Dave et al al JBCsecondrev wfigures

Snail1 and Zeb1 are E-cadherin-transcriptional repressors induced during epithelialmesenchymal-transition (EMT). In this article we have analyzed the factors controlling Zeb1 expression during EMT. In NMuMG cells treated with TGF-β, Snail1 RNA and protein are induced one hour after addition of the cytokine preceeding Zeb1 up-regulation that requires six to eight hours. Zeb1 gene expression is caused by increased RNA levels but also by enhanced protein stability and is markedly dependent on Snail1 since depletion of this protein prevents Zeb1 protein and RNA up-regulation. In addition to Snail1, depletion of Twist transcriptional factor retards Zeb1 stimulation by TGF-β 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-β, Snail1 is necessary for the rapid increase in Twist protein and later up-regulation of Twist 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 Zeb1 promoter although to different elements: whereas Ets1 interacts with the proximal promoter, Twist does it with a sequence 700 bp upstream the transcription start site. These results indicate that Snail1 controls Zeb1 expression at multiple levels and acts cooperatively with Twist in the induction of Zeb1 gene transcription.