Integrative eQTL-based analyses reveal the biology of breast cancer risk loci

Abstract

Germline determinants of gene expression in tumors are infrequently studied due to the complexity of transcript regulation caused by somatically acquired alterations. We performed expression quantitative trait locus (eQTL)-based analyses using the multi-level information provided in The Cancer Genome Atlas (TCGA). Of the factors we measured, cis-acting eQTLs accounted for 1.2% of the total variation of tumor gene expression, while somatic copy-number alteration and CpG methylation accounted for 7.3% and 3.3%, respectively. eQTL analyses of 15 previously reported breast cancer risk loci resulted in the discovery of three variants that are significantly associated with transcript levels (false discovery rate [FDR] < 0.1). Our trans-based analysis identified an additional three risk loci to act through ESR1, MYC, and KLF4. These findings provide a more comprehensive picture of gene expression determinants in breast cancer as well as insights into the underlying biology of breast cancer risk loci.

Figure 1

Effects of three determinants on gene expression in ER-positive breast cancer:cis-acting SNP loci, somatic copy number and CpG methylation. (a) Venn diagram shows the number of genes that are under regulation of one or multiple factors. (b) Pie chart shows the relative and absolute fraction of variance of gene expression explained by three factors. Please see also Figure S1, Figure S2, Table S1, Table S2, Table S3, Table S5.

Figure 2

Schematic of the hypothesis that risk alleles are cis-eQTLs of transcription factors. (A) A risk locus (blue triangle) cis-regulates a transcription factor (TF; red explosion). (B) the messenger RNA of the TF is translated into its active form and (C) binds to the target genes. (D) These target genes are associated with the risk allele, but not the TF because the TF is itself tightly regulated. (E) DNA sequences within DNaseI hypersensitive sites (yellow peak) are evaluated for TF binding motif enrichment. Please see also Table 1, Table S5.

Figure 3

Allelic imbalance (AI) of the ESR1, MYC, and KLF4 transcription factors by breast cancer risk genotypic status. Allelic specific expression measures of three TFs were derived from RNA-sequencing of 177 TCGA breast cancer samples. The association between ESR1, MYC and KLF4 and the corresponding risk loci of (A) 6q25 (rs2046210) (B) 8q24 (rs418269) and (C) 9q31 (rs471467) were evaluated using the F-test. Please see also Figure S3, Figure S4C.

Figure 4

Chromosome conformation capture (3C) demonstrates physical interactions between the 6q25 risk locus and the ESR1 promoter and the 9q21 risk locus and the KLF4 promoter. (A) The top panel shows screenshots of the restriction fragments used for the 6q25/ESR1 interaction. These fragments are separated by approximately 170 kb. The lower left panel shows the gel image of the ligation band. The 263 base pair band is visualized only in the sample with ligase (+Lg) and no band is seen in the negative control sample without ligase (-Lg). The lower right panel demonstrates sequence verification of the +Lg band, confirming this interaction. (B) The 9q21 risk locus and KLF4 physically interact over a distance of 640 kb. Please see also Figure 3, Table 1.