Functional annotation of the 2q35 breast cancer risk locus implicates a structural variant in influencing activity of a long-range enhancer element

Abstract

A combination of genetic and functional approaches has identified three independent breast cancer risk loci at 2q35. A recent fine-scale mapping analysis to refine these associations resulted in 1 (signal 1), 5 (signal 2), and 42 (signal 3) credible causal variants at these loci. We used publicly available in silico DNase I and ChIP-seq data with in vitro reporter gene and CRISPR assays to annotate signals 2 and 3. We identified putative regulatory elements that enhanced cell-type-specific transcription from the IGFBP5 promoter at both signals (30to 40-fold increased expression by the putative regulatory element at signal 2, 2-to 3-fold by the putative regulatory element at signal 3). We further identified one of the five credible causal variants at signal 2, a 1.4 kb deletion (esv3594306), as the likely causal variant; the deletion allele of this variant was associated with an average additional increase in IGFBP5 expression of 1.3-fold (MCF-7) and 2.2-fold (T-47D). We propose a model in which the deletion allele of esv3594306 juxtaposes two transcription factor binding regions (annotated by estrogen receptor alpha ChIP-seq peaks) to generate a single extended regulatory element. This regulatory element increases cell-typespecific expression of the tumor suppressor gene IGFBP5 and, thereby, reduces risk of estrogen receptor-positive breast cancer (odds ratio ¼ 0.77, 95% CI 0.74-0.81, p ¼ 3.1 3 10 À31 ).

Biological effects and regulation of IGFBP5 in breast cancer

“…Besides proximal promoter elements, sequences far upstream (telomeric) and downstream (centromeric) of the transcriptional start site (TSS) regulate IGFBP5 transcription ( Figure 5 ). The far upstream regulatory elements are located in the 2q35 protein-coding gene desert ( 23 – 27 ). This region has been recognized as a BC susceptibility locus, where several single nucleotide polymorphism (SNP) sites and a deletion variant (called enCNV or esv3594306) have been identified, potentially linking IGFBP5 to BC risk ( 25 – 27 , 129 ).…”

“…The far upstream regulatory elements are located in the 2q35 protein-coding gene desert ( 23 – 27 ). This region has been recognized as a BC susceptibility locus, where several single nucleotide polymorphism (SNP) sites and a deletion variant (called enCNV or esv3594306) have been identified, potentially linking IGFBP5 to BC risk ( 25 – 27 , 129 ).…”

Functional annotation of breast cancer risk loci: current progress and future directions

“…used CRISPR interference (CRISPRi) to introduce repressive histone modifications at an enhancer element (annotated by the most significant GWAS SNP at this locus) and demonstrated that this resulted in reduced levels of expression of two long noncoding RNAs ( CUPID1 and 2 ) and the presumed target gene CCND1 [ 22 ]. We have recently shown that targeting a catalytically inactive Cas9 fused to an activating VPR domain (CRISPRa) to an enhancer element at the 2q35 breast cancer risk locus increases expression of IGFBP5 (mapping ~400 kb distal) but neither of the neighbouring genes IGFBP2 and RPL37A (~460 kb and ~600 kb, respectively) [ 99 ]. A genome-wide framework for mapping gene regulation using CRISPRi has been developed; in this approach, using a high multiplicity of infection, random combinations of CRS were perturbed in the erythroleukaemia cell line K562 and expression of target genes (defined as K562-expressed genes within 1Mb of the CRS) was assayed using single-cell RNA-seq [ 100 ].…”

“…Locus-specific functional annotation studies for at least 17 loci (defined for these purposes as chromosomal regions) have been reported by BCAC investigators and collaborators at 1p11.2 [ 25 ], 2q33 [ 27 ], 2q35 [ 12 , 13 , 99 ], 4q24 [ 14 ], 5p15.33 [ 28 ], 5p12 [ 24 ], 5q11.2 [ 15 ], 6q25 [ 16 ], 8q24 [ 17 ], 9q31.2 [ 18 ], 10q21.1 [ 19 ], 10q26 [ 21 ], 11q13 [ 23 ], 12p11 [ 26 ], 12q24 [ 92 ], 17q22 [ 20 ] and 19p13 [ 29 ]. These analyses, published predominantly prior to the recent global fine-mapping analysis, begin with locus-specific fine-scale mapping to define independent signals and CCVs.…”

“…Other studies have followed up a subset of variants and genes using functional assays and, in some instances, report more robust evidence for a causal variant (or variants), a target gene (or genes) and a mechanism by which the causal variant influences the expression of the target gene to impact breast cancer risk. Target genes include well-documented breast cancer genes ( MAP3K1 at 5q11.2 [ 15 ], ESR1 at 6q25 [ 16 ], FGFR2 at 10q26 [ 21 ] and CCND1 at 11q13 [ 23 ]), TFs ( KLF4 at 9q31.2 [ 18 ], NRBF2 at 10q21.2 [ 19 ] and TBX3 at 12q24 [ 92 ]), a putative tumour suppressor gene ( IGFBP5 at 2q35 [ 12 , 13 , 99 ]), a methylcytosine dioxygenase ( TET2 at 4q24 [ 14 ]) and a ribonucleoprotein polymerase that maintains telomere ends (TERT at 5p12 [ 24 ]). The majority of studies propose a mechanism in which allele-specific binding of a TF (or TFs) influences the expression of the target gene; most commonly, it is the allele-specific binding of one of the three factors that define the ER+ transcriptome (ESR1, FOXA1 and GATA3) [ 33 – 35 ] that is implicated.…”