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

背景：在真兽类动物中，雌性需要X染色体失活(XCI)，以确保大多数X-连锁基因在雄性和雌性中转录水平相等。在雌性(XX)植入前胚胎中，从胚胎基因组激活开始，两条X染色体在转录上都是活跃的，直到一个长的非编码RNA(X非活性特定转录本：Xist)介导其中一条染色体的失活。我们知道，不同类型胎盘发育的物种在XCI启动的时间和调控方面有很大的多样性，但我们不知道这一过程是如何工作的。目的：1-确定XIST与不同胎盘表型胚胎中的RNADNA和蛋白质组分相互作用。2-整合相互作用数据，提取XIST与胎盘进化相关的功能机制/S。3-确定XIST功能复合体在不同胎盘和胚胎表型中的进化保守和差异的分子机制。新颖性：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如何调控XCI以及不同胎盘类型之间机制的进化这一基本问题。及时性：这将是第一次在体内解决lncRNAs的功能进化问题。体内下拉方法处于转录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：将来自小鼠、牛、羊和猪(即不同胎盘类型)的胚胎与可能的性别分类的精液进行培养。裂解产物将从雌性胚胎中产生，然后XIST RNA将被反义生物素化寡核苷酸(在体内)拉下。这些XIST络合物的组成将通过纳米LC MS/MS(蛋白质)、RNA-Seq(RNA)和ChIP-Seq(DNA)进行鉴定。得到的NGS数据将被计算分析，并将与高质量的哺乳动物基因组进行比较，以确定保护和变异的区域。

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.