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

背景：在真兽哺乳动物中，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）着床前胚胎中，两条x染色体在胚胎基因组激活时都具有转录活性，直到一条长非编码RNA （X-inactive specific transcript: XIST）介导其中一条染色体的失活。我们知道，具有不同胎盘发育类型的物种在XCI启动的时间和调节方面具有实质性的差异，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与之相互作用的RNA、DNA和蛋白质组分。2 .整合互作数据，提取XIST与胎盘进化相关的功能机制。鉴定不同胎盘和胚胎表型的XIST功能复合体的进化保守和分化分子机制。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如何调节XCI以及不同胎盘类型机制进化的基本问题。及时性：这将是首次在体内研究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：来自小鼠、牛、羊和猪的胚胎（即不同的胎盘类型）将在可能的情况下用性别分类的精液进行培养。裂解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拉低（体内）。这些XIST复合物的成分将通过纳米lc MS/MS（蛋白质），RNA-seq （RNA）和ChIP-Seq（DNA）进行鉴定。由此产生的NGS数据将进行计算分析，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.