Beyond pairwise DNA contacts: exploring higher-order genome structure using proximity ligation

项目摘要 人类基因组中大约两米长的DNA被复杂地包装成每个细胞核中的染色质和染色体。除了结构性作用外，该组织还具有重要的监管职能。特别是，人类基因组中枢纽的形成在调节不同细胞类型中的基因方面起着至关重要的作用。我们最近展示了使用原位Hi-C方法进行三维基因组测序来创建可靠的环图的能力。Hi-C通过确定全基因组范围内所有基因座对之间的物理接触频率来表征基因组的三维构型。拟议的项目将开发更复杂的技术，不仅可以识别涉及一对基因组位置的环，还可以识别涉及许多相互作用的DNA位置（通常是五个或更多个这样的位置）的枢纽。我们将研究这些枢纽在人类和小鼠胚胎干细胞分化过程中的动态变化。目标1将开发COLA，这是原位Hi-C协议的一种变体，可以在完整的细胞核中同时识别大量位点之间的接触。目标2将开发分裂池条形码，它可以识别无限数量的基因座之间的接触，但需要破坏细胞核。两种方法的结果将被整合并相互比较，在目标3中，将使用显微镜进行独立验证。拟议的项目将推进我们对染色质枢纽的决定因素和功能的理解，并为全面分析任何细胞类型中的高阶基因组结构提供技术框架。所有的方法和数据都将免费和迅速地向科学界公布。

Project Summary The roughly two meters of DNA in the human genome is intricately packaged to form the chromatin and chromosomes in each cell nucleus. In addition to its structural role, this organization has critical regulatory functions. In particular, the formation of hubs in the human genome plays an essential role in regulating genes in different cell types. We recently demonstrated the ability to create reliable maps of loops, using an in situ Hi-C method for three-dimensional genome sequencing. Hi-C characterizes the three-dimensional configuration of the genome by determining the frequency of physical contact between all pairs of loci, genome-wide. The proposed project will develop more sophisticated technologies that can identify not only loops - which involve a pair of genomic positions - but hubs, which can involve many interacting DNA positions (often five or more such positions). We will examine the dynamics of these hubs in the setting of differentiating embryonic stem (ES) cells in humans and mice. Aim 1 will develop COLA, a variant of the in situ Hi-C protocol that can identify contacts between large numbers of loci at once in intact nuclei. Aim 2 will develop split-pool barcoding, which can identify contacts between unlimited numbers of loci, but which requires disruption of the nucleus. The results of both methods will be integrated and compared to one another and, in Aim 3, will be independently validated using microscopy. The proposed project will advance our understanding of the determinants and functions of chromatin hubs, and present a technological framework for comprehensive analysis of higher-order genome structure in any cell type. All methods and data will be freely and rapidly released to the scientific community.