Generation of ESR1-2-mediated DNA loops in the 16p11.2 gene cluster. (A) Diagram of a genomic region interrogated by 3C-qPCR. Twenty reactions (number in blue) of 3C-qPCR were conducted in BamHI-digested DNA samples. Areas (red) selected for 3C-qPCR were frequently localized at the 5′-ends of genes. Inter- and intragenic regions (green) were also selected as negative controls for the assay. (B) Diminution of DNA loop formation by E2 treatment. After E2 stimulation, 3C-qPCR was conducted by using BamHI-treated DNA samples of MDECs (left) and MCF-7 cells (right). Data are shown in relative cross-linking frequencies compared with that of GAPDH. Mean ±SD (n = 3). (C) Proposed DNA loop model of the 16p11.2 gene cluster in normal and cancer cells. In normal breast epithelial cells, estrogen stimulation induces the formation of new DNA loops at ESR1-1 and diminishes the existing DNA loops at ESR1-2. In contrast, this free DNA movement is not present in MCF-7 breast cancer cells treated with estrogen (see detailed description in the text). (Red dot) ESR1-1 binding site; (green dot) ESR1-2 binding site.