Genetic and Epigenetic Regulation of Pvt1 Monoallelic Expression [Bisulfite-seq]

Each gene exists as two copies in the same nucleus of mammalian cells. Although most genes are equivalently controlled on both alleles, Random Monoallelic Expressing (RME) genes stably maintain the expression from only one of two alleles, but the mechanisms and functional consequences of RME remains unclear. Here we performed allele-specific RNA-seq on over 100 neural progenitor cell (NPC) clonal lines to reveal the extent of RME for genes across clonal lines. Interestingly out of 297 autosomal RME genes, Pvt1, a well-studied oncogenic long non-coding RNA, is monoallelically expressed with a skewed genetic bias towards the Castaneous allele in F1-hybrid NPCs. In the absence of genetic differences, allelic tracking by gene editing reveals Pvt1 undergoes balanced RME that is maintained across somatic cell generations. Monoallelic expression is maintained by a poised Pvt1 promoter with bivalent active histone marks, H3K4me3 and H3K27ac, and silencing histone marks, H3K4me3 and H3K27me3, demonstrating its ability to switch transcriptional states depending on the cellular context. Leveraging our large RNA-seq data set and the genetic skew, we use differential gene expression analysis on clonal lines based on their Pvt1 allelic status to reveal a transcription factor, Tfap2a, with differential binding at the Pvt1 promoter, providing a mechanism for the initiation and allelic choice for RME genes. Additionally, we demonstrate monoallelic Pvt1 expression results in an increase in Pvt1 expression leading to a growth advantage relative to Pvt1 biallelic expressing clonal lines These findings provide an example of how genetic differences can skew a stochastic process which results in an epigenetic phenomenon with a phenotypic consequence in early development. Overall design: Bisulfite sequencing of a neuronal progenitor cell (NPC) clonal line differentiated from a male F1-hybrid (129 x Cast) mESC.

在哺乳动物细胞的同一细胞核中，每个基因均以两份拷贝的形式存在。尽管大多数基因在两条等位基因上的调控模式等效，但随机单等位基因表达（Random Monoallelic Expressing, RME）基因可稳定维持仅一条等位基因的表达，然而RME的发生机制与功能效应目前仍不明确。本研究对超过100株神经前体细胞（neural progenitor cell, NPC）克隆株开展等位基因特异性RNA测序（RNA-seq），以解析不同克隆株中各类基因的RME发生范围。值得注意的是，在297个常染色体RME基因中，Pvt1——一种研究较为充分的致癌性长链非编码RNA——在F1杂交神经前体细胞中呈单等位基因表达，且对卡斯蒂（Castaneous）等位基因存在偏向性遗传倾斜。在无遗传差异的环境中，通过基因编辑进行等位基因追踪的实验显示，Pvt1呈现平衡型单等位基因表达，且该状态可在体细胞世代间稳定维持。Pvt1的单等位基因表达由处于待激活状态的Pvt1启动子介导，该启动子携带有二价活性组蛋白修饰H3K4me3与H3K27ac，同时还存在沉默型组蛋白修饰H3K4me3与H3K27me3，这表明其可根据细胞环境切换转录状态。依托本研究获得的大规模RNA测序数据集与遗传倾斜现象，我们根据各克隆株的Pvt1等位基因状态开展差异基因表达分析，发现转录因子Tfap2a在Pvt1启动子区域存在差异结合，这为RME基因的起始与等位基因选择提供了一种潜在机制。此外，本研究证实，单等位基因表达的Pvt1可提升其自身表达水平，相较于双等位基因表达的Pvt1克隆株，此类细胞具有生长优势。上述研究结果为遗传差异如何倾斜随机过程提供了实例——该过程可引发表观遗传现象，并在早期发育阶段产生表型效应。整体实验设计：对由雄性F1杂交（129 × Castaneous）小鼠胚胎干细胞（mouse embryonic stem cell, mESC）分化得到的神经前体细胞克隆株开展亚硫酸氢盐测序（Bisulfite sequencing）。