The mapping of DHSs using DNase-seq in the breast cancer cell line MCF-7 across the entire 8q24 cancer susceptibility region revealed a prominent site of DNase I sensitivity within the haplotype block that carries a breast and a prostate cancer predisposition variant, rs13281615 and rs620861, respectively (, Figure S1
). The strength of the signal for the DHS within this susceptibility block (S-DHS) is similar to that seen for promoter regions of the FAM84B
genes (). In addition, a strong DHS was observed in a conserved region 60kb upstream of the MYC
gene (MYC-DHS). The DNase-seq results were compared to results obtained in DNase-chip experiments (Figure S2
). All strong DHSs, for example at the MYC
promoter, were replicated. However, the S-DHS which maps to a repetitive element could not be detected by DNase-chip, as repetitive sequences are excluded from the array by design. Interestingly, the same DNase-seq approach applied to prostate cancer cell lines RWPE-1 and PC3 () did not detect a signal at the S-DHS location, suggesting that transcription factor occupancy in this region may differ between breast and prostate cancer cell lines. Signals at other locations, for example at the MYC
promoter are comparable in all three cell lines (). Figure S3A
shows an enlarged view of the region, confirming that the S-DHS is in an open chromatin state in MCF-7 cells, but in a closed state in two prostate cell lines. Despite being located within a LINE element, the sequence of S-DHS is unique enough to specifically align it to this genomic locus (see Figure S3
for details). To confirm that the open chromatin at the S-DHS is indicative of the binding of regulatory nuclear proteins, we examined chromatin immunoprecipitation data (ChIP-seq) for MCF-7 cells 
. Figure S5A and S5B
show that S-DHS sequences are bound by the two cohesin subunits Rad21 and SA1, by CTCF and the transcription factor FoxA1, making it highly likely that this region acts as a regulatory element in MCF-7 cells. Sequence alignments for ChIP-seq, as for the DNase-seq, are sufficiently specific to uniquely map the signals to this region.
Chromatin conformation at the 8q24 locus.
Next, we analysed the 1.2 kb S-DHS region for the presence of genetic variants that could be linked to variants previously reported to be associated with breast or prostate cancer. The 1000 Genomes database included 12 SNPs, of which only 5 were common (MAF >0.05) (Figure S2
). Of these, rs378854 showed complete LD (r2
1) with rs620861, the most strongly associated prostate cancer SNP reported by Al Olama et al. 
(independently confirmed by Yeager et al. 
), and also with rs445114, reported in Gudmundsson et al. 
, while the other variants displayed only low LD with this SNP. There was no strong connection to breast cancer in this region, as rs378854 only ranks as number 23 of all SNPs tested in this haplotype block 
. Previous work and LD analysis of this region (Figure S6
) supports the presence of two independent functional variants within this haplotype block, one for prostate cancer and one for breast cancer 
. Thus, we conclude that the closed state of chromatin within the S-DHS might be associated with risk of prostate cancer, through rs378854.
Motif prediction algorithms suggest that the minor, non-risk allele (A) of rs378854 creates either a YY1 or a C/EBPα transcription factor binding site. Using electrophoretic mobility shift assays (EMSA) with nuclear extracts from breast and prostate cancer cells (MCF-7 and PC3) we demonstrate that the oligonucleotide probe overlapping rs378854 is able to interact with several nuclear proteins (). The low mobility band (black arrow) is not affected by the SNP. In contrast, the high mobility band (red arrow) binds the minor allele more strongly in both cell types. A third, more variable complex of intermediate mobility is formed, which is not affected by the presence of the SNP. The specificity of binding was confirmed by competition assays with self, non-self and unrelated Oct-1 probe at different concentrations (). Competition with known transcription factor binding sites suggest that the high mobility band contains the transcription factor YY1 (). This is confirmed by a supershift observed after including a YY1 antibody in the reaction (open red arrow, ). The two upper complexes are sensitive to excess SP1 probe (), but only one of these complexes was supershifted by an SP1 antibody (black open arrow, ). SP1 and YY1 are known to interact physically and function co-operatively to modify chromatin structure 
. The SP1 binding site may therefore be able to enhance the allelic differences caused by YY1 binding to SNP rs378854. Using chromatin immunoprecipitation (ChIP) we also confirm that the identified YY1 site is occupied in vivo
in the prostate cancer cell line 1542-CP, but not in MCF-7 breast cancer cells (). In 1542-CP cells YY1 occupancy at rs378854 is higher than that observed for a positive control fragment from the promoter region of the glucocorticoid receptor, known to contain three independent YY1 binding sites 
. To test for allele-specific interaction between YY1 and rs378854 in 1542-CP, we attempted an allele-specific ChIP. Although there was an increase of binding towards the A allele in the majority of experiments, the effect was small and technical limitations prevent us from drawing definitive conclusions from this experiment.
Protein DNA interactions at the sequences overlapping rs378854.
The S-DHS was identified in two breast cancer cell lines, T47 and MCF-7 (Figure S7
). To examine the function of the S-DHS, we cloned a 395 bp fragment central to the S-DHS encompassing either the common (risk) or the minor (non-risk) allele of rs378854, into the pGL3-basic, pGL3-promoter and pGL3-enhancer vectors and assayed the ability of these allelic constructs to influence transcription in transient reporter assays. In the prostate cancer cell line PC3 the fragment containing the common (risk) allele has moderate ability to activate transcription in the context of the pGL3-enhancer construct, but for the minor allele there was statistically significant evidence for repression of transcription in the context of all constructs assayed (). Our observation are consistent with previous reports that YY1 can act as a potent repressor of transcription 
. In the breast cancer cell line MCF-7, the minor (protective) allele again displayed lower transcriptional activation than the common allele, but relative to the parental construct no repression was observed (). When the S-DHS was assayed in the pGL3-promoter vector the presence of the minor versus the common SNP had no effect in MCF-7 cells. Thus, rs378854 showed cell-type specific allelic effects on regulation of expression. The effect was evident in prostate cancer PC3 cells, with significant repressor activity of the protective minor allele. This may explain why rs378854 is associated with prostate, but not breast cancer susceptibility.
Relative transcriptional activation by the common and minor alleles of rs378854.
The S-DHS maps to a 1.2 MB region with very few annotated genes (). However, experiments for several 8q24 predisposition regions have indicated that this region is capable of undergoing long-range chromatin looping 
. We therefore used chromatin conformation capture (3C) to examine whether the DHS can physically interact with its neighbouring genes, MYC
. The pseudogene POU5F1P1
are not expressed in prostate cells at detectable levels 
and were therefore not included in this analysis. These experiments showed that in both the normal-like breast cell line HB2 and in the prostate cell line RWPE-1 the region surrounding the S-DHS interacts with a DHS 60 kb upstream of the MYC
gene (MYC-DHS) and with the MYC
promoters, located 360 kb, 420 kb and 480 kb 3′ of the bait sequence, respectively (). Similar results were obtained in MCF-7 cells (data not shown). There was no interaction with either the FAM84B
promoter or with a negative control sequence 400 kb 5′ of the DHS (). Our results suggest that in prostate cells both MYC
could be target genes of the S-DHS regulator element. We note that both cohesin and CTCF also bind to the MYC
promoters in MCF-7 cells 
, suggesting a mechanism by which an interaction may occur.
Expression of MYC
has been analysed extensively in human primary prostate and colon samples, but no correlation between its expression and the genotype of six different risk SNPs was identified 
. In contrast, our study revealed that in a set of 59 normal prostate samples mRNA expression level of PVT1
increased with the presence of the risk allele at rs378854 (p
0.025) (). The trend was similar for individuals of European-American and African-American origin, even though the frequency of the risk G allele was 0.66 in Europeans and 0.90 in African-Americans (data not shown). As PVT1
is located in a region of frequent genomic amplification, we examined copy-number variation (CNV) for intron 1 of PVT1
on DNA from all tissue samples used for expression studies. PVT1
CNV was not significantly associated with PVT1
mRNA expression and the association for rs378854 was not affected by the CNV adjustment (Figure S8
). At the same time, there was no evidence for association between MYC
expression and rs378854 in these samples (p
0.274). While this analysis will have to be repeated in larger sample sets, our results are consistent with a model in which disruption of YY1 binding at the common risk allele of rs378854 is associated with transcriptional activation of PVT1
. We also examined expression of miRNAs embedded within the PVT1
locus (hsa-mir-1204, hsa-mir-1205, hsa-mir-1206, hsa-mir-1207-3p and hsa-mir-1207-5p), but the expression was found to be very low or undetectable in both normal and tumour prostate samples and no clear pattern of association emerged (data not shown). However, we found hsa-mir-1208, located 50 kb downstream of PVT1
, to be expressed in all prostate samples tested. In samples homozygous for the risk allele the expression was borderline increased in normal samples (n
0.042), while being decreased in tumour samples (n
0.068), demonstrating significant interaction effect for hsa-mir-1208 expression dependent on tissue status (normal/tumour) and rs378854 genotype (n
0.020, Figure S9
). Similar analysis of hsa-mir-1208 expression in relation to previously reported rs6983267 did not reveal any significant associations in sets of normal and tumour samples, and in interaction (data not shown). The role of hsa-mir-1208 expression in prostate cancer and its long-distance regulation by rs378854 warrant further studies. In summary, we observe both a physical interaction between the risk SNP rs378854 and the PVT1
promoter and an association between genotype and PVT1
Association of PVT1 gene expression with rs378854 genotype in 59 normal prostate samples.