BACKGROUND
estrogens are key regulators of cell growth and differentiation in the mammary gland  <cit> , where they are involved in the pathogenesis and clinical outcome of breast cancer   <cit> .
these steroid hormones exert their effects in normal and transformed mammary epithelial cells by binding to specific receptors, erα and erβ, that mediate estrogen signaling by functioning as ligand-dependent transcription factors.
ligand-activated ers drive gene cascades comprising primary genes, whose transcription is directly controlled by the hormone through physical interaction of ers with regulatory sites in the genome  and/or with signal transduction effectors , as well as downstream genes whose activity depends upon the functions encoded by the primary responders  <cit> .
ers are able to bind dna at specific sites in the genome and thereby control gene activity by recruiting transcriptional mediators and co-regulators, as well as a host of other nuclear proteins with different roles in er-mediated control of gene activity  <cit> .
the two ers show 55% identity in their estrogen-binding domains  and approximately 97% similarity in the dna-binding domains   <cit> .
reflecting the high degree of similarity in their dbds, both receptors interact with the same conserved estrogen response element   as either homodimers or alpha/beta heterodimers  <cit> .
erβ, however, holds low trans-acting capability on ere-containing estrogen target genes and alpha/beta heterodimers are less efficient than erα homodimers in promoting target genes activity  <cit> .
the different behaviour of erα/erβ heterodimers respect to erα homodimers on transcriptional regulation of ere-containing genes might be explained by different co-factor recruitment, as erβ could prevent efficient co-activator binding to the erα moiety of the heterodimer, conversely inducing recruitment of co-repressors and/or driving assembly of co-regulatory complexes other than those involving erα only.
<cit> .
although the two receptors are quite similar in sequence and structure, in bc erβ has considerably different biological effects than erα  <cit> .
furthermore, the two ers show a remarkably different expression pattern in bcs, with higher erα and lower erβ levels observed in malignant cells compared to normal mammary epithelial or benign tumor cells  <cit> .
furthermore, while erα induces a mitogenic response to estrogen, when expressed alone the β subtype is not only unable to induce the same mitogenic response, but it reduces basal, hormone-independent cell proliferation .
finally, erβ was shown to change dramatically erα-positive bc cell behaviour in vivo, as its expression in the cell prevents tumorigenicity in mouse xenograft models by reducing tumor growth and angiogenesis  <cit> .
gene expression studies performed in bc cell lines expressing endogenous erα and recombinant erβ  <cit>  revealed multiple signaling pathways involving the α and/or β receptor subtypes  <cit> .
the two ers appear thus to share many target genes, although each of them may affect specific downstream targets.
for this reason, inhibition of hormone-responsive bc cell growth by erβ might be due to direct interference with erα activity on growth-promoting pathways as well as to the activity of erβ-specific target genes  <cit> .
recently, next-generation sequencing technologies combining chromatin immunoprecipitation  either with genomic dna hybridization to microarrays  or massively parallel sequencing , opened new venues for our understanding of physical and functional associations between transcription factors and chromatin in vivo.
these analytical strategies led to genome-wide mapping of erα-binding regions in intact chromatin of cultured cell lines  <cit> , revealing important new information relative to erα interaction with the genome.
carroll et al.
<cit> , for example, using chip-on-chip demonstrated that the forkhead factor foxa <dig> plays an important role as pioneering factor for erα binding to chromatin in bc cells, while cicatiello et al.
<cit>  identified novel gene regulation cascades mediating estrogen actions in hormone-responsive bc cells.
in contrast, although several studies focused on erβ interaction with the genome  <cit> , a thorough characterization of this important aspect of erβ biology in bc cells, essential to clarify the mechanisms mediating its control of estrogen-dependent gene pathways and the hormone-responsive phenotype, is still missing.
for this reason, we performed a comprehensive analysis of erβ and erα target sites in the genome of mcf- <dig> cells engineered to express both receptors to comparable levels, by integrating global mapping of in vivo er binding to the genome by chip-seq with comparative gene expression profiling in erβ+/erα+ vs erβ-/erα+ cells during early stimulation with 17-beta-estradiol , followed by in silico analyses of the erβ binding regions and responsive genes identified.
RESULTS
establishment and characterization of erβ-expressing mcf- <dig> cells
stabilized human bc cell lines expressing endogenous erα and erβ at comparable levels are not available.
for this reason, we first generated and characterized cell lines derived from erα-positive mcf- <dig> cells expressing full-length human erβ  at levels similar to those of endogenous erα.
this strategy was adopted to prevent artefacts due to erβ over-expression in the cell and to mimic what observed in primary breast tumors, where very high expression of this receptor has never been observed.
as suitable antibodies for efficient immunoprecipitation of chromatin-bound erβ are not available, the expressed proteins were tagged on either their c-  or n-terminus  with the tap epitope.
this approach allows to track tagged ers in different cell compartments and to efficiently immunoprecipitate and purify them in vitro by tandem affinity purification, to identify their molecular partners  <cit> , and ex vivo in chromatin immunoprecipitation assays .
preliminary tests were performed to verify whether the presence of the tap moiety could influence intracellular redistribution of erβ in response to 17β-estradiol  and its ability either to trans-activate an estrogen-responsive reporter gene or to interfere with erα activity on reporter gene transcription and bc cell proliferation.
to this end, er expression and nuclear translocation in response to e <dig> was determined in wt mcf- <dig>  ct-erβ and nt-erβ cells by subcellular fractionation followed by sds-page and immunoblotting .
in absence of hormone a larger fraction of both ers was found in the cytosol in all cases.
following estrogen stimulation, both receptors migrated to the nucleus, a crucial event to trigger target gene transcription via the genomic pathway of the estrogen signaling cascade.
an antibody against α-tubulin was used as control, and the absence of this protein in the nuclear fractions indicates that they were indeed free from cytosolic contaminants.
the erβ-expressing clones selected for this study showed a erβ/erα ratio < <dig>  as verified by immunoblotting analysis of the proteins in whole cell extracts and quantitative rtpcr of the corresponding rnas .
to control that the presence of the tap tag did not interfere with erβ activity, er-negative skbr- <dig> bc cells were transiently transfected with expression vectors encoding wt erβ, ct-erβ, nt-erβ, erα  or 'empty' vector , as controls, and ere-tk-luc  <cit> , a reporter gene where luciferase expression is driven by an estrogen-responsive minimal promoter.
exposure of transiently transfected cells to e <dig> induced reporter gene activation in the presence of erα, erβ, ct-erβ or nt-erβ, with the activity of both tagged erβ proteins slightly  lower than that of wt erβ .
we then tested whether the two recombinant forms of erβ were able to interfere with target gene activation by the endogenous erα resident in mcf- <dig> cells.
to this end, wt, ct-erβ+ and nt-erβ+ cells were transfected with ere-tk-luc and the response of the reporter gene to e <dig> was determined.
as shown in figure 1b , erβ-expressing cells showed in all cases a marked  reduction in reporter gene response to the hormone, when compared to wt mcf- <dig> cells, indicating that both tagged erβs are able to interfere with the activity of endogenous erα.
results show that cell lines stably expressing ct-erβ and nt-erβ display a marked reduction in proliferative response to the hormone, when compared to wt mcf- <dig> cells , in agreement with the known effects of erβ in erα-positive cells  <cit> .
furthermore, comparative rna expression profiling in exponentially growing ct-erβ and nt-erβ vs wt mcf- <dig> cells revealed extensive overlapping effects of the two tagged erβ proteins on the activity of estrogen target genes .
taken together, these observations confirmed that both tag-erβ expressing cell lines generated for this study show a well defined phenotype, with respect to the known activities of this er subtype in bc cells, and are thus suitable to investigate the genomic bases of erβ actions in this cell type.
as we could not exclude that the presence of the tap tag at either the n- or c- term of erβ may specifically influence its activity on cellular targets or pathways different from those investigated above, all experiments reported in this study were performed in both ct-erβ and nt-erβ cells and the data were combined for analysis, with the aim to focus on the most significant and reproducible actions of erβ independently from position of the tag in the receptor moiety.
effects of erβ on the estrogen-responsive mcf- <dig> cell transcriptome
expression of erβ is known to cause significant changes in the genomic response to estrogen in target cells.
to identify the genes whose estrogen regulation in hormone-responsive bc cells is perturbed by erβ, we performed gene expression profiling with microarrays in estrogen-starved, quiescent wt and tap-erβ+  mcf- <dig> cells following stimulation with 10-8m e <dig>  total rna was extracted from the three cell lines either before or after  <dig>   <dig> or  <dig> hrs stimulation, fluorescently labelled and analyzed on whole-genome microarrays.
samples extracted from the two erβ-expressing cell lines were pooled before analysis, to reduce the impact of clone-specific differences and to focus on the most significant effects of erβ, independent from tag position in the protein.
results obtained in these samples were then compared with those obtained under the same conditions in wt mcf- <dig> cells.
this analysis yielded  <dig> transcripts differentially regulated by the hormone in erβ+ vs erβ- cells , including  <dig> mrna whose regulation was detected only in wt mcf- <dig> cells,  <dig> regulated only in erβ+ cells ,  <dig> showing a similar pattern of response in both cell types  and  <dig> showing opposite responses to the hormone in erβ+ vs erβ- cells .
the full list of these differentially regulated transcripts is reported, with relevant information, in additional table s <dig>  taken together, these results indicate that the presence of erβ greatly influences the response of the mcf- <dig> cell genome to estrogen, by interfering with erα-mediated hormonal regulation of  <dig> genes  and promoting de novo regulation of  <dig> genes .
it should be noted that these analyses were performed with data obtained after  <dig> hrs of hormonal stimulation, a timing that allowed us to focus on early response genes, positioned upstream in the composite transcriptional cascade set in motion by the hormone in this cell type and more likely to include primary genomic targets of ers  <cit> .
it is thus possible that this analysis missed erβ-responsive genes showing significant changes in expression only at later times after hormonal stimulation.
however, analysis of the global effects of erβ on gene expression in these same cells, performed as described above in cultures exponentially growing under continuous hormonal stimulus, suggests that the number of regulated transcripts identified here is rather close to the total number of genes targeted by this er subtype in mcf- <dig> cells .
global mapping of erα and erβ binding to mcf- <dig> cell genome
the widespread effects of erβ on mcf- <dig> cell transcriptome are likely to result from multiple effects of this receptor in the cells, including direct regulation of primary response genes via genomic or non genomic mechanisms, and indirect gene regulation events mediated by the products of primary genes.
the primary erβ target genes are most likely to comprise also master regulators of complex cellular responses to the receptor, mediating its effects on the biological and clinical phenotype of bc cells.
to identify such primary genomic targets and investigate the mechanisms that allow their regulation by erβ, a global analysis of in vivo binding of this receptor to the genome was carried out in tap-erβ cells by chromatin immunoprecipitation coupled to massively parallel sequencing   <cit> , that allows detailed mapping of in vivo tf binding to the genome.
in parallel, we studied erα binding to the genome under the same conditions, to allow comparative analyses between the two er subtypes.
replicate chromatin samples were prepared from both ct-erβ and nt-erβ cells before and after stimulation for  <dig> minutes with 10-8m e <dig> and dna-bound proteins were immunoprecipitated either with antibodies against the n- and c-terminus of erα, or with iggs binding with high affinity the tap moiety of tagged erβ .
preliminary testing on several known erβ binding sites, including the promoter-near region of ps2/tff <dig> gene  <cit> , confirmed that the method selected to immunoprecipitate chromatin-bound ct-erβ and nt-erβ was efficient and specific .
the resulting dnas were used to generate chip-seq libraries for erα and erβ, respectively, that were then sequenced with the illumina genome analyzer.
the sequence tags obtained were then aligned to the human genome sequence and peaks enriched in the libraries generated after e2-treatment were identified using macs   <cit> .
this led to the identification of  <dig> binding sites for erβ and  <dig> sites for erα, of which  <dig> were shared by both receptors , with an average false discovery rate  of 3%.
the full list of these binding sites is available, with relevant information, in additional files  <dig> and  <dig>  interestingly, about half  erβ binding sites identified map within transcription units, mainly  in intronic regions.
this distribution is maintained also in  <dig> erβ-regulated transcription units , where  <dig> erβ binding sites located in the gene or within  <dig> kbps from it are distributed as follows:  <dig> in promoter regions,  <dig> in exons,  <dig> within one or more introns and the remaining either upstream of promoters  or downstream of the gene .
in both cases the erβ binding sites within genes did not show any preference with respect to exon or intron position nor for know intragenic regulatory elements .
it should be mentioned that the number of erβ binding sites identified is significantly higher that those mapped in mcf- <dig> cells by chip-on-chips  <cit> , possibly for technical differences due to erβ expression levels in the different mcf- <dig> cell-derived clones used, in immunoprecipitation efficiency and/or in dna analysis.
since only zhao et al.
<cit>  performed an unbiased search for erβ binding with whole-genome chips, we could confront our results only with those reported in that study.
this showed that 86% of high confidence erβ sites described in that study appear also in our dataset.
the binding sites identified here were then subjected to sequence analysis, searching first for the presence of eres , the characteristic er binding signature .
this analysis revealed that in all three cases  a high percentage of sites displayed one or more imperfectly palindromic ere , with a slightly higher positivity in erα sites .
as ers have been shown to bind both in vitro and in vivo to puggtca hemi-palindromes , we searched the sequence of the remaining  sites for perfect matches to this sequence.
results showed that almost half of them indeed contained one or more heres.
the percentages of sites not carrying a known er-binding element  were similar for both receptors .
we observed that erα and erβ binding sites were often found in close proximity to each other, a confounding factor when attempting to discern and analyze separately er subtype-specific sites and target genes.
this could be due to the limits of the chip-seq technology or of the algorithm used for peak selection.
to overcome this problem, and allow the identification of potential er subtype-specific sites, we used a cartographic approach to group nearby binding sites that might be the result, at least in part, of shortfalls of the mapping methods applied.
each binding peak was thus elongated in both directions by  <dig> bp and the overlapping ones obtained were merged into  <dig> erβ and  <dig> erα 'extended' binding regions.
these regions were intersected to define erα only, erβ only or erα+erβ binding regions.
in this way we could identify  <dig> erα-only and  <dig> erβ-only binding sites, comprised in these regions, none of which showed nearby binding of the other receptor.
these were named: er subtype 'prevalent' sites.
the binding peak sequences included in each of the three regions obtained  were then re-analyzed for the presence of ere or here elements.
in this way we could observe that sites within the erα+erβ regions showed now a much higher percentage of ere+ sequences , respect to those present in the erα-only or erβ-only regions .
since all three types of sites showed almost identical proportions of here+, this result suggests that perfectly or imperfectly palindromic eres are preferential binding sequences for erα-erβ heterodimers, while erα and erβ homodimers appear to be more flexible in dna recognition.
ere+ sequences were then analyzed in more detail with meme  <cit> , to investigate if the three classes of sites identified showed any difference in the relative base composition of their respective ere signatures.
for each list of sequences, the most significant position-specific probability matrix generated by meme was compared to the matrices present in the jaspar transcription factor binding profile database  <cit> , using the stamp tool-kit for dna motif comparison  <cit> .
as shown in figure 3c , this analysis revealed that the ere matrices derived from the three types of binding regions identified  are identical and, as a consequence, that erβ does not appear to display ere variant selectivity.
we then examined the ere- sequences to search for enriched binding motifs for other transcription factors that might play a role in er binding to chromatin in the absence of canonical eres.
ers are known to be able to bind chromatin indirectly, by physically interacting with dna-bound tfs , including sp <dig>  <cit>  or the ap1complex  <cit> , for gene trans-regulation.
tfbs enrichment respect to the whole genome was calculated thus in ere- sites with using the regionminer tool  <cit>  and only statistically significant  and highly enriched  matrices were further considered.
the results are summarized in the right panel of figure 3c, showing for comparison the over-representation values scored in here+ and ere+ sites by the same matrices selected in the ere- sites.
these numerical values, together with relevant information, are reported in additional table s <dig> .
the enriched matrices found in the ere- set of erβ prevalent sites include v$sp <dig> , v$sp <dig> , v$sp <dig> , v$sp <dig>  and v$gc.
<dig>  all belonging to the gc-box family targeted by sp <dig> and gcfc <dig>  factors, v$ctcf.
<dig> and v$ctcf.
<dig>  binding site matrices for the ccctc-binding factor ctcf, that is a known transcriptional repressor of c-myc  <cit> , v$nrf <dig> , binding nrf <dig> , a transcription factor that regulates the expression of nuclear-encoded mitochondrial genes  <cit> , v$zf <dig> , for the poz domain zinc finger protein zf <dig>  and v$znf <dig> , recognized by the zinc finger proteins znf <dig>   <dig>   <dig> and  <dig>  the majority of these tfs bind gc- and c-rich sequences that are structurally related to each other, suggesting the possibility that a significant portion of the sequence elements listed above might indeed be recognized by one or a limited number of tfs.
on the other hand, the v$gaga.
<dig> matrix was specifically enriched only in the erα prevalent ere- binding sites.
this sequence binds a transcription factor known to influence chromatin structure in drosophila  <cit>  and to bind throughout the genome  <cit> , but nothing is known about physical or functional interactions of this factor with ers or other nuclear receptors.
the results of this analysis point to tfs that could act as partners of erβ for binding to chromatin in the absence of canonical eres.
interestingly, the majority of these same matrices were found enriched also in the erβ binding sites comprising heres or eres , suggesting that one or more such dna matrices might cooperate with erβ for either dna binding or gene trans-regulation.
we performed a direct search for conserved motifs in the ere- binding sites of erβ with meme  <cit> , and the most significant position-specific probability matrices were compared to those present in the jaspar tf binding profile database  <cit> .
the results failed to provide any conclusive information, as each of several sequence motifs obtained with this analysis was found only in a small fraction of the binding sites analyzed.
identification of primary erβ target genes
to identify the genes directly controlled by erβ binding to the genome, and analyze the interplay between the two ers in gene regulation, we combined the chip-seq data with those relative to estrogen responsive genes differentially regulated by e <dig> in erβ+ vs erβ- mcf- <dig> cells under the same experimental conditions .
three sets of er binding regions  were used for this analysis: erβ  and erα  binding regions identified in tap-erβ cells, and all erα binding regions identified so far in wt mcf- <dig> .
the regions from set  <dig>  were intersected with those from set  <dig> , to define which of them was binding both receptor subtypes , only erβ  or only erα  in tap-erβ cells.
the 'homodimer erβ' and the 'heterodimer erα+erβ' groups were further filtered against set  <dig> binding regions , to identify the genomic sites recognized by erβ, with or without erα, but never by erα alone.
this allowed us to classify the sites comprised in these regions as follows: class  <dig>  including  <dig> sites occupied by erα in wt mcf- <dig> cells and by erβ in tap-erβ cells ; class  <dig>  showing  <dig> sites where erα can bind in wt mcf- <dig> cells and both receptors are detected in tap-erβ cells ; class  <dig>  with  <dig> sites binding only erβ and never, under any condition, erα ; class  <dig>  comprising  <dig> sites where both receptors are found in tap-erβ cells but none in wt mcf- <dig> cells ; class  <dig>  including  <dig> sites where erα binds only in tap-erβ but never in wt cells ; class  <dig>  composed of  <dig> sites that bind only erα both in wt and tap-erβ cells .
when combined with the results of the sequence analyses described above, this classification reveals that erβ-specific cis-acting regulatory elements are unlikely to exist in the genome, as all evidence point to the fact that the two er subtypes can recognize identical sequence features.
to identify among the genes differentially regulated by estrogen in erβ+ vs erβ- cells those representing direct targets for transcriptional regulation by dna-bound erβ in our cell model, we extracted from the list in additional table s <dig>  the genes bearing one or more erβ binding sites inside or within  <dig> kb of the tu, and termed them 'primary', to indicate that they are most likely to respond directly to the signal conveyed by the e2-activated receptor  <cit> .
of these  <dig> genes - listed in additional table s <dig> , whose kinetics of response to e <dig> in wt and tap-erβ cells is shown in figure  <dig>   <dig> show one erβ site of class  <dig> ,  <dig> a site of class  <dig> ,  <dig> a site of class  <dig>  and only  <dig> a class  <dig> site , while  <dig> showed multiple erβ sites belonging to different classes and were thus classified accordingly .
in the right panels of figure  <dig> are reported examples for each of the primary gene classes described above, showing the location of the receptor binding sites respect to the promoter and structural gene.
it is worth mentioning that when the gene expression data from wt mcf- <dig> cells stimulated with e <dig> for 8hrs  were combined with information concerning erα binding regions identified in wt mcf- <dig> cells under comparable conditions ,  <dig> putative primary erα target genes were identified -listed in additional table s <dig> , 71% of which  showed erβ binding in hormone-stimulated erβ+ cells.
this result supports the view that the two er subtypes tend to interact with the same targets in bc cells genome.
right: genome browser view of genomic loci representative of the different erα and erβ binding site categories identified.
chip-seq erα and chip-seq erβ indicate sites identified in this study, chip-seq erα <dig>  chip-seq erα <dig> and chip-on-chip indicates sites identified in mcf- <dig> cells by cicatiello et al.
<cit> , fullwood et al.
<cit> , and hurtado et al.
<cit> , respectively.
a functional analysis of the primary erβ target genes identified here with ontologizer  <cit>  showed that most primary erβ responsive genes are involved in key cellular processes, including control of cell proliferation, survival and differentiation status as well as cell motility, migration and adhesion, and can all greatly influence bc cell phenotype and response to estrogen - listed in additional table s <dig> .
when go analysis was performed on the erα target gene set from wt mcf- <dig> cells , results showed that the genes controlled directly by this er subtype appear to be involved in the same cellular processes described above for erβ -compare results reported in additional tables s <dig> and s <dig> , providing a further indication of the significant overlapping between gene pathways targeted by erβ and erα in bc cells.
focusing on the genes differentially regulated by e <dig> in erβ- vs erβ+ cells known for their involvement in cell proliferation, we observed that many of them encode for transcription factors and other key proteins controlling large gene networks of cell division cycle and cell survival and, in general, cell proliferation and differentiation pathways.
these include, in particular, cdk- <dig> , cebpa , dab <dig> , hes- <dig> , igfbp- <dig> , irs- <dig> and - <dig> , jak- <dig> , junb, mitf , myc, slit- <dig> , smarca- <dig> , sox- <dig> , tgfb- <dig> , tgfbi/lcd- <dig> , tgm- <dig> , tns- <dig>  and wisp- <dig> .
interestingly, the role of all these genes in tumor cell proliferation and differentiation is known and an involvement in hormone-mediated bc cell responses to erα has been reported for most of them, suggesting that discovery of an erβ direct effect on transcription of these genes provides a new molecular framework to elucidate the anti-proliferative and differentiative effects of this receptor subtype in hormone-responsive cells.
among the rnas encoded in the genome, micrornas  have emerged as master regulators of gene expression for their ability to influence mrna concentration and activity by post-transcriptional mechanisms.
recent results highlighted the role of mirna in bc cells response to estrogen  <cit>  and, in addition, several lines of evidence indicate extensive mirna deregulation in bc, including differential expression of mirnas in normal vs transformed mammary epithelial cells  <cit> .
for these reasons, we focused our attention on the tus encoding pre-mirnas, to test the possibility that erβ binding sites might be located in close proximity of these genes.
results show that  <dig> mirna-encoding loci  show one or more erβ site within  <dig> kb from the pre-mir sequence of the host gene - listed in additional table s <dig> .
distribution of these sites among the er binding classes described above was comparable to what observed for primary genes.
interestingly, in several cases erβ binds both up- and down-stream of the pre-mir locus, further suggesting that receptor docking might exert multiple regulatory actions on mirna biogenesis.
we tested the hypothesis that the observed distribution of sites near the pre-mirna loci occurred at random by applying a bootstrap approach.
to this end, we repeatedly sampled  <dig> times the same number of sites of the real erβ binding set, with the same length distribution, a similar distribution among chromosomes but randomly selected coordinates.
we then counted the number of pre-mirna loci and the number of randomly generated sites found within  <dig> kb of each other and compared their distributions with that of the experimentally detected ones.
the number of randomly generated sites within  <dig> kb of a pre-mirna never reached the value detected experimentally, while the number of mirna loci scoring an artificial site in close proximity was equal or above what measured only in  <dig> % of the cases.
these results can be explained also by the observation that in several cases erβ binds both up- and down-stream of the pre-mir locus, a result that support a functional significance of this observation.
in fact, the ratio between the number of erβ binding sites and the number of pre-mirna loci within  <dig> kb of each other is  <dig> , while this varied between  <dig>  and  <dig> for the randomly generated sets .
we thus conclude that although some of the erβ sites detected in or near pre-mirna loci may be the result of a random, non functional event, these is likely to represent rare events, as random distribution never reaches the enrichment level observed experimentally.
indeed, preliminary mirna profiling analyses carried out in wt mcf- <dig> and tap-erβ cells indicate that mature mirna expression undergo extensive deregulation in the presence of erβ , suggesting that at least some of the sites identified here might indeed be involved in erβ-mediated regulation of mirna biosynthesis in bc cells.
erβ binding to the mitochondrial genome
mitochondrial dna  is usually overlooked in whole-genome chip-seq analyses, since identification of enriched peaks is more difficult here due to a much higher noise, consequence of the high and variable number of mtdna copies in the cell.
erβ has been shown to localize in the mitochondria in different cell types  <cit>  including human bc cells  <cit> , and a role for estrogen in mitochondrial function, with implications on cell growth, has been established  <cit> .
we thus analyzed the sequence reads that aligned on the mtdna sequence with the same method used for whole genome data analysis, but applying a supplementary fold-intensity filter  to deal with the higher background noise.
this analysis revealed one erβ binding site in proximity of the mtdna d-loop, but no erα binding sites either in wt mcf- <dig> or in tap-erβ cells .
independent chip analysis confirmed this results, showing erβ binding to this same mtdna region upon activation by e <dig> or its selective agonist  <dig> -bis propionitrile  and lack of erα binding .
furthermore, the presence of erβ in mitochondria of tap-erβ cells was confirmed biochemically, by western blotting , an analysis that revealed also the presence of erα in the organelle.
when we analyzed the sequence of the erβ mitochondrial site with matinspector, we observed the presence of the matrix v$gata <dig> , bound by gata <dig>  a factor whose activity is strongly repressed by erα  <cit> , and v$hmga.
<dig>  bound by hmga <dig>  a non-histone chromosomal protein that is highly overexpressed in cancer cells  <cit>  and has been shown to interact with erα and to enhance its binding to dna  <cit> .
CONCLUSIONS
the results of this study indicate that in vivo erβ can interact with hormone-responsive bc cell chromatin either alone or complexed with erα, but in all cases the two receptors share the same genomic targets.
an observation that is in agreement with the conclusions of previous studies based on analysis of erα and erβ heterodimerization and binding to the ere  <cit> .
when both er subtypes are expressed in the same cell, the main action of erβ in the genome is thus achieved in combination with erα, by either heterodimerization or competition for binding to the same target sites in chromatin.
based on these observations, we propose that in hormone-responsive bc the final cellular response to estrogen is likely to depend upon the relative concentration of the two ers in the cell, their activation status and dna binding kinetics and the presence of other factors influencing their respective functions.

 estrogens are key regulators of cell growth and differentiation in the mammary gland, where they are involved in the pathogenesis and clinical outcome of breast cancer. these steroid hormones exert their effects in normal and transformed mammary epithelial cells by binding to specific receptors, erα and erβ, that mediate estrogen signaling by functioning as ligand-dependent transcription factors. ligand-activated ers drive gene cascades comprising primary genes, whose transcription is directly controlled by the hormone through physical interaction of ers with regulatory sites in the genome and/or with signal transduction effectors, as well as downstream genes whose activity depends upon the functions encoded by the primary responders. ers are able to bind dna at specific sites in the genome and thereby control gene activity by recruiting transcriptional mediators and co-regulators, as well as a host of other nuclear proteins with different roles in er-mediated control of gene activity. the two ers show 55% identity in their estrogen-binding domains and approximately 97% similarity in the dna-binding domains. reflecting the high degree of similarity in their dbds, both receptors interact with the same conserved estrogen response element as either homodimers or alpha/beta heterodimers. erβ, however, holds low trans-acting capability on ere-containing estrogen target genes and alpha/beta heterodimers are less efficient than erα homodimers in promoting target genes activity. the different behaviour of erα/erβ heterodimers respect to erα homodimers on transcriptional regulation of ere-containing genes might be explained by different co-factor recruitment, as erβ could prevent efficient co-activator binding to the erα moiety of the heterodimer, conversely inducing recruitment of co-repressors and/or driving assembly of co-regulatory complexes other than those involving erα only.. although the two receptors are quite similar in sequence and structure, in bc erβ has considerably different biological effects than erα. furthermore, the two ers show a remarkably different expression pattern in bcs, with higher erα and lower erβ levels observed in malignant cells compared to normal mammary epithelial or benign tumor cells. furthermore, while erα induces a mitogenic response to estrogen, when expressed alone the β subtype is not only unable to induce the same mitogenic response, but it reduces basal, hormone-independent cell proliferation. finally, erβ was shown to change dramatically erα-positive bc cell behaviour in vivo, as its expression in the cell prevents tumorigenicity in mouse xenograft models by reducing tumor growth and angiogenesis. gene expression studies performed in bc cell lines expressing endogenous erα and recombinant erβ revealed multiple signaling pathways involving the α and/or β receptor subtypes. the two ers appear thus to share many target genes, although each of them may affect specific downstream targets. for this reason, inhibition of hormone-responsive bc cell growth by erβ might be due to direct interference with erα activity on growth-promoting pathways as well as to the activity of erβ-specific target genes. recently, next-generation sequencing technologies combining chromatin immunoprecipitation either with genomic dna hybridization to microarrays or massively parallel sequencing, opened new venues for our understanding of physical and functional associations between transcription factors and chromatin in vivo. these analytical strategies led to genome-wide mapping of erα-binding regions in intact chromatin of cultured cell lines, revealing important new information relative to erα interaction with the genome. carroll et al., for example, using chip-on-chip demonstrated that the forkhead factor foxa plays an important role as pioneering factor for erα binding to chromatin in bc cells, while cicatiello et al. identified novel gene regulation cascades mediating estrogen actions in hormone-responsive bc cells. in contrast, although several studies focused on erβ interaction with the genome, a thorough characterization of this important aspect of erβ biology in bc cells, essential to clarify the mechanisms mediating its control of estrogen-dependent gene pathways and the hormone-responsive phenotype, is still missing. for this reason, we performed a comprehensive analysis of erβ and erα target sites in the genome of mcf- cells engineered to express both receptors to comparable levels, by integrating global mapping of in vivo er binding to the genome by chip-seq with comparative gene expression profiling in erβ+/erα+ vs erβ-/erα+ cells during early stimulation with 17-beta-estradiol, followed by in silico analyses of the erβ binding regions and responsive genes identified.  establishment and characterization of erβ-expressing mcf- cells stabilized human bc cell lines expressing endogenous erα and erβ at comparable levels are not available. for this reason, we first generated and characterized cell lines derived from erα-positive mcf- cells expressing full-length human erβ at levels similar to those of endogenous erα. this strategy was adopted to prevent artefacts due to erβ over-expression in the cell and to mimic what observed in primary breast tumors, where very high expression of this receptor has never been observed. as suitable antibodies for efficient immunoprecipitation of chromatin-bound erβ are not available, the expressed proteins were tagged on either their c- or n-terminus with the tap epitope. this approach allows to track tagged ers in different cell compartments and to efficiently immunoprecipitate and purify them in vitro by tandem affinity purification, to identify their molecular partners, and ex vivo in chromatin immunoprecipitation assays. preliminary tests were performed to verify whether the presence of the tap moiety could influence intracellular redistribution of erβ in response to 17β-estradiol and its ability either to trans-activate an estrogen-responsive reporter gene or to interfere with erα activity on reporter gene transcription and bc cell proliferation. to this end, er expression and nuclear translocation in response to e was determined in wt mcf- ct-erβ and nt-erβ cells by subcellular fractionation followed by sds-page and immunoblotting. in absence of hormone a larger fraction of both ers was found in the cytosol in all cases. following estrogen stimulation, both receptors migrated to the nucleus, a crucial event to trigger target gene transcription via the genomic pathway of the estrogen signaling cascade. an antibody against α-tubulin was used as control, and the absence of this protein in the nuclear fractions indicates that they were indeed free from cytosolic contaminants. the erβ-expressing clones selected for this study showed a erβ/erα ratio < as verified by immunoblotting analysis of the proteins in whole cell extracts and quantitative rtpcr of the corresponding rnas. to control that the presence of the tap tag did not interfere with erβ activity, er-negative skbr- bc cells were transiently transfected with expression vectors encoding wt erβ, ct-erβ, nt-erβ, erα or'empty' vector, as controls, and ere-tk-luc, a reporter gene where luciferase expression is driven by an estrogen-responsive minimal promoter. exposure of transiently transfected cells to e induced reporter gene activation in the presence of erα, erβ, ct-erβ or nt-erβ, with the activity of both tagged erβ proteins slightly lower than that of wt erβ. we then tested whether the two recombinant forms of erβ were able to interfere with target gene activation by the endogenous erα resident in mcf- cells. to this end, wt, ct-erβ+ and nt-erβ+ cells were transfected with ere-tk-luc and the response of the reporter gene to e was determined. as shown in figure 1b, erβ-expressing cells showed in all cases a marked reduction in reporter gene response to the hormone, when compared to wt mcf- cells, indicating that both tagged erβs are able to interfere with the activity of endogenous erα. results show that cell lines stably expressing ct-erβ and nt-erβ display a marked reduction in proliferative response to the hormone, when compared to wt mcf- cells, in agreement with the known effects of erβ in erα-positive cells. furthermore, comparative rna expression profiling in exponentially growing ct-erβ and nt-erβ vs wt mcf- cells revealed extensive overlapping effects of the two tagged erβ proteins on the activity of estrogen target genes. taken together, these observations confirmed that both tag-erβ expressing cell lines generated for this study show a well defined phenotype, with respect to the known activities of this er subtype in bc cells, and are thus suitable to investigate the genomic bases of erβ actions in this cell type. as we could not exclude that the presence of the tap tag at either the n- or c- term of erβ may specifically influence its activity on cellular targets or pathways different from those investigated above, all experiments reported in this study were performed in both ct-erβ and nt-erβ cells and the data were combined for analysis, with the aim to focus on the most significant and reproducible actions of erβ independently from position of the tag in the receptor moiety. effects of erβ on the estrogen-responsive mcf- cell transcriptome expression of erβ is known to cause significant changes in the genomic response to estrogen in target cells. to identify the genes whose estrogen regulation in hormone-responsive bc cells is perturbed by erβ, we performed gene expression profiling with microarrays in estrogen-starved, quiescent wt and tap-erβ+ mcf- cells following stimulation with 10-8m e total rna was extracted from the three cell lines either before or after or hrs stimulation, fluorescently labelled and analyzed on whole-genome microarrays. samples extracted from the two erβ-expressing cell lines were pooled before analysis, to reduce the impact of clone-specific differences and to focus on the most significant effects of erβ, independent from tag position in the protein. results obtained in these samples were then compared with those obtained under the same conditions in wt mcf- cells. this analysis yielded transcripts differentially regulated by the hormone in erβ+ vs erβ- cells, including mrna whose regulation was detected only in wt mcf- cells, regulated only in erβ+ cells, showing a similar pattern of response in both cell types and showing opposite responses to the hormone in erβ+ vs erβ- cells. the full list of these differentially regulated transcripts is reported, with relevant information, in additional table s taken together, these results indicate that the presence of erβ greatly influences the response of the mcf- cell genome to estrogen, by interfering with erα-mediated hormonal regulation of genes and promoting de novo regulation of genes. it should be noted that these analyses were performed with data obtained after hrs of hormonal stimulation, a timing that allowed us to focus on early response genes, positioned upstream in the composite transcriptional cascade set in motion by the hormone in this cell type and more likely to include primary genomic targets of ers. it is thus possible that this analysis missed erβ-responsive genes showing significant changes in expression only at later times after hormonal stimulation. however, analysis of the global effects of erβ on gene expression in these same cells, performed as described above in cultures exponentially growing under continuous hormonal stimulus, suggests that the number of regulated transcripts identified here is rather close to the total number of genes targeted by this er subtype in mcf- cells. global mapping of erα and erβ binding to mcf- cell genome the widespread effects of erβ on mcf- cell transcriptome are likely to result from multiple effects of this receptor in the cells, including direct regulation of primary response genes via genomic or non genomic mechanisms, and indirect gene regulation events mediated by the products of primary genes. the primary erβ target genes are most likely to comprise also master regulators of complex cellular responses to the receptor, mediating its effects on the biological and clinical phenotype of bc cells. to identify such primary genomic targets and investigate the mechanisms that allow their regulation by erβ, a global analysis of in vivo binding of this receptor to the genome was carried out in tap-erβ cells by chromatin immunoprecipitation coupled to massively parallel sequencing, that allows detailed mapping of in vivo tf binding to the genome. in parallel, we studied erα binding to the genome under the same conditions, to allow comparative analyses between the two er subtypes. replicate chromatin samples were prepared from both ct-erβ and nt-erβ cells before and after stimulation for minutes with 10-8m e and dna-bound proteins were immunoprecipitated either with antibodies against the n- and c-terminus of erα, or with iggs binding with high affinity the tap moiety of tagged erβ. preliminary testing on several known erβ binding sites, including the promoter-near region of ps2/tff gene, confirmed that the method selected to immunoprecipitate chromatin-bound ct-erβ and nt-erβ was efficient and specific. the resulting dnas were used to generate chip-seq libraries for erα and erβ, respectively, that were then sequenced with the illumina genome analyzer. the sequence tags obtained were then aligned to the human genome sequence and peaks enriched in the libraries generated after e2-treatment were identified using macs. this led to the identification of binding sites for erβ and sites for erα, of which were shared by both receptors, with an average false discovery rate of 3%. the full list of these binding sites is available, with relevant information, in additional files and interestingly, about half erβ binding sites identified map within transcription units, mainly in intronic regions. this distribution is maintained also in erβ-regulated transcription units, where erβ binding sites located in the gene or within kbps from it are distributed as follows: in promoter regions, in exons, within one or more introns and the remaining either upstream of promoters or downstream of the gene. in both cases the erβ binding sites within genes did not show any preference with respect to exon or intron position nor for know intragenic regulatory elements. it should be mentioned that the number of erβ binding sites identified is significantly higher that those mapped in mcf- cells by chip-on-chips, possibly for technical differences due to erβ expression levels in the different mcf- cell-derived clones used, in immunoprecipitation efficiency and/or in dna analysis. since only zhao et al. performed an unbiased search for erβ binding with whole-genome chips, we could confront our results only with those reported in that study. this showed that 86% of high confidence erβ sites described in that study appear also in our dataset. the binding sites identified here were then subjected to sequence analysis, searching first for the presence of eres, the characteristic er binding signature. this analysis revealed that in all three cases a high percentage of sites displayed one or more imperfectly palindromic ere, with a slightly higher positivity in erα sites. as ers have been shown to bind both in vitro and in vivo to puggtca hemi-palindromes, we searched the sequence of the remaining sites for perfect matches to this sequence. results showed that almost half of them indeed contained one or more heres. the percentages of sites not carrying a known er-binding element were similar for both receptors. we observed that erα and erβ binding sites were often found in close proximity to each other, a confounding factor when attempting to discern and analyze separately er subtype-specific sites and target genes. this could be due to the limits of the chip-seq technology or of the algorithm used for peak selection. to overcome this problem, and allow the identification of potential er subtype-specific sites, we used a cartographic approach to group nearby binding sites that might be the result, at least in part, of shortfalls of the mapping methods applied. each binding peak was thus elongated in both directions by bp and the overlapping ones obtained were merged into erβ and erα'extended' binding regions. these regions were intersected to define erα only, erβ only or erα+erβ binding regions. in this way we could identify erα-only and erβ-only binding sites, comprised in these regions, none of which showed nearby binding of the other receptor. these were named: er subtype'prevalent' sites. the binding peak sequences included in each of the three regions obtained were then re-analyzed for the presence of ere or here elements. in this way we could observe that sites within the erα+erβ regions showed now a much higher percentage of ere+ sequences, respect to those present in the erα-only or erβ-only regions. since all three types of sites showed almost identical proportions of here+, this result suggests that perfectly or imperfectly palindromic eres are preferential binding sequences for erα-erβ heterodimers, while erα and erβ homodimers appear to be more flexible in dna recognition. ere+ sequences were then analyzed in more detail with meme, to investigate if the three classes of sites identified showed any difference in the relative base composition of their respective ere signatures. for each list of sequences, the most significant position-specific probability matrix generated by meme was compared to the matrices present in the jaspar transcription factor binding profile database, using the stamp tool-kit for dna motif comparison. as shown in figure 3c, this analysis revealed that the ere matrices derived from the three types of binding regions identified are identical and, as a consequence, that erβ does not appear to display ere variant selectivity. we then examined the ere- sequences to search for enriched binding motifs for other transcription factors that might play a role in er binding to chromatin in the absence of canonical eres. ers are known to be able to bind chromatin indirectly, by physically interacting with dna-bound tfs, including sp or the ap1complex, for gene trans-regulation. tfbs enrichment respect to the whole genome was calculated thus in ere- sites with using the regionminer tool and only statistically significant and highly enriched matrices were further considered. the results are summarized in the right panel of figure 3c, showing for comparison the over-representation values scored in here+ and ere+ sites by the same matrices selected in the ere- sites. these numerical values, together with relevant information, are reported in additional table s. the enriched matrices found in the ere- set of erβ prevalent sites include v$sp, v$sp, v$sp, v$sp and v$gc. all belonging to the gc-box family targeted by sp and gcfc factors, v$ctcf. and v$ctcf. binding site matrices for the ccctc-binding factor ctcf, that is a known transcriptional repressor of c-myc, v$nrf, binding nrf, a transcription factor that regulates the expression of nuclear-encoded mitochondrial genes, v$zf, for the poz domain zinc finger protein zf and v$znf, recognized by the zinc finger proteins znf and the majority of these tfs bind gc- and c-rich sequences that are structurally related to each other, suggesting the possibility that a significant portion of the sequence elements listed above might indeed be recognized by one or a limited number of tfs. on the other hand, the v$gaga. matrix was specifically enriched only in the erα prevalent ere- binding sites. this sequence binds a transcription factor known to influence chromatin structure in drosophila and to bind throughout the genome, but nothing is known about physical or functional interactions of this factor with ers or other nuclear receptors. the results of this analysis point to tfs that could act as partners of erβ for binding to chromatin in the absence of canonical eres. interestingly, the majority of these same matrices were found enriched also in the erβ binding sites comprising heres or eres, suggesting that one or more such dna matrices might cooperate with erβ for either dna binding or gene trans-regulation. we performed a direct search for conserved motifs in the ere- binding sites of erβ with meme, and the most significant position-specific probability matrices were compared to those present in the jaspar tf binding profile database. the results failed to provide any conclusive information, as each of several sequence motifs obtained with this analysis was found only in a small fraction of the binding sites analyzed. identification of primary erβ target genes to identify the genes directly controlled by erβ binding to the genome, and analyze the interplay between the two ers in gene regulation, we combined the chip-seq data with those relative to estrogen responsive genes differentially regulated by e in erβ+ vs erβ- mcf- cells under the same experimental conditions. three sets of er binding regions were used for this analysis: erβ and erα binding regions identified in tap-erβ cells, and all erα binding regions identified so far in wt mcf-. the regions from set were intersected with those from set, to define which of them was binding both receptor subtypes, only erβ or only erα in tap-erβ cells. the'homodimer erβ' and the'heterodimer erα+erβ' groups were further filtered against set binding regions, to identify the genomic sites recognized by erβ, with or without erα, but never by erα alone. this allowed us to classify the sites comprised in these regions as follows: class including sites occupied by erα in wt mcf- cells and by erβ in tap-erβ cells; class showing sites where erα can bind in wt mcf- cells and both receptors are detected in tap-erβ cells; class with sites binding only erβ and never, under any condition, erα; class comprising sites where both receptors are found in tap-erβ cells but none in wt mcf- cells; class including sites where erα binds only in tap-erβ but never in wt cells; class composed of sites that bind only erα both in wt and tap-erβ cells. when combined with the results of the sequence analyses described above, this classification reveals that erβ-specific cis-acting regulatory elements are unlikely to exist in the genome, as all evidence point to the fact that the two er subtypes can recognize identical sequence features. to identify among the genes differentially regulated by estrogen in erβ+ vs erβ- cells those representing direct targets for transcriptional regulation by dna-bound erβ in our cell model, we extracted from the list in additional table s the genes bearing one or more erβ binding sites inside or within kb of the tu, and termed them'primary', to indicate that they are most likely to respond directly to the signal conveyed by the e2-activated receptor. of these genes - listed in additional table s, whose kinetics of response to e in wt and tap-erβ cells is shown in figure show one erβ site of class, a site of class, a site of class and only a class site, while showed multiple erβ sites belonging to different classes and were thus classified accordingly. in the right panels of figure are reported examples for each of the primary gene classes described above, showing the location of the receptor binding sites respect to the promoter and structural gene. it is worth mentioning that when the gene expression data from wt mcf- cells stimulated with e for 8hrs were combined with information concerning erα binding regions identified in wt mcf- cells under comparable conditions, putative primary erα target genes were identified -listed in additional table s, 71% of which showed erβ binding in hormone-stimulated erβ+ cells. this result supports the view that the two er subtypes tend to interact with the same targets in bc cells genome. right: genome browser view of genomic loci representative of the different erα and erβ binding site categories identified. chip-seq erα and chip-seq erβ indicate sites identified in this study, chip-seq erα chip-seq erα and chip-on-chip indicates sites identified in mcf- cells by cicatiello et al., fullwood et al., and hurtado et al., respectively. a functional analysis of the primary erβ target genes identified here with ontologizer showed that most primary erβ responsive genes are involved in key cellular processes, including control of cell proliferation, survival and differentiation status as well as cell motility, migration and adhesion, and can all greatly influence bc cell phenotype and response to estrogen - listed in additional table s. when go analysis was performed on the erα target gene set from wt mcf- cells, results showed that the genes controlled directly by this er subtype appear to be involved in the same cellular processes described above for erβ -compare results reported in additional tables s and s, providing a further indication of the significant overlapping between gene pathways targeted by erβ and erα in bc cells. focusing on the genes differentially regulated by e in erβ- vs erβ+ cells known for their involvement in cell proliferation, we observed that many of them encode for transcription factors and other key proteins controlling large gene networks of cell division cycle and cell survival and, in general, cell proliferation and differentiation pathways. these include, in particular, cdk-, cebpa, dab, hes-, igfbp-, irs- and -, jak-, junb, mitf, myc, slit-, smarca-, sox-, tgfb-, tgfbi/lcd-, tgm-, tns- and wisp-. interestingly, the role of all these genes in tumor cell proliferation and differentiation is known and an involvement in hormone-mediated bc cell responses to erα has been reported for most of them, suggesting that discovery of an erβ direct effect on transcription of these genes provides a new molecular framework to elucidate the anti-proliferative and differentiative effects of this receptor subtype in hormone-responsive cells. among the rnas encoded in the genome, micrornas have emerged as master regulators of gene expression for their ability to influence mrna concentration and activity by post-transcriptional mechanisms. recent results highlighted the role of mirna in bc cells response to estrogen and, in addition, several lines of evidence indicate extensive mirna deregulation in bc, including differential expression of mirnas in normal vs transformed mammary epithelial cells. for these reasons, we focused our attention on the tus encoding pre-mirnas, to test the possibility that erβ binding sites might be located in close proximity of these genes. results show that mirna-encoding loci show one or more erβ site within kb from the pre-mir sequence of the host gene - listed in additional table s. distribution of these sites among the er binding classes described above was comparable to what observed for primary genes. interestingly, in several cases erβ binds both up- and down-stream of the pre-mir locus, further suggesting that receptor docking might exert multiple regulatory actions on mirna biogenesis. we tested the hypothesis that the observed distribution of sites near the pre-mirna loci occurred at random by applying a bootstrap approach. to this end, we repeatedly sampled times the same number of sites of the real erβ binding set, with the same length distribution, a similar distribution among chromosomes but randomly selected coordinates. we then counted the number of pre-mirna loci and the number of randomly generated sites found within kb of each other and compared their distributions with that of the experimentally detected ones. the number of randomly generated sites within kb of a pre-mirna never reached the value detected experimentally, while the number of mirna loci scoring an artificial site in close proximity was equal or above what measured only in % of the cases. these results can be explained also by the observation that in several cases erβ binds both up- and down-stream of the pre-mir locus, a result that support a functional significance of this observation. in fact, the ratio between the number of erβ binding sites and the number of pre-mirna loci within kb of each other is, while this varied between and for the randomly generated sets. we thus conclude that although some of the erβ sites detected in or near pre-mirna loci may be the result of a random, non functional event, these is likely to represent rare events, as random distribution never reaches the enrichment level observed experimentally. indeed, preliminary mirna profiling analyses carried out in wt mcf- and tap-erβ cells indicate that mature mirna expression undergo extensive deregulation in the presence of erβ, suggesting that at least some of the sites identified here might indeed be involved in erβ-mediated regulation of mirna biosynthesis in bc cells. erβ binding to the mitochondrial genome mitochondrial dna is usually overlooked in whole-genome chip-seq analyses, since identification of enriched peaks is more difficult here due to a much higher noise, consequence of the high and variable number of mtdna copies in the cell. erβ has been shown to localize in the mitochondria in different cell types including human bc cells, and a role for estrogen in mitochondrial function, with implications on cell growth, has been established. we thus analyzed the sequence reads that aligned on the mtdna sequence with the same method used for whole genome data analysis, but applying a supplementary fold-intensity filter to deal with the higher background noise. this analysis revealed one erβ binding site in proximity of the mtdna d-loop, but no erα binding sites either in wt mcf- or in tap-erβ cells. independent chip analysis confirmed this results, showing erβ binding to this same mtdna region upon activation by e or its selective agonist -bis propionitrile and lack of erα binding. furthermore, the presence of erβ in mitochondria of tap-erβ cells was confirmed biochemically, by western blotting, an analysis that revealed also the presence of erα in the organelle. when we analyzed the sequence of the erβ mitochondrial site with matinspector, we observed the presence of the matrix v$gata, bound by gata a factor whose activity is strongly repressed by erα, and v$hmga. bound by hmga a non-histone chromosomal protein that is highly overexpressed in cancer cells and has been shown to interact with erα and to enhance its binding to dna.  the results of this study indicate that in vivo erβ can interact with hormone-responsive bc cell chromatin either alone or complexed with erα, but in all cases the two receptors share the same genomic targets. an observation that is in agreement with the conclusions of previous studies based on analysis of erα and erβ heterodimerization and binding to the ere. when both er subtypes are expressed in the same cell, the main action of erβ in the genome is thus achieved in combination with erα, by either heterodimerization or competition for binding to the same target sites in chromatin. based on these observations, we propose that in hormone-responsive bc the final cellular response to estrogen is likely to depend upon the relative concentration of the two ers in the cell, their activation status and dna binding kinetics and the presence of other factors influencing their respective functions. 
the identification of the genomic targets of erβ in hormone-responsive bc cells is thus a critical step to elucidate the roles of this receptor in estrogen signaling and tumor cell biology. results indicate that the vast majority of the genomic targets of erβ can bind also erα, suggesting that the overall action of erβ on the genome of hormone-responsive bc cells depends mainly on the relative concentration of both ers in the cell. these putative primary erβ target genes control cell proliferation, death, differentiation, motility and adhesion, signal transduction and transcription, key cellular processes that might explain the biological and clinical phenotype of tumors expressing this er subtype. by chip-seq we identified erβ and erα binding sites in estrogen-stimulated cells, comprising sites occupied by either erβ, erα or both er subtypes. a search for tf binding matrices revealed that the majority of the binding sites identified comprise one or more estrogen response element and the remaining show binding matrixes for other tfs known to mediate er interaction with chromatin by tethering, including ap e2f and sp of genes differentially regulated by estrogen in erβ+ vs erβ- cells, showed one or more erβ site within kb. the two receptors can be found co-expressed and play specific, often opposite, roles, with erβ being able to modulate the effects of erα on gene transcription and cell proliferation. expression of full-length erβ in hormone-responsive, erα-positive mcf- cells resulted in a marked reduction in cell proliferation in response to estrogen and marked effects on the cell transcriptome. estrogen receptors alpha and beta are transcription factors that mediate estrogen signaling and define the hormone-responsive phenotype of breast cancer.
 estrogen receptors alpha and beta are transcription factors that mediate estrogen signaling and define the hormone-responsive phenotype of breast cancer. the two receptors can be found co-expressed and play specific, often opposite, roles, with erβ being able to modulate the effects of erα on gene transcription and cell proliferation. erβ is frequently lost in bc, where its presence generally correlates with a better prognosis of the disease. the identification of the genomic targets of erβ in hormone-responsive bc cells is thus a critical step to elucidate the roles of this receptor in estrogen signaling and tumor cell biology.  expression of full-length erβ in hormone-responsive, erα-positive mcf- cells resulted in a marked reduction in cell proliferation in response to estrogen and marked effects on the cell transcriptome. by chip-seq we identified erβ and erα binding sites in estrogen-stimulated cells, comprising sites occupied by either erβ, erα or both er subtypes. a search for tf binding matrices revealed that the majority of the binding sites identified comprise one or more estrogen response element and the remaining show binding matrixes for other tfs known to mediate er interaction with chromatin by tethering, including ap e2f and sp of genes differentially regulated by estrogen in erβ+ vs erβ- cells, showed one or more erβ site within kb. these putative primary erβ target genes control cell proliferation, death, differentiation, motility and adhesion, signal transduction and transcription, key cellular processes that might explain the biological and clinical phenotype of tumors expressing this er subtype. erβ binding in close proximity of several mirna genes and in the mitochondrial genome, suggests the possible involvement of this receptor in small non-coding rna biogenesis and mitochondrial genome functions.  results indicate that the vast majority of the genomic targets of erβ can bind also erα, suggesting that the overall action of erβ on the genome of hormone-responsive bc cells depends mainly on the relative concentration of both ers in the cell. 