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

摘要 长链非编码RNA（lncRNA）构成哺乳动物转录组的大部分，并且具有重要的生物学功能。 在不同的生物过程中发挥作用，包括在早期发育过程中蛋白质编码基因的沉默。在 滋养层，lncRNA已经显示出通过它们与 高度保守的染色质修饰酶，称为Polycomb抑制复合物（PRC）。具体地说， lncRNA Xist、Airn和Kcnq 1ot 1引导PRC分离基因组结构域，每个结构域跨越数百万个碱基 对，其中特定的基因被沉默。事实上，滋养层中lncRNA的缺陷沉默导致 不适当的基因表达可能是不孕症的主要原因， 胚胎的正常发育依赖于滋养层。尽管滋养层细胞在早期 胚胎发生，维持它的分子机制，特别是通过调控基因网络， lncRNA沉默，未知。我们实验室最近的工作表明，在滋养层干细胞（TSCs）中， 被Xist、Airn和Kcnq1ot 1沉默的基因组结构域含有分布不均匀的PRC， 某些区域比其他区域更容易沉默。虽然建立这一点的基本特征 不均匀性还不完全清楚，我们和其他人提出，染色质相关的因素至少是 部分负责。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， 滋养层，我建议研究如何染色质构象，染色质和lncRNA之间的接触， 与CGIs结合的序列特异性转录因子合作， lncRNA沉默结构域中的沉默。我的研究将强调调节表观遗传网络的特定因素 滋养层的细胞此外，我的工作将为lncRNA的基因调控提供新的范例， 由此特异性DNA结合蛋白控制由lncRNA在一个区域上诱导的沉默的强度， 区域基础。反过来，这些数据可能会为如何治疗出生缺陷或预防不孕症提供新的见解。 这是由lncRNA沉默基因的剂量改变引起的。 我的长期目标是追求在染色质和表观遗传学研究的职业生涯，作为一个主要的研究者在一个 大型研究机构。我建议的培训活动旨在为我实现这一目标做好准备， 立即获得染色质和表观遗传学博士后奖学金。为此，我的研究 该计划将为我提供定量基因组学方面的宝贵培训，以及发展和 通过计算和分子细胞生物学严格测试假设。我找了一个合作者 还有一个共同赞助人，他的计算专业知识与我的主要赞助人互补。我的技术 培训将伴随着口头交流、科学写作和评价方面的专业发展， 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.