Functional analysis of novel TALE homeodomain transcription factor Prep2

在TALL同源结构域超家族蛋白中，HTH/MEIS/Prep通过与另一种TALL同源结构域蛋白PBX形成复合物来调控HOX转录因子。本项目分离了新的脊椎动物Prep家族成员Prep2，并对其功能进行了分析。Prep2的氨基酸序列与Prep1有很高的相似性，但Prep2在同源结构域下游有很长的酸性氨基酸，这一未来与几种脊椎动物的Prep2是共同的。从斑马鱼胚胎的实验结果来看，这种酸性拉伸具有掩盖Prep2转录抑制功能的功能。Prep2在早期鸡胚毛胚层中表达，与MEIS的表达具有互补性。在早期胚胎中，视黄酸诱导MEIS的表达。另一方面，维甲酸对Prep2的表达没有影响，提示不同的机制控制着Prep2和Meis在早期胚胎中的表达。MEIS…More和Prep2在肢芽中也表现出部分互补的表达谱。MEIS是其伙伴PBX在近侧肢芽中的核定位所必需的。Prep2在肢芽的更远端区域表达，其功能是促进PBX在与MEIS表达区域不同的区域内的核定位。Prep2与Pbx1形成异二聚体，类似于Prep1或Meis。在转染系统中，Prep2即使在没有Pbx1的情况下也会迁移到细胞核中。另一方面，Prep1绝对需要Pbx1进行核定位。如果PBX存在，Prep2与PBX形成复合体，该复合体迁移到细胞核中。与Prep1-Pbx1复合体不同，Prep2或Prep2-PBX复合体不能刺激靶基因转录。在斑马鱼胚胎中单独注射Prep2或Pbx4基因对胚胎发育没有影响。然而，联合注射Prep2和Pbx4基因可导致眼和头部结构畸形。这表明Prep2-Pbx4在胚胎中具有功能。获得了靶向敲除小鼠Prep1和Prep2。Prep1KO纯合子胚胎在8-9dpc之间停止发育，没有表现出明显的形态变化。另一方面，Prep2KO纯合子动物是活的和可生育的。Prep1似乎与生理功能相结合，而不是与形态途径相结合。较少

Among TALE homeodomain superfamily protein, Hth/Meis/Prep are known to control Hox transcription factor through making complex with another TALE homeodomain protein Pbx. In this project novel vertebrate Prep family member Prep2 was isolated and the function was analyzed. The amino acid sequence of Prep2 exhibited highly similarity to Prep1 however Prep2 has long acidic amino acid stretch down stream of the homeodomain and this future was common to Prep2 from several vertebrates. From the results of the experiment using Zebrafish embryos, this acidic stretch carries the function to mask the transcriptional repressive function of Prep2. Prep2 was expressed in the anterior mosodermal layer of the early chicken embryo and this expression was complementary to the Meis expression. In the early embryo, Meis expression was induced by retinoic acid. On the other hand retinoic acid had no effect on Prep2 expression indicating different mechanism controls Prep2 and Meis in the early embryos. Meis … More and Prep2 also showed partially complementary expression profile in the limb bud. Meis was required for the nuclear localization of its partner Pbx in the proximal limb bud. Prep2 was expressed in more distal region of the limb bud and functioned to facilitate nuclear localization of Pbx in different domain from the Meis expression domain. Prep2 forms heterodimer with Pbx1 like Prep1 or Meis. In the transfection system, Prep2 migrate into the nucleus even in the absence from Pbx1. On the other hand Prep1 absolutely requires Pbx1 for nuclear localization. If Pbx was present Prep2 form complex with Pbx and the complex migrate into the nucleus. Unlike to the Prep1-Pbx1 complex, Prep2 or Prep2-Pbx complex did not stimulate target gene transcription. Injection of Prep2 mRNA or Pbx4 mRNA alone in to the Zebrafish embryo has no effect on the embryonic development. However co-injection of Prep2 mRNA and Pbx4 mRNA induced malformation of eye and head structure. This indicates Prep2-Pbx4 has a function in the embryo. Targeted knockout mouse of Prep1 and Prep2 were generated. Prep1KO homozygote embryos cease development between 8-9dpc exhibiting no obvious morphological changes. On the other hand homozygote animal of Prep2KO was viable and fertile. Prep1 seems to engage to the physiological function rather than morphological pathway. Less