Reid Christine D, Steiner Aaron B, Yaklichkin Sergey, Lu Qun, Wang Shouwen, Hennessy Morgan, Kessler Daniel S
Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine Smilow Center for Translational Research, Room 9-104, 3400 Civic Center Blvd Philadelphia, PA 19104, USA.
Dev Biol. 2016 Jun 1;414(1):34-44. doi: 10.1016/j.ydbio.2016.04.006. Epub 2016 Apr 13.
In the vertebrate blastula and gastrula the Nodal pathway is essential for formation of the primary germ layers and the organizer. Nodal autoregulatory feedback potentiates signaling activity, but mechanisms limiting embryonic Nodal ligand transcription are poorly understood. Here we describe a transcriptional switch mechanism mediated by FoxH1, the principle effector of Nodal autoregulation. FoxH1 contains a conserved engrailed homology (EH1) motif that mediates direct binding of groucho-related gene 4 (Grg4), a Groucho family corepressor. Nodal-dependent gene expression is suppressed by FoxH1, but enhanced by a FoxH1 EH1 mutant, indicating that the EH1 motif is necessary for repression. Grg4 blocks Nodal-induced mesodermal gene expression and Nodal autoregulation, suggesting that Grg4 limits Nodal pathway activity. Conversely, blocking Grg4 function in the ectoderm results in ectopic expression of Nodal target genes. FoxH1 and Grg4 occupy the Xnr1 enhancer, and Grg4 occupancy is dependent on the FoxH1 EH1 motif. Grg4 occupancy at the Xnr1 enhancer significantly decreases with Nodal activation or Smad2 overexpression, while FoxH1 occupancy is unaffected. These results suggest that Nodal-activated Smad2 physically displaces Grg4 from FoxH1, an essential feature of the transcriptional switch mechanism. In support of this model, when FoxH1 is unable to bind Smad2, Grg4 occupancy is maintained at the Xnr1 enhancer, even in the presence of Nodal signaling. Our findings reveal that FoxH1 mediates both activation and repression of Nodal gene expression. We propose that this transcriptional switch is essential to delimit Nodal pathway activity in vertebrate germ layer formation.
在脊椎动物囊胚和原肠胚中，Nodal信号通路对于初级胚层和组织者的形成至关重要。Nodal的自调节反馈增强了信号活性，但限制胚胎Nodal配体转录的机制却知之甚少。在此，我们描述了一种由FoxH1介导的转录开关机制，FoxH1是Nodal自调节的主要效应因子。FoxH1包含一个保守的En蛋白同源（EH1）基序，该基序介导与Groucho家族共抑制因子groucho相关基因4（Grg4）的直接结合。Nodal依赖性基因表达受到FoxH1的抑制，但FoxH1 EH1突变体可增强该表达，这表明EH1基序对于抑制作用是必需的。Grg4可阻断Nodal诱导的中胚层基因表达和Nodal自调节，提示Grg4限制了Nodal信号通路的活性。相反，在外胚层中阻断Grg4的功能会导致Nodal靶基因的异位表达。FoxH1和Grg4占据Xnr1增强子，且Grg4的占据依赖于FoxH1的EH1基序。随着Nodal激活或Smad2过表达，Xnr1增强子处的Grg4占据显著减少，而FoxH1的占据不受影响。这些结果表明，Nodal激活的Smad2可将Grg4从FoxH1上物理性置换下来，这是转录开关机制的一个关键特征。支持该模型的是，当FoxH1无法结合Smad2时，即使存在Nodal信号，Grg4在Xnr1增强子处的占据仍会维持。我们的研究结果表明，FoxH1介导了Nodal基因表达的激活和抑制。我们提出，这种转录开关对于在脊椎动物胚层形成过程中界定Nodal信号通路的活性至关重要。
