The long non-coding RNA Paupar regulates the expression of both local and distal genes [Pax6 KD]. Mus musculus

Although some long noncoding RNAs (lncRNAs) have been shown to regulate gene expression in cis, it remains unclear whether lncRNAs can directly regulate transcription in trans by interacting with chromatin genome-wide independently of their sites of synthesis. Here, we describe the genomically local and more distal functions of Paupar, a vertebrate-conserved and central nervous system-expressed lncRNA transcribed from a locus upstream of the gene encoding the Pax6 transcription factor. Knockdown of Paupar disrupts the normal cell cycle profile of neuroblastoma cells and induces neuronal differentiation. Paupar acts in a transcript-dependent manner both locally, to regulate Pax6, as well as distally by binding and regulating genes on multiple chromosomes, in part through physical association with Pax6 protein. Paupar binding sites are enriched near promoters and can function as transcriptional regulatory elements whose activity is modulated by Paupar transcript levels. Our findings demonstrate that a lncRNA can function in trans at transcriptional regulatory elements distinct from its site of synthesis to control large-scale transcriptional programmes. Overall design: N2A cells transfected with a non-targeting control vector were compared to N2A cells transfected with a Pax6 knockdown construct. Three biological replicates of each condition were analysed on Affymetrix GeneChip Mouse Gene 1.0 ST Arrays.

尽管已有研究证实部分长链非编码RNA（long noncoding RNAs，lncRNAs）可通过顺式作用调控基因表达，但目前仍不明确的是，lncRNAs能否脱离自身合成位点，在全基因组范围内与染色质相互作用，进而通过反式作用直接调控转录。本研究对Paupar的基因组局部及远端调控功能进行了系统描述：Paupar是一种在脊椎动物中保守、于中枢神经系统中表达的lncRNA，其转录位点位于编码Pax6转录因子的基因上游区域。敲低Paupar会扰乱神经母细胞瘤细胞的正常细胞周期谱，并诱导其向神经元方向分化。Paupar以转录本依赖的方式发挥调控功能：既可以在局部调控Pax6基因，也能通过结合并调控多条染色体上的基因实现远端调控，其部分功能依赖于与Pax6蛋白的物理结合。Paupar的结合位点在启动子区域附近显著富集，且可作为转录调控元件发挥功能，其活性受Paupar转录本水平的调控。本研究结果证实，lncRNAs可在脱离自身合成位点的转录调控元件处通过反式作用，调控大规模的转录程序。整体实验设计：将转染非靶向对照载体的N2A细胞与转染Pax6敲低载体的N2A细胞进行对照分析，每组设置3次生物学重复，所有样本均采用Affymetrix GeneChip Mouse Gene 1.0 ST基因芯片进行检测分析。