Department of Biochemistry, Institute of Cell Differentiation and Aging, College of Medicine, Hallym University, Chuncheon 24252, Korea.
Department of Molecular Medicine, School of Medicine, Gachon University, Incheon 21999, Korea.
Cells. 2021 Oct 17;10(10):2779. doi: 10.3390/cells10102779.
Inhibition of the bone morphogenetic proteins (BMPs) is the primary step toward neuroectoderm formation in vertebrates. In this process, the Spemann organizer of the dorsal mesoderm plays a decisive role by secreting several extracellular BMP inhibitors such as Chordin (Chrd). Chrd physically interacts with BMP proteins and inhibits BMP signaling, which triggers the expression of neural-specific transcription factors (TFs), including Foxd4l1.1. Thus, Chrd induces in a BMP-inhibited manner and promotes neuroectoderm formation. However, the regulatory feedback mechanism of Foxd4l1.1 on mesodermal genes expression during germ-layer specification has not been fully elucidated. In this study, we investigated the regulatory mechanism of Foxd4l1.1 on (a mesodermal gene). We demonstrate that Foxd4l1.1 inhibits expression during neuroectoderm formation in two ways: First, Foxd4l1.1 directly binds to FRE (Foxd4l1.1 response elements) within the promoter region to inhibit transcription. Second, Foxd4l1.1 physically interacts with Smad2 and Smad3, and this interaction blocks Smad2 and Smad3 binding to activin response elements (AREs) within the promoter. Site-directed mutagenesis of FRE within the promoter completely abolished repressor activity of the Foxd4l1.1. RT-PCR and reporter gene assay results indicate that Foxd4l1.1 strongly inhibits mesoderm- and ectoderm-specific marker genes to maintain neural fate. Altogether, these results suggest that Foxd4l1.1 negatively regulates transcription by dual mechanism. Thus, our study demonstrates the existence of precise reciprocal regulation of transcription during neuroectoderm and mesoderm germ-layer specification in embryos.
抑制骨形态发生蛋白（BMPs）是脊椎动物神经外胚层形成的首要步骤。在这个过程中，背侧中胚层的 Spemann 组织者通过分泌几种细胞外 BMP 抑制剂（如 Chordin，Chrd）发挥决定性作用。Chrd 与 BMP 蛋白物理相互作用并抑制 BMP 信号转导，从而触发神经特异性转录因子（TFs）的表达，包括 Foxd4l1.1。因此，Chrd 以 BMP 抑制的方式诱导并促进神经外胚层的形成。然而，Foxd4l1.1 在胚胎中胚层特化过程中对中胚层基因表达的调控反馈机制尚未完全阐明。在这项研究中，我们研究了 Foxd4l1.1 对（一种中胚层基因）的调控机制。我们证明 Foxd4l1.1 通过两种方式抑制神经外胚层形成过程中的表达：首先，Foxd4l1.1 直接结合到 启动子区域内的 FRE（Foxd4l1.1 反应元件）以抑制转录。其次，Foxd4l1.1 与 Smad2 和 Smad3 物理相互作用，这种相互作用阻止 Smad2 和 Smad3 与 启动子内的激活素反应元件（AREs）结合。FRE 在内的启动子的定点突变完全消除了 Foxd4l1.1 的抑制活性。RT-PCR 和报告基因检测结果表明，Foxd4l1.1 强烈抑制中胚层和外胚层特异性标记基因以维持神经命运。总之，这些结果表明 Foxd4l1.1 通过双重机制负调控的转录。因此，我们的研究表明，在胚胎中，Foxd4l1.1 对神经外胚层和中胚层的精确相互调节。
