Probing the dynamics of chromosome organization in single cells

项目摘要 哺乳动物细胞将两米长的线性DNA包装成一个直径平均只有几厘米的细胞核。 微米这种包装必须以这样一种方式完成，即DNA的转录，复制， 并且修复可以忠实地发生。“Hi-C”技术的发展使得能够绘制 在大细胞群体中，染色体-染色体在全基因组范围内的相互作用频率。你好-- C生成了详细的数据集，揭示了关键的组织特征，包括 相互作用频率升高，基因组广泛分为活性和非活性 “隔间。这些特征的破坏与癌症和发育疾病有关。然而，在这方面， 部分由于技术上的限制，关于核爆炸的原因和后果， 3D基因组组织，特别是在单细胞中。例如，目前尚不完全清楚， 域实际上是在单个细胞中形成的，或者相反，它代表了许多单个状态的平均值， 因为单细胞Hi-C方法提供了非常稀疏的数据集， 基因组组织的大部分特征是不可见的。此外，这些材料的物理性质 基因组特征尚未在单细胞中进行检查，目前还不知道这些区域是否产生了 不同的扩散环境，可以影响反式作用因子的活动。最后，发现 影响核结构的其他蛋白质受到高财政和资源成本的限制 Hi-C的。我们以前的工作建立了一个强大的新平台，用于研究3D基因组组织， 单细胞显微镜下。我们介绍了一种可编程的荧光原位杂交（FISH） 一种使用生物信息学设计的寡核苷酸探针组来创建复杂杂交的方法 固定样本中的模式。我们利用这项技术开创了单分子超分子的应用， 分辨率显微镜研究染色体结构的纳米级，介绍了一种技术， 视觉上区分同源染色体，也已经开发出一种方法，方便 固定细胞和组织中荧光信号的多重放大。我们将建立在这个基础上， 通过开发和应用单细胞成像技术和利用先进的光学 直接解决这些未解决的问题。具体而言，我们将引入一个广泛的， 单细胞成像策略能够以前所未有的方式解决3D基因组组织的特征 详细使用多重FISH（目标1）并研究3D组织的生物物理特性 活细胞成像（目标2）。我们将通过高通量、无偏见的 筛选方法以鉴定3D基因组结构的新调节子（目标3）。我们详细的 核结构的表征及其生物物理后果也将提供一个增强的 这是一个理解3D基因组组织的改变如何导致人类疾病的框架。

Project Summary Mammalian cells package two meters of linear DNA into a nucleus whose diameter is on average only a few microns. This packaging must be done in such a way that DNA transactions such as transcription, replication, and repair can faithfully occur. The development of the `Hi-C' technique has enabled the mapping of chromosome-chromosome interaction frequency on the genome-wide scale in large populations of cells. Hi- C has generated detailed datasets that have revealed key organizational features, including `domains' of elevated interaction frequency and the broad partitioning of the genome into active and inactive `compartments.' Disruption of these features is associated with cancer and developmental disease. However, in part due to technical limitations, pressing open questions remain about the causes and consequences of 3D genome organization, particularly in single cells. For instance, it is not fully clear whether features such as domains actually form in individual cells, or instead represent the averaging of many individual states that are present in the population of cells profiled because single-cell Hi-C methods provide very sparse datasets in which most features of genome organization remain invisible. Furthermore, the physical properties of these genomic features have yet to be examined in single cells, and it is not currently known if these regions create distinct diffusive environments that can impact the activity of trans-acting factors. Finally, the discovery of additional proteins that influence nuclear architecture has been limited by the high financial and resource cost of Hi-C. Our previous work established a powerful new platform for investigating 3D genome organization in single cells using microscopy. We introduced a programmable fluorescent in situ hybridization (FISH) approach that uses sets of bioinformatically designed oligonucleotide probes to create complex hybridization patterns in fixed samples. We have harnessed this technology to pioneer the use of single-molecule super- resolution microscopy to study chromosome structure on the nanoscale, introduced a technique capable of visually distinguishing homologous chromosomes, and also have developed an approach that facilitates the multiplexed amplification of fluorescent signals in fixed cells and tissues. We will build upon this foundational work by developing and applying single-cell imaging technologies and leveraging advanced optical approaches to directly address these unsolved questions. Specifically, we will introduce a broadly enabling single-cell imaging strategy capable of resolving features of 3D genome organization with unprecedented detail using multiplexed FISH (Objective 1) and investigate the biophysical properties of 3D organizational features with live-cell imaging (Objective 2). We will augment these efforts with high-throughput, unbiased screening approaches to identify new regulators of 3D genome structure (Objective 3). Our detailed characterization of nuclear architecture and its biophysical consequences will also provide an enhanced framework for understanding how alterations in 3D genome organization can lead to human disease.