Single-molecule systems for decoding combinatorial chromatin modifications

项目摘要： 基因组DNA被包装到染色质中的方式调节基因活性，这一过程被称为 “后生学”。表观遗传控制在许多（如果不是大多数）人类疾病中被破坏。此外，新兴 “表观遗传疗法”可能会纠正表观遗传缺陷。然而，目前的研究工具 染色质和表观遗传机制是不精确的，阻碍了理解基因和 基因组调控 拟议的项目将开发新的工具，用于绘制染色质和转录因子的相互作用， 单分子精确度，从而为功能基因组学建立全新的能力。染色质 由DNA包裹的组蛋白组成。组蛋白经历许多化学修饰（乙酰化， 甲基化，磷酸化），其功能仍然知之甚少。在一项原理验证研究中，我们 在表面上捕获单个染色质分子（核小体）， 荧光标记抗体和全内反射荧光（TIRF）显微镜，以及 测序了相关的DNA基于这些令人鼓舞的结果，我们现在建议建立一个强大的 用于同时检测数百个组蛋白和DNA修饰的实验系统 数百万个核小体。然后，我们将调整这些系统，用于分析罕见的细胞类型和单个 细胞，以及用于表征组合转录因子-调节元件相互作用。 总之，我们提出了研究染色质和转录因子组合的创新系统 具有单分子精确度和全基因组覆盖的相互作用。成功实施这一 这项技术将改变我们研究染色质结构的能力，并加快确定染色质结构的进展。 控制我们基因组健康和疾病的序列和结构。

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.