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

2021 
Abstract Smad transcription factors are the main downstream effectors of the Transforming growth factor β superfamily (TGFβ) 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 TGFβ-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 TGFβ- 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 TGFβ-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. Synopsis Using structural biology approaches, we have elucidated some of the rules that help define the formation of MH1 domain dimers of BMP activated Smad proteins. We also propose a new hypothesis on how dimers and monomers can influence the composition of Smad complexes as well as the recognition of specific cis-regulatory elements genome-wide. Abbreviations: ChIP-seq, Chromatin immunoprecipitation followed by high-throughput sequencing; Smad proteins, Homologous proteins to the Caenorhabditis elegans SMA (“small” worm phenotype and MAD family (“Mothers Against Decapentaplegic” of genes in Drosophila MH1 domain, Mad homology 1; BEST, Band-Selective Excitation Short-Transient-type experiments; TROSY, Transverse relaxation optimized spectroscopy
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