Systematic elucidation of allele specific proteome at Imprint Control Regions

印记控制区域等位基因特异性蛋白质组的系统阐明

• 批准号: 10576890
• 负责人: Satya K. Kota
• 金额: $ 35.6万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-09 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10576890
• 关键词: 14q32; Affect; Alleles; Bioinformatics; Biotin; Biotinylation; CRISPR/Cas technology; Cells; Chimeric Proteins; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; DNA Methylation; DNA Sequence; Data; Defect; Development; Disease; Elements; Embryo; Embryonic Development; Endocrine; Enhancers; Epigenetic Process; Evaluation; Gametogenesis; Gene Expression; Gene Expression Profile; Generations; Genes; Genome; Genomic Imprinting; Genotype; Growth; Health; Homeostasis; Human; Human Chromosomes; Hybrids; Individual; Knowledge; Label; Lead; Ligase; Longevity; Maintenance; Mammals; Mass Spectrum Analysis; Maternal uniparental disomy; Methodology; Methylation; Modeling; Molecular; Mus; Names; Nature; Neurologic; Parents; Paternal uniparental disomy; Pathologic; Pattern; Play; Postembryonic; Process; Protein Dynamics; Protein Region; Proteins; Proteome; Proteomics; RNA; Rare Diseases; Regulation; Research; Research Personnel; Research Proposals; Role; Structure; Syndrome; Technology; Testing; Tissues; Training; Transgenic Organisms; Uniparental Disomy; Untranslated RNA; X Inactivation; autosome; design; diagnostic strategy; dosage; embryo tissue; embryonic stem cell; epigenome editing; experience; genome editing; human model; imprint; mammalian genome; maternal imprint; mutant; novel diagnostics; novel therapeutics; paternal imprint; protein complex; recruit; stem cell model

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.

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，起源的父母- 哺乳动物基因组中一小部分基因（<150）的特异性表达。 基因在胚胎发育期间和胚胎发育后是必不可少的， 印记基因导致复杂的罕见表观遗传疾病，通常涉及多个组织， 在印迹结构域内，命名为“印迹障碍”（ID）的DNA序列元件 印记控制区（ICRs）调节基因的印记表达，并且与基因的印记表达有差异。 在配子发生过程中，当母亲和父亲的基因组 仍然在不同的分区。ICRs如何带来印记基因的表达和什么反式作用 需要占据亲本ICR等位基因的蛋白因子和复合物来建立 以及基因表达的印记模式的维持仍然是未知的。的 本研究的主要目的是阐明亲本等位基因的蛋白质组装， 印迹控制区，以了解如何顺式调节反式因子保持父母的， 原始表达式。我们将通过采用相对较新的方法来实现这一点， 基因座特异性基因组靶向和细胞内蛋白质标记。在第一个目标中， 将利用转基因互交杂种ES系来解读母本或父本ICR 等位基因特异性蛋白复合物。在第二个目标，我们将测试如何消耗顺式作用 RNA和ICR增强子功能的相关丧失影响母体细胞上的蛋白复合物 最后，在第三个目标中，我们将集中于定量ES中的分子蛋白质组学变化， 细胞与单亲二体，以了解ICR功能的印记疾病。我们一起努力， 我们的研究将i）阐明亲本ICR等位基因特异性蛋白复合物，ii）定义ICR衍生的 它们的蛋白质复合物定位/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复合物。从这个研究结果 将导致对印迹控制区的反式蛋白因子的更好理解， 将以公正的方式提供ICR功能顺式调节的机制细节。