Assessing parent-of-origin transcriptional effects in Xenopus laevis

描述(由申请人提供)：父母起源效应是表观遗传现象，表现为杂合子之间的表型差异，取决于起源的等位父母。基因组印迹是导致母本起源效应的主要表观遗传现象。有些印迹基因只在母本中表达，而另一些则在父本中表达。印记基因在许多复杂的特征中扮演着重要的角色，特别是在早期发育中。尽管基因印迹的分类分布仍然不确定，但一个被广泛接受的观点认为，在动物中，它仅限于哺乳动物，而且大多数研究都是在小鼠模型上进行的。在评估更广泛种类的物种中的印迹方面存在重大的技术挑战。一个主要的限制因素是缺乏多个在核苷酸水平上存在显著差异的自交系，因为 鉴定印迹的最直接方法是在这些品系之间产生倒数杂交。此外，对等位基因特异性表达的转录组范围调查直到最近才变得可行，用于分析此类数据的分析和统计工具仍处于初级阶段。我们已经开创了几种分析等位基因特异性表达的技术。在与非洲爪蛙生物学专家的合作下，我们对非洲爪蛙近交系之间的混合杂交进行了初步的RNA测序实验，这些实验强烈表明非洲爪蛙中存在表现出亲本来源印记的基因。这个项目的目标是仔细和彻底地评估这一引人注目的假设。为了做到这一点，我们将执行一种“放大测序”的方法，它包括一个“宽而浅”轮，我们筛选候选基因，另一个“深而窄”轮，我们详细地关注一组候选基因的等位基因偏向。父母的识别 两栖动物的起源印记将代表着在理解这种表观遗传机制的进化起源及其在发育中的作用方面的重大概念进步。此外，非洲爪哇长期以来一直是脊椎动物分子、细胞和发育生物学体内研究的重要工具。这是唯一的脊椎动物模型系统，允许高通量的在体基因功能和生化分析。非洲爪哇作为印迹研究的模型，将允许开发屏幕，以确定对疾病重要的新靶点，并设计涉及印记基因的新疗法。

DESCRIPTION (provided by applicant): Parent-of-origin effects are epigenetic phenomena that appear as phenotypic differences between heterozygotes depending on the allelic parent of origin. The primary epigenetic phenomenon that can lead to the parent-of-origin effects is genomic imprinting. Some imprinted loci are expressed from the maternal copy only, while others from the paternal copy. Imprinted genes play an important part in a number of complex traits, notably in early development. Though the taxonomic distribution of gene imprinting remains uncertain, a widely-accepted view holds that among animals it is limited to mammals, and most studies had been performed in mouse models. There are significant technical challenges in assessment of imprinting in a wider variety of species. A major limiting factor is lack of multiple inbred lines with significant nucleotide-level differences between them, since the most direct way to identify imprinting involves generation of reciprocal crosses between such lines. In addition, transcriptome-wide surveys of allele-specific expression have only recently become feasible, and the analytical and statistical tools for analysis of such data are still in thir infancy. We have pioneered several technologies for the analysis of allele-specific expression. In collaboration with experts in the biology of the African clawed frog, Xenopus laevis, we have performed preliminary RNA sequencing experiments on mixed crosses between inbred lines that strongly suggest that there are genes in X. laevis that exhibit parent-of-origin imprinting. The goal of this project is to carefully and thoroughly assess this striking hypothesis. In order t do that, we will execute a "zoom in sequencing" approach which consists of a "wide but shallow" round where we screen for candidate genes, complemented by a "deep but narrow" round where we focus in detail on the allelic bias of a set of candidate genes. Identification of parent of-origin imprinting in an amphibian would represent a significant conceptual advance in understanding the evolutionary origins of that epigenetic mechanism and its role in development. Moreover, Xenopus has long been an important tool for in vivo studies in molecular, cell, and developmental biology of vertebrate animals. It is the only vertebrate model system that allows for high-throughput in vivo analyses of gene function and biochemistry. Xenopus as a model for the study of imprinting would allow for the development of screens to identify new targets important for disease and for the design of new therapies involving imprinted genes.