Profiling the long noncoding RNA interaction network in the regulatory elements of target genes by chromatin in situ reverse transcription sequencing

Long noncoding RNAs (lncRNAs) can regulate the activity of target genes by participating in the organization of chromatin architecture. We have devised a “chromatin-RNA in situ reverse transcription sequencing” (CRIST-seq) approach to profile the lncRNA interaction network in gene regulatory elements by combining the simplicity of RNA biotin labeling with the specificity of the CRISPR/Cas9 system. Using gene-specific gRNAs, we describe a pluripotency-specific lncRNA interacting network in the promoters of Sox2 and Pou5f1 , two critical stem cell factors that are required for the maintenance of pluripotency. The promoter-interacting lncRNAs were specifically activated during reprogramming into pluripotency. Knockdown of these lncRNAs caused the stem cells to exit from pluripotency. In contrast, overexpression of the pluripotency-associated lncRNA activated the promoters of core stem cell factor genes and enhanced fibroblast reprogramming into pluripotency. These CRIST-seq data suggest that the Sox2 and Pou5f1 promoters are organized within a unique lncRNA interaction network that determines the fate of pluripotency during reprogramming. This CRIST approach may be broadly used to map lncRNA interaction networks at target loci across the genome.

长链非编码RNA（lncRNAs）可通过参与染色质结构的组织来调控靶基因的活性。我们设计了一种“染色质 - RNA原位逆转录测序”（CRIST - seq）方法，通过将RNA生物素标记的简便性与CRISPR/Cas9系统的特异性相结合，来描绘基因调控元件中的lncRNA相互作用网络。利用基因特异性的向导RNA（gRNAs），我们描述了在Sox2和Pou5f1启动子中的多能性特异性lncRNA相互作用网络，Sox2和Pou5f1是维持多能性所需的两个关键干细胞因子。在重编程为多能性的过程中，与启动子相互作用的lncRNAs被特异性激活。敲低这些lncRNAs会导致干细胞退出多能性状态。相反，多能性相关lncRNA的过表达激活了核心干细胞因子基因的启动子，并增强了成纤维细胞重编程为多能性的能力。这些CRIST - seq数据表明，Sox2和Pou5f1启动子在一个独特的lncRNA相互作用网络内进行组织，该网络决定了重编程过程中多能性的命运。这种CRIST方法可广泛用于绘制全基因组目标位点的lncRNA相互作用网络。