BACKGROUND
esophageal cancer  comprises of heterogeneous groups of tumors that differ in pathogenesis and etiological and pathological features.
ec ranks among the ten most frequent cancers worldwide with regionally dependent incidence rates and histological subtypes  <cit> .
statistics indicate that ec mortality rates are very similar to incidence rates due to the relatively late stage of diagnosis, the poor efficacy of treatment  <cit> , and the poor prognosis of ec result in a five year survival rate of 5-20%  <cit> .
the most recurrent histological subtype is esophageal squamous cell carcinoma , followed by adenocarcinoma   <cit> .
escc has a worse prognosis than adc due to the primary escc tumor being in contact with the tracheobronchial tree in 75% of cases, while adc is found below the tracheal bifurcation in 94% of cases  <cit> .
the striking 3-4: <dig> male predominance of escc was previously ascribed to the different patterns of smoking and drinking between males and females.
however, more recently bodelon et al.
reported that current users of estrogen and progestin therapy show reduced risk of escc  <cit> .
previous research supports this finding as several groups have reported estrogen induced gene regulation in esophageal squamous cell carcinoma  and barrett’s esophageal adenocarcinoma   <cit> .
moreover, wang et al.
specifically demonstrated that serum level of estradiol of escc patients from the high risk areas were significantly lower compared to healthy controls from both high and low risk areas and suggested the use of estrogen analogues as promising targets for the prevention and treatment of escc  <cit> .
additionally, published scientific data shows that estrogen induces an inhibitory effect on esophageal carcinoma by activating the estrogen receptor   <cit> .
the activated er functions as a transcription factor that binds to a specific tfbs known as the estrogen response element   <cit> .
there are two er subtypes, erα and erβ, that are encoded on human chromosomes 6q <dig>   <cit>  and chromosome 14q22- <dig>  <cit> , respectively.
both erα and erβ bind to the same eres, but erα does so with an approximately twofold higher affinity  <cit> .
additionally, erβ is known to bind to erα suppressing erα function  <cit> .
the inverse biological effect associated with the two er subtypes has been confirmed to exist in escc  <cit> .
this collation of research findings suggests that the estrogen based therapies which have improved survival rates of cancer types such as: prostate cancer  <cit> , lung cancer  <cit> , brain and spinal cord tumors  <cit> , and breast cancer  <cit> , may also improve the outcome of escc.
our current study aims at identifying estrogen responsive genes by using escc as a model.
potentially, such genes could be affected by estrogen.
we propose a methodology that provides insight into the underlying regulation of estrogen responsive escc genes.
we mapped eres to the promoters of  <dig> escc genes using the dragon ere finder version  <dig>    <cit> .
the  <dig> escc genes were divided into two groups: 1) genes whose promoters contain predicted eres, and 2) genes lacking predicted eres.
these two gene groups were further divided into those known to be experimentally confirmed as estrogen responsive and those that are not.
to accomplish this the  <dig> escc genes were cross checked against two databases housing estrogen responsive genes, namely kberg  <cit>  and ertargetdb  <cit>  databases.
at the time of analysis the kberg database contained  <dig> experimentally confirmed estrogen-responsive genes.
the ertargetdb database contained:   <dig> genes with  <dig> experimentally verified ere direct binding sites and  <dig> experimentally verified ere tethering sites;   <dig> genes identified via chip-on-chip assay for estrogen binding and   <dig> genes from gene expression microarrays, all of which were included in this study.
however, this study excludes the  <dig> computationally predicted estrogen responsive genes included the ertargetdb, database.
thus this study defines estrogen responsive genes as genes that can be modulated by an external estrogen source.
we classified the  <dig> escc genes into the following four categories :
c1/escc genes with predicted eres in their promoters and known as estrogen responsive,
c2/escc genes with predicted eres in their promoters but not known as estrogen responsive,
c3/escc genes having no predicted eres in their promoters, but known as estrogen responsive,
c4/escc genes having no predicted eres in their promoters and not known as estrogen responsive.
we used these categories to develop a methodology for the identification of co-localized tfbss  that characterize the promoters of the known estrogen responsive gene sets ) as opposed to the background set .
these significant ctfbss were mapped to the promoter sequences of the candidate estrogen responsive escc genes in class c <dig>  the genes in class c <dig> whose promoters contained such ctfbss were singled out as novel putative estrogen responsive genes in escc .
to the best of our knowledge our study provided the first computational large-scale analysis of the transcription potential of estrogen responsive escc genes and suggests important regulatory potential of these genes.
although we used escc as a model, the developed system biology based methodology has a potential to identify hormone responsive genes using other hormone-affected diseases, and provides a framework for identifying hormone responsive genes based on complex diseases.
RESULTS
the prediction and identification of putative estrogen responsive genes in escc
a sequential two-step process was used to predict and verify estrogen responsive genes in escc:
eres were mapped to the promoters of escc genes, and
based on the experimental evidence the genes in  were classified as being estrogen responsive or not.
the  <dig> escc genes were extracted from the dragon database of genes implicated in esophageal cancer   <cit> .
the  <dig> putative promoters of these escc genes  were extracted from the fantom <dig> cage tag data  <cit>  and analyzed for the presence of eres via the dragon ere finder version  <dig>    <cit> .
eres were mapped to  <dig> promoter sequences that correspond to  <dig> escc genes.
<dig> escc genes had no eres mapped to the promoter sequences.
lists of genes that have been experimentally validated to be responsive to estrogen as indicated in the kberg  <cit>  and ertargetdb  <cit>  databases were used to confirm which escc genes are responsive to estrogen .
of the  <dig> genes with predicted eres,  <dig> %  are known to be estrogen responsive , while  <dig> %  were new candidate estrogen responsive genes .
eres did not map to  <dig> escc genes of which  <dig> %  are known to be estrogen responsive  .
tfbs analysis of estrogen responsive genes in escc
tfbs analysis entailed the following three steps:  mapping the tfbss matrix models to the promoters of all escc genes,  determining the ctfbss significantly over-represented in class  relative to class c <dig> , and  mapping significantly over-represented ctfbss determined in  to promoters of genes in class c <dig>  in , we required that at least four of the  <dig> ctfbss map the promoters of each gene in class c <dig>  this threshold corresponds to the maximum difference in the number of genes with these ctfbss in the positive set ) as compared to the background set  .
all class c <dig> genes that have such ctfbss in their promoters  we considered as new candidate estrogen responsive escc genes since they have in their promoters both: a/mapped eres, and b/ctfbss characteristic of escc genes that are responsive to estrogen.
this increases confidence that these  <dig> escc genes are responsive to estrogen since due to the similar regulatory potential with estrogen-responsive genes, these genes have higher chance to express when estrogen-responsive genes are expressing and additionally they have ere that potentially bind ers.
tfbs matrices mapped to the promoters of  <dig> escc genes
the transfac mammalian matrix models of tfbss  were mapped to the promoters of estrogen responsive genes in escc using matchtm <cit> .
of the  <dig> matrices mapped,  <dig> mapped to the promoters of the  <dig> escc genes at  <dig>  positions, not considering strand .
ctfbss significantly over-represented in class  as opposed to class c4
we developed a methodology to identify the ctfbss significantly over-represented in the known estrogen responsive gene set ) relative to the background set  .
each tfbs was ranked using a method that ensures that the top ranked tfbss were not only over-represented but also more likely to be co-localized within the promoters.
in order to reduce the search space for the potentially significant co-localized tfbss, a heuristic approach was applied where the  <dig> tfbss with the lowest p-value  were selected for subsequent analysis.
every possible combination of ctfbss that includes some of the  <dig> tfbs were determined.
the significant ctfbss with a p-value  <  <dig>  were selected.
we identified  <dig> significant ctfbss consisting of  <dig> doublet ctfbs,  <dig> triplet ctfbs,  <dig> 4-element ctfbs,  <dig> 5-element ctfbs and  <dig> 6-element ctfbs .
the  <dig> tfbss that make these ctfbss are determined by the following transfac identifiers v$elk1_ <dig>  v$cets1p54_ <dig>  v$yy1_ <dig>  v$gata3_ <dig>  v$taxcreb_ <dig>  v$freac4_ <dig>  v$areb6_ <dig>  v$creb_q <dig>  v$e2a_q <dig> and v$ebox_q6_ <dig>  of the  <dig> ctfbss, eight combinations were completely absent in the background set .
the most significant ctfbss  was not present in class c <dig>  but mapped  <dig> times to the promoters of genes in class c <dig> and  <dig> times to the promoters of genes in class c <dig>
<dig> significant ctfbss consisting of  <dig> doublet ctfbs,  <dig> triplet ctfbs,  <dig> 4-element ctfbs,  <dig> 5-element ctfbs and  <dig> 6-element ctfbs were identified.
<dig> ctfbss used to increase confidence in a subset of the new candidate estrogen responsive genes in class c2
we mapped the  <dig> significant ctfbss to the promoters of the genes in class c <dig>  c <dig>  c <dig> and c <dig> thereby generating  <dig>   <dig>   <dig> and  <dig> predictions of ctfbss, respectively.
this result for the mapping of ctfbss to the promoters of all categories indicates that multiple ctfbss are present in the promoters of genes.
moreover, these multiple ctfbss are more dominant in genes from class  known to be responsive to estrogen, as well as genes with ere predictions in their promoters .
consequently, we applied a threshold that each gene promoter must contain at least four of the significant ctfbss, as this threshold defines the maximum difference in the number of genes that contain such ctfbss between the known estrogen responsive gene set ) relative to the background set  .
it was determined that at least four of the significant ctfbss were present in  <dig> %  of the genes in class c <dig> ,  <dig> %  of the genes in class c <dig> ,  <dig> %  of the genes in class c <dig>  and  <dig> %  of the genes in class c <dig>  .
an overview of the regulatory effects of the ctfbss on the  <dig> genes in class c2a is shown in figure  <dig>  this figure illustrates each association in class c2a, in the form of a color dot in a heat map format using tmev  <cit> .
the heat map clearly depicts gene clusters based on the ctfbss common to the promoters of multiple genes in class c2a.
moreover, a review of the recently published scientific literature reveals that 47%  of the c2a genes have now been shown experimentally to be estrogen responsive.
these  <dig> genes include muc5b <cit> , mmp <dig> <cit> , loxl <dig> <cit> , actn <dig> <cit> , dnmt <dig> <cit> , gpr <dig> <cit> , muc <dig> <cit> , wnt7b <cit> , bmp <dig> <cit> , gpx <dig> <cit> , cdc25b <cit> , nfκb <dig> <cit> , prdm <dig> <cit> , mdm <dig> <cit>  and timp <dig> <cit> .
discussion
in this study, we propose a methodology aimed at providing an insight into the underlying transcription regulatory potential related to genes’ response to estrogen in escc.
in this systems biology study, we combined information obtained from several databases, genomic sequences of promoters of relevant genes, and analysis of transcription regulation potential of these genes to infer if the genes are estrogen responsive.
two computational components are used to suggest escc genes responsive to estrogen: 1) the ere prediction , and 2) predicted ctfbss that characterize the promoters of known estrogen responsive escc genes .
these ctfbss were mapped to the promoters of escc genes not being known to be responsive to estrogen, but having ere predictions in their promoters.
in this way we increased the confidence that the escc genes with ere predictions are responsive to estrogen since they, in addition to eres, also contain ctfbss characteristic of estrogen responsive escc genes.
escc genes predicted to be responsive to estrogen
carroll et al.
has reported that er binds selectively to a limited number of sites, majority of which are distant from the transcriptional start sites of regulated genes and that direct er binding requires the presence of forkhead factor  binding in close proximity  <cit> .
however, several computational approaches has been undertaken to identify target genes based on the presence of eres in the proximal promoter regions  <cit> .
bourdeau et al.
in particular screened for eres that were conserved in the human and mouse genome and identified  <dig> gene proximal eres of which several were validated as genuine er interaction sites  <cit> .
this analysis has also been restricted to the proximal promoter region due to computational limitations and regulatory tfs binding closer to the transcription start site.
eres were mapped to the promoters of  <dig> escc genes using the dragon ere finder version  <dig>  .
bajic et al.
have demonstrated that this ere locator predicts known ere and estrogen responsive genes at a sensitivity of  <dig> .
we further identified which of the escc genes are known to be responsive to estrogen using the kberg  <cit>  and ertargetdb  <cit>  databases.
of the  <dig> predicted estrogen responsive genes,  <dig> %  are known to be estrogen responsive, while  <dig> %  were novel putative estrogen responsive genes.
these  <dig> genes lay the foundation for increasing insights into the molecular events triggered by estrogen via an ere dependant mode of regulation in escc.
eres did not map to  <dig> escc genes of which  <dig> %  are known to be responsive to estrogen.
the promoters of these  <dig> gene did not contain an ere motif, but the genes are known to be responsive to estrogen.
the response to estrogen of these genes may be through the interactions of ers with other transcription factors forming complexes that do not require the presence of eres  <cit> .
it is also possible that the ere models are not sufficiently good to predict eres in these promoter regions.
our analysis generated four gene categories : class c <dig> , class c <dig> , class c <dig> , and class c <dig> .
we found that the four gene categories had a different number of enriched pathways using kyoto encyclopedia of genes and genomes  .
however, in each category the more general kegg pathway “pathways in cancer”  enriched with genes forming the gene sets.
other more specialized and equally important pathways show enrichment with genes forming certain categories.
category  <dig> genes are highly enriched in the pathways such as “transcriptional misregulation in cancer” , “small cell lung cancer” , “melanoma” .
category  <dig> genes are highly enriched in the pathways e.g.
“p <dig> signaling pathway” , “bladder cancer” , “small cell lung cancer” .
category  <dig> genes are highly enriched for many pathways, e.g.
“prostate cancer” ,“colorectal cancer” , “small cell lung cancer” , “chronic myeloid leukemia” , “endometrial cancer” , etc.
category  <dig> genes is additionally highly enriched in the “bladder cancer”  pathway.
these categories were used to identify the ctfbss that characterize the promoters of the  <dig>  escc gene ) known to be responsive to estrogen.
the ctfbss that characterize the promoters of escc genes known to be responsive to estrogen
since gene expression is driven by the cohesive action of multiple tfs binding to specific tfbss, common ctfbss may define co-regulated genes  <cit> .
we identified ctfbss significantly over-represented in the promoters of genes known to be responsive to estrogen ) as compared to the background set .
when comparing the  <dig>  known estrogen responsive genes ) to the background set , we selected the  <dig> tfbss  to be used in subsequent analysis.
every possible combination of ctfbss made of these  <dig> tfbss were determined.
the significant ctfbss with a p-value  <  <dig>  were selected.
<dig> significant ctfbss were identified, eight of which were not present in the background set .
the most significant ctfbs comprised of the following transfac identifiers: v$taxcreb_ <dig>  v$areb6_ <dig>  v$creb_q <dig> and v$e2a_q <dig>  the above mentioned ctfbs was not present in class c <dig>  but mapped to the promoters of  <dig> % of genes in class c <dig>   <dig> % of genes in class c <dig>  and  <dig> % of genes in class c <dig> .
v$areb6_ <dig> is known to bind areb <dig>   <cit> ; v$taxcreb_ <dig> binds creb, deltacreb and tax/creb complex  <cit> ; v$creb_q <dig> possibly binds creb <dig>  cremalpha, deltacreb, atf- <dig>  atf- <dig>  atf- <dig>  atf- <dig>  atf-a, and atf-2-xbb4; and v$e2a_q <dig> possibly binds e2a, tcf <dig>  tcf <dig>  tff <dig>  ascl <dig>  myf <dig>  myf <dig>  myf <dig>  and myf <dig>  none of the above mentioned tfs has been linked to estrogen, but play a role in the progression of cancer  <cit> .
further details of these tfbss and their associated tfs can be viewed in additional file  <dig>
even though we have identified ctfbss that characterize the promoter regions of the known estrogen responsive genes in escc, it is unclear whether the tfs that bind the tfbss function as transcriptional activators or transcriptional repressors in the estrogen responsive escc genes  <cit> .
nonetheless, these significant ctfbss are over-represented in the promoters of known estrogen responsive genes and thus can be used to identify genes that are likely co-regulated with genes responsive to estrogen.
identification of candidate estrogen responsive escc genes with eres and ctfbss mapped to the promoters
the  <dig> significantly over-represented ctfbss were used to increase confidence in a subset of the new candidate estrogen responsive genes in class c <dig>  it was determined that at least four of the significant ctfbss were present in  <dig> %  of the genes in class c <dig> ,  <dig> %  of the genes in class c <dig> ,  <dig> %  of the genes in class c <dig>  and  <dig> %  of the genes in class c <dig>  .
the  <dig> ctfbss were determined based on class , but the findings show that the genes with the ctfbss are concentrated in class c <dig> , since class c <dig> has  <dig> % more genes with a ctfbss in the promoter sequence as compared to class c <dig>  this result indicates that class c1a gene promoters with eres also contain distinctive ctfbss that may define multiple co-regulated genes responsive to estrogen.
these co-regulated genes may define estrogen responsive genes that function in an ere-dependent manner.
thus, the  <dig> genes with putative eres in class c2a that have at least four of the ctfbss may be an additional fraction of these co-regulated genes.
these results increase confidence in the new candidate estrogen responsive genes in class c2a since they contain both eres and ctfbss characteristic of escc genes that are responsive to estrogen.
we found  <dig> tfs that interact with er via  <dig> significant  binding sites using biogrid  <cit>  and the transfac  <cit>  databases , of which at least one binding site is in close proximity to the ere of the  <dig> genes identified as estrogen responsive.
we additionally found  <dig>  tfs to be self-regulating.
interestingly, one group  <cit>  has reported that their unbiased sequence interrogation of the genuine chromatin binding sites suggests that direct er binding requires the presence of foxa <dig> binding in close proximity, as knockdown of foxa <dig> expression blocked the association of er with the chromatin and estrogen induced gene expression.
we do not know if this estrogenic response requirement is restricted to breast cancer cells, but  <dig> % of the  <dig> genes we have identified as estrogen responsive has the ere in close proximity to the foxa <dig> binding site.
further, we provide an overview of the potentially co-regulated gene in class c2a in the form of a heat map .
figure  <dig> clusters class c2a genes based on the presence of common ctfbss mapped to the gene promoters.
multiple clusters of genes in the heat map show that different groups of genes have different specific combinations of ctfbss, making them more likely to be co-regulated.
akap <dig> , loxl <dig> , and timp <dig>  cluster together and contain the highest number of combinations that are common to their promoters.
we further ranked the  <dig> genes based on the number of ctfbss present in each promoter .
akap <dig>  loxl <dig>  timp <dig>  cdc25b, muc <dig>  crlf <dig>  vim, mmp <dig> and muc5b are identified as the top nine ranked genes.
a further literature survey disclosed that akap <dig> belongs to the dbl family of proto-oncogenes that function as a rho family guanine nucleotide exchange factor.
it is known to bind and influence the activity of glucocorticoid receptors  and ers  <cit> .
it has been experimentally demonstrated that akap <dig> interacts with the ligand activated er to form a tertiary complex with either rhoa or rho related gtpase cdc <dig> .
it has been demonstrated that these complexes bind to ere sites thereby driving genes expression induced by estrogen  <cit> .
interestingly, rhoa is also known to be up-regulated in escc  <cit> .
moreover, the p <dig> mapk inhibitor sb <dig> abrogates erβ activity by akap <dig> indicating that akap <dig> activates erβ via the p <dig> mapk pathway  <cit> .
pathway analysis using david  <cit>  indicates that four of our putative estrogen response genes  are involved in the mapk signaling pathway.
cdc25b belongs to the cdc <dig> family of phosphatases that activates cyclin dependent kinases by removal of inhibitory phosphates.
this gene is also known to bind and influence the activity of nuclear receptors such as progesterone receptor  and er.
it has been experimentally demonstrated that cdc25b interacts with the ligand activated er in a hormone-dependent er transactivation manner.
also, the p300/cbp-associated factor and creb binding protein were shown to interact and synergize with cdc25b and further enhance its co-activation activity  <cit> .
these findings link akap <dig> and cdc25b, two of the top  <dig> ranked putative estrogen response genes, to estrogen activity and highlight their functioning as co-factors in the ers transcriptional activity.
because these genes are putative estrogen responsive genes, this finding may be indicative of a cascading event that may be an important step in regulating hormone-dependent er transactivation.
recent publications show that muc5b <cit> , mmp <dig> <cit> , loxl <dig> <cit> , actn <dig> <cit> , dnmt <dig> <cit> , gpr <dig> <cit> , muc <dig> <cit> , wnt7b <cit> , bmp <dig> <cit> , gpx <dig> <cit> , cdc25b <cit> , nfκb <dig> <cit> , prdm <dig> <cit> , mdm <dig> <cit>  and timp <dig> <cit>  are responsive to estrogen.
these findings further increase confidence that the  <dig> new candidate estrogen responsive escc genes may indeed be estrogen responsive.
CONCLUSIONS
our study proposes a methodology that provides insight into the regulatory potential of estrogen responsive genes and identifies  <dig> new candidate estrogen responsive genes using escc as the framework.
akap <dig>  loxl <dig>  timp <dig>  cdc25b, muc <dig>  crlf <dig>  vim, mmp <dig> and muc5b were identified as the top nine ranked genes, of which akap <dig>  <cit>  and cdc25b  <cit>  have independently been identified in other studies as essential components of er complexes that are required to drive estrogen induced gene expression.
moreover, estrogen responsiveness of 47%  of genes predicted by our method is supported by experimental findings in recent publications.
these insights into the transcription regulation potential associated with estrogen response provide information of potential interest to those with interest in studying estrogen effects in escc and in design estrogen-based ec therapies.
this study is the first to use a cancer disease model as the framework to identify hormone responsive genes.
although we used escc and estrogen for this purpose, the methodology, however, can be extended analogously to use other diseases as the model and other hormones.

 esophageal cancer comprises of heterogeneous groups of tumors that differ in pathogenesis and etiological and pathological features. ec ranks among the ten most frequent cancers worldwide with regionally dependent incidence rates and histological subtypes. statistics indicate that ec mortality rates are very similar to incidence rates due to the relatively late stage of diagnosis, the poor efficacy of treatment, and the poor prognosis of ec result in a five year survival rate of 5-20%. the most recurrent histological subtype is esophageal squamous cell carcinoma, followed by adenocarcinoma. escc has a worse prognosis than adc due to the primary escc tumor being in contact with the tracheobronchial tree in 75% of cases, while adc is found below the tracheal bifurcation in 94% of cases. the striking 3-4: male predominance of escc was previously ascribed to the different patterns of smoking and drinking between males and females. however, more recently bodelon et al. reported that current users of estrogen and progestin therapy show reduced risk of escc. previous research supports this finding as several groups have reported estrogen induced gene regulation in esophageal squamous cell carcinoma and barrett’s esophageal adenocarcinoma. moreover, wang et al. specifically demonstrated that serum level of estradiol of escc patients from the high risk areas were significantly lower compared to healthy controls from both high and low risk areas and suggested the use of estrogen analogues as promising targets for the prevention and treatment of escc. additionally, published scientific data shows that estrogen induces an inhibitory effect on esophageal carcinoma by activating the estrogen receptor. the activated er functions as a transcription factor that binds to a specific tfbs known as the estrogen response element. there are two er subtypes, erα and erβ, that are encoded on human chromosomes 6q and chromosome 14q22-, respectively. both erα and erβ bind to the same eres, but erα does so with an approximately twofold higher affinity. additionally, erβ is known to bind to erα suppressing erα function. the inverse biological effect associated with the two er subtypes has been confirmed to exist in escc. this collation of research findings suggests that the estrogen based therapies which have improved survival rates of cancer types such as: prostate cancer, lung cancer, brain and spinal cord tumors, and breast cancer, may also improve the outcome of escc. our current study aims at identifying estrogen responsive genes by using escc as a model. potentially, such genes could be affected by estrogen. we propose a methodology that provides insight into the underlying regulation of estrogen responsive escc genes. we mapped eres to the promoters of escc genes using the dragon ere finder version. the escc genes were divided into two groups: 1) genes whose promoters contain predicted eres, and 2) genes lacking predicted eres. these two gene groups were further divided into those known to be experimentally confirmed as estrogen responsive and those that are not. to accomplish this the escc genes were cross checked against two databases housing estrogen responsive genes, namely kberg and ertargetdb databases. at the time of analysis the kberg database contained experimentally confirmed estrogen-responsive genes. the ertargetdb database contained: genes with experimentally verified ere direct binding sites and experimentally verified ere tethering sites; genes identified via chip-on-chip assay for estrogen binding and genes from gene expression microarrays, all of which were included in this study. however, this study excludes the computationally predicted estrogen responsive genes included the ertargetdb, database. thus this study defines estrogen responsive genes as genes that can be modulated by an external estrogen source. we classified the escc genes into the following four categories: c1/escc genes with predicted eres in their promoters and known as estrogen responsive, c2/escc genes with predicted eres in their promoters but not known as estrogen responsive, c3/escc genes having no predicted eres in their promoters, but known as estrogen responsive, c4/escc genes having no predicted eres in their promoters and not known as estrogen responsive. we used these categories to develop a methodology for the identification of co-localized tfbss that characterize the promoters of the known estrogen responsive gene sets ) as opposed to the background set. these significant ctfbss were mapped to the promoter sequences of the candidate estrogen responsive escc genes in class c the genes in class c whose promoters contained such ctfbss were singled out as novel putative estrogen responsive genes in escc. to the best of our knowledge our study provided the first computational large-scale analysis of the transcription potential of estrogen responsive escc genes and suggests important regulatory potential of these genes. although we used escc as a model, the developed system biology based methodology has a potential to identify hormone responsive genes using other hormone-affected diseases, and provides a framework for identifying hormone responsive genes based on complex diseases.  the prediction and identification of putative estrogen responsive genes in escc a sequential two-step process was used to predict and verify estrogen responsive genes in escc: eres were mapped to the promoters of escc genes, and based on the experimental evidence the genes in were classified as being estrogen responsive or not. the escc genes were extracted from the dragon database of genes implicated in esophageal cancer. the putative promoters of these escc genes were extracted from the fantom cage tag data and analyzed for the presence of eres via the dragon ere finder version. eres were mapped to promoter sequences that correspond to escc genes. escc genes had no eres mapped to the promoter sequences. lists of genes that have been experimentally validated to be responsive to estrogen as indicated in the kberg and ertargetdb databases were used to confirm which escc genes are responsive to estrogen. of the genes with predicted eres, % are known to be estrogen responsive, while % were new candidate estrogen responsive genes. eres did not map to escc genes of which % are known to be estrogen responsive. tfbs analysis of estrogen responsive genes in escc tfbs analysis entailed the following three steps: mapping the tfbss matrix models to the promoters of all escc genes, determining the ctfbss significantly over-represented in class relative to class c, and mapping significantly over-represented ctfbss determined in to promoters of genes in class c in, we required that at least four of the ctfbss map the promoters of each gene in class c this threshold corresponds to the maximum difference in the number of genes with these ctfbss in the positive set ) as compared to the background set. all class c genes that have such ctfbss in their promoters we considered as new candidate estrogen responsive escc genes since they have in their promoters both: a/mapped eres, and b/ctfbss characteristic of escc genes that are responsive to estrogen. this increases confidence that these escc genes are responsive to estrogen since due to the similar regulatory potential with estrogen-responsive genes, these genes have higher chance to express when estrogen-responsive genes are expressing and additionally they have ere that potentially bind ers. tfbs matrices mapped to the promoters of escc genes the transfac mammalian matrix models of tfbss were mapped to the promoters of estrogen responsive genes in escc using matchtm. of the matrices mapped, mapped to the promoters of the escc genes at positions, not considering strand. ctfbss significantly over-represented in class as opposed to class c4 we developed a methodology to identify the ctfbss significantly over-represented in the known estrogen responsive gene set ) relative to the background set. each tfbs was ranked using a method that ensures that the top ranked tfbss were not only over-represented but also more likely to be co-localized within the promoters. in order to reduce the search space for the potentially significant co-localized tfbss, a heuristic approach was applied where the tfbss with the lowest p-value were selected for subsequent analysis. every possible combination of ctfbss that includes some of the tfbs were determined. the significant ctfbss with a p-value < were selected. we identified significant ctfbss consisting of doublet ctfbs, triplet ctfbs, 4-element ctfbs, 5-element ctfbs and 6-element ctfbs. the tfbss that make these ctfbss are determined by the following transfac identifiers v$elk1_ v$cets1p54_ v$yy1_ v$gata3_ v$taxcreb_ v$freac4_ v$areb6_ v$creb_q v$e2a_q and v$ebox_q6_ of the ctfbss, eight combinations were completely absent in the background set. the most significant ctfbss was not present in class c but mapped times to the promoters of genes in class c and times to the promoters of genes in class c significant ctfbss consisting of doublet ctfbs, triplet ctfbs, 4-element ctfbs, 5-element ctfbs and 6-element ctfbs were identified. ctfbss used to increase confidence in a subset of the new candidate estrogen responsive genes in class c2 we mapped the significant ctfbss to the promoters of the genes in class c c c and c thereby generating and predictions of ctfbss, respectively. this result for the mapping of ctfbss to the promoters of all categories indicates that multiple ctfbss are present in the promoters of genes. moreover, these multiple ctfbss are more dominant in genes from class known to be responsive to estrogen, as well as genes with ere predictions in their promoters. consequently, we applied a threshold that each gene promoter must contain at least four of the significant ctfbss, as this threshold defines the maximum difference in the number of genes that contain such ctfbss between the known estrogen responsive gene set ) relative to the background set. it was determined that at least four of the significant ctfbss were present in % of the genes in class c, % of the genes in class c, % of the genes in class c and % of the genes in class c. an overview of the regulatory effects of the ctfbss on the genes in class c2a is shown in figure this figure illustrates each association in class c2a, in the form of a color dot in a heat map format using tmev. the heat map clearly depicts gene clusters based on the ctfbss common to the promoters of multiple genes in class c2a. moreover, a review of the recently published scientific literature reveals that 47% of the c2a genes have now been shown experimentally to be estrogen responsive. these genes include muc5b, mmp, loxl, actn, dnmt, gpr, muc, wnt7b, bmp, gpx, cdc25b, nfκb, prdm, mdm and timp. discussion in this study, we propose a methodology aimed at providing an insight into the underlying transcription regulatory potential related to genes’ response to estrogen in escc. in this systems biology study, we combined information obtained from several databases, genomic sequences of promoters of relevant genes, and analysis of transcription regulation potential of these genes to infer if the genes are estrogen responsive. two computational components are used to suggest escc genes responsive to estrogen: 1) the ere prediction, and 2) predicted ctfbss that characterize the promoters of known estrogen responsive escc genes. these ctfbss were mapped to the promoters of escc genes not being known to be responsive to estrogen, but having ere predictions in their promoters. in this way we increased the confidence that the escc genes with ere predictions are responsive to estrogen since they, in addition to eres, also contain ctfbss characteristic of estrogen responsive escc genes. escc genes predicted to be responsive to estrogen carroll et al. has reported that er binds selectively to a limited number of sites, majority of which are distant from the transcriptional start sites of regulated genes and that direct er binding requires the presence of forkhead factor binding in close proximity. however, several computational approaches has been undertaken to identify target genes based on the presence of eres in the proximal promoter regions. bourdeau et al. in particular screened for eres that were conserved in the human and mouse genome and identified gene proximal eres of which several were validated as genuine er interaction sites. this analysis has also been restricted to the proximal promoter region due to computational limitations and regulatory tfs binding closer to the transcription start site. eres were mapped to the promoters of escc genes using the dragon ere finder version. bajic et al. have demonstrated that this ere locator predicts known ere and estrogen responsive genes at a sensitivity of. we further identified which of the escc genes are known to be responsive to estrogen using the kberg and ertargetdb databases. of the predicted estrogen responsive genes, % are known to be estrogen responsive, while % were novel putative estrogen responsive genes. these genes lay the foundation for increasing insights into the molecular events triggered by estrogen via an ere dependant mode of regulation in escc. eres did not map to escc genes of which % are known to be responsive to estrogen. the promoters of these gene did not contain an ere motif, but the genes are known to be responsive to estrogen. the response to estrogen of these genes may be through the interactions of ers with other transcription factors forming complexes that do not require the presence of eres. it is also possible that the ere models are not sufficiently good to predict eres in these promoter regions. our analysis generated four gene categories: class c, class c, class c, and class c. we found that the four gene categories had a different number of enriched pathways using kyoto encyclopedia of genes and genomes. however, in each category the more general kegg pathway “pathways in cancer” enriched with genes forming the gene sets. other more specialized and equally important pathways show enrichment with genes forming certain categories. category genes are highly enriched in the pathways such as “transcriptional misregulation in cancer”, “small cell lung cancer”, “melanoma”. category genes are highly enriched in the pathways e.g. “p signaling pathway”, “bladder cancer”, “small cell lung cancer”. category genes are highly enriched for many pathways, e.g. “prostate cancer”,“colorectal cancer”, “small cell lung cancer”, “chronic myeloid leukemia”, “endometrial cancer”, etc. category genes is additionally highly enriched in the “bladder cancer” pathway. these categories were used to identify the ctfbss that characterize the promoters of the escc gene ) known to be responsive to estrogen. the ctfbss that characterize the promoters of escc genes known to be responsive to estrogen since gene expression is driven by the cohesive action of multiple tfs binding to specific tfbss, common ctfbss may define co-regulated genes. we identified ctfbss significantly over-represented in the promoters of genes known to be responsive to estrogen ) as compared to the background set. when comparing the known estrogen responsive genes ) to the background set, we selected the tfbss to be used in subsequent analysis. every possible combination of ctfbss made of these tfbss were determined. the significant ctfbss with a p-value < were selected. significant ctfbss were identified, eight of which were not present in the background set. the most significant ctfbs comprised of the following transfac identifiers: v$taxcreb_ v$areb6_ v$creb_q and v$e2a_q the above mentioned ctfbs was not present in class c but mapped to the promoters of % of genes in class c % of genes in class c and % of genes in class c. v$areb6_ is known to bind areb; v$taxcreb_ binds creb, deltacreb and tax/creb complex; v$creb_q possibly binds creb cremalpha, deltacreb, atf- atf- atf- atf- atf-a, and atf-2-xbb4; and v$e2a_q possibly binds e2a, tcf tcf tff ascl myf myf myf and myf none of the above mentioned tfs has been linked to estrogen, but play a role in the progression of cancer. further details of these tfbss and their associated tfs can be viewed in additional file even though we have identified ctfbss that characterize the promoter regions of the known estrogen responsive genes in escc, it is unclear whether the tfs that bind the tfbss function as transcriptional activators or transcriptional repressors in the estrogen responsive escc genes. nonetheless, these significant ctfbss are over-represented in the promoters of known estrogen responsive genes and thus can be used to identify genes that are likely co-regulated with genes responsive to estrogen. identification of candidate estrogen responsive escc genes with eres and ctfbss mapped to the promoters the significantly over-represented ctfbss were used to increase confidence in a subset of the new candidate estrogen responsive genes in class c it was determined that at least four of the significant ctfbss were present in % of the genes in class c, % of the genes in class c, % of the genes in class c and % of the genes in class c. the ctfbss were determined based on class, but the findings show that the genes with the ctfbss are concentrated in class c, since class c has % more genes with a ctfbss in the promoter sequence as compared to class c this result indicates that class c1a gene promoters with eres also contain distinctive ctfbss that may define multiple co-regulated genes responsive to estrogen. these co-regulated genes may define estrogen responsive genes that function in an ere-dependent manner. thus, the genes with putative eres in class c2a that have at least four of the ctfbss may be an additional fraction of these co-regulated genes. these results increase confidence in the new candidate estrogen responsive genes in class c2a since they contain both eres and ctfbss characteristic of escc genes that are responsive to estrogen. we found tfs that interact with er via significant binding sites using biogrid and the transfac databases, of which at least one binding site is in close proximity to the ere of the genes identified as estrogen responsive. we additionally found tfs to be self-regulating. interestingly, one group has reported that their unbiased sequence interrogation of the genuine chromatin binding sites suggests that direct er binding requires the presence of foxa binding in close proximity, as knockdown of foxa expression blocked the association of er with the chromatin and estrogen induced gene expression. we do not know if this estrogenic response requirement is restricted to breast cancer cells, but % of the genes we have identified as estrogen responsive has the ere in close proximity to the foxa binding site. further, we provide an overview of the potentially co-regulated gene in class c2a in the form of a heat map. figure clusters class c2a genes based on the presence of common ctfbss mapped to the gene promoters. multiple clusters of genes in the heat map show that different groups of genes have different specific combinations of ctfbss, making them more likely to be co-regulated. akap, loxl, and timp cluster together and contain the highest number of combinations that are common to their promoters. we further ranked the genes based on the number of ctfbss present in each promoter. akap loxl timp cdc25b, muc crlf vim, mmp and muc5b are identified as the top nine ranked genes. a further literature survey disclosed that akap belongs to the dbl family of proto-oncogenes that function as a rho family guanine nucleotide exchange factor. it is known to bind and influence the activity of glucocorticoid receptors and ers. it has been experimentally demonstrated that akap interacts with the ligand activated er to form a tertiary complex with either rhoa or rho related gtpase cdc. it has been demonstrated that these complexes bind to ere sites thereby driving genes expression induced by estrogen. interestingly, rhoa is also known to be up-regulated in escc. moreover, the p mapk inhibitor sb abrogates erβ activity by akap indicating that akap activates erβ via the p mapk pathway. pathway analysis using david indicates that four of our putative estrogen response genes are involved in the mapk signaling pathway. cdc25b belongs to the cdc family of phosphatases that activates cyclin dependent kinases by removal of inhibitory phosphates. this gene is also known to bind and influence the activity of nuclear receptors such as progesterone receptor and er. it has been experimentally demonstrated that cdc25b interacts with the ligand activated er in a hormone-dependent er transactivation manner. also, the p300/cbp-associated factor and creb binding protein were shown to interact and synergize with cdc25b and further enhance its co-activation activity. these findings link akap and cdc25b, two of the top ranked putative estrogen response genes, to estrogen activity and highlight their functioning as co-factors in the ers transcriptional activity. because these genes are putative estrogen responsive genes, this finding may be indicative of a cascading event that may be an important step in regulating hormone-dependent er transactivation. recent publications show that muc5b, mmp, loxl, actn, dnmt, gpr, muc, wnt7b, bmp, gpx, cdc25b, nfκb, prdm, mdm and timp are responsive to estrogen. these findings further increase confidence that the new candidate estrogen responsive escc genes may indeed be estrogen responsive.  our study proposes a methodology that provides insight into the regulatory potential of estrogen responsive genes and identifies new candidate estrogen responsive genes using escc as the framework. akap loxl timp cdc25b, muc crlf vim, mmp and muc5b were identified as the top nine ranked genes, of which akap and cdc25b have independently been identified in other studies as essential components of er complexes that are required to drive estrogen induced gene expression. moreover, estrogen responsiveness of 47% of genes predicted by our method is supported by experimental findings in recent publications. these insights into the transcription regulation potential associated with estrogen response provide information of potential interest to those with interest in studying estrogen effects in escc and in design estrogen-based ec therapies. this study is the first to use a cancer disease model as the framework to identify hormone responsive genes. although we used escc and estrogen for this purpose, the methodology, however, can be extended analogously to use other diseases as the model and other hormones. 
we were able to map these ctfbss to promoters of of the new candidate estrogen responsive escc genes, thereby increasing confidence that these escc genes are responsive to estrogen since their promoters contain both: a/mapped eres, and b/at least four ctfbss characteristic of escc genes that are responsive to estrogen. eres mapped to the promoter sequence of % of escc genes of which % are known to be estrogen responsive, while % are new candidate estrogen responsive genes. we believe that our results provide useful information for those interested in genes responsive to hormones and in the design of hormone-based therapies. our study develops a systems biology methodology using esophageal squamous cell carcinoma as a model to identify estrogen responsive genes. by analyzing transcription factor binding sites in the promoters of the known estrogen responsive escc genes under study, we found that their regulatory potential may be characterized by significantly over-represented co-localized tfbss. to the best of our knowledge our study is the first to use a cancer disease model as the framework to identify hormone responsive genes. moreover, several groups have reported the importance of estrogen induced gene regulation in esophageal cancer. recent publications confirm that 47% of these predicted genes are indeed responsive to estrogen. the efficient design of estrogen therapies requires as complete as possible list of genes responsive to estrogen. putative estrogen responsive elements mapped to the promoter region of the escc genes were used to initially identify candidate estrogen responsive genes. of these genes, % are known to be estrogen responsive. eres did not map to escc genes.
 estrogen therapy has positively impact the treatment of several cancers, such as prostate, lung and breast cancers. moreover, several groups have reported the importance of estrogen induced gene regulation in esophageal cancer. this suggests that there could be a potential for estrogen therapy for ec. the efficient design of estrogen therapies requires as complete as possible list of genes responsive to estrogen. our study develops a systems biology methodology using esophageal squamous cell carcinoma as a model to identify estrogen responsive genes. these genes, on the other hand, could be affected by estrogen therapy in escc.  based on different sources of information we identified genes implicated in escc. putative estrogen responsive elements mapped to the promoter region of the escc genes were used to initially identify candidate estrogen responsive genes. eres mapped to the promoter sequence of % of escc genes of which % are known to be estrogen responsive, while % are new candidate estrogen responsive genes. eres did not map to escc genes. of these genes, % are known to be estrogen responsive. by analyzing transcription factor binding sites in the promoters of the known estrogen responsive escc genes under study, we found that their regulatory potential may be characterized by significantly over-represented co-localized tfbss. we were able to map these ctfbss to promoters of of the new candidate estrogen responsive escc genes, thereby increasing confidence that these escc genes are responsive to estrogen since their promoters contain both: a/mapped eres, and b/at least four ctfbss characteristic of escc genes that are responsive to estrogen. recent publications confirm that 47% of these predicted genes are indeed responsive to estrogen.  to the best of our knowledge our study is the first to use a cancer disease model as the framework to identify hormone responsive genes. although we used escc as the disease model and estrogen as the hormone, the methodology can be extended analogously to other diseases as the model and other hormones. we believe that our results provide useful information for those interested in genes responsive to hormones and in the design of hormone-based therapies. 