Dissecting mechanisms of gene silencing by the lncRNA Kcnq1ot1 in mouse trophoblast stem cells

摘要 一种保守的长链非编码RNA（lncRNA）称为Kcnq 1 ot 1，是哺乳动物正常发育所必需的。 Kcnq 1 ot 1基因沉默的缺陷导致Beckwith-Wiedemann综合征，这是一种出生缺陷， 高死亡率-通过体外受精怀孕的儿童死亡率增加。尽管其关键 尽管Kcnq 1 ot 1在发育中的作用，但其用于沉默基因的机制仍然知之甚少。虽然 已知Kcnq 1 ot 1通过募集称为Polycomb Repressive的组蛋白修饰酶来沉默基因， 复合物和G9 a，目前尚不清楚如何在Kcnq 1 ot 1序列元件促进这些相互作用。作为一个 可能的线索是，另一种对发育至关重要的抑制性lncRNA，Xist，已被证明与 RNA结合蛋白（RBP），反过来，招募Polycomb沉默酶。在我自己的研究中， 发现许多相同的RBP与Kcnq 1 ot 1的结合水平显著高于与其他RNA的结合水平 这意味着它们对Kcnq 1 ot 1的功能也很重要。我研究的目的 是通过鉴定转录物中的功能序列结构域， 识别结合这些区域的RBP，并确定它们参与招募Polycomb和G9 a 沉默酶如果成功，我希望不仅能阐明保守的Kcnq 1 ot 1的机制， 在小鼠和人类中，但进一步我们对lncRNA在早期发育中的功能的一般理解。从长远来看， 这些信息最终将有助于优化体外受精和发现基因 与不孕症有关我提议的活动将为我提供RNA生物学和基因组学方面的关键培训， 分子生物学，发育生物学，教学和指导，口头交流技能和网络， 和科学写作，其中每一个都将是我未来的职业生涯作为一个主要的研究者在一个关键 本科院校。

ABSTRACT A conserved long non-coding RNA (lncRNA) called Kcnq1ot1 is essential for proper mammalian development. Defects in proper gene silencing by Kcnq1ot1 result in Beckwith-Wiedemann syndrome, a birth defect with a high mortality rate whose prevalence is increased in children conceived by in vitro fertilization. Despite its critical role in development, the mechanisms that Kcnq1ot1 uses to silence genes remain poorly understood. While it is known that Kcnq1ot1 silences genes by recruiting histone-modifying enzymes called the Polycomb Repressive Complexes and G9a, it is not clear how sequence elements in Kcnq1ot1 facilitate these interactions. As one possible clue, another repressive lncRNA that is essential for development, Xist, has been shown to interact with RNA-binding proteins (RBPs), that in turn, recruit the Polycomb silencing enzymes. In my own studies, I have found that many of these same RBPs associate with Kcnq1ot1 at levels significantly higher than with other RNAs in the transcriptome, implying that they too are important for the function of Kcnq1ot1. The objective of my study is to further develop the mechanism of Kcnq1ot1 by identifying functional sequence domains in the transcript, identify the RBPs that bind these regions, and determine their involvement in recruiting the Polycomb and G9a silencing enzymes. If successful, I hope to not only elucidate the mechanism of Kcnq1ot1, which is conserved in mice and humans, but further our general understanding of lncRNA function in early development. Long term, this information could eventually contribute to optimization of in vitro fertilization and discovery of genes implicated in infertility. The activities I propose will provide me with critical training in RNA biology and genomics, molecular biology, developmental biology, teaching and mentorship, oral communication skills and networking, and scientific writing, each of which will be critical for my future career as a principal investigator at a primarily undergraduate institution.