Reconstruction of 3D Genome Architecture from Chromatin Conformation Capture Data

描述(申请人提供)：人们普遍认为真核细胞染色质的三维(3D)结构在核和细胞功能中起着关键作用。的确， 这种关系构成了ENCODE联盟刚刚发表的(09/06/12)《自然》系列论文的关键“线索”之一。然而，直到几年前，即使是在中等分辨率下观察/推断3D结构也是有问题的，部分原因是基因组高度浓缩。最近设计的分子技术正在改变这种情况。特别是，新的基因组规模分析的发展，如染色质构象捕获(CCC)，使染色质“接触”的激发成为可能。这些技术已经以前所未有的分辨率提供了对染色质组织的洞察，并允许探索这种组织对各种生物的下游影响。值得注意的是，基因调控和致癌基因融合被认为受到3D组织的强烈影响。因此，获得基因组结构的高分辨率3D重建是一项引人注目的生物学探索。然而，大多数CCC数据的分析都集中在一维(1D)接触水平上，而针对评估3D重建的准确性和可重复性以及利用这种结构来分析随后的生物过程的工作略少。这项建议要解决的最重要的假设是，染色质接触数据可以可靠地用于确定基因组的3D结构，并评估下游与生物功能的关系。为了验证这一假设，我们将开发新的和改进现有的重建算法，并将对它们的性能和操作特征进行系统评估。唯一开发的基因组规模的算法使用约束优化，由于高维的原因，计算负担很大，并且可能陷入局部最优。因此，我们将研究替代算法。我们的初步工作表明，在数据和约束输入的合理扰动下，重复性很差。我们将制定衡量标准 来量化3D协议。虽然可以使用计算和统计工具评估扰动下的重复性，但评估准确性需要使用有针对性的验证性测试，正如我们的湿实验室合作者将部署的那样。此外，我们将应用我们的3D重建纲要来探索与功能元件接近有关的一系列生物学问题。

DESCRIPTION (provided by applicant): It is widely recognized that the three dimensional (3D) architecture of eukaryotic chromatin plays critical roles in nuclear and cellular function. Indeed, such relationships constitute one of the key "threads" of the just published (09/06/12) suite of Nature papers from the ENCODE consortium. However, until a few years ago, observing / inferring, 3D structure at even modest resolutions was problematic, in part because genomes are highly condensed. Recently devised molecular techniques are changing this situation. In particular, the development of novel genome-scale assays, such as chromatin conformation capture (CCC), has enabled elicitation of chromatin "contacts". These techniques have already provided insight into chromatin organization at unprecedented resolutions, and permitted exploration of the downstream influence of such organization on a variety of biological. Notably, gene regulation and cancer-driving gene fusions are believed to be strongly influenced by 3D organization. Accordingly, obtaining high resolution 3D reconstructions of genome architecture is a compelling biological quest. However, most analysis of CCC data has focused on the one dimensional (1D) contact level, with appreciably less effort directed toward evaluating accuracy and reproducibility of 3D reconstructions, and deploying such structures to analyze consequent biological processes. The overarching hypothesis to be addressed in this proposal is that chromatin contact data can be reliably used to determine 3D structures of genomes and to assess downstream relationships with biological function. To test this hypothesis we will develop new, and refine existing, reconstruction algorithms, and will undertake a systematic evaluation of their performance and operating characteristics. The only genome-scale algorithms developed employ constrained optimization which, on account of high-dimensionality, is computationally burdensome and can be trapped in local optima. Accordingly, we will investigate alternate algorithms. Our preliminary work indicates that reproducibility under plausible perturbations to data and constraint inputs is qualitatively poor. We will devise metrics to quantify 3D agreement. While reproducibility under perturbations can be assessed using computational and statistical tools, appraising accuracy requires using targeted confirmatory assays, as will be deployed by our wet-lab collaborators. Further, we will apply our compendium of 3D reconstructions to explore a series of biologic questions relating to proximities of functional elements.