TGFβ promotes widespread enhancer chromatin opening and operates on genomic regulatory domains

The Transforming Growth Factor-β (TGFβ) signaling pathway controls transcription by regulating enhancer activity. How TGFβ-regulated enhancers are selected and what chromatin changes are associated with TGFβ-dependent enhancers regulation are still unclear. Here we report that TGFβ treatment triggers fast and widespread increase in chromatin accessibility in about 80% of the enhancers of normal mouse mammary epithelial-gland cells, irrespective of whether they are activated, repressed or not regulated by TGFβ. This enhancer opening depends on both the canonical and non-canonical TGFβ pathways. Most TGFβ-regulated genes are located around enhancers regulated in the same way, often creating domains of several co-regulated genes that we term TGFβ regulatory domains (TRD). CRISPR-mediated inactivation of enhancers within TRDs impairs TGFβ-dependent regulation of all co-regulated genes, demonstrating that enhancer targeting is more promiscuous than previously anticipated. The area of TRD influence is restricted by topologically associating domains (TADs) borders, causing a bias towards co-regulation within TADs.

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All studies must disclose on these points even when the disclosure is negative. Sequencing data (raw data and processed files) are available at NCBI GEO under the accession code GSE140552. SMAD2/3 ChIP-seq data was downloaded as a raw data from: GSE121254. CTCF ChIP-seq data was downloaded as a raw data from GSE74826 . HiC data was downloaded as a raw data from GSE96033. Other data will be made available through a Source data file, includding processed data used to construct the figure plots and non-sequencing source data.
Samples size for each experiment is indicated in the figure legends. The sample size was chosen based on previous experience in the lab, for each experiment to yield high statistics power. For ChIP-seq, ATAC-seq and RNAse-seq n=2 biological replicates were normally performed. ENCODE requires 2 biological replicates for their ChIP-seq analysis (see for example https://www.nature.com/articles/s41586-020-2023-4 No data were excluded from the analyses RT-qPCR experiments were performed in at least 3 biologically independent replicates using two or three technical replicates. Fluorescence microscopy analysis were also performed in at least 3 biologically independent replicates. ChIP-qPCR were performed in at least 2 biologically independent replicates with three technical replicates each one. Western blot experiments were performed in at least 3 biological independent replicates. All replicates were reported in the manuscript.  Confirm that both raw and final processed data have been deposited in a public database such as GEO. Confirm that you have deposited or provided access to graph files (e.g. BED files) for the called peaks.

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Files in database submission Ku, A.A., Hu, H., Zhao, X. et al. Integration of multiple biological contexts reveals principles of synthetic lethality that affect reproducibility. Nat Commun 11, 2375Commun 11, (2020 NMuMG was validated as a mouse cell line through our Next Generation Sequencing experiments. Moreover, both the epithelial phenotype and the kinetic of the induction to the mesenchymal phenotype with TGFbeta were assessed in our experiments many times through immunofluorescence, western blotting and RT-qPCR analysis of epithelial to mesenchymal transition markers assays (SNAI1, VIM, FN1, CDH1). All these characteristics are typical and some of them unique of NMuMG cells. MCF7, RPE-1 and HEK293T cell lines were no authenticated.