Single-molecule systems for decoding combinatorial chromatin modifications

项目总结： 基因活性受基因组DNA被包装到染色质中的方式的调节--这个过程被称为 “表观遗传学”。表观遗传控制在许多人类疾病中被破坏，如果不是大多数的话。此外，新兴的 “表观遗传疗法”有可能纠正表观遗传缺陷。然而，目前用于研究的工具 染色质和表观遗传机制是不精确的，阻碍了对基因和 基因组调控。 拟议的项目将开发新的工具来绘制染色质和转录因子与 单分子精确度，从而为功能基因组学建立了全新的能力。染色质是 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)，其功能仍然知之甚少。在一项原则证明研究中，我们 捕获表面上的单个染色质分子(核小体)，用 荧光标记抗体和全内反射荧光(TIRF)显微镜 对相关的DNA进行了测序。基于这些令人鼓舞的结果，我们现在建议建立健全的 同时检测数百个组蛋白和DNA修饰的实验系统 数以百万计的单个核小体。然后我们将调整这些系统来分析稀有细胞类型和单个 细胞，并用于表征组合转录因子-调节元件的相互作用。 总之，我们提出了研究组合染色质和转录因子的创新系统 与单分子精确度和全基因组覆盖的相互作用。这一点的成功实施 技术将改变我们研究染色质结构的能力，并加快定义染色质结构的进展 在健康和疾病中控制我们基因组的序列和结构。

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.