Smad2 and Smad3 have differential sensitivity in relaying TGF b signaling and inversely regulate early lineage specification

The transforming growth factor beta (TGFß) related signaling is one of the most important signaling pathways regulating early developmental events. Smad2 and Smad3 are structurally similar and it is mostly considered that they are equally important in mediating TGFß signals. Here, we show that Smad3 is an insensitive TGFß transducer as compared with Smad2. Smad3 preferentially localizes within the nucleus and is thus sequestered from membrane signaling. The ability of Smad3 in oligomerization with Smad4 upon agonist stimulation is also impaired given its unique linker region. Smad2 mediated TGFß signaling plays a crucial role in epiblast development and patterning of three germ layers. However, signaling unrelated nuclear localized Smad3 is dispensable for TGFß signaling-mediated epiblast specification, but important for early neural development, an event blocked by TGFß/Smad2 signaling. Both Smad2 and Smad3 bind to the conserved Smads binding element (SBE), but they show nonoverlapped target gene binding specificity. We conclude that Smad2 and Smad3 possess differential sensitivities in relaying TGFß signaling and have distinct roles in regulating early developmental events. Overall design: GFP, GFP-Smad2 and GFP-Smad3 constitutively expressed Smad3-/- mouse ESCs were differentiated to day6 neuroepithelia and collected for Chip-Seq with an anti-GFP antibody.

转化生长因子β（transforming growth factor beta, TGF-β）相关信号通路是调控早期发育事件的关键信号通路之一。Smad2与Smad3结构相似，既往研究普遍认为二者在介导TGF-β信号转导中具有同等重要的作用。本研究发现，相较于Smad2，Smad3是敏感性更低的TGF-β信号转导分子。Smad3更倾向于定位于细胞核内，因此被隔离于膜信号通路之外。由于其独特的连接区域，Smad3在激动剂刺激下与Smad4发生寡聚化的能力同样受损。Smad2介导的TGF-β信号通路在上皮外胚层发育与三胚层模式建成中发挥关键作用。然而，不参与信号转导的核定位Smad3，对于TGF-β信号介导的上皮外胚层特化并非必需，但却对早期神经发育至关重要——而这一过程会被TGF-β/Smad2信号通路所阻断。Smad2与Smad3均可结合保守的Smads结合元件（Smads binding element, SBE），但二者的靶基因结合特异性不存在重叠。综上，Smad2与Smad3在传递TGF-β信号时具有不同的敏感性，并在调控早期发育事件中发挥截然不同的作用。实验整体设计：构建分别组成型表达GFP、GFP-Smad2及GFP-Smad3的Smad3基因敲除（Smad3-/-）小鼠胚胎干细胞系，将其诱导分化至第6天的神经上皮细胞，随后使用抗GFP抗体进行染色质免疫沉淀测序（Chip-Seq）。