Mechanistic Analysis of Parent-of-Origin-Specific Gene Expression in the Placenta

描述（由申请人提供）：胎盘是真兽目哺乳动物中调节母亲和胎儿之间气体交换以及营养物质和废物运输的专门和必要结构。胎盘中的基因表达受到严格控制，这种微调的基因调控是胎盘正常发育和功能的先决条件。一种被称为基因组印记的特殊形式的基因调控对胎盘功能至关重要。基因组印记导致基因的母系或父系遗传等位基因的优先表达。印迹基因被假设为调节胎儿组织的生长速率，并且基因组印迹位点的失调与异常胎盘生长和胎盘组织血管化的变化有关。胎盘中的基因组印记与另一个有趣的表观遗传过程相似，即印记X染色体失活。印记X失活导致XX女性早期胚胎的所有细胞中父系遗传的遗传X染色体的优先表观遗传沉默，以使X连锁基因的剂量与XY男性的剂量相等。虽然已经描述了许多基因组印记基因座，但一个等位基因以亲本来源特异性方式的优先表达如何发生仍然知之甚少。印迹X失活和印迹常染色体基因表达都与长链非编码RNA（lncRNA）有关。LncRNA是非蛋白质编码转录物，其被假设为将染色质修饰复合物募集到限定的基因组区域，促进转录或导致发育期间的基因沉默。印记基因表达依赖于亲本种系中的差异表观遗传标记，其随后表现为后代中一个或另一个亲本等位基因的表达。LncRNA表达可以作为这种差异表观遗传种系标记的工具，以在胚胎中建立亲本来源特异性基因表达模式。我的工作假设是，未发现的lncRNA触发区域，父母的起源特定的表观遗传沉默在胚胎发育过程中的胎盘。在这里，我建议利用一种新的链和等位基因特异性RNA测序方法和计算管道，我已经设计，以确定新的父母的起源特异性lncRNA。我将使用滋养层干细胞作为RNA的来源。这些细胞经历印记X-失活，并且还在常染色体上显示稳健的印记基因表达，并且代表产生胎盘的干细胞群体的体外模型。通过这些实验，我们希望确定胎盘中基因组印记的启动子，并为进一步了解母体特异性基因调控的分子机制铺平道路。

DESCRIPTION (provided by applicant): The placenta is a specialized and essential structure that regulates gas exchange and the transport of nutrients and waste between the mother and the fetus in eutherian mammals. Gene expression in the placenta is tightly controlled, and this finely-tuned gene regulation is a pre-requisite for proper placental development and function. A specialized form of gene regulation known as genomic imprinting is critical for placental function. Genomic imprinting results in the preferential expression of either the maternally- or paternally-inherited allele of a gene. Imprinted genes are hypothesized to regulate the growth rate of fetal tissues, and dysregulation of genomically imprinted loci has been linked to abnormal placental growth and changes in vascularization of the placental tissues. Genomic imprinting in the placenta parallels another intriguing epigenetic process, imprinted X-chromosome inactivation. Imprinted X-inactivation results in the preferential epigenetic silencing of the paternally-inherited inherited X-chromosome in all cells of the early embryo in XX females to equalize the dosage of X-linked genes to that of XY males. While many genomically imprinted loci have been described, how the preferential expression of one allele in a parent-of-origin-specific manner occurs remains poorly understood. Both imprinted X-inactivation and imprinted autosomal gene expression have been linked to long non-coding RNAs (lncRNAs). LncRNAs are non protein-coding transcripts that are hypothesized to recruit chromatin modifying complexes to defined genomic regions, either facilitating transcription or leading to gene silencing during development. Imprinted gene expression relies on differential epigenetic marking in the parental germlines, which is then manifested in expression of one or the other parental allele in the offspring. LncRNA expression can act as an instrument of this differential epigenetic germline marking to establish parent-of-origin-specific gene expression patterns in the embryo. My working hypothesis is that undiscovered lncRNAs trigger regional, parent-of-origin specific epigenetic silencing in the placenta during embryogenesis. Here, I propose to utilize a novel strand- and allele-specific RNA-sequencing approach and a computational pipeline that I have devised in order to identify novel parent-of-origin-specific lncRNAs. I will ue trophoblast stem cells as a source of the RNA. These cells undergo imprinted X- inactivation and also display robust imprinted gene expression on autosomes, and represent an in vitro model of the stem cell population that gives rise to the placenta. Through these experiments, we hope to identify initiators of genomic imprinting in the placenta, and pave the way for further understanding of the molecular mechanisms underlying parent-of-origin specific gene regulation.