Dynamic Chromatin Modification Sustains Epithelial-Mesenchymal Transition following Inducible Expression of Snail-1 (gene expression)

Epithelial-Mesenchymal Transition (EMT) is thought to contribute to cancer metastasis, but its underlying mechanisms are not well understood. To define early steps in this cellular transformation, we analyzed human mammary epithelial cells with tightly regulated expression of Snail-1, a master regulator of EMT. Following Snail-1 induction, epithelial markers were repressed within 6 hours and mesenchymal genes induced at 24 hours. Snail-1 binding to its target promoters was transient (6-48 hours) despite continued protein expression and it was followed by both transient and long-lasting chromatin changes. To generate a potent reversible EMT-inducing stimulus, we created a Snail-1 retroviral expression construct, using a fused estrogen receptor (ER) response element to mediate regulation by exogenous 4-hydroxy-tamoxifen (4-OHT). Since Snail-1 protein stability and nuclear localization are suppressed by GSK3-beta-mediated phosphorylation, we substituted the six targeted amino acids (ER-Snail-1(6SA)), thus conferring constitutive activity to the induced protein (Zhou et al., 2004, Pubmed ID 15448698). Infection of non-transformed, immortalized human mammary epithelial MCF10A cells with ER-Snail-1(6SA), followed by treatment with 4-OHT, triggered morphological and biomarker characteristics of EMT. At 3, 6, 12, 24, 72 and 120 hours after beginning exposure to 4-OHT in ethanol (or, for controls, ethanol only) we extracted RNA and did gene expression analysis using microarrays. We perfomed three replicates of each.