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.

中文翻译：

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SMAD2 和 SMAD3 在 FOXH1 先驱导向的 TGF-β 信号传导中的不同作用的结构基础。

TGF-β 受体磷酸化 SMAD2 和 SMAD3 转录因子，然后与 SMAD4 形成异源三聚体复合物，并与上下文特异性转录因子协同激活靶基因。在这里，我们提供了生化和结构证据，表明 SMAD2 与 DNA 的结合取决于 E3 插入片段的构象，E3 插入片段是 SMAD2 独有的结构元素，之前被认为使 SMAD2 无法结合 DNA。基于这一发现，我们通过定义 SMAD2 和 SMAD3 的不同作用，以及叉头先驱因子 FOXH1 作为小鼠中内胚层前体分化基因调控的伙伴，进一步描述了 TGF-β 信号转导。FOXH1 预先结合到这些基因座中的目标位点，并独立于 TGF-β 信号招募 SMAD3，而 SMAD2 在基础状态下主要保留在细胞质中，并设置为结合 SMAD4 并在目标启动子处加入 SMAD3:FOXH1 以响应 Nodal TGF-β 信号。结果支持一个模型，在该模型中，SMAD3 和 FOXH1 启动子中胚层分化基因启动子的信号独立结合用于激活，信号驱动的 SMAD2:SMAD4 与预加载 SMAD3:FOXH1 的启动子结合以激活转录。