting for  <dig>  and  <dig> % of induced sites, respectively.
our data accords with another chip-chip analysis of stat <dig> binding which reported that  <dig> % of ifnγ-induced stat <dig> sites in hela cells treated for 30 min with ifnγ  are occupied in uninduced cells  <cit> .
further analysis suggests that basal tf binding detected here is physiologically relevant.
of the genes with basal stat <dig> or irf <dig> binding, we assessed  <dig> by microarray and/or rt-pcr and all were expressed in untreated cells .
in contrast, of  <dig> randomly selected isgs that lacked basal tf binding, only  <dig> were basally expressed.
indeed, constitutive expression of psmb <dig> and tap <dig> requires constitutive irf <dig> binding  <cit> .
in addition, several loci with basal tf binding are in paralogous gene clusters suggesting conservation of high affinity binding sites during gene duplication .
a high fraction  of the  <dig> irf <dig> basally occupied sites possessed irf <dig> binding motifs.
thus, our data supports the notion that basal binding of stat <dig> and irf <dig> is physiologically relevant.
remote ifnγ activated enhancers are common at isgs
in ifnγ treated cells, 54%  of stat <dig> and 44%  of irf <dig> peaks were within 5 kb of the transcription start site  of all  <dig> known genes on the array .
adding other databases, including predicted genes, raised the fraction to 64% for stat <dig> and 57% for irf <dig> .
of equal numbers of randomly generated sites, the proportion at <5 kb from gene starts was much lower .
thus stat <dig> binding is slightly skewed to promoter proximal sites, while irf <dig> binding is slightly biased toward remote sites .fig.
<dig> proximal vs. distal stat <dig> and irf <dig> binding.
a the percentage of ifnγ-induced stat <dig> or irf <dig> binding sites  or randomly generated controls at proximal  or distal  sites relative to the tss of known genes.
b irf <dig> to stat <dig> ratio at proximal and remote sites
prior analysis of chromatin modification and looping at ciita, and partial looping analysis at  <dig> locus support the idea that remote sites are functionally important  <cit> .
to further test this notion we performed additional assessment of socs <dig>  a key negative regulator of ifnγ signaling that is responsive to ifns and other immune signaling pathways  <cit> .
socs <dig> responds to ifnγ in hela cells .
chip-chip data exposed  <dig> ifnγ-induced stat <dig> and/or irf <dig> peaks ± 100 kb of the socs <dig> tss .
chip-qpcr analysis verified binding at the socs <dig> promoter , and at + <dig>  + <dig>  − <dig>  − <dig>  − <dig> and −72 kb .
a negative region at −63 kb was also validated.
in favor of functional relevance of proximal and remote sites, we detected constitutive histone h <dig> acetylation  and/or h4ac at psocs <dig> , − <dig> and −55 kb , and ifnγ induced acetylation at psocs <dig> and the  <dig> remote sites but not at the irrelevant −63 kb site .
these constitutive and inducible events paralleled recruitment of the hats cbp and/or p <dig> .
h3k4me <dig> also marks enhancers  <cit> , and constitutive h3k4me <dig> was detected at psocs <dig>  −3 kb, −55 kb, matching constitute histone acetylation, and also at the −72 kb enhancer , which contacts the promoter .
ifnγ treatment did not further increase methylation at these sites, but did induce h3k4me <dig> at +50 kb, +15 kb and −72 kb .
finally, we detected constitutive pol ii recruitment at psocs <dig> and −55 kb but not at the other tf binding or negative control sites .
after ifnγ treatment, pol ii recruitment increased at psocs <dig>  +15 kb and −55 kb .
association with the +15 kb element may reflect ifnγ-induced promoter looping .fig.
<dig> stat <dig> and irf <dig> binding at socs <dig>  a rt-qpcr for socs <dig> mrna in hela cells treated with ifnγ for  <dig> or 6 h. data are in arbitrary units relative to β-actin levels .
b chip-chip maps of stat <dig> and irf <dig> binding across the socs <dig> locus treated as in a. black arrowheads indicate tf binding sites of interest, with distances from the tss  in kb.
the tf-free −63 kb site  is used as a negative control in c. c chip-qpcr analysis of the basal and ifnγ-induced histone modifications or recruitment of the indicated factors.
marks  show significant differences  in the indicated comparisons
chromosome conformation capture  revealed both constitutive and ifnγ-induced contacts between the promoter and remote stat <dig> and/or irf <dig> sites at ciita  <cit> .
to examine looping at socs <dig>  we studied six ecori  or four ncoi  fragments .
of a total of  <dig> possible interactions, we studied  <dig> previously  and assessed an additional  <dig> in this study.
as expected, no interaction was observed between fragments containing the promoter and irrelevant sites at − <dig>  − <dig>  or +70 kb, either before or after cytokine exposure.
however, between suspected functional elements, we detected a total of  <dig> loops of all  <dig> putative interactions in the basal state, and each of these loops was enhanced after ifnγ treatment and was accompanied by a new interaction between the + <dig> and −72 kb enhancers that lie 122 kb apart .
our data suggest that the socs <dig> locus is basally present in a mega looping complex that becomes more compact and involves more inter-element interactions after ifnγ treatment.
together, the chip and 3c data show that stat <dig> and irf <dig> binding is linked to extensive chromatin modifications and looping.fig.
<dig> basal and ifnγ-induced looping at socs <dig>  a a schematic view of the socs <dig> locus.
circles indicate the socs <dig> promoter , putative remote enhancers , and negative control sites , with distances from the tss  indicated above in kb, while fragments used in 3c assays with primers  are shown below.
b cross linking frequencies between the promoter and remote sites across the socs <dig> locus.
quantitative 3c was performed with chromatin from hela cells left untreated or exposed to ifnγ for 6 h. bar graphs show the crosslinking frequency of a selected number of interactions.
underlined interactions were published previously  <cit> .
marked interactions  are significantly different at the indicated comparisons .
c summary of looping events.
interacting sites and dna strands are colored as in a. stat1/irf <dig>  and hac/h3k4me <dig>  are also depicted
unusual irf <dig> distribution at mhc loci
as noted earlier, irf <dig> exceeded stat <dig> sites by ~twofold, but this varied at some regions, most notably at the mhc class i locus where the ratio was  <dig> : <dig> .
the ratio was particularly skewed at the extended  versus classic  mhc class i region.
<dig> of all mhc class i irf <dig> sites were within 5 kb of known gene starts and  <dig> within 5 kb of pseudogenes , giving a total of 77%  promoter proximal sites, which is higher than the 44% at all loci .
however, whereas 66%  of irf <dig> sites were promoter-proximal in the classical mhc class i region, this dropped to only 6%  at the extended mhc class i region, and was low even after including pseudogenes , leaving an unusually high fraction of remote irf <dig> sites .
thus, irf <dig> seems to play a broader role than stat <dig> at the mhc class i cluster, primarily at proximal elements in the classic region, but at remote elements in the extended region.
stat <dig> and irf <dig> induce ciita, the master regulator of mhc class ii expression .
the number of stat <dig> and irf <dig> sites was typically very low in the mhc class ii region .
out of  <dig> mhc class ii genes,  <dig>  were resistant to ifnγ in hela cells,  <dig>  responded only after 24 h, a time of maximum production of ciita  <cit> , and  <dig>  were es-indisgs.
with the exception of dob, none of the resistant or late-induced genes exhibited stat <dig> or irf <dig> promoter binding.
however, of the  <dig> es-indisgs, two had promoter proximal irf <dig> binding while dma had irf <dig> binding fairly near  its promoter.
thus irf <dig> may cooperate with ciita at a subset of mhc class ii promoters.
others reported ciita-independent induction of mhc class ii genes , which may, therefore, involve irf <dig>
stat <dig> and irf <dig> binding is enriched at robustly induced isgs
as discussed, isgs fell into  <dig> classes depending on whether ifnγ caused induction, no effect , or repression, and whether induction/repression were early or late, and strong or weak .
we plotted the distribution of stat <dig> and irf <dig> binding sites relative to all  <dig> gene classes.
binding sites were assigned to the nearest gene class, designated as proximal or distal when ≤ <dig> or >5 kb from the tss, respectively, and were compared to  <dig> randomly chosen sites equaling number of stat1 + irf <dig> sites .
we also calculated the tf enrichment ratio  in which the % distribution of tfs at proximal and distal locations was normalized to the % distribution of random sites .
a binding frequency twice that of random sites  was assigned as an arbitrary minimum threshold.fig.
<dig> enrichment of stat/irf <dig> at strong early induced isgs.
a histogram shows the percent of stat <dig>  irf <dig>  or an equal number of randomly generated sites at proximal  or distal  sites of isgs , irgs, and other genes.
the number of sites in each category is indicated below each bar.
“random frequency”: distribution of  <dig> randomly generated sites.
b distribution of tfs normalized to that of random controls.
unshaded region indicates ter > twofold
stat <dig> and irf <dig> binding sites were most highly associated with robustly induced ifnγ targets .
this applied when stat <dig> or irf <dig> were considered together, separately, and at proximal or remote locations .
consistent with this finding, weakly induced genes  had fewer binding events and lower ters .
of  <dig> other genes , a total of only  <dig> had  <dig> stat <dig> and  <dig> irf <dig> proximal peaks, mostly  located at the mhc and rt-qpcr confirmed no induction at 10/ <dig> of these genes .
ifnγ enhancers loop over large distances at ciita  <cit>  and socs <dig> , so proximal and distal enhancers nearest to other genes may target neighboring isgs.
in summary, the data indicate a clear bias of stat <dig> and irf <dig> binding at rapidly and robustly induced isgs, but not other gene classes.
isolated or dual stat <dig> and irf <dig> recruitment is directed by binding motifs
next we compared the fraction of isolated or dual stat1/irf <dig> binding events.
of a total of  <dig> discrete tf binding regions, 16%  exhibited stat <dig> binding alone , of which almost half  were proximal; 61%  exhibited only irf <dig> binding , of which 42%  were proximal; and 23%  showed overlap , of which slightly more than half  were proximal .
randomly generated sites showed negligible overlap , but dual stat1/irf <dig> binding represented more than half  of all stat <dig> sites and about a quarter  of irf <dig> peaks .
fewer overlapping irf <dig> peaks reflect their twofold excess relative to stat <dig> peaks.
thus, stat <dig> preferentially binds with irf <dig> at ifnγ enhancers, whereas most irf <dig> sites are not co-localized with stat <dig> fig.
<dig> stat <dig> and irf <dig> binding correlates with binding motifs.
a percent distribution of isolated stat <dig> or irf <dig> or dual stat1 + irf <dig> binding at proximal  or remote  sites of known gene promoters.
b tf binding sites were classified into  <dig> subclasses, then mapped motifs using cisgenome’s “known motif mapping” program .
sets of equal numbers of randomly generated “peaks” were used to define the background occurrence of stat <dig> and irf <dig> motifs.
asterisk indicates significant difference between true and random peaks .
c ratio of stat1/irf <dig> motifs at different categories of peaks
jasper analysis of irf <dig> and stat <dig> peak regions revealed that the cognate binding motif was observed at a statistically significant level relative to equal numbers of random peaks .
60% of isolated stat <dig> peaks had a stat <dig> motif, and only 30% had an irf <dig> motif, while 70% of isolated irf <dig> peaks possessed an irf <dig> motif, but only 20% had a stat <dig> motif.
a strong correlation existed between stat1/irf <dig> binding and the presence of the corresponding motifs .
indeed 40% of dual stat1/irf <dig> sites had both binding motifs, whereas there were none at equal numbers of randomly generated sites .
dual sites which have only a stat <dig> or irf <dig> binding motif may reflect protein–protein interaction or dna looping as seen at the socs <dig> and ciita loci   <cit> .
in summary, dna sequence directs isolated or dual stat1/irf <dig> binding in ifnγ treated cells.
dual stat <dig> and irf <dig> targeted enhancers distinguish responsive from resistant isgs
comparing inducible isgs in our array study with ~all known isgs in a large database  revealed resistant isgs  in hela cells .
there were far fewer stat1/irf <dig> binding events at res-isgs vs ind-isgs, and the ter  at res-isgs was low, and similar to that at other genes .
there was near ubiquitous association of both stat <dig> and irf <dig> at es-indisg promoters, but they were virtually absent at res-isg promoters .
to quantify the types of tf binding events , we plotted the frequency of genes with at least one binding event within or beyond 5 kb , and the density of each type of binding event per gene .
isolated tf binding did not discriminate the two gene classes, whereas there was significantly more dual stat <dig> and irf <dig> binding at esisgs, at both proximal and distal sites .
thus, cooperation between stat <dig> and irf <dig> plays a central role in mediating ifnγ responsiveness.fig.
<dig> stat/irf <dig> recruitment at es-indisgs vs res-isgs.
a map of isolated and dual stat <dig> and irf <dig> binding ± 100 kb of the tss of es-indisgs  and resisgs , after removing ciita targets, in hela cells treated for 6 h with ifnγ.
red arrow indicated tss and direction of transcription.
genes are ranked according to fold induction, indicated in brackets.
b histograms show the percentage of es-indisgs or resisgs with proximal  or distal  binding of stat <dig> and/or irf <dig>  asterisk p <  <dig> , fisher exact test.
c average number of tf binding events at proximal and remote sites at resisgs or es-indisgs.
error bar: sem; asterisk p <  <dig> , anova followed by fisher test
degree of tf binding and responsiveness in hela predicts isg responsiveness in other cell types
many studies have analyzed ifn-gene responsiveness, but a comprehensive analysis of which isgs show broad or cell-type specific expression and, more importantly, the mechanism underlying such variability, has not been attempted.
to assess variability in isg induction, we compiled expression data on isgs from  <dig> different human cell lines or primary cells, including  <dig> listed in additional file 1: table s <dig>  plus hela cells  and brg1-reconstituted sw <dig> cells  <cit> .
across all  <dig> cell lines there were a total of  <dig> isgs, the majority  were exclusively induced in only one cell type, 28% were induced in 2– <dig> cell types, and 11% were induced in most  cell types .
only  <dig> genes were induced in every context and these included stat <dig> and irf <dig>  in line with their apical role in ifnγ signaling.fig.
<dig> link between isg responsiveness in hela cells, responsiveness in other cell types, and stat1/irf <dig> binding.
a percentage of ubiquitous , partially shared , or exclusive  human isgs based on their responsiveness to ifnγ in the indicated number of cell lines.
b percentage of hela es-indisgs which respond in only hela or in more cell types .
for full lists of isgs and es-indisgs see additional file 1: tables s <dig>  s <dig>  c level to which ub, psh or ex es-indisgs are induced in hela cells.
d average number of tf binding at promoter  and remote  sites of the indicated types of es-indisgs.
error bar indicates sem; asterisk p <  <dig> , anova followed by fisher test
we assessed the relationship between broad responsiveness, degree of induction, and stat1/irf <dig> binding.
hela chip-chip data provided stat <dig> and irf <dig> binding information for  <dig> es-indisgs present in all  <dig> expression array datasets.
of these, 3/ <dig> were induced exclusively in hela, 10/ <dig> were induced in 2– <dig> lines and 11/ <dig> were induced in 5– <dig> lines .
of note, genes induced exclusively in hela were up-regulated to a much lower extent than ubiquitously ifnγ-responsive targets .
greater induction of ubiquitously responsive loci was paralleled by a higher density of tf binding at promoter proximal sites .
thus, the level of induction is linked to the degree of stat <dig> and irf <dig> recruitment, and there is an unexpected link between the strength of isg induction in one context  and competency to respond to ifnγ in other contexts.
snps modulate stat <dig> and irf <dig> binding in vitro
defects in ifnγ signaling are linked to a wide range of disorders .
several studies focused on the association between genetic variants and the risk of ifnγ related disorders, but at gene promoters or coding regions of isgs rather than ifnγ responsive enhancers.
within the 16 mb of dna around isgs studied here, there are a total of  <dig>  ×  <dig> dbsnps .
of these,  <dig> dbsnps lay within the  <dig> stat1/irf <dig> peaks.
only  <dig> of these  <dig> dbsnps were listed on the gwas database.
gwas snps do not define all disease associated snps  because gwas genotyping arrays provide low genomic coverage  <cit>  and therefore the  <dig> dbsnps may encompass other da-snps not mapped yet.
none of the  <dig> da-snps  overlapped with a stat1/irf <dig> motif, but  <dig> of the  <dig> dbsnps overlapped with  <dig> stat <dig> and  <dig> irf <dig> motifs .
we studied which of these  <dig> snps affect stat1/irf <dig> binding.
first, we utilized the cisgenome “known motif mapping” program to predict which of the variants may modulate stat1/irf <dig> binding motifs .
cisgenome compares the position weight matrix  in the jaspar core database and creates likelihood scores for the reference or variant allele.
we calculated the fold change in likelihood scores  to assess the predicted relative effect.
at a cutoff of  <dig> -fold, the variant alleles of 34/ <dig> dbsnps were predicted to modulate the binding affinity of  <dig> irf <dig> motifs and  <dig> stat <dig> motifs .
to test these predictions in vitro, we developed an elisa-based dna affinity assay .
canonical stat <dig> or irf <dig> motif-containing biotinylated 33-mers were immobilized on streptavidin-coated 96-well plates.
cell lysates from hela cells exposed to ifnγ for 6 h were mixed with either no or various amounts of wt , mutated , or dbsnp  competitor probes, then added to the immobilized biotinylated probe, and the amount of bound tf determined using anti-stat <dig> or anti-irf <dig> antibody.
we tested  <dig> or  <dig> snps affecting irf <dig> or stat <dig> sites, respectively .
wt irf <dig> and stat <dig> probes exhibited strong binding with low ic50s of  <dig>  ±  <dig>  or  <dig>  ±  <dig>  pmol/well, respectively, whereas control mutated probes had minimal/no effect .
3/ <dig> of the irf <dig> snps decreased affinity  and 1/ <dig> created a strong irf <dig> site  .
the single stat <dig> snp that we tested created a putative binding site, and indeed the t allele of rs <dig> showed high affinity binding .
our chip-chip data indicated that this snp lies within an isolated remote irf <dig> peak, suggesting that the t allele would convert this regulatory element to a dual stat1/irf <dig> enhancer.
in summary, these data show close concordance between the predicted and actual effects of snps on stat <dig> and irf <dig> binding.
thus, it is likely that most of the  <dig> predicted functional snps do in fact alter binding.fig.
<dig> snps modulate stat <dig> and irf <dig> binding in vitro.
a irf <dig>  and: b stat <dig> binding assays.
graphs show stat <dig> and irf <dig> binding signal to immobilized probes in the presence of different concentrations of competitor probes with either the variant or reference allele.
100% binding is that obtained in the absence of competitor.
arrows highlight the affected base .
as indicated, rs <dig> was also assessed in vivo
rs <dig> affects irf <dig> binding in vivo
next we asked if the t allele of rs <dig>  which creates an irf <dig> site in vitro , has this effect in vivo.
this snp lies ~1 kb upstream of the hla-a locus, within an ifnγ-responsive irf <dig> chip-chip peak in hela cells .
to test whether it affects irf <dig> binding in vivo we employed the ebv-transformed lymphoblastic cell line gm <dig>  which is heterozygous for rs <dig> , implying that irf <dig> should only bind to one  allele.
treatment with ifnγ for 6 h induced a  <dig> -fold increase in the total irf <dig> chip-qpcr signal .
snapshot sequencing revealed that this ifnγ-dependent increase was due solely to elevated binding to the t allele .
thus, in silico prediction, an in vitro binding assay, and in vivo allele specific chip all show that the g to t switch creates an irf <dig> binding site .fig.
<dig> rs <dig> modulates irf <dig> binding in vivo.
a chromosomal location of rs <dig> and the alleles , and a genome browser view of the snp, which lies upstream of hla-a and within an ifnγ-induced irf <dig> chip-chip peak in hela cells.
b chip-qpcr of basal and ifnγ-induced irf <dig> recruitment at rs <dig> in gm <dig> ebv transformed lymphocytes.
c electropherogram on left shows snapshot sequencing of chip dna, with peak quantification plotted on the right
discussion
stat <dig> and irf <dig> drive the induction of ifn induced genes, but the extent to which they act collectively is unclear.
we report that most stat <dig> binding  occurs together with irf <dig>  but most irf <dig> binding  is isolated .
binding occurs where there are cognate binding motifs , suggesting that most chip signals reflect direct recruitment.
both proximal and remote stat <dig> and irf <dig> binding is observed at robustly induced isgs, but not at other loci .
in line with the importance of tf occupancy for responsiveness  <cit> , every responsive locus exhibits a mixture of stat <dig> and irf <dig> bound enhancers .
moreover, dual bound enhancers distinguish induced vs resistant isgs, whereas single bound enhancers are found with similar frequency at responsive or non-responsive isgs .
this is not to say, however, that single bound enhancers are irrelevant.
for example, while multiple remote socs <dig> enhancers recruit both tfs, the +50 kb element or promoter are targeted only by stat <dig> or irf <dig>  respectively, yet both are involved in 3d looping .
similarly, while dual stat1/irf <dig> binding occurs at the active ciita promoter, the −50 kb and +59 kb enhancers recruit only stat <dig> or irf <dig>  respectively, yet contribute to 3d looping and are essential for responsiveness  <cit> .
indeed, all the responsive genes we surveyed exhibit a mix of stat1-only, irf1-only, and stat1/irf <dig> dual enhancers .
together, these results suggest that ifnγ-responsiveness requires cooperation between enhancers that bind both or either tf, but that stat1- or irf1-only enhancers are insufficient for gene induction.
irrespective, it is clear that responsive isgs integrate information from both stat <dig> and irf <dig>
previously, we showed that there is a pre-existing 3d structure at the silent ciita locus, generated through looping between enhancers that subsequently recruit stat <dig> and irf <dig> upon ifnγ treatment  <cit> .
this was true even in the absence of brg <dig>  a chromatin remodeling enzyme that is critical to allow stable tf recruitment and thus ifnγ-responsiveness.
subsequent genome-wide analyses indicate that enhancer looping in the poised but silent state is common at inducible loci  <cit> .
we observed the same phenomenon at the ifnγ responsive socs <dig> locus .
potentially, these contacts are mediated by pioneer factors that mark responsive enhancers, but their identity at isgs is unknown.
the data here and other studies show that stat <dig> and irf <dig> can bind some sites in the basal state  <cit> , so in theory, low/unstable binding  could poise isg enhancers.
it would thus be interesting to perform looping studies at isgs in stat1/irf <dig> deficient cells.
it is of note that the degree to which isgs were induced in hela cells predicted whether they were likely to respond to ifnγ in other cells .
thus, the chromatin at these genes is accessible in many contexts.
the factors that mediate this broad poised, open state may also initiate the basal looping at isgs.
over 90% of the disease markers identified in gwas studies lie within the non-protein-coding regions of the genome  <cit> .
these markers correlate with gene expression , and lie within gene regulatory regions .
there is thus considerable interest in identifying snps that influence tf binding and, therefore, gene regulation.
we identified  <dig> snps within stat <dig> or irf <dig> motifs, and in silico assessment predicted that  <dig> may alter binding.
in vitro quantification confirmed these predictions in 5/ <dig> cases, arguing that most of/all the other predictions are accurate.
the availability of a cell line heterozygous for one such snp allowed us to test whether the prediction held up in vivo.
indeed, the t allele of rs <dig>  which lies just upstream of the hla-a locus, bound irf <dig> whereas the g allele did not, as observed in silico and in vitro.
these data serve as proof of principle that in silico prediction is a reliable tool to anticipate the effect of snps on stat <dig> and irf <dig> binding.
CONCLUSIONS
this study provides strong evidence for widespread cooperation between stat <dig> and irf <dig> at isgs, and suggests that in silico predictions reliably predict the effect of nucleotide variants on binding in vivo.

 ifnγ is a pleiotropic cytokine that plays essential roles in antiviral and anticancer immune responses. ifnγ binds to its receptor complex and activates receptor-associated jak kinases, which phosphorylate a substantial fraction of cytoplasmic signal transducer and activator of transcription. phosphorylated stat forms homodimers that translocate to the nucleus and bind ifnγ activation sites. stat recruits histone acetyltransferases and other transcriptional co-activators to acetylate chromatin and facilitate transcription. genomic studies showed that stat binds at promoter proximal and distal sites, suggesting a role in remote gene regulation. indeed, ifnγ induces long range interactions between stat1-bound enhancers and target promoters. interferon regulatory factor is a primary target gene of stat like stat irf also acts as a transcription factor, binding to irf-e motifs and interferon-stimulated response elements. access of both stat and irf to target enhancers requires the swi/snf chromatin remodeling complex to counter prc which uses the histone methyl transferase ezh to deposit h3k27me and block the induction of many other cytokine and cytokine responsive loci. irf functions at the transcription initiation level by facilitating rna pol ii recruitment to isgs promoters. irf also binds to remote enhancers of the ciita locus that loop together to form a 3d interconnected hub with the promoter. indeed, chip-chip and chip-seq studies show that irf binds many remote enhancers, and analysis of transcription factors in k cells revealed that stat1-irf co-binding is a recurring pattern in ifnγ treated cells. notably, stat is essential but not sufficient for gene induction, and both stat and irf are required for the ifnγ-induced expression of ciita, gbp and gp. in addition, stat complexes with irf at the lmp promoter and maintains its constitutive expression. here, we studied the extent of stat and irf cooperation in hela cells within isg-rich chromosomal segments encompassing ~10% of all known isgs. most of these loci responded to ifnγ in hela cells, leaving ~20% resistant isgs. irf binding sites outnumbered stat sites to a large fraction of stat1/irf binding occurred at remote sites and looping studies confirmed the functional role of putative enhancers at the socs locus. most stat binding occurred at or near to irf sites, but irf often bound isolated from stat dual stat and irf but not isolated irf or stat binding was linked to isg responsiveness. finally, several variants affecting stat1/irf motifs induce or impair binding.  diverse gene responses to ifnγ to define patterns of tf binding around isgs, we employed tiling arrays to focus on 16 mb distributed across distinct chromosomal segments with a high density of isgs. nine segments were 1 mb genomic regions on six chromosomes centered on specific ifnγ target genes. two others included a 2 mb segment centered on ciita, and a 5 mb segment covering the complete classical  mb mhc locus and an additional  mb 5′ region including much of the so-called extended mhc class i region. within these regions 25% of the genes are known isgs, ~5× more than the genome-wide isg frequency and ~15-fold above the average isgs density per mb. the total number of refseq genes and ucsc known genes in the 16 mb regions is. of these, 95% were represented on the illumina- human wg-6v array used to assess gene expression. the frequency of refseq genes across the genome is ~6/mb, but most of the chromosomal segments in our study were gene dense, especially at the mhc, psme and ifitm clusters. there are also pseudogenes across the 16 mb, with most located at the mhc cluster. pseudogenes are not represented on the illumina genome wide array we used to study expression.fig.  ifnγ target subclasses. a human chromosome ideograms, drawn using ncbi map viewer, show locations of the studied chromosomal segments. b the genes within these regions were classified as “ifnγ stimulated genes”, “ifnγ-repressed genes” or “other genes” according to our expression array data and on prior studies. known isgs were either induced or resistant in hela cells. genes were further subcategorized according to their robustness and timing of induction/repression. other genes were termed “not-linked to ifnγ”, or “potential isgs” if they were either isg paralogs or exhibited ifnγ-induced tf binding at their promoter. italics indicate the number of ciita targets in each class. c to validate array data, mrna was isolated from hela cells left untreated or exposed to ifnγ for 6 h. rt-pcr was performed on induced or resistant isgs, selected based on array data 6 h after ifnγ treatment. pie diagram shows the percentage of validated genes signaling pathway target loci show cell-specific responsiveness, but the exact tf binding patterns that distinguish induction versus resistance in a specific cell type are unclear. thus, we compared the pattern of stat and irf binding at different gene types. for this we compiled a database of ~ all known isgs using our own and prior transcriptome data. as summarized in fig. 1b, isgs fell into classes depending on whether ifnγ caused induction, no effect, or repression, and whether induction/repression were early or late, and strong or weak. the microarray expression data was validated using reverse transcription and quantitative pcr, which confirmed 83% of indisgs and 87% of resisgs. of all known isgs on the array, genes were es-indisgs, were ls-indisgs, were ew-indisgs or lw-indisgs, and were resisgs. es-indisgs were distributed at an average density of /mb within the studied regions. the highest density was observed at the ifit and gbp clusters with an average of es-indisgs/mb. no genes were repressed at the early 6 h time point, while and were strongly or weakly repressed at later times, respectively, suggesting indirect regulation of irgs. the remaining genes that were not ifnγ-responsive either in this or any prior study were termed “other genes”. in summary, known isgs fall into induced and resistant subclasses in hela cells, providing a useful system to define stat and irf binding patterns linked to responsiveness. validation of stat and irf chip-chip analyses chip-chip was used to locate stat and irf sites at promoter proximal and distal sites of the genes of each category. stat and irf chips were performed on chromatin from hela cells that were either untreated or exposed to ifnγ for 6 h. hybridization intensities were normalized to internal standards and values from quadruplicate spots were averaged. significantly different intensities between chip dna and input dna samples in three biological replicates were determined using the wilcoxon rank sum test. peaks representing the significantly enriched dna regions where the ratio of chip to input dna was ≥  -fold were visualized in the ucsc browser and plotted on a log scale. only stat and irf peaks were identified in untreated cells, rising to and post-ifnγ treatment, respectively. browser views are shown in additional file 2: figure s and can be visualized at http://research.lunenfeld.ca/ifny. chip-qpcr validated 91% and 96% of stat and irf chip-chip peaks, respectively. we compared stat binding at 6 h with ifnγ-induced stat binding after 30 min, also assessed in hela cells. in the 16 mb of dna assessed here, the latter study detected stat sites, of which overlapped with the stat sites we detected.fig.  stat and irf binding patterns. arbitrarily selected chip-chip stat, irf sites were re-examined by chip-qpcr on chromatin from hela cells with no or 6 h of ifnγ treatment. 91% of peaks were validated in both cases. the frequency of consensus motifs identified by jasper within stat or irf peaks is indicated basal tf binding unphosphorylated stat has roles in regulating isgs days after ifn treatment, but its role in untreated cells is less clear, although stat nuclear cytoplasmic shuttling occurs even in untreated cells. basal stat binding is linked to the nuclear localization of unphosphorylated stat and contributes to the constitutive expression of some targets. irf is also expressed to low levels in unstimulated hela cells and it cooperates with stat to maintain low basal expression levels of lmp. in addition, there is also some stat phosphorylation in untreated cells that contributes to basal activity, as shown elegantly by knockin studies in mice. we detected stat and irf binding sites in untreated cells, accounting for and % of induced sites, respectively. our data accords with another chip-chip analysis of stat binding which reported that % of ifnγ-induced stat sites in hela cells treated for 30 min with ifnγ are occupied in uninduced cells. further analysis suggests that basal tf binding detected here is physiologically relevant. of the genes with basal stat or irf binding, we assessed by microarray and/or rt-pcr and all were expressed in untreated cells. in contrast, of randomly selected isgs that lacked basal tf binding, only were basally expressed. indeed, constitutive expression of psmb and tap requires constitutive irf binding. in addition, several loci with basal tf binding are in paralogous gene clusters suggesting conservation of high affinity binding sites during gene duplication. a high fraction of the irf basally occupied sites possessed irf binding motifs. thus, our data supports the notion that basal binding of stat and irf is physiologically relevant. remote ifnγ activated enhancers are common at isgs in ifnγ treated cells, 54% of stat and 44% of irf peaks were within 5 kb of the transcription start site of all known genes on the array. adding other databases, including predicted genes, raised the fraction to 64% for stat and 57% for irf. of equal numbers of randomly generated sites, the proportion at <5 kb from gene starts was much lower. thus stat binding is slightly skewed to promoter proximal sites, while irf binding is slightly biased toward remote sites.fig.  proximal vs. distal stat and irf binding. a the percentage of ifnγ-induced stat or irf binding sites or randomly generated controls at proximal or distal sites relative to the tss of known genes. b irf to stat ratio at proximal and remote sites prior analysis of chromatin modification and looping at ciita, and partial looping analysis at locus support the idea that remote sites are functionally important. to further test this notion we performed additional assessment of socs a key negative regulator of ifnγ signaling that is responsive to ifns and other immune signaling pathways. socs responds to ifnγ in hela cells. chip-chip data exposed ifnγ-induced stat and/or irf peaks ± 100 kb of the socs tss. chip-qpcr analysis verified binding at the socs promoter, and at + + − − − and −72 kb. a negative region at −63 kb was also validated. in favor of functional relevance of proximal and remote sites, we detected constitutive histone h acetylation and/or h4ac at psocs, − and −55 kb, and ifnγ induced acetylation at psocs and the remote sites but not at the irrelevant −63 kb site. these constitutive and inducible events paralleled recruitment of the hats cbp and/or p. h3k4me also marks enhancers, and constitutive h3k4me was detected at psocs −3 kb, −55 kb, matching constitute histone acetylation, and also at the −72 kb enhancer, which contacts the promoter. ifnγ treatment did not further increase methylation at these sites, but did induce h3k4me at +50 kb, +15 kb and −72 kb. finally, we detected constitutive pol ii recruitment at psocs and −55 kb but not at the other tf binding or negative control sites. after ifnγ treatment, pol ii recruitment increased at psocs +15 kb and −55 kb. association with the +15 kb element may reflect ifnγ-induced promoter looping.fig.  stat and irf binding at socs a rt-qpcr for socs mrna in hela cells treated with ifnγ for or 6 h. data are in arbitrary units relative to β-actin levels. b chip-chip maps of stat and irf binding across the socs locus treated as in a. black arrowheads indicate tf binding sites of interest, with distances from the tss in kb. the tf-free −63 kb site is used as a negative control in c. c chip-qpcr analysis of the basal and ifnγ-induced histone modifications or recruitment of the indicated factors. marks show significant differences in the indicated comparisons chromosome conformation capture revealed both constitutive and ifnγ-induced contacts between the promoter and remote stat and/or irf sites at ciita. to examine looping at socs we studied six ecori or four ncoi fragments. of a total of possible interactions, we studied previously and assessed an additional in this study. as expected, no interaction was observed between fragments containing the promoter and irrelevant sites at − − or +70 kb, either before or after cytokine exposure. however, between suspected functional elements, we detected a total of loops of all putative interactions in the basal state, and each of these loops was enhanced after ifnγ treatment and was accompanied by a new interaction between the + and −72 kb enhancers that lie 122 kb apart. our data suggest that the socs locus is basally present in a mega looping complex that becomes more compact and involves more inter-element interactions after ifnγ treatment. together, the chip and 3c data show that stat and irf binding is linked to extensive chromatin modifications and looping.fig.  basal and ifnγ-induced looping at socs a a schematic view of the socs locus. circles indicate the socs promoter, putative remote enhancers, and negative control sites, with distances from the tss indicated above in kb, while fragments used in 3c assays with primers are shown below. b cross linking frequencies between the promoter and remote sites across the socs locus. quantitative 3c was performed with chromatin from hela cells left untreated or exposed to ifnγ for 6 h. bar graphs show the crosslinking frequency of a selected number of interactions. underlined interactions were published previously. marked interactions are significantly different at the indicated comparisons. c summary of looping events. interacting sites and dna strands are colored as in a. stat1/irf and hac/h3k4me are also depicted unusual irf distribution at mhc loci as noted earlier, irf exceeded stat sites by ~twofold, but this varied at some regions, most notably at the mhc class i locus where the ratio was:. the ratio was particularly skewed at the extended versus classic mhc class i region. of all mhc class i irf sites were within 5 kb of known gene starts and within 5 kb of pseudogenes, giving a total of 77% promoter proximal sites, which is higher than the 44% at all loci. however, whereas 66% of irf sites were promoter-proximal in the classical mhc class i region, this dropped to only 6% at the extended mhc class i region, and was low even after including pseudogenes, leaving an unusually high fraction of remote irf sites. thus, irf seems to play a broader role than stat at the mhc class i cluster, primarily at proximal elements in the classic region, but at remote elements in the extended region. stat and irf induce ciita, the master regulator of mhc class ii expression. the number of stat and irf sites was typically very low in the mhc class ii region. out of mhc class ii genes, were resistant to ifnγ in hela cells, responded only after 24 h, a time of maximum production of ciita, and were es-indisgs. with the exception of dob, none of the resistant or late-induced genes exhibited stat or irf promoter binding. however, of the es-indisgs, two had promoter proximal irf binding while dma had irf binding fairly near its promoter. thus irf may cooperate with ciita at a subset of mhc class ii promoters. others reported ciita-independent induction of mhc class ii genes, which may, therefore, involve irf stat and irf binding is enriched at robustly induced isgs as discussed, isgs fell into classes depending on whether ifnγ caused induction, no effect, or repression, and whether induction/repression were early or late, and strong or weak. we plotted the distribution of stat and irf binding sites relative to all gene classes. binding sites were assigned to the nearest gene class, designated as proximal or distal when ≤ or >5 kb from the tss, respectively, and were compared to randomly chosen sites equaling number of stat1 + irf sites. we also calculated the tf enrichment ratio in which the % distribution of tfs at proximal and distal locations was normalized to the % distribution of random sites. a binding frequency twice that of random sites was assigned as an arbitrary minimum threshold.fig.  enrichment of stat/irf at strong early induced isgs. a histogram shows the percent of stat irf or an equal number of randomly generated sites at proximal or distal sites of isgs, irgs, and other genes. the number of sites in each category is indicated below each bar. “random frequency”: distribution of randomly generated sites. b distribution of tfs normalized to that of random controls. unshaded region indicates ter > twofold stat and irf binding sites were most highly associated with robustly induced ifnγ targets. this applied when stat or irf were considered together, separately, and at proximal or remote locations. consistent with this finding, weakly induced genes had fewer binding events and lower ters. of other genes, a total of only had stat and irf proximal peaks, mostly located at the mhc and rt-qpcr confirmed no induction at 10/ of these genes. ifnγ enhancers loop over large distances at ciita and socs, so proximal and distal enhancers nearest to other genes may target neighboring isgs. in summary, the data indicate a clear bias of stat and irf binding at rapidly and robustly induced isgs, but not other gene classes. isolated or dual stat and irf recruitment is directed by binding motifs next we compared the fraction of isolated or dual stat1/irf binding events. of a total of discrete tf binding regions, 16% exhibited stat binding alone, of which almost half were proximal; 61% exhibited only irf binding, of which 42% were proximal; and 23% showed overlap, of which slightly more than half were proximal. randomly generated sites showed negligible overlap, but dual stat1/irf binding represented more than half of all stat sites and about a quarter of irf peaks. fewer overlapping irf peaks reflect their twofold excess relative to stat peaks. thus, stat preferentially binds with irf at ifnγ enhancers, whereas most irf sites are not co-localized with stat fig.  stat and irf binding correlates with binding motifs. a percent distribution of isolated stat or irf or dual stat1 + irf binding at proximal or remote sites of known gene promoters. b tf binding sites were classified into subclasses, then mapped motifs using cisgenome’s “known motif mapping” program. sets of equal numbers of randomly generated “peaks” were used to define the background occurrence of stat and irf motifs. asterisk indicates significant difference between true and random peaks. c ratio of stat1/irf motifs at different categories of peaks jasper analysis of irf and stat peak regions revealed that the cognate binding motif was observed at a statistically significant level relative to equal numbers of random peaks. 60% of isolated stat peaks had a stat motif, and only 30% had an irf motif, while 70% of isolated irf peaks possessed an irf motif, but only 20% had a stat motif. a strong correlation existed between stat1/irf binding and the presence of the corresponding motifs. indeed 40% of dual stat1/irf sites had both binding motifs, whereas there were none at equal numbers of randomly generated sites. dual sites which have only a stat or irf binding motif may reflect protein–protein interaction or dna looping as seen at the socs and ciita loci. in summary, dna sequence directs isolated or dual stat1/irf binding in ifnγ treated cells. dual stat and irf targeted enhancers distinguish responsive from resistant isgs comparing inducible isgs in our array study with ~all known isgs in a large database revealed resistant isgs in hela cells. there were far fewer stat1/irf binding events at res-isgs vs ind-isgs, and the ter at res-isgs was low, and similar to that at other genes. there was near ubiquitous association of both stat and irf at es-indisg promoters, but they were virtually absent at res-isg promoters. to quantify the types of tf binding events, we plotted the frequency of genes with at least one binding event within or beyond 5 kb, and the density of each type of binding event per gene. isolated tf binding did not discriminate the two gene classes, whereas there was significantly more dual stat and irf binding at esisgs, at both proximal and distal sites. thus, cooperation between stat and irf plays a central role in mediating ifnγ responsiveness.fig.  stat/irf recruitment at es-indisgs vs res-isgs. a map of isolated and dual stat and irf binding ± 100 kb of the tss of es-indisgs and resisgs, after removing ciita targets, in hela cells treated for 6 h with ifnγ. red arrow indicated tss and direction of transcription. genes are ranked according to fold induction, indicated in brackets. b histograms show the percentage of es-indisgs or resisgs with proximal or distal binding of stat and/or irf asterisk p < , fisher exact test. c average number of tf binding events at proximal and remote sites at resisgs or es-indisgs. error bar: sem; asterisk p < , anova followed by fisher test degree of tf binding and responsiveness in hela predicts isg responsiveness in other cell types many studies have analyzed ifn-gene responsiveness, but a comprehensive analysis of which isgs show broad or cell-type specific expression and, more importantly, the mechanism underlying such variability, has not been attempted. to assess variability in isg induction, we compiled expression data on isgs from different human cell lines or primary cells, including listed in additional file 1: table s plus hela cells and brg1-reconstituted sw cells. across all cell lines there were a total of isgs, the majority were exclusively induced in only one cell type, 28% were induced in 2– cell types, and 11% were induced in most cell types. only genes were induced in every context and these included stat and irf in line with their apical role in ifnγ signaling.fig.  link between isg responsiveness in hela cells, responsiveness in other cell types, and stat1/irf binding. a percentage of ubiquitous, partially shared, or exclusive human isgs based on their responsiveness to ifnγ in the indicated number of cell lines. b percentage of hela es-indisgs which respond in only hela or in more cell types. for full lists of isgs and es-indisgs see additional file 1: tables s s c level to which ub, psh or ex es-indisgs are induced in hela cells. d average number of tf binding at promoter and remote sites of the indicated types of es-indisgs. error bar indicates sem; asterisk p < , anova followed by fisher test we assessed the relationship between broad responsiveness, degree of induction, and stat1/irf binding. hela chip-chip data provided stat and irf binding information for es-indisgs present in all expression array datasets. of these, 3/ were induced exclusively in hela, 10/ were induced in 2– lines and 11/ were induced in 5– lines. of note, genes induced exclusively in hela were up-regulated to a much lower extent than ubiquitously ifnγ-responsive targets. greater induction of ubiquitously responsive loci was paralleled by a higher density of tf binding at promoter proximal sites. thus, the level of induction is linked to the degree of stat and irf recruitment, and there is an unexpected link between the strength of isg induction in one context and competency to respond to ifnγ in other contexts. snps modulate stat and irf binding in vitro defects in ifnγ signaling are linked to a wide range of disorders. several studies focused on the association between genetic variants and the risk of ifnγ related disorders, but at gene promoters or coding regions of isgs rather than ifnγ responsive enhancers. within the 16 mb of dna around isgs studied here, there are a total of  ×  dbsnps. of these, dbsnps lay within the stat1/irf peaks. only of these dbsnps were listed on the gwas database. gwas snps do not define all disease associated snps because gwas genotyping arrays provide low genomic coverage and therefore the dbsnps may encompass other da-snps not mapped yet. none of the da-snps overlapped with a stat1/irf motif, but of the dbsnps overlapped with stat and irf motifs. we studied which of these snps affect stat1/irf binding. first, we utilized the cisgenome “known motif mapping” program to predict which of the variants may modulate stat1/irf binding motifs. cisgenome compares the position weight matrix in the jaspar core database and creates likelihood scores for the reference or variant allele. we calculated the fold change in likelihood scores to assess the predicted relative effect. at a cutoff of -fold, the variant alleles of 34/ dbsnps were predicted to modulate the binding affinity of irf motifs and stat motifs. to test these predictions in vitro, we developed an elisa-based dna affinity assay. canonical stat or irf motif-containing biotinylated 33-mers were immobilized on streptavidin-coated 96-well plates. cell lysates from hela cells exposed to ifnγ for 6 h were mixed with either no or various amounts of wt, mutated, or dbsnp competitor probes, then added to the immobilized biotinylated probe, and the amount of bound tf determined using anti-stat or anti-irf antibody. we tested or snps affecting irf or stat sites, respectively. wt irf and stat probes exhibited strong binding with low ic50s of  ±  or  ±   pmol/well, respectively, whereas control mutated probes had minimal/no effect. 3/ of the irf snps decreased affinity and 1/ created a strong irf site. the single stat snp that we tested created a putative binding site, and indeed the t allele of rs showed high affinity binding. our chip-chip data indicated that this snp lies within an isolated remote irf peak, suggesting that the t allele would convert this regulatory element to a dual stat1/irf enhancer. in summary, these data show close concordance between the predicted and actual effects of snps on stat and irf binding. thus, it is likely that most of the predicted functional snps do in fact alter binding.fig.  snps modulate stat and irf binding in vitro. a irf and: b stat binding assays. graphs show stat and irf binding signal to immobilized probes in the presence of different concentrations of competitor probes with either the variant or reference allele. 100% binding is that obtained in the absence of competitor. arrows highlight the affected base. as indicated, rs was also assessed in vivo rs affects irf binding in vivo next we asked if the t allele of rs which creates an irf site in vitro, has this effect in vivo. this snp lies ~1 kb upstream of the hla-a locus, within an ifnγ-responsive irf chip-chip peak in hela cells. to test whether it affects irf binding in vivo we employed the ebv-transformed lymphoblastic cell line gm which is heterozygous for rs, implying that irf should only bind to one allele. treatment with ifnγ for 6 h induced a -fold increase in the total irf chip-qpcr signal. snapshot sequencing revealed that this ifnγ-dependent increase was due solely to elevated binding to the t allele. thus, in silico prediction, an in vitro binding assay, and in vivo allele specific chip all show that the g to t switch creates an irf binding site.fig.  rs modulates irf binding in vivo. a chromosomal location of rs and the alleles, and a genome browser view of the snp, which lies upstream of hla-a and within an ifnγ-induced irf chip-chip peak in hela cells. b chip-qpcr of basal and ifnγ-induced irf recruitment at rs in gm ebv transformed lymphocytes. c electropherogram on left shows snapshot sequencing of chip dna, with peak quantification plotted on the right discussion stat and irf drive the induction of ifn induced genes, but the extent to which they act collectively is unclear. we report that most stat binding occurs together with irf but most irf binding is isolated. binding occurs where there are cognate binding motifs, suggesting that most chip signals reflect direct recruitment. both proximal and remote stat and irf binding is observed at robustly induced isgs, but not at other loci. in line with the importance of tf occupancy for responsiveness, every responsive locus exhibits a mixture of stat and irf bound enhancers. moreover, dual bound enhancers distinguish induced vs resistant isgs, whereas single bound enhancers are found with similar frequency at responsive or non-responsive isgs. this is not to say, however, that single bound enhancers are irrelevant. for example, while multiple remote socs enhancers recruit both tfs, the +50 kb element or promoter are targeted only by stat or irf respectively, yet both are involved in 3d looping. similarly, while dual stat1/irf binding occurs at the active ciita promoter, the −50 kb and +59 kb enhancers recruit only stat or irf respectively, yet contribute to 3d looping and are essential for responsiveness. indeed, all the responsive genes we surveyed exhibit a mix of stat1-only, irf1-only, and stat1/irf dual enhancers. together, these results suggest that ifnγ-responsiveness requires cooperation between enhancers that bind both or either tf, but that stat1- or irf1-only enhancers are insufficient for gene induction. irrespective, it is clear that responsive isgs integrate information from both stat and irf previously, we showed that there is a pre-existing 3d structure at the silent ciita locus, generated through looping between enhancers that subsequently recruit stat and irf upon ifnγ treatment. this was true even in the absence of brg a chromatin remodeling enzyme that is critical to allow stable tf recruitment and thus ifnγ-responsiveness. subsequent genome-wide analyses indicate that enhancer looping in the poised but silent state is common at inducible loci. we observed the same phenomenon at the ifnγ responsive socs locus. potentially, these contacts are mediated by pioneer factors that mark responsive enhancers, but their identity at isgs is unknown. the data here and other studies show that stat and irf can bind some sites in the basal state, so in theory, low/unstable binding could poise isg enhancers. it would thus be interesting to perform looping studies at isgs in stat1/irf deficient cells. it is of note that the degree to which isgs were induced in hela cells predicted whether they were likely to respond to ifnγ in other cells. thus, the chromatin at these genes is accessible in many contexts. the factors that mediate this broad poised, open state may also initiate the basal looping at isgs. over 90% of the disease markers identified in gwas studies lie within the non-protein-coding regions of the genome. these markers correlate with gene expression, and lie within gene regulatory regions. there is thus considerable interest in identifying snps that influence tf binding and, therefore, gene regulation. we identified snps within stat or irf motifs, and in silico assessment predicted that may alter binding. in vitro quantification confirmed these predictions in 5/ cases, arguing that most of/all the other predictions are accurate. the availability of a cell line heterozygous for one such snp allowed us to test whether the prediction held up in vivo. indeed, the t allele of rs which lies just upstream of the hla-a locus, bound irf whereas the g allele did not, as observed in silico and in vitro. these data serve as proof of principle that in silico prediction is a reliable tool to anticipate the effect of snps on stat and irf binding.  this study provides strong evidence for widespread cooperation between stat and irf at isgs, and suggests that in silico predictions reliably predict the effect of nucleotide variants on binding in vivo. 
we show that irf binds at proximal or distant isg sites twice as often as stat increasing to sixfold at the mhc class i locus. dual binding sites at remote or proximal enhancers distinguished isgs that were responsive to ifnγ versus cell-specific resistant isgs, which showed fewer and mainly single binding events. we show that in silico prediction of snp effects accurately reflects altered binding both in vitro and in vivo. surprisingly, inducibility in one cell type predicted isg-responsiveness in other cells. these data reveal broad cooperation between stat and irf explain cell type specific differences in isg-responsiveness, and identify genetic variants that may participate in the pathogenesis of immune disorders.
 stat and irf collaborate to induce interferon-γ stimulated genes, but the extent to which they act alone or together is unclear. the effect of single nucleotide polymorphisms on in vivo binding is also largely unknown.  we show that irf binds at proximal or distant isg sites twice as often as stat increasing to sixfold at the mhc class i locus. stat almost always bound with irf while most irf binding events were isolated. dual binding sites at remote or proximal enhancers distinguished isgs that were responsive to ifnγ versus cell-specific resistant isgs, which showed fewer and mainly single binding events. surprisingly, inducibility in one cell type predicted isg-responsiveness in other cells. several dbsnps overlapped with stat and irf binding motifs, and we developed methodology to rapidly assess their effects. we show that in silico prediction of snp effects accurately reflects altered binding both in vitro and in vivo.  these data reveal broad cooperation between stat and irf explain cell type specific differences in isg-responsiveness, and identify genetic variants that may participate in the pathogenesis of immune disorders. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. http://dx.doi.org/ /501100000024canadian institutes of health researchmop 111004bremner rod http://dx.doi.org/ /501100000015canadian cancer society research institute703079bremner rod issue-copyright-statement© the author 2017 