Functional analysis of novel TALE homeodomain transcription factor Prep2

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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