Control of gene silencing by long noncoding RNAs in trophoblast stem cells

摘要 长的非编码RNA(LncRNAs)构成了哺乳动物转录本的很大一部分，并具有重要的 在不同的生物过程中的作用，包括在早期发育过程中蛋白质编码基因的沉默。在……里面 滋养层细胞，lncRNAs已经被证明通过它们与 高度保守的染色质修饰酶称为多梳抑制复合体(PRCs)。具体来说， LncRNAs Xist、Airn和Kcnq1ot1将pRC定向到分开的基因组结构域，每个基因组结构域跨越数百万个碱基 配对，其中特定的基因是沉默的。事实上，滋养细胞中有缺陷的lncRNA沉默导致 基因的不适当表达可能是不孕症的主要原因，因此植入前 胚胎的正常发育依赖于滋养层细胞。尽管滋养细胞在早期很重要 胚胎发生，维持胚胎发生的分子机制，特别是通过调节基因网络 LncRNA沉默，都是未知的。我们实验室最近的工作表明，在滋养层干细胞(TSCs)中， Xist、Airn和Kcnq1ot1沉默的基因组结构域含有非均匀分布的pRC，具有 某些地区比其他地区受到更大程度的沉默。虽然确立这一点的基本特征 非均匀性还不完全清楚，我们和其他人提出，染色质相关因子至少是 对此负有部分责任。在我们最近的研究中，三维(3D)基因组结构和特定的CpG岛 (CGI)DNA元素似乎是重要的贡献者。因此，使用TSCs作为体外模型用于 滋养层细胞，我建议研究染色质的构象，染色质和lncRNA之间的联系， 与CGI结合的序列特异性转录因子协同决定区域和远距离 LncRNA沉默结构域中的沉默。我的研究将强调调控表观遗传网络的特定因素 来维持滋养层细胞。此外，我的工作将为lncRNAs的基因调控提供新的范式， 从而特定的DNA结合蛋白通过以下方式控制LncRNAs在区域上诱导的沉默的强度- 以地区为基础。反过来，这些数据可能会为如何治疗出生缺陷或预防不孕不育提供新的见解 这是由于改变了剂量的lncRNA沉默的基因造成的。 我的长期目标是从事染色质和表观遗传学研究，成为一家 主要的研究机构。我建议的培训活动是为了让我为这个目标做好准备，以及下一个目标 立即获得染色质和表观遗传学的博士后奖学金。为此，我的研究 PLAN将为我提供宝贵的定量基因组学培训，以及开发和 通过计算和分子细胞生物学严格测试假说。我得到了一位合作者的帮助 和一位共同赞助人，他的计算专长与我的主要赞助人相辅相成。我的技术 培训将伴随着口头交流、科学写作和评估方面的专业发展， 和网络，这将有助于实现我成为一名独立调查员的目标。

ABSTRACT Long noncoding RNAs (lncRNAs) make up a large portion of mammalian transcriptomes and have essential roles in diverse biological processes, including silencing of protein-coding genes during early development. In the trophoblast, lncRNAs have been shown to have augmented silencing potency through their relationship with highly conserved chromatin-modifying enzymes called Polycomb Repressive Complexes (PRCs). Specifically, the lncRNAs Xist, Airn, and Kcnq1ot1 direct PRCs to separate genomic domains that each span millions of base pairs, in which specific genes are silenced. Indeed, defective silencing by lncRNAs in the trophoblast leads to inappropriate expression of genes that can be a major cause of infertility, whereby the preimplantation embryo depends on the trophoblast for proper development. Despite the importance of the trophoblast in early embryogenesis, the molecular mechanisms that sustain it, particularly through regulation of gene networks by lncRNA silencing, are unknown. Recent work from our lab has shown that, in trophoblast stem cells (TSCs), the genomic domains silenced by Xist, Airn, and Kcnq1ot1 harbor PRCs that are distributed non-uniformly, with certain regions subject to greater levels of silencing than others. While the underlying features that establish this non-uniformity are not fully known, we and others have proposed that chromatin-associated factors are at least partly responsible. In our recent study, three-dimensional (3D) genome architecture and specific CpG island (CGI) DNA elements appeared to be important contributors. Thus, using TSCs as an ex vivo model for the trophoblast, I propose to investigate how chromatin conformation, contacts between chromatin and lncRNAs, and sequence-specific transcription factors bound to CGIs cooperate to dictate both regional and long-distance silencing in lncRNA-silenced domains. My studies will highlight specific factors that regulate epigenetic networks that sustain the trophoblast. Furthermore, my work will provide new paradigms for gene regulation by lncRNAs, whereby specific DNA-binding proteins control the intensity of silencing induced by lncRNAs on a region-by- region basis. In turn, these data may provide new insights on how to treat birth defects or prevent the infertility that results from altered dosage of lncRNA-silenced genes. My long-term goal is to pursue a career in chromatin and epigenetics research as a principle investigator at a major research institution. The training activities I propose are designed to prepare me for this goal, with the next immediate step to obtain a postdoctoral fellowship in chromatin and epigenetics. To these ends, my research plan will provide me with invaluable training in quantitative genomics and the skills needed to develop and rigorously test hypotheses via computational and molecular cell biology. I have enlisted the help of a collaborator and a co-sponsor whose computational expertise is complementary to that of my primary sponsor. My technical training will be accompanied by professional development in oral communication, scientific writing and evaluation, and networking, which will be instrumental to attaining my goal of becoming an independent investigator.