TGF-ß and RAS jointly unmask primed enhancers to drive metastasis.

Epithelial-to-mesenchymal transitions (EMTs) and extracellular matrix (ECM) remodeling are distinct yet important processes during carcinoma invasion and metastasis. Transforming growth factor ß (TGF-ß) and RAS, signaling through SMAD and RAS-responsive element-binding protein 1 (RREB1), jointly trigger expression of EMT and fibrogenic factors as two discrete arms of a common transcriptional response in carcinoma cells. Here, we demonstrate that both arms come together to form a program for lung adenocarcinoma metastasis and identify chromatin determinants tying the expression of the constituent genes to TGF-ß and RAS inputs. RREB1 localizes to H4K16acK20ac marks in histone H2A.Z-loaded nucleosomes at enhancers in the fibrogenic genes interleukin-11 (IL11), platelet-derived growth factor-B (PDGFB), and hyaluronan synthase 2 (HAS2), as well as the EMT transcription factor SNAI1, priming these enhancers for activation by a SMAD4-INO80 nucleosome remodeling complex in response to TGF-ß. These regulatory properties segregate the fibrogenic EMT program from RAS-independent TGF-ß gene responses and illuminate the operation and vulnerabilities of a bifunctional program that promotes metastatic outgrowth.

Structural basis for distinct roles of SMAD2 and SMAD3 in FOXH1 pioneer-directed TGF-ß signaling.

TGF-ß receptors phosphorylate SMAD2 and SMAD3 transcription factors, which then form heterotrimeric complexes with SMAD4 and cooperate with context-specific transcription factors to activate target genes. Here we provide biochemical and structural evidence showing that binding of SMAD2 to DNA depends on the conformation of the E3 insert, a structural element unique to SMAD2 and previously thought to render SMAD2 unable to bind DNA. Based on this finding, we further delineate TGF-ß signal transduction by defining distinct roles for SMAD2 and SMAD3 with the forkhead pioneer factor FOXH1 as a partner in the regulation of differentiation genes in mouse mesendoderm precursors. FOXH1 is prebound to target sites in these loci and recruits SMAD3 independently of TGF-ß signals, whereas SMAD2 remains predominantly cytoplasmic in the basal state and set to bind SMAD4 and join SMAD3:FOXH1 at target promoters in response to Nodal TGF-ß signals. The results support a model in which signal-independent binding of SMAD3 and FOXH1 prime mesendoderm differentiation gene promoters for activation, and signal-driven SMAD2:SMAD4 binds to promoters that are preloaded with SMAD3:FOXH1 to activate transcription.

TGIF1 homeodomain interacts with Smad MH1 domain and represses TGF-ß signaling.

TGIF1 is a multifunctional protein that represses TGF-ß-activated transcription by interacting with Smad2-Smad4 complexes. We found that the complex structure of TGIF1-HD bound to the TGACA motif revealed a combined binding mode that involves the HD core and the major groove, on the one hand, and the amino-terminal (N-term) arm and the minor groove of the DNA, on the other. We also show that TGIF1-HD interacts with the MH1 domain of Smad proteins, thereby indicating that TGIF1-HD is also a protein-binding domain. Moreover, the formation of the HD-MH1 complex partially hinders the DNA-binding site of the complex, preventing the efficient interaction of TGIF1-HD with DNA. We propose that the binding of the TGIF1 C-term to the Smad2-MH2 domain brings both the HD and MH1 domain into close proximity. This local proximity facilitates the interaction of these DNA-binding domains, thus strengthening the formation of the protein complex versus DNA binding. Once the protein complex has been formed, the TGIF1-Smad system would be released from promoters/enhancers, thereby illustrating one of the mechanisms used by TGIF1 to exert its function as an active repressor of Smad-induced TGF-ß signaling.

TPPU_DSF: A Web Application to Calculate Thermodynamic Parameters Using DSF Data.

Here we present TPPU_DSF (https://maciasnmr.net/tppu_dsf/). This is a free and open-source web application that opens, converts, fits, and calculates the thermodynamic parameters of protein unfolding from standard differential scanning fluorimetry (DSF) data in an automated manner. The software has several applications. In the context of screening compound libraries for protein binders, obtaining thermodynamic parameters provides a more robust approach to detecting hits than the changes in the melting temperature (Tm) alone, thereby helping to increase the number of positive hits in screening campaigns. Moreover, changes in ΔGuo indicate protein response to binding at lower compound concentrations than those in the Tm, thereby reducing the costs associated with the amounts of protein and compounds required for the assays. Also, by adding thermodynamic information to the Tm comparison, the software can contribute to the optimization of protein constructs and buffer conditions, a common practice before structural and functional projects.

HTSDSF Explorer, A Novel Tool to Analyze High-throughput DSF Screenings.

The identification of new drugs for novel therapeutic targets requires the screening of libraries containing tens of thousands of compounds. While experimental screenings are assisted by high-throughput technologies, in target-based biophysical assays, such as differential scanning fluorimetry (DSF), the analysis steps must be calculated manually, often combining several software packages. To simplify the determination of the melting temperature (Tm) of the target and the change induced by ligand binding (ΔTm), we developed the HTSDSF explorer, a versatile, all-in-one, user-friendly application suite. Implemented as a server-client application, in the primary screenings, HTSDSF explorer pre-analyzes and displays the Tm and ΔTm results interactively, thereby allowing the user to study hundreds of conditions and select the primary hits in minutes. This application also allows the determination of preliminary binding constants (KD) through a series of subsequent dose-response assays on the primary hits, thereby facilitating the ranking of validated hits and the advance of drug discovery efforts.

Unveiling the dimer/monomer propensities of Smad MH1-DNA complexes.

Smad transcription factors are the main downstream effectors of the Transforming growth factor ß superfamily (TGFß) signalling network. The DNA complexes determined here by X-ray crystallography for the Bone Morphogenetic Proteins (BMP) activated Smad5 and Smad8 proteins reveal that all MH1 domains bind [GGC(GC)|(CG)] motifs similarly, although TGFß-activated Smad2/3 and Smad4 MH1 domains bind as monomers whereas Smad1/5/8 form helix-swapped dimers. Dimers and monomers are also present in solution, as revealed by NMR. To decipher the characteristics that defined these dimers, we designed chimeric MH1 domains and characterized them using X-ray crystallography. We found that swapping the loop1 between TGFß- and BMP- activated MH1 domains switches the dimer/monomer propensities. When we scanned the distribution of Smad-bound motifs in ChIP-Seq peaks (Chromatin immunoprecipitation followed by high-throughput sequencing) in Smad-responsive genes, we observed specific site clustering and spacing depending on whether the peaks correspond to BMP- or TGFß-responsive genes. We also identified significant correlations between site distribution and monomer or dimer propensities. We propose that the MH1 monomer or dimer propensity of Smads contributes to the distinct motif selection genome-wide and together with the MH2 domain association, help define the composition of R-Smad/Smad4 trimeric complexes.