Direct activation of a mouse Hoxd11 axial expression enhancer by Gdf11/Smad signalling
Direct activation of a mouse Hoxd11 axial expression enhancer by Gdf11/Smad signalling
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DOI：
10.1016/j.ydbio.2013.08.025
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发表时间：
2013-11-01
影响因子：
2.7
通讯作者：
Paul, Yu-Lee
中科院分区：
文献类型：
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作者：
Gaunt, Stephen J.;George, Martin;Paul, Yu-Lee
在转基因小鼠胚胎中以类似 Hoxd11 的轴向表达模式表达的 Hoxd11/lacZ 报告基因在 Gdf11（一种 TGF-β 生长/分化因子）存在下培养时，会在尾芽片段中受到刺激。当瞬时转染 HepG2 细胞培养物时，相同的构建体也会受到 Gdf11 的刺激。 HepG2 细胞中报告基因的刺激得到增强，因为它仅包含荧光素酶或 lacZ 报告基因上游或下游的 332 bp Hoxd11 增强子区域 VIII。该增强子含有从鱼到小鼠保守的三个元件，其中一个具有 Smad3/4 结合元件的序列。该基序的突变会抑制 Gdf11 在 HepG2 细胞测定中增强报告基因活性的能力。染色质免疫沉淀实验显示 Smad2/3 蛋白与 Hoxd11 区域 VIII 增强子结合的直接证据。 HepG2 细胞中 Gdf11 对 Hoxd11 的作用至少部分受到 SIS3（Smad3 的特异性抑制剂）的抑制。 SIS3还在培养的转基因尾芽中对Hoxd11/lacZ表达产生部分抑制，表明Smad3可能在Hoxd11的胚胎表达中发挥类似的作用。转基因小鼠实验表明，Smad 结合基序对于 Hoxd11/lacZ 报告基因在胚胎尾芽、后中胚层和神经外胚层中的轴向表达至关重要。 (C) 2013 Elsevier Inc. 保留所有权利。
A Hoxd11/lacZ reporter, expressed with a Hoxd11-like axial expression pattern in transgenic mouse embryos, is stimulated in tailbud fragments when cultured in presence of Gdf11, a TGF-beta growth/differentiation factor. The same construct is also stimulated by Gdf11 when transiently transfected into cultures of HepG2 cells. Stimulation of the reporter in HepG2 cells is enhanced where it contains only the 332 bp Hoxd11 enhancer region VIII upstream or downstream of a luciferase or lacZ reporter. This enhancer contains three elements conserved from fish to mice, one of which has the sequence of a Smad3/4 binding element. Mutation of this motif inhibits the ability of Gdf11 to enhance reporter activity in the HepG2 cell assay. Chromatin immunoprecipitation experiments show direct evidence of Smad2/3 protein binding to the Hoxd11 region VIII enhancer. The action of Gdf11 upon Hoxd11 in HepG2 cells is inhibited, at least in part, by SIS3, a specific inhibitor of Smad3. SIS3 also produces partial inhibition of Hoxd11/lacZ expression in cultured transgenic tailbuds, indicating that Smad3 may play a similar role in the embryonic expression of Hoxd11. Transgenic mouse experiments show that the Smad binding motif is essential for the axial expression of Hoxd11/lacZ reporter in the embryo tailbud, posterior mesoderm and neurectoderm. (C) 2013 Elsevier Inc. All rights reserved.