Abstract
TGF-β 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-β 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-β 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-β 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.
Highlights
Transforming growth factor β (TGF-β) signaling is crucial for metazoan development, tissue homeostasis, wound healing, and immune surveillance (David and Massagué 2018)
SMAD2 and SMAD3 are similar in amino acid sequence (91% identity) (Supplemental Fig. S1A) except for a 10-residue extension of the loop connecting the first two α-helices, and the 30-amino acid E3 insert, which is spliced in SMAD2β, an isoform that closely resembles SMAD3 (Fig. 1A)
SMAD2 and SMAD3 cooperate as mediators of gene expression, with SMAD2 serving as a conditional DNA binding protein and classic signal-driven transcriptional regulator, and SMAD3 with the pioneer factor FOXH1 binding to target promoters and marking these sites for incorporation of signal-driven SMAD2:SMAD4 complexes (Fig. 7H)
Summary
Transforming growth factor β (TGF-β) signaling is crucial for metazoan development, tissue homeostasis, wound healing, and immune surveillance (David and Massagué 2018). SMAD proteins have intrinsic DNA-binding activity, their binding to target regulatory regions requires other transcription factors as DNA-binding partners, as observed in progenitor cells of diverse lineages (Chen et al 1997; Germain et al 2000; Hata et al 2000; Qing et al 2000; Seoane et al 2004; Mullen et al 2011; Trompouki et al 2011). SMAD proteins consist of an N-terminal DNA-binding domain (MH1 domain) and a C-terminal region including the linker and the MH2 domain that contacts partner transcription factors like FOXH1, coactivators, and corepressors (Shi and Massagué 2003; Aragon et al 2011; Macias et al 2015; Miyazono et al 2018). The SMAD2β isoform lacking the E3 insert is a minor species in most tissues
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