The transcriptional factor Snail1 is a repressor of E-cadherin (CDH1) gene expression essential for triggering epithelial-mesenchymal transition. Snail1 represses CDH1, directly binding its promoter and inducing the synthesis of the Zeb1 repressor. In this article, we show that repression of CDH1 by Snail1, but not by Zeb1, is dependent on the activity of Polycomb repressive complex 2 (PRC2). Embryonic stem (ES) cells null for Suz12, one of the components of PRC2, show higher levels of Cdh1 mRNA than control ES cells. In tumor cells, interference of PRC2 activity prevents the ability of Snail1 to downregulate CDH1 and partially derepresses CDH1. Chromatin immunoprecipitation assays demonstrated that Snail1 increases the binding of Suz12 to the CDH1 promoter and the trimethylation of lysine 27 in histone H3. Moreover, Snail1 interacts with Suz12 and Ezh2, as shown by coimmunoprecipitation experiments. In conclusion, these results demonstrate that Snail1 recruits PRC2 to the CDH1 promoter and requires the activity of this complex to repress E-cadherin expression.

Expression of Snail transcriptional factor is a determinant in the acquisition of a mesenchymal phenotype by epithelial tumor cells. However, the regulation of the transcription of this gene is still unknown. We describe here the characterization of a human SNAIL promoter that contains the initiation of transcription and regulates the expression of this gene in tumor cells. This promoter was activated in cell lines in response to agents that induce Snail transcription and the mesenchymal phenotype, as addition of the phorbol ester PMA or overexpression of integrin-linked kinase (ILK) or oncogenes such as Ha-ras or v-Akt. Although other regions of the promoter were required for a complete stimulation by Akt or ILK, a minimal fragment (À78/ þ 59) was sufficient to maintain the mesenchymal specificity. Activity of this minimal promoter and SNAIL RNA levels were dependent on ERK signaling pathway. NFjB/p65 also stimulated SNAIL transcription through a region located immediately upstream the minimal promoter, between À194 and À78. These results indicate that Snail transcription is driven by signaling pathways known to induce epithelial to mesenchymal transition, reinforcing the role of Snail in this process.

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Snail1 and Zeb1 are E-cadherin-transcriptional repressors induced during epithelial mesenchymal transition (EMT).In this article we have analyzed the factors controlling Zeb1 expression during EMT. In NMuMG cells treated with TGF-␤, Snail1 RNA and protein are induced 1 h after addition of the cytokine preceding Zeb1 up-regulation that requires 6 -8 h. Zeb1 gene expression is caused by increased RNA levels but also by enhanced protein stability and is markedly dependent on Snail1 because depletion of this protein prevents Zeb1 protein and RNA up-regulation. In addition to Snail1, depletion of the Twist transcriptional factor retards Zeb1 stimulation by TGF-␤ or decreases Zeb1 expression in other cellular models indicating that this factor is also required for Zeb1 expression. Accordingly, Snail1 and Twist cooperate in the induction of Zeb1: cotransfection of both cDNAs is required for the maximal expression of ZEB1 mRNA. Unexpectedly, the expression of Snail1 and Twist shows a mutual dependence although to a different extent; whereas Twist depletion retards Snail1 up-regulation by TGF-␤, Snail1 is necessary for the rapid increase in Twist protein and later up-regulation of Twist1 mRNA induced by the cytokine. Besides this effect on Twist, Snail1 also induces the nuclear translocation of Ets1, another factor required for Zeb1 expression. Both Twist and Ets1 bind to the ZEB1 promoter although to different elements: whereas Ets1 interacts with the proximal promoter, Twist does it with a 700-bp sequence upstream of the transcription start site. These results indicate that Snail1 controls Zeb1 expression at multiple levels and acts cooperatively with Twist in the ZEB1 gene transcription induction. Epithelial to mesenchymal transition (EMT)5 defines a process during which cells lose their epithelial characteristics and acquire typical properties of mesenchymal cells. This transition requires complex changes in cell shape that happen concomitantly to gene expression reprogramming (1). The main hallmark of EMT is the down-regulation of the adherens junction protein E-cadherin due to transcriptional repression. Overexpression of Snail1 in epithelial cells causes a complete EMT and down-regulates E-cadherin through its binding to the Ecadherin promoter (2, 3); moreover, up-regulation of Snail1 RNA is observed in many cellular systems when EMT is induced (4). Besides Snail1, other cellular factors such as the Snail1-related Slug (Snail2) (5), the basic helix-loop-helix protein E12/E47 (6), or two members of the Zeb family, Zeb1/ ␦EF-1 and Zeb2/Sip1 (7-9), are capable of repressing E-cadherin (CDH1) promoter activity and RNA levels. Curiously, all of these factors bind to the same elements in the CDH1 gene: three E-boxes with a core 5Ј-CACCTG-3Ј sequence placed in the proximal promoter. Different results indicate that expression of some of these genes is interdependent; for instance, it has been shown that overexpression of Snail1 increases the levels of ZEB1 mRNA (10). A relevant role for Zeb1 in the definitive repression o...