Systematic elucidation of allele specific proteome at Imprint Control Regions

项目摘要 基因组印记是一种表观遗传过程，导致单等位基因、起源亲本- 哺乳动物基因组中一小部分基因(&lt；150)的特异性表达。印迹 基因在胚胎发育过程中和胚胎后发育中是必不可少的，并且在剂量不平衡方面存在缺陷 印记基因导致复杂的罕见表观遗传病，通常涉及多个组织， 命名印迹障碍(ID)。在印迹结构域中，DNA序列元素名为 印迹控制区(ICR)调控基因的印迹表达，并具有差异性 以配子发生过程中的DNA甲基化为特征的，此时母体和父体基因组 仍然处于不同的分区。ICR是如何带来印记基因表达的，以及什么是反式作用 需要占据亲本ICR等位基因的蛋白质因子和复合体才能建立 而基因表达印迹模式的维持仍然大部分是未知的。这个 本研究的主要目的是阐明双亲等位基因上的蛋白质组合。 印记控制区，以了解顺式调节如何通过反式因子维持亲本 原点表达式。我们将通过采用相对较新的方法实现这一点 基因定位和细胞内蛋白质标记。在第一个目标中，多重 转基因正反交ES系将被用于破译母本或父本的ICR 等位基因特异性蛋白质复合体。在第二个目标中，我们将测试Cis的耗尽如何起作用 RNAs和相关ICR增强子功能的丧失影响母体上的蛋白质复合体 最后，在第三个目标中，我们将重点定量研究胚胎干细胞的分子蛋白质组变化 具有单亲二体的细胞以了解印迹疾病中的ICR功能。一起， 我们的研究将一)阐明亲本ICR等位基因特异性蛋白复合体，二)定义ICR衍生 蛋白质复合体定位/印迹功能所需的顺式元件和iii)定义 单亲染色体二体中的差异蛋白质复合体。这项研究的发现 将使我们更好地理解印迹控制区和 将以公正的方式提供ICR职能的独联体监管的机械性细节。

Project Summary Genomic imprinting is an epigenetic process resulting in the monoallelic, parent-of-origin- specific expression of a small subset of genes (<150) in the mammalian genome.Imprinted genes are essentialduring and post-embryonic development and defects in dosage imbalance of imprinted genes result in complex rare epigenetic diseases generally involving multiple tissues, named Imprinting Disorders(IDs).Within an imprinted domain, DNA sequence elements named Imprinting Control Regions(ICRs) regulate imprinted expression of genes and are differentially marked by DNA methylation during gametogenesis when maternal and paternal genomes are still in distinct partitions.How ICRs bring about imprinted gene expression and what trans acting protein factors and complexes that occupy parental ICR alleles are required for establishment and maintenance of imprinted patterns of gene expression still remain mostly unknown. The main objective of this proposalis to elucidate the protein assemblies on parental alleles of imprint control regions to understand how cis regulation by trans factors maintain the parent-of- origin expression. We will accomplish this by employing the relatively new methodologies of locus specific genome targeting and intracellular protein labeling. In the first aim, multiple transgenic reciprocal hybrid ES lines will be utilized to decipher the maternal or paternal ICR allele specific protein complexes. In the second aim we will test how depletion of cis acting RNAs and associated loss of ICR enhancer function affects the protein complexes on maternal allele.Finally, in the third aim, we will focus to quantitate the molecular proteomic changes in ES cells with uniparental disomies to understand ICR functions in imprinted disorders. Together, our studies will i) illuminate parental ICR allele specific protein complexes, ii) define ICR derived cis elements required for their protein complex localization/imprinting function and iii) define differential protein complexes in uniparental chromosomal disomies. Findings from this study will lead to a greater understanding of the trans protein factors at Imprint Control Regions and will provide mechanistic details into cis regulation of ICR functions in an unbiased manner.