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状DNA包装成一个平均直径只有几个的细胞核 微米。这种打包必须以这样一种方式进行：DNA交易，例如转录、复制 而修复可以忠实地发生。Hi-C技术的发展使测绘成为可能 在大的细胞群体中，染色体-染色体相互作用在基因组范围内的频率。嗨- C生成了详细的数据集，揭示了关键的组织特征，包括 相互作用频率的提高和基因组分为活跃和不活跃的广泛划分 “车厢。”这些特征的破坏与癌症和发育性疾病有关。然而， 在一定程度上，由于技术上的限制，关于肺炎的原因和后果的迫切问题仍然存在。 3D基因组组织，特别是在单细胞中。例如，目前还不完全清楚诸如 属性域实际上是在单个单元格中形成的，或者表示多个独立状态的平均值 存在于被剖析的细胞群体中，因为单细胞Hi-C方法在 基因组组织的哪些大部分特征仍然是看不见的。此外，这些物质的物理性质 基因组特征尚未在单细胞中进行检查，目前尚不清楚这些区域是否会产生 可以影响反式作用因子活性的独特扩散环境。最后，发现了 影响核结构的额外蛋白质受到了高昂的财务和资源成本的限制 Hi-C。我们以前的工作为研究3D基因组组织建立了一个强大的新平台 用显微镜观察单个细胞。我们介绍了一种可编程的荧光原位杂交(FISH) 一种使用生物信息设计的寡核苷酸探针集来创建复杂杂交的方法 固定样本中的图案。我们已经利用这项技术开创了单分子超级... 分辨率显微镜在纳米尺度上研究染色体结构，引入了一种能够 在视觉上区分同源染色体，并开发了一种方法，使 固定细胞和组织中荧光信号的多路放大。我们将以这一基础为基础 通过开发和应用单细胞成像技术并利用先进的光学 直接解决这些悬而未决的问题的方法。具体地说，我们将引入一个广泛的授权 能够以前所未有的方式解析3D基因组组织特征的单细胞成像策略 使用多路FISH(目标1)进行详细描述，并调查3D组织的生物物理属性 活细胞成像的特征(目标2)。我们将以高吞吐量、不偏不倚的方式加强这些努力 确定3D基因组结构的新调节因子的筛选方法(目标3)。我们的详细信息 对核结构及其生物物理后果的表征也将提供增强的 了解3D基因组组织的变化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.