A conserved enhancer element that drives FGF4 gene expression in the embryonic myotomes is synergistically activated by GATA and bHLH proteins
A conserved enhancer element that drives FGF4 gene expression in the embryonic myotomes is synergistically activated by GATA and bHLH proteins
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DOI：
10.1016/j.ydbio.2004.03.012
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发表时间：
2004-06-15
影响因子：
2.7
通讯作者：
Basilico, C
中科院分区：
文献类型：
--
作者：
Iwahori, A;Fraidenraich, D;Basilico, C
FGF 4是胚胎发生过程中表达的成纤维细胞生长因子（FGF）家族的最早成员，在胚胎发生过程中，它在植入后发育和肢体生长和图案形成中起重要作用。Fgf 4基因在特定发育阶段（包括胚泡的ICM、肌节和肢芽AER）中的表达受3' UTR中的不同增强子元件（Hom）调节。我们以前确定了Hom 3a区域作为负责肌节和AER中Fgf 4表达的主要DNA元件，并表明保守的E-box是肌原性bHLH转录因子MYF 5和MYOD的靶点。为了进一步定义决定Hom 3a活性的顺式和反式作用元件，我们对Hom 3a区域驱动转基因小鼠肌节中lacZ表达的能力进行了突变分析。我们确定了一个最小的226 nt的增强子，它包含四个元件，包括E-box，这是驱动肌节中基因表达所必需的。其中一个元件是转录因子加塔家族的结合位点，我们在这里表明，加塔1-4和6可以与MYF 5或MYOD协同作用，激活由Hom 3a增强子的一部分（包括加塔位点和E盒）驱动的报告质粒的转录。与这一发现一致，我们可以显示MYF 5/MYOD和加塔-3或加塔-4之间的直接相互作用，由MYF 5/MYOD的N-末端和bHLH结构域以及加塔-3/4的C-末端zing finger结构域介导。为了进一步研究Hom 3a增强子在指导Fgf 4表达中的作用以及Fgf 4在肢体和肌肉发育中的功能，我们产生了其中Fgf 4 Hom 3a区域已经缺失的突变小鼠（Delta 3a）。在E11.5的Delta 3a/Delta 3a胚胎切片的原位杂交分析显示肌节和AER中Fgf 4 mRNA的表达显著降低。然而，这些小鼠发育正常，没有显示肢体或肌肉缺陷，杂合小鼠也是如此，其中一个Fgf 4等位基因携带Hom 3a缺失，另一个是无效等位基因（Delta 3a/Fgf 4（-））。总之，这些结果表明Hom 3a是指导肌节和肢芽AER中Fgf 4表达的主要DNA增强子元件，并且其在肌节中的活性至少部分地由结合Hom 3a增强子中进化保守序列的加塔和bHLH肌原性因子的协同作用引起。然而，在小鼠胚胎的肌节或AER中Fgf 4的表达似乎对肌肉或肢体发育不是必需的。(C)2004年爱思唯尔公司All rights reserved.
FGF4 is the earliest member of the fibroblast growth factor (FGF) family expressed during embryogenesis where it plays essential roles in post-implantation development and limb growth and patterning. The expression of the Fgf4 gene in specific developmental stages, including the ICM of the blastocyst, the myotomes, and the limb bud AER, is regulated by distinct enhancer elements (Hom) in the 3' UTR. We previously identified the Hom3a region as the major DNA element responsible for Fgf4 expression in the myotomes and AER, and showed that a conserved E-box is a target for the myogenic bHLH transcription factors MYF5 and MYOD. To further define the cis- and trans-acting elements that determine Hom3a activity, we conducted a mutational analysis of the ability of the Hom3a region to drive lacZ expression in the myotomes of transgenic mice. We identified a minimal enhancer of 226nt that contains four elements, including the E-box, necessary to drive gene expression in the myotomes. One of these elements is a binding site for the GATA family of transcription factors, and we show here that GATA 1-4 and 6 can synergize with MYF5 or MYOD to activate transcription of a reporter plasmid driven by a portion of the Hom3a enhancer including the GATA site and the E-box. In line with this finding, we could show a direct interaction between MYF5/MYOD and GATA-3 or GATA-4, mediated by the N-terminal and bHLH domains of MYF5/MYOD and the C-terminal zing finger domain of GATA-3/4. To further study the role of the Hom3a enhancer in directing Fgf4 expression and the function of FGF4 in limb and muscle development, we generated mutant mice in which the Fgf4 Hom3a region had been deleted (Delta3a). In situ hybridization analysis of sections from Delta3a/ Delta3a embryos at E11.5 showed a drastically reduced expression of Fgf4 mRNA in the myotomes and AER. However, these mice developed normally and show no limb or muscle defects, and the same was true of heterozygous mice in which one Fgf4 allele carried the Hom3a deletion and the other was a null allele (Delta3a/Fgf4(-)). Together, these results show that Hom3a is the major DNA enhancer element directing Fgf4 expression in myotomes and limb bud AER, and that its activity in the myotomes results at least in part from the synergistic action of GATA and bHLH myogenic factors that bind to evolutionary conserved sequences in the Hom3a enhancer. However, expression of Fgf4 in the myotomes or AER of murine embryos does not appear to be essential for muscle or limb development. (C) 2004 Elsevier Inc. All rights reserved.