Role of regulatory sequence at the H19 imprinting control region

H19 印记控制区调控序列的作用

• 批准号: 7689758
• 负责人: Folami Y Ideraabdullah
• 金额: $ 5.01万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7689758
• 关键词: Address; Affect; Alleles; Angelman Syndrome; Assisted Reproductive Technology; Beckwith-Wiedemann Syndrome; Binding; Binding Sites; Biological Assay; CCCTC-binding factor; Characteristics; Child; Chromatin; Chromosomes; Complex; CpG dinucleotide; Defect; Development; Disease; Epigenetic Process; Fetal Liver; Functional RNA; Gene Expression; Genes; Genetic; Genetic Transcription; Genome; Genomic Imprinting; Genomics; Goals; H19 gene; Hepatocyte; Higher Order Chromatin Structure; Human; Incidence; Individual; Insulin-Like Growth Factor II; Introns; Knock-out; Knockout Mice; Lead; Link; Maintenance; Mammalian Cell; Mammals; Mediating; Methylation; Modeling; Molecular Conformation; Mus; Mutation; Oocytes; Parents; Patients; Pattern; Phenotype; Physiological; Play; Prader-Willi Syndrome; Precipitation; Regulatory Element; Research; Role; Site; Staging; Study models; Testing; Transcript; Transcription Initiation Site; Transcriptional Regulation; Uniparental Disomy; Work; cancer type; histone modification; human disease; imprint; microdeletion; mouse model; next generation; promoter; transcription factor

DESCRIPTION (provided by applicant): Defects in parent of origin dependent, or "imprinted", gene expression are associated with a number of human diseases including Beckwith-Wiedemann Syndrome (BWS), Prader-Willi Syndrome, Angelman Syndrome and several types of cancer. The purpose of this study is to examine the genetic and epigenetic mechanisms underlying loss of imprinting (LOI) at H19 and IGF2 that has been observed in patients with BWS. LOI in these patients was attributed to microdeletions in the imprinting control region (ICR) of H19 and Igf2. Previous studies have shown that binding sites for the insulator protein CTCF present at the ICR are necessary for imprinting control. Furthermore, the formation of higher-order chromatin at the H19/lgf2 locus has been implicated in imprinting control. This work will address the role of the spacing and pattern of CTCF sites in imprinting. To achieve the proposed aims, mice will be generated carrying the following microdeletions at the H19/lgf2 ICR: (i) 0.8 kb between CTCF sites 2 and 3; and 1.3 kb between sites 1 and 4. The effects of these mutations on imprinting at H19 and Igf2 will be determined using allele-specific assays to test expression levels of H19 and Igf2 and methylation at the ICR and H19 promoter. It will also be determined whether these mutations interfere with CTCF binding or posttranslational histone modifications by allele-specific chromatin immuno-precipitation assays. Whether the mutations affect the formation of a higher-order chromatin structure at the H19/lgf2 locus will be tested by chromosome conformation capture (3C) assays. As the number and spacing of CTCF sites present at the ICR differs between mice and humans, it is necessary to generate a model to directly test the effect of mutations observed in patients. Therefore, mice that carry the human ICR (hlC1) in place of the mouse ICR will be generated and tested for whether hlC1 can regulate imprinting of mouse H19 and Igf2. Finally, a recent study revealed that the H19/lgf2 ICR is transcribed. Here, the level and pattern of ICR transcription will be determined and how it relates to the level and pattern of imprinted expression at H19 and Igf2. Moreover, the role of CTCF in ICR transcription will be investigated by studying ICR transcription in conditional CTCF knockout oocytes. Research pertaining to the association between epigenetics and human disease is a relatively new and fast growing field. An increasing number of diseases are being attributed to epigenetic defects, however, the underlying mechanism is still poorly understood. The research proposed here will open new doors to our understanding of the genetic and epigenetic mechanisms underlying genomic imprinting and thereby have significant impact on the study and treatment of the associated human diseases.

描述（由申请人提供）： 起源依赖性或“印记”基因表达的亲本缺陷与许多人类疾病相关，包括Beckwith-Wiedemann综合征（BWS）、Prader-Willi综合征、Angelman综合征和几种类型的癌症。本研究的目的是研究在BWS患者中观察到的H19和IGF 2印迹缺失（LOI）的遗传和表观遗传机制。这些患者的LOI归因于H19和Igf 2印迹控制区（ICR）的微缺失。以前的研究表明，在ICR存在的绝缘子蛋白CTCF的结合位点是必要的印迹控制。此外，在H19/lgf 2基因座处的高阶染色质的形成已涉及印迹控制。这项工作将解决的作用，空间和模式的CTCF网站的印记。为了实现所提出的目的，将产生在H19/lgf 2 ICR处携带以下微缺失的小鼠：（i）CTCF位点2和3之间的0.8kb;以及位点1和4之间的1.3kb。这些突变对H19和Igf 2印迹的影响将使用等位基因特异性测定法测定，以检测H19和Igf 2的表达水平以及ICR和H19启动子的甲基化。还将通过等位基因特异性染色质免疫沉淀测定来确定这些突变是否干扰CTCF结合或翻译后组蛋白修饰。将通过染色体构象捕获（3C）试验检测突变是否影响H19/lgf 2基因座处高阶染色质结构的形成。由于小鼠和人类之间ICR处存在的CTCF位点的数量和间隔不同，因此有必要生成模型以直接测试在患者中观察到的突变的影响。因此，将产生携带人ICR（hlCl）代替小鼠ICR的小鼠，并测试hlCl是否可以调节小鼠H19和Igf 2的印迹。最后，最近的一项研究表明，H19/lgf 2 ICR是转录的。在这里，将确定ICR转录的水平和模式以及它如何与H19和Igf 2处的印迹表达的水平和模式相关。此外，CTCF在ICR转录中的作用将通过研究条件性CTCF敲除卵母细胞中的ICR转录来研究。关于表观遗传学和人类疾病之间的关联的研究是一个相对较新和快速增长的领域。越来越多的疾病被归因于表观遗传缺陷，然而，潜在的机制仍然知之甚少。本文提出的研究将为我们理解基因组印记的遗传和表观遗传机制打开新的大门，从而对相关人类疾病的研究和治疗产生重大影响。