ESR1 Is Co-Expressed with Closely Adjacent Uncharacterised Genes Spanning a Breast Cancer Susceptibility Locus at 6q25.1

Approximately 80% of human breast carcinomas present as oestrogen receptor α-positive (ER+ve) disease, and ER status is a critical factor in treatment decision-making. Recently, single nucleotide polymorphisms (SNPs) in the region immediately upstream of the ER gene (ESR1) on 6q25.1 have been associated with breast cancer risk. Our investigation of factors associated with the level of expression of ESR1 in ER+ve tumours has revealed unexpected associations between genes in this region and ESR1 expression that are important to consider in studies of the genetic causes of breast cancer risk. RNA from tumour biopsies taken from 104 postmenopausal women before and after 2 weeks treatment with an aromatase (oestrogen synthase) inhibitor was analyzed on Illumina 48K microarrays. Multiple-testing corrected Spearman correlation revealed that three previously uncharacterized open reading frames (ORFs) located immediately upstream of ESR1, C6ORF96, C6ORF97, and C6ORF211 were highly correlated with ESR1 (Rs = 0.67, 0.64, and 0.55 respectively, FDR<1×10−7). Publicly available datasets confirmed this relationship in other groups of ER+ve tumours. DNA copy number changes did not account for the correlations. The correlations were maintained in cultured cells. An ERα antagonist did not affect the ORFs' expression or their correlation with ESR1, suggesting their transcriptional co-activation is not directly mediated by ERα. siRNA inhibition of C6ORF211 suppressed proliferation in MCF7 cells, and C6ORF211 positively correlated with a proliferation metagene in tumours. In contrast, C6ORF97 expression correlated negatively with the metagene and predicted for improved disease-free survival in a tamoxifen-treated published dataset, independently of ESR1. Our observations suggest that some of the biological effects previously attributed to ER could be mediated and/or modified by these co-expressed genes. The co-expression and function of these genes may be important influences on the recently identified relationship between SNPs in this region and breast cancer risk.


Introduction
Breast cancer is the most common malignancy in women, accounting for more than 400,000 deaths per year worldwide [1]. Approximately 80% of human breast carcinomas present as oestrogen receptor a-positive (ER+ve) disease and ER status is arguably the most clinically important biological factor in all oncology [2]. The major molecular features of breast cancer segregate differentially between ER+ve and ER2ve tumours [3,4]. Tumours which express ERa have been termed luminal type [3,5] and are associated with response to antioestrogen therapy and improved survival, although the mechanisms by which oestrogen receptor dictates tumour status are poorly understood.
Recent genome wide studies have identified SNPs around C6ORF97, an open reading frame (ORF) immediately upstream of the gene encoding ER (ESR1) to be associated with increased risk of breast cancer. Zheng et al. found that heterozygosity at rs2046210, a SNP in the region between C6ORF97 and ESR1, increased breast cancer risk by an odds ratio of 1.59 in a Chinese population and that this risk was also present in a European population, albeit to a weaker extent [6]. Easton and colleagues confirmed the risk associated with this SNP and reported an at least partly independent risk associated with a second adjacent SNP (rs3757318) in intron 7 of C6ORF97 [7]. Using ancestry-shift refinement mapping, Stacey et al. closed in on the identification of the pathogenic variant and found that the risk allele of a novel SNP in this region (rs77275268), disrupts a partially methylated CpG sequence within a known CTCF binding site [8]. More recently, two further studies have confirmed an association with the region [9,10]. Our studies have revealed unexpected relationships in the expression patterns in breast carcinomas between ESR1, C6ORF97 and the two genes immediately upstream (C6ORF211 and C6ORF96 [RMND1]).
Oestrogenic ligands, predominantly oestradiol, are the key mitogens for ER+ve breast cancer. In recent years, high throughput genomic technologies have revealed significant numbers of genes that are expressed in response to oestradiol stimulation in vitro [11][12][13] and downregulated in response to oestrogen deprivation in tumours [14][15][16]. Similarly, the transcriptional targets of ERa have been characterised in detail using genome wide chromatin interaction mapping in MCF7 cells [17,18]. Key oestrogen responsive genes such as TFF1 and GREB1 have been shown to be highly responsive to oestradiol stimulation in cell culture models through the binding of ERa to their promoters [19,20]. Additional genes have been found in hierarchical clustering analyses of ER+ve and ER2ve tumours as part of the so-called ''luminal epithelial'' gene set characterized by the expression of genes typically expressed in the cells that line the ducts of normal mammary glands including GATA3 and FOXA1 [12]. However, the correlates of ESR1 within an exclusively ER+ve group and the inherent heterogeneity within an exclusively ER+ subgroup remain poorly defined.
Modern, non-steroidal aromatase inhibitors (AIs) are widely used, effective treatments for ER+ve breast cancer [21,22] and are also excellent pharmacological probes for oestrogen-dependent processes in vivo because of their specificity and highly effective suppression of oestrogen synthesis. In this study, we found that the expression of genes in the region immediately upstream of ESR1 associate strongly with ESR1 expression in ER+ve primary breast cancers before and after AI treatment and uncover evidence that these associations might impact upon the biological and clinical importance of ERa.

ESR1 expression is correlated with three open reading frames on chromosome 6 in tumours
To investigate correlates of ESR1, expression profiles were derived from pairs of 14-guage core cut biopsies before and after 2 weeks' treatment with 1 mg/d anastrozole, an AI, from 104 patients with ER+ve primary breast cancer [23]. Genes whose expression correlated with expression of ESR1 levels pre-treatment were identified (Spearman corrected for multiple testing at false discovery rate ,1610 27 , Table 1 pre-treatment). The mRNA species most highly correlated with ESR1 were chromosome 6 ORF 97 (C6ORF97, Rs = 0.67) (Figure 1a), followed by C6ORF211. Other notable inclusions amongst the top 20 most correlated genes included well-established ER-associated genes such as FOXA1, MYB and GATA3, plus C6ORF96, also known as RMND1 (Required for Meiotic Nuclear Division 1 homolog). The mean pre-treatment expression of the three ORFs was highly correlated with ESR1 (Rs = 0.70, Figure 1b). After 2 weeks' AI treatment, the top three genes correlating with ESR1 were C6ORF96, C6ORF97 and C6ORF211 (Rs.0.7 for all, Table 1 two weeks post-treatment). These three ORFs are all located less than 0.5 MB upstream of the ESR1 start site on the q arm of chromosome 6 ( Figure 1e). The expression of other genes located within a 50 MB region surrounding ESR1 were not correlated with ESR1 expression (Rs,0.25) (Table S1).
The correlation was present in all of five published microarray data sets of ER+ve breast cancer in which the C6orfs were included on the array ( Table 2). The expression of the three ORFs was lower in ER2ve than ER+ve tumours in the Wang dataset [24] (p = 0.002). No significant correlation was found in the ER2ve subgroup of this dataset. This may be a characteristic of ER2ve tumours or, alternatively, the measurement error associated with low levels of ESR1 transcript could preclude detection of a significant correlation in microarray data.

Correlation between ESR1 and the C6orfs is not explained by amplification
Amplification of the ESR1 locus has been reported inconsistently [25,26]. To determine whether the ESR1/C6ORFs correlation may be the result of underlying genomic co-amplification or deletion events, copy number (CN) status of ESR1 and the C6orfs was examined using array CGH analysis (resolution 40-60 kb) [27] on DNA from the 44 tumour samples from which adequate further tissue was available. One tumour was shown to be amplified and eight showed gains at ESR1, C6ORF96, C6ORF97 and C6ORF211, while four showed losses at all four loci. One was measured as having loss of C6ORF96, C6ORF211 and part of C6ORF97. While there was some correlation between CN and transcription of the four genes ( Figure S1), CN alterations did not explain the correlation between ESR1 and the C6orfs. In fact, when samples with identified CN changes were removed from the dataset, the correlation between ESR1 and mean C6orf expression levels strengthened rather than weakened (Rs = 0.83) (Figure 1c), suggesting that transcriptional co-regulation rather than genomic changes is more likely to underlie ESR1/C6ORF co-expression.

Change in ESR1 expression upon aromatase inhibitor treatment is correlated with change in C6orf expression
To assess whether the correlation in ESR1/C6ORF expression seen in pre-treatment biopsies is reflected in a concordant change in expression of these genes upon treatment, the relationship between the magnitude of change of each of these genes was investigated. Change in expression of ESR1 induced by aromatase inhibitor treatment over 2 weeks was strongly correlated with change in the C6orfs (Rs = 0.70) (Figure 1d). Given that this short duration of treatment, which has no measurable impact on cellularity or tumour size, is unlikely to facilitate DNA copy number changes throughout the sample this supports the probability that the co-regulation of these genes is at a transcriptional level.

Expression of ESR1 and the C6orfs are correlated in MCF7 and BT-474 cells in vitro
To determine whether the ESR1/C6ORF correlations were maintained in vitro, transcript levels of ERa and the three C6orfs were measured in oestrogen-deprived MCF7 cells and lapatinibtreated BT-474 cells over a 48-and 96-hour period, respectively. These treatments are both known to have significant effects on the

Author Summary
Recent genome-wide analysis has revealed that the way in which genes are arranged on chromosomes and the conformation of these chromosomes are crucial for the regulation of gene expression. Reflecting this arrangement, clusters of genes which are regulated together have been discovered. We have identified a previously unreported transcriptional activity hub spanning ESR1, the gene encoding the important breast cancer biomarker oestrogen receptor. Genetic variants immediately upstream of ESR1 have recently been linked to breast cancer risk. We found that three open reading frames within this region are tightly co-expressed with ESR1. We investigated the function of these genes and discovered that one of these co-expressed genes, C6ORF211, affects proliferation in cultured cells and is correlated with proliferation in breast tumours. Another of the genes, C6ORF97, is negatively correlated with proliferation in breast tumours and predicts for outcome on the anti-oestrogen drug tamoxifen. These findings suggest that the genes could contribute to the phenotype associated with oestrogenreceptor positivity. In addition, they may be involved in the mechanism by which genetic variation in this region of the genome contributes to breast cancer susceptibility.
expression of ESR1. Lapatinib has been shown to increase ERa in BT-474 cells [28,29], potentially via loss of Akt and de-repression of FOXO3a. This provides a useful model for manipulation to test the correlation between ESR1 and the C6orfs in vitro. Conversely, absence of oestradiol leads to a short-term reduction in ER expression [30]. Expression of all four genes followed a similar timecourse of expression and was highly correlated (Figure 2a and 2b).
ICI 182,780 (ICI) is a steroidal pure anti-oestrogen which causes ERa expression to be suppressed and downregulated [31,32]. Treatment of MCF7 cells with ICI did not affect ORF expression or their correlation with ESR1 ( Figure 2c). To confirm that the observed correlation was not being influenced by RNA transcribed prior to the addition of ICI, we also measured newly synthesised nascent RNA using PCR amplicons designed to cross an exon/ intron boundary [33]. This analysis revealed that nascent transcripts for ESR1 and the C6orfs remained correlated in both the presence and absence of ICI. The observation that transcription of the genes remains strongly correlated in the presence of ICI suggests that transcriptional regulation by ERa is not the main driver of the ESR1/C6ORF co-expression.

Knockdown of C6ORF211 by siRNA induces a reduction in proliferation in MCF7cells
The effect of reducing expression of each C6orf on cell proliferation was determined by transfecting siRNA SMART-POOLs directed against each ORF into MCF7 cells. In cells grown in both E2-containing media and without E2, all three siRNAs reduced transcript levels of their target ORF to ,30% of levels in cells transfected with the control non-targeting siRNA pool. Levels of ESR1, and the non-targeted ORFs were unaffected by the SMARTpool's ( Figure S2) while ESR1-SMARTpool siRNA led to a reduction in levels of all three C6orfs ( Figure  S3). Immunoblotting with a polyclonal antibody raised against a polypeptide of the predicted product of C6ORF211 showed an 86% reduction at the protein level ( Figure S4). Cells transfected with C6ORF211 siRNA showed a mean 36% reduction in cell number (p,0.0001) over four separate repeat experiments ( Figure 3A). C6ORF211 knockdown had no effect on oestrogendependent proliferation ( Figure 3B). Deconvolution of the SMARTPOOL showed that the four constituent siRNAs had a reproducible anti-proliferative effect when compared with scrambled control siRNA ( Figure S5). No consistent alteration in proliferation was observed in cells transfected with siRNAs directed against C6ORF96 or C6ORF97 ( Figure 3A).

C6ORF211 correlates with proliferation and clinical outcome in tumours
To determine whether the association between C6ORF211 expression and proliferation seen in cultured cells is reflected in tumours, the relationship between C6ORF211 expression and a metagene composed of known proliferation-associated genes [34] was investigated. In baseline biopsies, levels of C6ORF211 but not ESR1 correlated significantly with proliferation (C6ORF211, Rs = 0.23, p = 0.04; ESR1, Rs = 20.01, p = ns) (Figure 4a), suggesting that C6ORF211 is more strongly associated with proliferation than ESR1. Correlations were also observed with a number of wellknown proliferation-associated genes (Table S2). The relationship with proliferation was validated in data from a set of 354 ER+ve tumours [35]   To determine whether the relationship of the ORFs with proliferation is related to clinical outcome, recurrence free survival (RFS) in tamoxifen-treated patients was investigated for association with C6ORF97 and C6ORF211 expression. Despite the fact that in the Loi dataset ESR1 was not predictive of a significant difference in survival over 5 years [36], the lowest quartile of C6ORF97 was associated with significantly higher risk of recurrence (HR = 3.1, p = 0.0014) (Figure 4d). A similar trend was observed in untreated ER+ve tumours from the Wang dataset [24], although this was not significant (HR = 1.6, p = 0.16) ( Figure S6a). C6ORF211 was not significantly associated with RFS ( Figure S6b and S6c).

Discussion
Our observation of a previously unreported transcriptional activity hub in the ESR1/C6ORF region of 6q25.1 has implications for recently identified associations between SNPs in the ESR1 region and breast cancer risk, as well as broader implications for the biological and clinical importance of ERa in established breast cancer. A number of SNPs, including rs3757318 within intron 7 of C6ORF97 [7], have been associated with breast cancer risk but the causative variant and mechanism remain undefined [6][7][8][9][10]. In an attempt to identify the pathogenic variant, Stacey and colleagues recently reported that GG homozygotes at rs9397435, located immediately downstream of C6ORF97, may express higher mean levels of ESR1 and that the rs9397435 [G] allele conferred significant risk of both hormone receptor positive and hormone receptor negative breast cancer in European and Taiwanese patients [8]. The association of a SNP in this region with ER expression is consistent with findings from our own group which have revealed that the variant genotype of SNP rs2046210 is associated with increased ERa expression as measured by immunohistochemistry [37]. The findings reported in this paper suggest that, due to their high degree of correlation with ESR1, levels of C6ORF97, C6ORF96 and C6ORF211 are also likely to correlate with the rs2046210 and rs9397435 genotype. Consequently, these genes may be involved in the pathogenesis of the variant SNPs and could explain the apparent anomaly noted by Stacey and colleagues in that the SNPs predispose to both hormone receptor positive and negative disease.
To date, analysis of ESR1 co-expressed genes has focussed on genes which are also downstream targets of the oestradiolactivated transcription factor activity of ERa such as FOXA1, TFF1 and GATA3. High throughput technologies have identified numerous classical and novel ERa-dependent targets of oestradiol [11,17]. This association with the expression of ORFs has, however, not been reported other than by ourselves in abstract form [38].
The transcriptional correlation between ESR1 and these ORFs is highly statistically significant in our dataset, and in all of the publicly available datasets we examined. In our own patient cohort, we showed that two weeks' treatment with anastrozole induces a concomitant change in ESR1 and the C6orfs and a yet stronger correlation in their expression. Genomic amplification does not account for the correlations. This suggests that transcriptional co-regulation rather than major genomic rearrangement is likely to underlie their co-expression. To our knowledge, a transcriptional activity hub surrounding a major cancer related gene has not previously been identified.
The observation that the four transcripts remain correlated over a short timecourse in MCF7 and BT474 cells further supports the idea that the co-regulation of these genes is likely to occur at a transcriptional level. Given that ERa can autoregulate its own transcription by binding to an oestrogen responsive element (ERE) in its promoter [17,39], the possibility that ERa could co-regulate itself and the C6orfs provides an attractive potential explanation for the correlation. We tested this hypothesis by treating MCF7 cells with the ERa antagonist ICI in the absence of E2. Our finding that the nascent transcripts of ESR1 and the three C6orfs remain correlated in the presence of ICI (Figure 2c) suggests that this co-regulation is not dependent on ERa transcriptional activation.
Regulation of the steady-state level of ERa in breast cancer cells is a complex phenomenon that includes transcriptional and posttranscriptional mechanisms [40][41][42]. C6ORF96 is transcribed off the opposite DNA strand to ESR1 (Figure 1e), therefore excluding the possibility that ESR1 and the ORFs are transcribed as a single polycistronic mRNA. Recent genome-wide mapping experiments have revealed the importance of chromatin organisation for gene expression [18,43] suggesting that 3-D chromatin arrangement could represent a potential explanation for C6ORF/ESR1 coexpression. However, analysis of the data produced by Fullwood and colleagues [18] shows that C6ORF96, C6ORF97 and C6ORF211 are not encompassed by an ERa-bound long-range chromatin loop. Nevertheless, it remains possible that a loop driven by an alternative transcription factor could explain the transcriptional activity in this area.
At the nucleotide level, all three ORFs show some homology with ESR1, suggesting they may have arisen from gene duplication events [44]. C6ORF97 encodes a 715 amino acid coiled-coil domain-containing protein that is conserved across 11 species [45] while C6ORF211 is a member of the UPF0364 protein family of unknown function and is also conserved across multiple species [45]. Confocal analysis revealed that the protein encoded by C6ORF211 was expressed mainly in the cytoplasm and did not colocalize with ER ( Figure S7). In a proteomic screen it has been found to interact with SAP18, a Sin3A-associated cell growth inhibiting protein [46]. This reported interaction with a growth inhibitory protein could explain our observation that knockdown of C6ORF211 induces suppression of proliferation in cultured cells. This association is mirrored in tumours, where a proliferation metagene correlates significantly with C6ORF211. Conversely, C6ORF97 expression correlates negatively with expression of the proliferation metagene and high C6ORF97 predicts for improved disease-free survival in a tamoxifen-treated published dataset, independently of ESR1 ( Figure 4d). As high ESR1 has previously been shown to be associated with improved outcome on endocrine therapy [47], this raises the possibility that, given the observed correlation of C6ORF97 with ESR1, some of this association with outcome could be attributable to C6ORF97.
The high degree of correlation between ESR1 and the C6orfs has significant potential implications for our interpretation of ER levels and therapy of ER+ve breast cancers. As a transducer of mitogenic oestrogen signalling, disruption of ER represents a key target of therapies for ER+ve breast cancer, including tamoxifen and fulvestrant. Our data shows that C6ORF211 and C6ORF97 may contribute to the proliferative phenotype of ER+ve tumours, yet these proteins are unlikely to be affected by therapies targeted directly at ERa. Consequently, these proteins may represent potential targets for synergistic therapies in patients with high levels of C6orf expression or targets for breast cancer prevention. In addition, along with further research these relationships could shed light on recent associations between breast cancer risk and SNPs in the region.

Patient samples
Core-cut tumor biopsies (14-gauge) were obtained from 112 postmenopausal women with stage I to IIIB ER+ early breast cancer before and after two-weeks' anastrozole treatment in a neoadjuvant trial [23]. This study received approval from an institutional review board at each site and was conducted in accordance with the 1964 Declaration of Helsinki [48] and International Conference on Harmonization/Good Clinical Practice guidelines. Written informed consent was obtained from each patient before participation. Tissue was stored in RNAlater at 220uC. Two 4 mm sections from the core were stained with Gene expression analysis and data pre-processing RNA amplification, labelling and hybridization on HumanWG-6 v2 Expression BeadChips were performed according to the manufacturer's instructions (http://www.illumina.com) at a single Illumina BeadStation facility. Tumor RNA of sufficient quality and quantity was available to generate expression data from 104 pre-treatment biopsies. Data was extracted using BeadStudio software and normalized with variance-stabilizing transformation (VST) and Robust Spline Normalisation method (RSN) in the Lumi package [49]. Probes that were not detected in any samples (detection p value .1%) were discarded from further analysis.

Data analysis
Multiple correlation analysis was performed in BRB-Array Tools (http://linus.nci.nih.gov/BRB-ArrayTools.html). A statistical significance level for each gene for testing the hypothesis that the Spearman's correlation between expression of ESR1 and other genes was zero was calculated and p-values were then used in a multivariate permutation test [50] from which false discovery rates were computed. Other statistical analyses were performed in SPSS for Windows (SPSS Inc., Chicago, IL), S-PLUS (TIBCO Software Inc., Palo Alto, CA) and Graphpad Prism (Graphpad Software Inc., La Jolla, CA).
Multivariable analysis was performed in a forward stepwise fashion, the most significant additional variable (satisfying p,0.05) being added at each stage. Cases with missing values for any of the variables in the model were excluded from analysis.
Data from the 72 genes comprising the proliferation metagene was retrieved from tumours from the Wang and Loi datasets and proliferation metagene scores were calculated as described previously [54]. Spearman correlation between the proliferation metagene and ESR1 and the C6orfs was calculated in Graphpad Prism. Survival analysis was carried out in these datasets using the quartiled expression of the C6orfs and the endpoints of recurrence free survival or time to relapse, according to the original publication.

DNA extraction
Five 8 mm sections from frozen core biopsies were mounted onto Superfrost glass slides, stained with nuclear fast red, and microdissected with a sterile needle under a stereomicroscope to obtain a percentage of tumor cells .75% as described previously [55]. Genomic DNA was extracted as described previously [55]. The concentration of the DNA was measured with Picogreen according to the manufacturer's instructions (Invitrogen).

Array CGH analysis
The 32K bacterial artificial chromosome (BAC) re-array collection (CHORI) tiling path aCGH platform used for this study was constructed in the Breakthrough Breast Cancer Research Centre [55]. DNA labelling, array hybridisations, image acquisition and filtering were performed as described in Natrajan et al. [56]. Data were smoothed using the circular binary segmentation (cbs) algorithm [27]. A categorical analysis was applied to the BACs after classifying them as representing gain, loss or no-change according to their smoothed Log2 ratio values as defined [56].

Cell culture
MCF7 cells were routinely maintained in phenol red free RPMI1640 (Invitrogen, Paisley, UK) supplemented with 10% foetal bovine serum and oestradiol (1 nM). Cells were passaged weekly and medium replenished every 48-72 hours. In the case of BT474, cell monolayers were cultured in phenol red containing medium supplemented with 10% foetal bovine serum. Cell lines were shown to be free of mycoplasma by routine testing. Real-time quantitative PCR Total RNA from treated MCF7 and BT-474 cells was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. All RNA quantification was performed using the Agilent 2100 Bioanalyzer with RNA Nano LabChip Kits (Agilent Technologies, Wokingham, Berkshire, UK). RNA was reverse transcribed using SuperScript III (Invitrogen), and random primers. Twenty nanograms of resulting cDNA of each sample was analyzed in triplicates by qRT-PCR using the ABI Perkin-Elmer Prism 7900HT Sequence detection system (Applied Biosystems). Taqman gene expression assays (Applied Biosystems) were used to quantitate processed transcripts of ESR1 (Hs01046818_m1), C6ORF96 (Hs00215537_m1), C6ORF97 (Hs01563344_m1), C6ORF211 (Hs00226188_m1), which were normalized to two housekeeping genes, FKBP15 (Hs00391480_m1) and TBP (Hs00427620_m1). These housekeepers were selected from a previously published list of appropriate reference genes for breast cancer [57]. Custom assays using primers designed to span intron-exon boundaries were used to measure nascent RNA (Table S3). Gene expression was quantified using a standard curve generated from serial dilutions of reference cDNA from a pooled breast cancer cell line RNA.

Immunoblots
Cell monolayers were washed with cold PBS twice and collected by scraping. Cell pellets were lysed in extraction buffer, resolved by SDS-PAGE and transferred to nitrocellulose membranes as described previously [30]. Membranes were blocked and probed with a polyclonal antibody directed against the predicted peptide (amino acids 368-382) of C6orf211 (Eurogentec, Southampton, UK) and anti b-actin (Sigma-Aldrich, Poole, UK) using the methods described previously [58]. Quantification of immunoblots was performed using the NIH ImageJ software, and immunoblots were normalized to actin.

Immunofluorescence and confocal studies
Cells were grown on glass coverslips in standard growth medium. Cells were fixed and incubated in the presence of primary antibodies as described previously [58]. Coverslips were washed with PBS and cells were incubated in the presence of appropriate Alexa Fluor 555 (red) or Alexa Fluor 488 (green)labeled secondary antibodies (Molecular Probes, Invitrogen, Paisley, UK) diluted 1:1000 for 1 hr. Cells were washed in PBS and nuclei (DNA) were counterstained with 4,6-diamidino-2phenylindole (DAPI; Invitrogen) diluted 1:10000. Coverslips were mounted onto glass slides using Vectashield mounting medium (Vector Laboratories, Peterborough, UK). Images were collected sequentially in three channels on a Zeiss LSM710 (Carl Zeiss Ltd, Welwyn Garden City, UK) laser scanning confocal microscope at the same magnification (663 oil immersion objective).

Cell proliferation assays
Cell lines were depleted of steroids for 3 days by culturing in DCCmedium [59], seeded into 12-well plates at a density of 1610 4 cells/ well for MCF7 and 4610 4 cells per well for BT474, monolayers were allowed to acclimatize for 24 h before treatment with drug combinations indicated for 6 d with daily changes. Cell number was determined using a Z1 Coulter Counter (Beckman Coulter). Results were confirmed in a minimum of three independent experiments, and each experiment was performed in triplicate.

Effect of oestradiol and ICI182780 on ORF RNA expression
Wt-MCF7 cells were stripped of steroid for 3 days as described above. Cells were subsequently seeded into 12 well plates at a density of 1610 5 cells/well. After 24 hours monolayers were treated with vehicle (0.01% v/v ethanol), oestradiol (1 nM) or ICI182780 (10 nM) for the time intervals indicated. RNA was extracted using RNeasy Mini kit (Qiagen) and subjected to qRT-PCR as described.

SiRNA knockdown of ORFs
Wt-MCF7 cells were stripped of steroid for 24 hours in DCCmedium. Stripped cells were subsequently seeded into 12 well plates at a density of 2610 4 cells/well for proliferation assays or 1610 5 cells/well for RNA expression analysis. After 24 hours monolayers were transfected with 100 nM of either siRNA against C6ORF96, C6ORF97, C6ORF211 or control siRNA using DharmaFECT 1 reagent (Dharmacon, Thermo Fisher Scientific, UK). Medium was then replenished the following day and cells were allowed to acclimatise for a further 24 hours. After 24 hours samples were taken for RNA expression analysis. For analysis of oestrogen-dependent proliferation, the monolayers were treated with increasing concentrations of oestradiol (0.01, 0.1 or 1 nM) 48 hours post transfection. The remaining plates were treated daily with the treatments indicated for 6 days before carrying out cell counts as described above.  Figure S5 Validation of proliferation changes induced by individual siRNAs. WT-MCF7 cells were stripped of steroid for 24 hours in DCC-medium. Stripped cells were seeded into 12 well plates at a density of 20,000 cells/well for proliferation assays or 100,000 cells/well for RNA expression analysis. After 24 hours monolayers were transfected with 100 nM of single siRNAs against C6ORF211 or control siRNA (SMARTPool). Medium was replenished the following day and cells were allowed to acclimatise for a further 24 hours. Monolayers were subsequently treated with fresh DCC medium. The remaining plates were treated with DCC medium for 6 days. Proliferation in response to individual siRNA knockdown were established by change in cell number using a coulter counter (Beckman Scientific UK). Data presented is expressed as absolute cell number or fold change over siControl (SMARTpool). All data is from triplicate wells, each well read twice. confocal analysis was carried out using a polyclonal antibody directed against the predicted peptide (amino acids 368-381). MCF-7 cells were plated onto coverslips and stained. a. Nuclei were visualized using DAPI and stained with antibodies against C6ORF211 (b) and oestrogen receptor (c). An overlay of all three images is shown in (d). Found at: doi:10.1371/journal.pgen.1001382.s007 (0.07 MB DOC) Author Contributions