Regulatory Roles and Dynamics of Nuclear long-noncoding RNAs in Pluripotency

人类基因组计划最大的惊喜之一是在基因组中观察到的大量转录。 非编码基因组，由数千个长的非编码RNA（lncRNA）组成。在以前的工作中，我们 鉴定了一些lncRNA，它们在人类重编程和维持 多能性。我们之前的建议，与Meissner博士和Gnirke博士的建议合作，现在已经 鉴定了一百多个特异于多能状态的lncRNA， 重新编程和富集以驻留在关键发育调节器附近。我们还发现了一种新兴的 lncRNA作为调控因子在塑造核结构中的机制主题， 多能基因表达程序。基于这些研究，我们现在建议采取下一个飞跃 解决以下紧迫问题： 目的1：寻找与多能性和发育相关的多能特异性表达的lncRNA位点， 调节因子在功能上有助于维持多能性？人们很容易认为， 位于关键多能性附近，发育调节因子可以顺式发挥作用， 因子或抑制分化基因。在这里，我们将系统地应用转录抑制（CRISPR-i） 在一百个lncRNA基因座上筛选维持多能性所需的基因座。我们将进一步 并行进行许多单独的验证测试和RNase-H介导的lncRNA消耗。最后， 我们将确定lncRNA缺失对多能性和分化基因表达程序的影响。 功能（LOF）的研究。 目的2：核lncRNA的全基因组RNA-DNA和RNA-蛋白定位特性是什么， 促进适当的多能性基因表达程序？我们修改了提案，以便更深入地关注 FIRRE和CISTR-ACT是两种lncRNA，如在先前的提议中所确定的，它们高度共享关键特性。 与拟议的研究有关：需要有稳定性，促进核结构的共同机制 与人类疾病密切相关。通过对鉴定的蛋白质伴侣进行功能丧失研究， 可以开始确定RNA/DNA序列，染色质环境，三个- 空间邻近和蛋白质相互作用对lncRNA核定位的影响。 目的3：什么是FIRRE和CISTR-ACT介导的核组织的动力学。我们现在有 将CRISPR-显示适配为活细胞成像（CLING）。通过这种方法，我们可以监控3D交互 最多5条染色体。我们将研究的WT和LOF和GOF状态 多能性和重编程期间的FIRRE和CISTR-ACT染色体动力学。总的来说，这些 研究将确定lncRNA与DNA和蛋白质的分子相互作用，以及它们如何促进三种 和多能基因组的四维结构。

One of the biggest surprises of the human genome project was the vast amount of transcription observed in the noncoding genome, comprised of thousands of long noncoding RNAs (lncRNA). In previous work, we identified a few lncRNAs that contribute important functional roles in human reprograming and maintenance of pluripotency. Our previous proposal, in collaboration with the Drs. Meissner's and Gnirke's proposals, has now identified more than a hundred lncRNAs that are specific to the pluripotent state, dynamically regulated during reprograming and enriched to reside near key developmental regulators. We have also identified an emerging mechanistic theme of lncRNAs as regulatory factors in shaping nuclear architecture and, in turn, driving pluripotent gene expression programs. Based on these studies we now propose to take the next leap forward towards addressing the following pressing questions: Aim 1: Do pluripotent specific expressed lncRNA loci that are in proximity to pluripotency and developmental regulators functionally contribute to the maintenance of pluripotency? It is tempting to think that lncRNAs that reside near key pluripotency and developmental regulators may function in cis to either activate pluripotency factors or repress differentiation genes. Here we will systematically apply a transcriptional inhibition (CRISPR-i) screen on a hundred of these lncRNA loci for those that are required to maintain pluripotency. We will further perform numerous individual validation tests and RNase-H mediated depletion of lncRNAs in parallel. Finally, we will determine influences on pluripotent and differentiation gene-expression programs upon lncRNA loss-of function (LOF) using massively-parallel RNA-sequencing technologies. Aim 2: What are the genome wide RNA-DNA and RNA-Protein localization properties of nuclear lncRNAs that facilitate proper pluripotency gene-expression programs? We have revised the proposal for a deeper focus on FIRRE and CISTR-ACT, two lncRNAs that, as determined in the previous proposal, share key properties highly relevant to the proposed study: required for stemness, shared mechanism of facilitating nuclear architecture and strongly linked to human disease. By performing loss of function studies on identified protein partners we can begin to determine the underlying influences of RNA/DNA sequences, chromatin environments, three- dimensional proximity and protein interactions on lncRNA nuclear localization. Aim 3: What are the dynamics of FIRRE and CISTR-ACT mediated nuclear organization. We now have adapted CRISPR-Display to Live Cell Imaging (CLING). With this approach we can monitor the 3D interactions of up to 5 chromosomes through time in a living cell. We will investigate the WT and LOF and GOF states of FIRRE and CISTR-ACT chromosomal dynamics in pluripotency and during reprograming. Collectively, these studies will identify the molecular interactions of lncRNAs with DNA and Protein and how they facilitate three and four dimensional organization of the pluripotent genome.