Single-molecule systems for decoding combinatorial chromatin modifications

用于解码组合染色质修饰的单分子系统

• 批准号: 9360134
• 负责人: BRADLEY Evan BERNSTEIN
• 金额: $ 66.31万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-28 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9360134
• 关键词: Acetylation; Address; Alleles; Antibodies; Binding; CRISPR/Cas technology; Cells; Chemicals; Chromatin; Chromatin Structure; Complex; DNA; DNA Methylation; DNA Modification Process; Defect; Dependency; Detection; Disease; Elements; Enhancers; Epigenetic Process; Fluorescence Microscopy; Genes; Genome; Genomic DNA; Genomics; Goals; Health; Histones; Human Genome; Individual; Label; Location; Maps; Methods; Methylation; Modification; Nucleosomes; Phosphorylation; Positioning Attribute; Procedures; Process; Regulation; Regulator Genes; Regulatory Element; Research Personnel; Sampling; Solid; Structure; Surface; System; Systems Analysis; Techniques; Technology; base; cell type; chromatin modification; combinatorial; embryonic stem cell; functional genomics; genome-wide; histone modification; human disease; imaging system; imprint; innovation; new technology; novel; promoter; single molecule; technology validation; tool; transcription factor

Project Summary: Gene activity is modulated by the way genomic DNA is packaged into chromatin – a process termed ‘epigenetics’. Epigenetic controls are disrupted in many, if not most, human diseases. Moreover, emerging ‘epigenetic therapies’ could potentially correct epigenetic defects. However, current tools for studying chromatin and epigenetic mechanisms are imprecise, hindering progress towards understanding gene and genome regulation. The proposed project will develop novel tools for mapping chromatin and transcription factor interactions with single-molecule precision, thereby establishing entirely new capabilities for functional genomics. Chromatin is made up of histones wrapped by DNA. Histones are subject to many chemical modifications (acetylation, methylation, phosphorylation) whose functions remain poorly understood. In a proof-of-principle study, we captured individual chromatin molecules (nucleosomes) on a surface, probed their modifications with fluorescently-labeled antibodies and Total Internal Reflection Fluorescence (TIRF) microscopy, and sequenced the associated DNA. Based on these encouraging results, we now propose to establish robust experimental systems for detecting multiple histone and DNA modifications concurrently on hundreds of millions of individual nucleosomes. We will then adapt these system for analyzing rare cell types and single cells, and for characterizing combinatorial transcription factor – regulatory element interactions. In summary, we propose innovative systems for investigating combinatorial chromatin and transcription factor interactions with single-molecule precision and genome-wide coverage. Successful implementation of this technology would transform our ability to study chromatin structure, and hasten progress towards defining the sequences and structures that control our genome in health and disease.

项目摘要： 基因活性是通过将基因组DNA包装到染色质的方式来调节的。 “表观遗传学”。在许多（即使不是大多数人）的人类疾病中，表观遗传控制被破坏。而且，新兴 “表观遗传疗法”可能会纠正表观遗传缺陷。但是，目前的研究工具 染色质和表观遗传机制是暗示的，阻碍了理解基因和 基因组调节。 拟议的项目将开发用于绘制染色质和转录因子相互作用的新工具 单分子精度，从而为功能基因组学建立了全新的功能。染色质是 由由DNA包裹的组蛋白组成。组蛋白受到许多化学修饰（乙酰化， 甲基化，磷酸化），其功能保持不足。在原理证明的研究中，我们 在表面上捕获的单个染色质分子（核小体），证明了它们的修饰 荧光标记的抗体和总内反射荧光（TIRF）显微镜和 测序相关的DNA。基于这些令人鼓舞的结果，我们现在建议建立强大的 实验系统，用于在数百个上同时检测多个Hisstone和DNA修饰 数百万个单个核小体。然后，我们将适应这些系统以分析稀有细胞类型和单一细胞类型 细胞，并用于表征组合转录因子 - 调节元件相互作用。 总而言之，我们提出了用于研究组合染色质和转录因子的创新系统 与单分子精度和全基因组覆盖率的相互作用。成功实施 技术将改变我们研究染色质结构的能力，并加速进步 控制我们的健康和疾病基因组的序列和结构。