Role of regulatory sequence at the H19 imprinting control region

描述(由申请人提供)： 亲本依赖或“印记”基因表达缺陷与许多人类疾病有关，包括Beckwith-Wiedemann综合征(BWS)、Prader-Willi综合征、Angelman综合征和几种癌症。这项研究的目的是检验在BWS患者中观察到的H19和IGF2印迹丢失(LOI)的遗传和表观遗传学机制。这些患者的LOI归因于H19和Igf2印迹控制区(ICR)的微缺失。先前的研究表明，ICR上存在的绝缘蛋白CTCF的结合位点是印迹控制所必需的。此外，H19/lgf2位点高阶染色质的形成与印迹控制有关。这项工作将解决CTCF点的间距和图案在印记中的作用。为了实现所提出的目标，将产生在H19/lgf2 ICR上携带以下微缺失的小鼠：(I)CTCF2和3之间的0.8kb，以及1和4之间的1.3kb。这些突变对H19和Igf2印记的影响将通过等位基因特异性分析来确定，以测试H19和Igf2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分析来确定这些突变是否干扰CTCF结合或翻译后组蛋白修饰。这些突变是否会影响H19/lgf2基因座高阶染色质结构的形成，将通过染色体构象捕获(3C)分析进行测试。由于ICR上存在的CTCF位点的数量和间距在小鼠和人类之间不同，因此有必要建立一个模型来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(Hlc1)代替小鼠ICR的小鼠，并测试hlc1是否能调节小鼠H19和Igf2的印迹。最后，最近的一项研究表明，H19/lgf2 ICR是转录的。在这里，将确定ICR转录的水平和模式，以及它与H19和Igf2印记表达的水平和模式的关系。此外，将通过研究条件性CTCF基因敲除卵母细胞中的ICR转录来研究CTCF在ICR转录中的作用。关于表观遗传学和人类疾病之间的联系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，其潜在的机制仍然知之甚少。这里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。

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.