Transcriptional regulators ensuring specific gene expression and decision making at high TGFβ doses

TGF{beta}-signaling regulates cancer progression by controlling cell division, migration and death. These outcomes are mediated by gene expression changes, but the mechanisms of decision making towards specific fates remain unclear. Here, we combine SMAD transcription factor imaging, genome-wide RNA sequencing and morphological assays to quantitatively link signaling, gene expression and fate decisions in mammary epithelial cells. Fitting genome-wide kinetic models to our time-resolved data, we find that the majority of TGF{beta} target genes can be explained as direct targets of SMAD transcription factors, whereas the remainder show signs of complex regulation, involving delayed regulation and strong amplification at high TGF{beta} doses. Knockdown experiments followed by global RNA sequencing revealed transcription factors interacting with SMADs in feedforward loops to control delayed and dose-discriminating target genes, thereby reinforcing the specific epithelial-to-mesenchymal transition at high TGF{beta} doses. We identified early repressors, preventing premature activation, and a late activator, boosting gene expression responses for a sufficiently strong TGF{beta} stimulus. Taken together, we present a global view of TGF{beta}-dependent gene regulation and describe specificity mechanisms reinforcing cellular decision making.