Complex regulation of Eomes levels mediated through distinct functional features of the Meteor long non-coding RNA locus (RNA-Seq). Complex regulation of Eomes levels mediated through distinct functional features of the Meteor long non-coding RNA locus (RNA-Seq)

Long non-coding RNAs (lncRNAs) are diverse transcription products emanating from thousands of loci in mammalian genomes, implicated in a wide array of cellular processes including transcriptional regulation, differentiation, cellular reprogramming, and many others. While a large majority of functional lncRNAs have been proposed to act in cis to their sites of transcription, an in-depth understanding of the functional features within such lncRNA loci, as well as their mechanisms of action, is currently lacking. Further insights are hindered by the heavy dependencies of observed phenotypes on the perturbation techniques employed. Here, we study Meteor, a lncRNA transcribed nearby the gene encoding for Eomes, a pleiotropic transcription factor with highly regulated spatiotemporal expression patterns throughout early development and in adult tissues. Using a wide array of perturbation techniques, we show that transcription elongation through the Meteor locus is required for Eomes activation in naive mouse embryonic stem cells (mESCs), with Meteor repression linked to a reduction in cells potentiated to differentiate to the mesoderm lineage. We further demonstrate that a distinct functional feature of the locus – namely, the underlying DNA element – is required for suppressing Eomes expression following neuronal differentiation of mESCs. Our results demonstrate the complex regulation that can be conferred by a single lncRNA locus, and emphasize the importance of careful selection of perturbation techniques for the study of lncRNA loci. Overall design: RNA-seq of either mESCs or NPCs of various genotypes (WT, Meteor promoter KO, Meteor promoter inversion) or perturbations (Ctrl, Meteor CRISPR/Cas9 KD).

长链非编码RNA（long non-coding RNAs, lncRNAs）是一类源自哺乳动物基因组中数千个基因座的多样化转录产物，参与广泛的细胞生物学过程，包括转录调控、细胞分化、细胞重编程等。尽管绝大多数功能性长链非编码RNA被认为在其转录位点附近的顺式（cis）区域发挥功能，但目前对于这类长链非编码RNA基因座内部的功能特征及其作用机制仍缺乏深入认知。此外，观测到的表型高度依赖于所使用的扰动技术，这进一步阻碍了相关研究的推进。
本研究聚焦于Meteor——一种转录于Eomes编码基因附近的长链非编码RNA；Eomes是一种多效性转录因子，在早期发育及成年组织中均受到严格的时空表达调控。本研究采用多种扰动技术，证实幼稚型小鼠胚胎干细胞（naive mouse embryonic stem cells, mESCs）中，Meteor基因座的转录延伸对于Eomes的激活是必需的；而Meteor的沉默会降低具备分化为中胚层谱系潜能的细胞比例。本研究进一步证实，该基因座的一项独特功能特征——即其下方的DNA元件——对于小鼠胚胎干细胞向神经元分化过程中抑制Eomes的表达至关重要。本研究结果揭示了单个长链非编码RNA基因座所能介导的复杂调控机制，并强调了在长链非编码RNA基因座研究中谨慎选择扰动技术的重要性。
实验整体设计：对不同基因型（野生型（wild type, WT）、Meteor启动子敲除（knockout, KO）、Meteor启动子倒位）或经不同扰动处理（对照组Ctrl、Meteor CRISPR/Cas9敲低（knockdown, KD））的小鼠胚胎干细胞或神经前体细胞（neural progenitor cells, NPCs）进行RNA测序。