Establishment and maintenance of repressed chromatin

抑制染色质的建立和维持

• 批准号: 7215569
• 负责人: ERIC E BOUHASSIRA
• 金额: $ 35.98万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-05-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7215569
• 关键词: Acute Erythroblastic Leukemia; Binding Sites; Cells; Chimeric Proteins; Chromatin; Chromatin Structure; Complex; Condition; DNA Methylation; Development; Disease; Doctor of Philosophy; Enterobacteria phage P1 Cre recombinase; Epigenetic Process; Erythroid Cells; Gene Expression; Gene Silencing; Generations; Genes; Genetic; Genetic Transcription; Genome; Globin; Goals; Hemoglobinopathies; Heritability; Histone Code; Histones; Human; In Vitro; Internet; Lac Repressors; Lentivirus Vector; Location; Maintenance; Measures; Mediating; Methods; Methylation; Modification; Mus; Mutate; Nuclear; Nucleotides; Pathway interactions; Patients; Play; Post-Translational Protein Processing; Primer Extension; RNA Interference; Reverse Transcriptase Polymerase Chain Reaction; Role; Site; Small Interfering RNA; System; Tars; Technology; Testing; Today; Transcript; Transgenes; Vision; base; bisulfite; chromatin immunoprecipitation; design; gene therapy; human embryonic stem cell; insight; mouse model; recombinase; transcription factor

DESCRIPTION (provided by applicant): Epigenetic control of chromatin structure has emerged as a critical layer in the complex web of interactions that regulates gene expression. Factors such as DNA methylation and histone post-translational modifications are believed to limit access of transcription factors and others effectors to their binding sites and thus, play a central role in the overall functional organization of the genome. The overall goal of this project is to further our understanding of how epigenetic information is established and maintained on globin transgenes. The significance of this goal is double. Firstly, dissecting these mechanisms will provide us with new insights into how the globin genes are regulated in their native locus. Secondly, understanding transgene silencing will help us design better gene therapy cassettes. Our experimental system will be recombinase-mediated cassette exchange (RMCE) and other Cre-based technology in mouse erythroleukemia cells and in human erythroid cells derived from human ES cells. In Aim 1, we will study the establishment of silent chromatin and try to answer the question "Do transcriptional interferences cause silencing?" We will use various methods to block transcriptional interference and the RNAi pathway. In Aim 2, we will focus on the maintenance of silent chromatin and on the question "Does DNA methylation induce a specific histone code?" We will characterize heritable and non-heritable chromatin structures using bisulfite sequencing, CHIP assays, RNAi anc targeted chromatin modifications with the help of Lac repressor fusion proteins. In Aim 3, we will extend these studies to silencing of human globin transgenes in human primary erythroid cells produced in vitro by differentiation of human ES cells

描述（由申请人提供）：染色质结构的表观遗传控制已成为调节基因表达的复杂相互作用的临界层。据信，诸如DNA甲基化和组蛋白后修饰等因素可以限制转录因子和其他效应子对其结合位点的访问，因此在基因组的整体功能组织中起着核心作用。该项目的总体目标是进一步了解我们如何在Globin转基因上建立和维护表观遗传信息。这个目标的意义是双重的。首先，剖析这些机制将为我们提供新的见解，了解如何在其本地基因座中调节球蛋白基因。其次，了解转基因沉默将有助于我们设计更好的基因治疗盒。 我们的实验系统将是重组酶介导的盒式盒式交换（RMCE）和其他基于CRE的技术在小鼠红血症细胞中以及来自人类ES细胞的人红细胞细胞中。 在AIM 1中，我们将研究静音染色质的建立，并尝试回答“转录干扰是否会导致沉默？”的问题。我们将使用各种方法来阻止转录干扰和RNAi途径。在AIM 2中，我们将重点关注静音染色质的维持，以及“ DNA甲基化会诱导特定组蛋白代码？”的问题。我们将使用Bisulfite测序，芯片测定法表征可遗传和非可遗产的染色质结构，RNAi ANC借助LAC抑制剂融合蛋白​​靶向染色质修饰。在AIM 3中，我们将这些研究扩展到通过分化人类ES细胞在体外产生的人类原发性红细胞细胞中人类球蛋白转基因的沉默