Division of Life Science, Graduate School of Science and Engineering, Saitama University, Shimo-Okubo, Sakura-ku, Saitama City, Saitama 338-8570, Japan.
Division of Life Science, Graduate School of Science and Engineering, Saitama University, Shimo-Okubo, Sakura-ku, Saitama City, Saitama 338-8570, Japan; Saitama University Brain Science Institute, Saitama University, Shimo-Okubo, Sakura-ku, Saitama City, Saitama 338-8570, Japan.
Exp Cell Res. 2018 Mar 1;364(1):28-41. doi: 10.1016/j.yexcr.2018.01.023. Epub 2018 Jan 31.
Zebrafish pou5f3 (previously named pou2), a close homologue of mouse Oct4, encodes a PouV-family transcription factor. pou5f3 has been implicated in diverse aspects of developmental regulation during embryogenesis. In the present study, we addressed the molecular function of Pou5f3 as a transcriptional regulator and the mechanism by which pou5f3 expression is transcriptionally regulated. We examined the influence of effector genes on the expression of the luciferase gene under the control of the upstream 2.1-kb regulatory DNA of pou5f3 (Luc-2.2) in HEK293T and P19 cells. We first confirmed that Pou5f3 functions as a transcriptional activator both in cultured cells and embryos, which confirmed autoregulation of pou5f3 in embryos. It was further shown that Luc-2.2 was activated synergistically by pou5f3 and sox3, which is similar to the co-operative activity of Oct4 and Sox2 in mice, although synergy between pou5f3 and sox2 was less obvious in this zebrafish system. The effects of pou5f3 deletion constructs on the regulation of Luc-2.2 expression revealed different roles for the three subregions of the N-terminal region in Pou5f3 in terms of its regulatory functions and co-operativity with Sox3. Electrophoretic mobility shift assays confirmed that Pou5f3 and Sox3 proteins specifically bind to adjacent sites in the 2.1-kb DNA and that there is an interaction between the two proteins. The synergy with sox3 was unique to pou5f3-the other POU factor genes examined did not show such synergy in Luc-2.2 regulation. Finally, functional interaction was observed between pou5f3 and sox3 in embryos in terms of the regulation of dorsoventral patterning and convergent extension movement. These findings together demonstrate co-operative functions of pou5f3 and sox3, which are frequently coexpressed in early embryos, in the regulation of early development.
斑马鱼 pou5f3（以前称为 pou2）是 mouse Oct4 的密切同源物，编码 PouV 家族转录因子。pou5f3 参与胚胎发生过程中发育调控的多个方面。在本研究中，我们研究了 Pou5f3 作为转录调节因子的分子功能，以及 pou5f3 表达的转录调控机制。我们检查了效应基因对在 HEK293T 和 P19 细胞中受 pou5f3 上游 2.1kb 调控 DNA（Luc-2.2）控制的荧光素酶基因表达的影响。我们首先证实 Pou5f3 在培养细胞和胚胎中均起转录激活剂的作用，这证实了胚胎中 pou5f3 的自身调控。进一步表明，Luc-2.2 被 Pou5f3 和 sox3 协同激活，这类似于小鼠中 Oct4 和 Sox2 的协同活性，尽管在这个斑马鱼系统中 pou5f3 和 sox2 之间的协同作用不太明显。pou5f3 缺失构建体对 Luc-2.2 表达调控的影响揭示了 N 端区域的三个亚区在 Pou5f3 的调节功能及其与 Sox3 的协同作用方面的不同作用。电泳迁移率变动分析证实，Pou5f3 和 Sox3 蛋白特异性结合到 2.1kb DNA 的相邻位点，并且两种蛋白之间存在相互作用。与 sox3 的协同作用是 pou5f3 所特有的-检查的其他 POU 因子基因在 Luc-2.2 调节中没有显示出这种协同作用。最后，在 Dorsoventral 模式形成和会聚延伸运动的调节方面，观察到 pou5f3 和 sox3 在胚胎中的功能相互作用。这些发现共同证明了在早期胚胎中经常共表达的 pou5f3 和 sox3 的协同功能，在早期发育的调节中。
