HEB and E2A function as SMAD/FOXH1 cofactors. Xenopus tropicalis

Nodal signaling, mediated through SMAD transcription factors, is necessary for pluripotency maintenance and endoderm commitment. We have identified a new motif, termed SMAD Complex Associated (SCA) that is bound by SMAD2/3/4 and FOXH1 in human embryonic stem cells (hESCs) and derived endoderm. We demonstrate that two bHLH proteins - HEB and E2A - bind the SCA motif at regions overlapping SMAD2/3 and FOXH1. Further, we show that HEB and E2A associate with SMAD2/3 and FOXH1, suggesting they form a complex at critical target regions. This association is biologically important, as E2A is critical for mesendoderm specification, gastrulation, and Nodal signal transduction in Xenopus tropicalis embryos. Taken together, E2A is a novel Nodal signaling cofactor that associates with SMAD2/3 and FOXH1 and is necessary for mesendoderm differentiation. Overall design: ChIP-seq of Smad2/3 and Input in X.tropicalis, stage 10.5 embryo.由SMAD转录因子介导的Nodal信号通路，对多能性维持与内胚层细胞定型至关重要。我们鉴定出一种全新的基序，命名为SMAD复合物结合基序（SMAD Complex Associated, SCA），该基序在人类胚胎干细胞（human embryonic stem cells, hESCs）及其诱导的内胚层中可被SMAD2/3与FOXH1结合。我们通过实验证实，两类碱性螺旋-环-螺旋蛋白（basic Helix-Loop-Helix, bHLH）——HEB与E2A——可在与SMAD2/3及FOXH1结合区域重叠的位点结合SCA基序。此外，我们发现HEB与E2A可与SMAD2/3及FOXH1发生相互作用，提示二者可在关键靶区域形成复合物。这种相互作用具有重要的生物学意义：在热带爪蟾（Xenopus tropicalis）胚胎中，E2A对中内胚层定型、原肠胚形成以及Nodal信号转导均至关重要。综上，E2A是一种全新的Nodal信号通路辅因子，可与SMAD2/3及FOXH1结合，且对中内胚层分化不可或缺。实验整体设计：针对10.5期热带爪蟾胚胎开展Smad2/3的染色质免疫共沉淀测序（Chromatin Immunoprecipitation sequencing, ChIP-seq）实验，并设置Input对照。