Department of Biochemistry, Institute of Cell Differentiation and Aging, College of Medicine, Hallym University, Chuncheon, Gangwon-Do, 24252, Republic of Korea.
Department of Biochemistry, Institute of Cell Differentiation and Aging, College of Medicine, Hallym University, Chuncheon, Gangwon-Do, 24252, Republic of Korea; Department of Molecular Medicine, School of Medicine, Gachon University, Incheon, 21999, Republic of Korea.
Biochem Biophys Res Commun. 2021 Jun 25;559:168-175. doi: 10.1016/j.bbrc.2021.04.048. Epub 2021 May 1.
Transforming growth factor (TGF)β/activin superfamily regulates diverse biological processes including germ layer specification and axis patterning in vertebrates. TGFβ/activin leads to phosphorylation of Smad2 and Smad3, followed by regulation of their target genes. Activin treatment also induces the essential organizer gene chordin (chrd). The involvement of Smad2/3 in chrd expression has been unclear as to whether Smad2/3 involvement is direct or indirect and whether any cis-acting response elements for Smad2/3 are present in the proximal or distal regions of its promoter. In the present study, we isolated the -2250 bps portion of the chrd promoter, showing that it contained Smad2/3 direct binding sites at its distal portion, separate from the proximal locations of other organizer genes, goosecoid and cerberus. The pattern of transcription activation for the promoter (-2250 bps) was indistinguishable from that of the endogenous chrd in gastrula Xenopus embryos. Reporter gene assays and site-directed mutagenesis analysis of the chrd promoter mapped two active activin/Smad response elements (ARE1 and ARE2) for Smad2 and Smad3. For a differential chrd induction, Smad2 acted on both ARE1 and ARE2, but Smad3 was only active for ARE2. Collectively, the results demonstrate that the distal region of chrd promoter contains the direct binding cis-acting elements for Smad2 and Smad3, which differentially modulate chrd transcription in gastrula Xenopus embryos.
转化生长因子-β（TGF-β）/激活素超家族调节多种生物学过程，包括脊椎动物的胚层特化和轴模式形成。TGF-β/激活素导致 Smad2 和 Smad3 的磷酸化，随后调节它们的靶基因。激活素处理还诱导必需的组织者基因 chordin（chrd）。Smad2/3 参与 chrd 表达的情况尚不清楚，Smad2/3 的参与是直接的还是间接的，以及其启动子近端或远端是否存在 Smad2/3 的顺式作用反应元件。在本研究中，我们分离了 chrd 启动子的-2250 bps 部分，表明它在远端部分包含 Smad2/3 的直接结合位点，与其他组织者基因 goosecoid 和 cerberus 的近端位置分开。启动子（-2250 bps）的转录激活模式与原肠胚期 Xenopus 胚胎中内源性 chrd 的模式无法区分。报告基因检测和 chrd 启动子的定点突变分析表明，Smad2 和 Smad3 有两个有效的激活素/Smad 反应元件（ARE1 和 ARE2）。对于差异诱导 chrd，Smad2 作用于 ARE1 和 ARE2，但 Smad3 仅对 ARE2 起作用。总之，这些结果表明，chrd 启动子的远端区域包含 Smad2 和 Smad3 的直接结合顺式作用元件，它们在原肠胚期 Xenopus 胚胎中差异调节 chrd 转录。
