An inquiry into the function of Ronin in embryogenesis and pluripotent stem cells

项目总结 长期以来的观点认为，干细胞和发育中基因调控的复杂性是通过 明智地组织了数百万个增强剂元素。最近发现的一类新的DNA结合面- 被称为THAPs(Thanatos相关区域包含凋亡相关蛋白)的Tors可能提供新的 对干细胞基因调控的洞察。这个家族中的一个关键成员，罗宁(塔普11)，是我们发现的 在胚胎干细胞中。罗宁在胚胎发育的最早阶段表达，是必不可少的。 在体外和体内维持干细胞的多能性。因为罗宁缺乏明显的交易- 激活活性，但作为一种与DNA结合的核蛋白，具有很强的蛋白质二聚活性，我们 认为它可能不会以转录调节的经典方式发挥作用(例如，具有酸性活性- 转而可能通过促进罗宁结合之间的通讯来增强基因的表达 通过DNA循环的站点。当我们发现大多数(83%)的 罗宁介导的环事件涉及启动子到启动子的连接，而不是增强子到启动子的连接 与典型转录因子的相互作用。在这项研究中，我们将(I)确定Ro-Ro的影响。 NIN介导的启动子对干细胞转录和基因调控的影响；(Ii)确定其影响 启动子依赖于发育转变，特别是从幼稚到启动的多能性状态；以及 (3)询问其他THAP是否也参与发育特定基因聚集性。

PROJECT SUMMARY A long-held view is that gene regulatory complexity in stem cells and development is achieved through the judicious organization of millions of enhancer elements. A recently identified new class of DNA binding fac- tors called THAPs (Thanatos-associated domain-containing apoptosis-associated proteins) may provide new insights into gene regulation in stem cells. One key member of this family, Ronin (Thap11), was discovered by us in embryonic stem cells. Ronin is expressed during the earliest stages of embryonic development and is essential for the maintenance of stem cell pluripotency, both in vitro and in vivo. Since Ronin lacks appreciable transac- tivation activity but functions as a DNA-binding nuclear protein with strong protein dimerization activity, we considered that it may not act in the classical manner of a transcriptional regulator (e.g., having an acidic activa- tion domain) but instead might augment gene expression by facilitating communication among Ronin binding sites through DNA looping. This working hypothesis gained traction when we found that most (83%) of the looping events mediated by Ronin involve promoter-to-promoter links rather than the enhancer-to-promoter interactions seen with typical transcription factors. In this research study we will (i) establish the effects of Ro- nin-mediated promoter tethering on transcription and gene regulation in stem cells; (ii) determine the influence of promoter tethering on developmental transitions especially from the naïve to primed pluripotent state; and (iii) ask if other THAPs participate in development specific gene clustering as well.