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

描述(申请人提供)：胎盘是一种特殊的和基本的结构，在真核哺乳动物中调节母亲和胎儿之间的气体交换和营养物质和废物的运输。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迹的特殊形式的基因调控对胎盘功能至关重要。基因组印迹导致一个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度，而基因印迹基因的失调与胎盘生长异常和胎盘组织血管形成的变化有关。胎盘中的基因组印记与另一个有趣的表观遗传过程--印记X染色体失活--平行。印记X失活导致XX雌性早期胚胎所有细胞中父系遗传的X染色体优先表观遗传沉默，以使X连锁基因的剂量与XY雄性相同。虽然已经描述了许多基因组印记的基因座，但一个等位基因如何以亲本特有的方式优先表达仍然知之甚少。印记X失活和印记常染色体基因表达都与长非编码RNA(LncRNAs)有关。LncRNAs是一种非蛋白质编码的转录本，被认为是将染色质修饰复合体招募到特定的基因组区域，促进转录或导致发育过程中的基因沉默。印记基因的表达依赖于亲本种系中不同的表观遗传标记，然后在后代中表现为一个或另一个亲本等位基因的表达。LncRNA的表达可以作为这种不同的表观遗传种系标记的工具，以建立胚胎中亲本特有的基因表达模式。我的工作假设是，在胚胎发生过程中，未被发现的lncRNA会在胎盘中触发区域性的、双亲起源的特定表观遗传学沉默。在这里，我建议利用一种新的链和等位基因特异的RNA测序方法和我设计的计算管道来识别新的亲本特异的lncRNA。我将利用滋养层干细胞作为RNA的来源。这些细胞经历印记X失活，并在常染色体上表现出强大的印记基因表达，并代表了形成胎盘的干细胞群体的体外模型。通过这些实验，我们希望在胎盘中识别基因组印记的启动子，并为进一步了解亲本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.