cis regulated role of lncRNA in transcriptional or developmental process (ChIRP-Seq). Mus musculus

We report that long noncoding RNAs contribute to transcription and developmental process. Thousands of lncRNAs have been identified in the whole genome, and tend to located closely to protein-coding genes. To study position relationship between lncRNA and protein-coding genes, we classified all of lncRNA to several subgroups based on the genome position with their coding neighbors. XH, the head to head subgroup is associated with transcription and development in GO analysis. Here, we knockdown serveral XH lncRNA by shRNA in embryonic stem cells and induce nondirectional differnetiation by removing LIF or neural differnetiation by RA. Knockdown of XH lncRNAs led to uniform downregulation of nearby coding genes, and form regulatory circuits with its nearby coding genes to fine-tune embryonic lineage development. In addition, we also knockout one lncRNA-Evx1as and its nearby protein-coding gene-EVX1 by CRISPR, and get similar results as knockdown.We propose that XH lncRNA may function primarily as 'cis-regulators' of the expression of nearby protein-coding genes, and tend to participate in transcriptional or development regulations as their coding neighbors. Overall design: All RNA-seq(s) were designed to reveal the differentially expressed genes between wild-type and XH lncRNA knockdown/knockout ESCs during differentiation.

本研究报道长链非编码RNA（long noncoding RNAs，lncRNAs）参与转录调控与发育进程。目前已在全基因组范围内鉴定出数千种lncRNAs，且其通常定位于蛋白编码基因邻近区域。为探究lncRNA与蛋白编码基因的位置关联，我们依据lncRNA与其编码相邻基因的基因组位置，将所有lncRNA划分为若干亚组。基因本体（Gene Ontology，GO）分析显示，头对头亚组（head to head subgroup，XH）与转录及发育过程显著相关。我们通过短发夹RNA（short hairpin RNA，shRNA）在胚胎干细胞中敲降数种XH型lncRNA，并分别通过移除白血病抑制因子（leukemia inhibitory factor，LIF）诱导非定向分化，或通过视黄酸（retinoic acid，RA）诱导神经分化。实验结果表明，敲降XH型lncRNA会导致其邻近编码基因整体下调，并与其邻近编码基因形成调控环路，以精细调控胚胎谱系发育。此外，我们还通过成簇规律间隔短回文重复序列（Clustered Regularly Interspaced Short Palindromic Repeats，CRISPR）技术敲除了一种lncRNA——Evx1as及其邻近蛋白编码基因EVX1，得到了与敲降实验一致的结果。我们推测，XH型lncRNA主要作为邻近蛋白编码基因的顺式调控因子（cis-regulators）发挥功能，并倾向于与其邻近编码基因共同参与转录或发育调控。整体实验设计：所有RNA测序（RNA sequencing，RNA-seq）实验均用于揭示分化过程中野生型与XH型lncRNA敲降/敲除胚胎干细胞之间的差异表达基因。