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Dissecting the molecular mechanisms of lncRNA function in X chromosome inactivation across mammalian gestation evolution

剖析哺乳动物妊娠进化中 X 染色体失活中 lncRNA 功能的分子机制

基本信息

批准号：
1941968
负责人：
金额：
--
依托单位：
University of Leeds
依托单位国家：
英国
项目类别：
Studentship
财政年份：
2017
资助国家：
英国
起止时间：
2017 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=studentship-1941968
关键词：
Dissecting molecular mechanisms lncRNA function

项目摘要

Background:In eutherian mammals, X-chromosome inactivation (XCI) is required in females to ensure equal transcription levels of most X-linked genes for both males and females. In female (XX) preimplantation embryos, both X-chromosomes are transcriptionally active from embryonic genome activation until a long non-coding RNA (X-inactive specific transcript: XIST) mediates the inactivation of one. Weknow that species with different types of placental development have substantial diversity in the timing and regulation of XCI initiation but we do not know how this process works. Objectives:1-To determine the RNA, DNA and protein components that XIST interacts with in embryos from different placental phenotypes.2-To integrate interaction data and extract the mechanism/s of function of XIST as it relates to placental evolution.3-To identify evolutionary conserved and divergent molecular mechanisms in the XIST functional complex across different placental and embryo phenotypes. Novelty:This cutting edge project combines reproductive biology, RNA biology and computational evolutionary biology to address the fundamental questions of how the lncRNA XIST regulates XCI and the evolution of the mechanism across different placental types. Timeliness:This will be the first time the functional evolution of lncRNAs will be addressed in vivo. The in vivo pull-down methods are at the cutting edge of transcriptomic and proteomic technologies. Our recentlyfounded LeedsOmics will provide the bioinformatics training required. Experimental Approaches:Embryos from mouse, bovine, sheep and pig (i.e. different placenta types) will be cultured with sex-sorted semen where possible. Lysates will be generated from the female embryos and then XIST RNA will be pulled-down with antisense biotinylated oligos (in vivo). The components of these XIST complexes will be identified by nano-LC MS/MS (proteins), RNA-seq (RNA) and ChIP-Seq(DNA). Resulting NGS data will be analysed computationally and will be compared to high quality mammal genomes to identify regions of conservation and variation.

背景：在真兽目哺乳动物中，雌性需要X染色体失活（XCI）以确保雄性和雌性大多数X连锁基因的转录水平相等。在雌性（XX）植入前胚胎中，两条X染色体从胚胎基因组激活开始都是转录活性的，直到一条长的非编码RNA（X失活特异性转录本：XIST）介导其中一条失活。我们知道不同类型胎盘发育的物种在XCI启动的时间和调节方面有很大的差异，但我们不知道这个过程是如何工作的。目的：1-确定XIST在不同胎盘表型胚胎中相互作用的RNA，DNA和蛋白质组分。2-整合相互作用数据并提取XIST功能的机制，因为它与胎盘进化有关。3-确定XIST功能复合体中不同胎盘和胚胎表型的进化保守和不同的分子机制。新奇：这个前沿项目结合了生殖生物学，RNA生物学和计算进化生物学，以解决lncRNA XIST如何调节XCI以及不同胎盘类型机制进化的基本问题。时间：这将是第一次在体内解决lncRNA的功能进化。体内下拉方法是转录组学和蛋白质组学技术的前沿。我们最近成立的LeedsOmics将提供所需的生物信息学培训。实验方法：在可能的情况下，将小鼠、牛、绵羊和猪（即不同胎盘类型）的胚胎与性别分选的精液一起培养。将从雌性胚胎中产生裂解物，然后用反义生物素化寡核苷酸（体内）拉下XIST RNA。将通过nano-LC MS/MS（蛋白质）、RNA-seq（RNA）和ChIP-Seq（DNA）鉴定这些XIST复合物的组分。将对所得NGS数据进行计算分析，并将其与高质量的哺乳动物基因组进行比较，以确定保守和变异区域。