ESR1-mediated concurrent silencing of a 14-gene cluster on chromosome 16p11.2. (A) Progressive repression of the 14-gene unit in mammosphere-derived epithelial cells treated with 17β-estradiol (E2, 70 nM) or diethylstilbestrol (DES, 70 nM). Total RNA was isolated and subject to quantitative RT-PCR analysis from three independent sets of MDEC samples. Expression data are summarized in heat maps (see also detailed results in Supplemental Fig. S2). (B) ESR1-mediated epigenetic repression of the gene cluster in MCF-7 cells. Six hours before E2 stimulation, cells were pretreated with 5-aza-2′-deoxycytidine (DAC, 1 μM), trichostatin A (TSA, 1 μM), and/or ESR1 antagonist ICI182780 (ICI, 1 μM). Total RNA was subjected to RT-qPCR analysis, and expression data are summarized in heat maps (see also detailed results in Supplemental Fig. S3). (C) Coherent repression of the 16p11.2 gene cluster in differentiated cells pre-exposed to endocrine disruptors. Progenitors were exposed to E2 (70 nM), diethylstilbestrol (DES, 70 nM), daidzein (10 μM), 1,3,5-tris(4-hydroxyphenyl)- 4-propyl-1H-pyrazole (PPT, 0.1 nM), 4-nonylphenol (NP, 1 μM), N-butyl-benzyl phthalate (BBP, 10 μM), di(2-ethylhexyl)-phthalate (DEHP, 10 μM), 4,4′-dichloro-biphnyl (PCB, 0.1 nM), and bisphenol A (BPA, 4 nM) for 3 wk. After the pre-exposure, the differentiated cells in the absence of estrogenic exposure were subjected to RT-qPCR analysis. Data was calculated from three independent sets of MDEC samples and presented in heat maps (see also detailed results in Supplemental Fig. S4). (D) Concurrent silencing of 16p11.2 homologs (n = 10) in a rat model pre-exposed to bisphenol A (BPA). After birth, rats were exposed to sesame oil (Ctrl) or BPA (Treated; 250 μg/kg BW) during the prepubertal period (∼21 d). Then, total RNA of mammary gland obtained from 50-d-old animals prepubertally exposed to BPA were collected for expression analysis. Results of four independent sets of rat samples are shown in heat maps (see also detailed results in Supplemental Fig. S5)