Fig. 5

Molecular analysis of signal 1 CCVs at 12q24. a Chromatin interactions in MCF7 cells. Topologically associating domains (TADs) are shown as horizontal gray bars above GENCODE-annotated coding (blue) and non-coding (green) genes. The PCHi-C baits are depicted as black boxes. Risk signals 1–4 are numbered, and the CCVs within each signal are shown as colored vertical lines. ENCODE ChIP-seq data for available histone marks are depicted as gray boxes. The ATAC-seq track is shown as a dark blue histogram. ESR1, GATA3, and FOXA1 binding are shown as black histograms. Peaky defined MPPC values (from PCHi-C BaitID: 596031) are plotted with the prioritized CCVs overlaid as red vertical lines. CHiCAGO-scored interactions are shown as black arcs. The dashed red outline highlights signal 1 CCVs and the dashed gray outline the target gene (TBX3). b The 12q24 enhancer was repressed by targeting dCas9-KRAB to the enhancer in MCF7 cells with two different CRISPRi single-guide (sg) RNAs (SgEnh1 and SgEnh2). PgCON contains a non-targeting control sgRNA. Gene expression of TBX3, TBX5, and MED13L was measured by qPCR and normalized to GUSB. Error bars represent the SEM (n = 3). p values were determined by two-way ANOVA followed by Dunnett’s multiple-comparison test (**p < 0.01). c Luciferase reporter assays following transient transfection of MCF7 cells. The 12q24 enhancer containing either the risk or protective (Prot.) haplotype was cloned into TBX3 promoter-driven luciferase constructs (TBX3 prom). Error bars represent the SEM (n = 3). p values were determined by two-way ANOVA followed by Dunnett’s multiple-comparison test (**p < 0.01). d Allele-specific DNase I hypersensitivity at CCV rs1391721 in heterozygous MCF7 cells. The depth of reads containing the risk (red) and protective (blue) alleles are shown. e EMSAs for signal 1 CCVs to detect allele-specific binding of nuclear proteins. Labeled oligonucleotide duplexes were incubated with MCF7 nuclear extract. Red arrowheads show the bands of different mobility detected between risk (R) and protective (P) alleles. f Position weight matrix of the GATA3 binding site from JASPAR, with homology to the risk (a) and protective (g) alleles of rs1391721 colored below. g Allele-specific GATA3 ChIP-PCR results assessed at CCV rs1391721 in heterozygous MCF7 cells. Error bars represent the SEM (n = 3). p values were determined by a two-tailed Student’s t test (**p < 0.01). h Allelic discrimination plot of the GATA3 ChIP in MCF7 cells. Genomic DNA extracted from homozygous T47D and Hs578T breast cancer cells were used as controls