Local regulation of gene expression by lncRNA promoters, transcription, and splicing. Mus musculus

Mammalian genomes are pervasively transcribed to produce thousands of spliced long noncoding RNAs (lncRNAs), whose functions remain poorly understood. Because recent evidence has implicated several specific lncRNA loci in the local regulation of gene expression, we sought to determine whether such local regulation is a property of many lncRNA loci. We used genetic manipulations to dissect 12 genomic loci that produce lncRNAs and found that 5 of these loci influence the expression of a neighboring gene in cis. Surprisingly, however, none of these effects required the specific lncRNA transcripts themselves and instead involved general processes associated with their production, including enhancer-like activity of gene promoters, the process of transcription, and the splicing of the transcript. Interestingly, such effects are not limited to lncRNA loci: we found similar effects on local gene expression at 4 of 6 protein-coding loci. These results demonstrate that ‘crosstalk’ among neighboring genes is a prevalent phenomenon that can involve multiple mechanisms and cis regulatory signals, including a novel role for RNA splice sites. These mechanisms may coordinate the expression of neighboring genes and explain the function and evolution of some genomic loci that produce lncRNAs. Overall design: We developed a genetic approach based on classical cis-trans tests to distinguish between (i) primary effects on the expression of nearby genes resulting from direct local functions of the lncRNA locus and (ii) secondary effects on nearby genes resulting from indirect downstream consequences of the lncRNA acting elsewhere in the cell. We generated clonal cell lines carrying heterozygous genetic modifications at lncRNA loci and compared the expression of nearby genes within 1 megabase on the cis and trans alleles (i.e., on the modified and unmodified homologous chromosomes) in the same cells. Changes in neighboring gene expression that involve only the cis allele likely result from local functions, while changes that involve both the cis and trans alleles likely result as downstream effects of non-local functions. We performed genetic modifications in 129/Castaneus F1 hybrid mouse embryonic stem cells (mESCs) that contain ~1 polymorphic site every 140 basepairs (bp), enabling us to distinguish the two alleles using RNA sequencing, and we checked for consistency between knockouts on each genetic background to control for potential haplotype-specific effects. We initially characterized allele-specific gene expression using RNA sequencing combined with hybrid selection, and we further characterized a subset of clones in selected loci using global run-on sequencing (GRO-seq), chromatin immunoprecipitation (ChIP-Seq), and ATAC-Seq.

哺乳动物基因组会广泛转录产生数千种剪接型长链非编码RNA（long noncoding RNAs，lncRNAs），其功能至今仍未得到充分阐释。近期研究已证实多个特定lncRNA位点参与基因表达的局部调控，本研究旨在探究此类局部调控是否为多数lncRNA位点共有的特性。
我们通过遗传操作对12个产生lncRNAs的基因组位点进行剖析，发现其中5个位点可顺式调控邻近基因的表达。然而令人意外的是，这些调控效应均不依赖于特定的lncRNA转录本本身，而是涉及与转录本生成相关的通用过程，包括基因启动子的增强子样活性、转录过程以及转录本的剪接。
有趣的是，此类调控效应并非仅限于lncRNA位点：我们在6个蛋白质编码基因位点中的4个上也观察到了类似的局部基因表达调控效应。
本研究结果表明，邻近基因间的“交叉对话”是一种普遍存在的现象，可涉及多种机制和顺式调控信号，包括RNA剪接位点的全新功能。这些机制可协同调控邻近基因的表达，同时也为部分产生lncRNAs的基因组位点的功能与进化提供了解释。
整体实验设计：我们基于经典的顺式-反式测验开发了一种遗传学分析方法，用以区分两类效应：（1）由lncRNA位点直接局部功能所导致的邻近基因表达初级效应；（2）由lncRNA在细胞其他部位发挥功能所产生的间接下游效应，进而对邻近基因造成的次级影响。
我们构建了在lncRNA位点携带杂合遗传修饰的克隆细胞系，并比较了同一细胞中顺式和反式等位基因（即经修饰与未修饰的同源染色体）上1兆碱基范围内邻近基因的表达水平。仅影响顺式等位基因的邻近基因表达变化，大概率源自该位点的局部功能；而同时影响顺式和反式等位基因的变化，则大概率由非局部功能引发的下游效应导致。
我们在129品系/栗色小家鼠（Castaneus）F1杂交小鼠胚胎干细胞（mouse embryonic stem cells，mESCs）中开展遗传操作，该细胞系每140个碱基对（basepairs，bp）即存在约1个多态性位点，可通过RNA测序区分两个等位基因；同时我们验证了不同遗传背景下敲除实验的结果一致性，以控制潜在的单倍型特异性效应。
我们最初通过结合杂交筛选的RNA测序对等位基因特异性基因表达进行了表征，并进一步通过全局run-on测序（global run-on sequencing，GRO-seq）、染色质免疫沉淀测序（chromatin immunoprecipitation sequencing，ChIP-Seq）以及转座酶可及性染色质测序（assay for transposase-accessible chromatin sequencing，ATAC-Seq）对选定位点的部分克隆进行了深入分析。