Figure 2.

Analysis of allelic binding and effects on allelic expression of IGFBP5. (A) ChIP-seq read density for a 3Kb region overlapping the ERα-bound looping enhancer was plotted for ERα (38), H3K27Ac, H3K27me3, and ENCODE layered H3K27Ac (21) (panels as labeled). The blue bar indicates the location of the intergenic enhancer copy number variation. (B) ERα binding activity at the ERE (orange bar, 199bp upstream of enCNV breakpoint) was assayed by Chromatin Immunoprecipitation (ChIP)-qPCR for the variant (red) and wildtype (blue) alleles, and a negative control region (in ACTB, purple), in heterozygous MCF7 cells with oestrogen treatment (vehicle and oestrogen indicated in light and dark shades for each site, respectively). Allelic detection primers were designed as indicated on inset map. Error bars represent SD of biological triplicates. *P < 0.004; **P < 0.002. (C) Investigation of allele-specific expression of IGFBP5 was conducted by allelic amplification of intronic marker SNP, rs7565131. Briefly, nuclear RNA from oestrogen or vehicle treated cells was isolated. Total IGFBP5 nuclear RNA was determined by detection of 3’UTR sequence (total bar height; error bars represent SD of biological triplicates). Allelic expression was evaluated by detection of allele-specific products by a modified MAMA(25)-qPCR. Relative abundance (total signal %) indicated by color (rs7565131-A and C as red and blue, respectively). Error bars with hats represent SD of biological triplicates. ***P = 0.027; ****P = 0.014.