Distance-Hi-C: Creating Photo Activated X-linkers To Define Nuclear Architecture

Distance-Hi-C：创建光激活 X 链接器来定义核架构

• 批准号: 9144434
• 负责人: JOHN T LIS
• 金额: $ 71.51万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-15 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9144434
• 关键词: 3-Dimensional; Animals; Architecture; Binding; Binding Sites; Biology; Biotin; Cell Line; Cell Nucleus; Cells; Chromatin; Chromatin Loop; Chromosomes; Code; Complex; Cross-Linking Reagents; Crosslinker; DNA; DNA crosslink; Data; Development; Digoxigenin; Disease; Distal; Drosophila genome; Drosophila genus; Elements; Enhancers; Ficusin; Fixatives; Formaldehyde; Gene Activation; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Genome; Genomics; Goals; Health; Heat-Shock Response; Histones; Homeostasis; Human; Human Cell Line; In Vitro; Intercalating Agents; K562 Cells; Lasers; Lead; Length; Life; Ligation; Malignant Neoplasms; Measures; Mechanics; Methodology; Methods; Modeling; Molecular Conformation; Names; Nature; Nuclear; Organism; Process; Proteins; Protocols documentation; Psoralens; RNA Polymerase II; Reagent; Regulator Genes; Regulatory Element; Resolution; Sampling; Signal Transduction; Site; Specificity; System; Technology; Testing; Time; Transcriptional Activation; Transgenes; Transgenic Organisms; Validation; base; cost; crosslink; design; ds-DNA; effective therapy; flexibility; genetic information; genome-wide; insight; nano; promoter; protein crosslink; research study; transcription factor; two-photon

 DESCRIPTION (provided by applicant): Nuclear organization of DNA is complex and consists of multiple layers. At the lowest resolution, large sections of chromosomes are packed in territories, at a higher level chromosomal regions are organized in topologically-associated domains that provide a framework for interactions of transcription factors (TFs) bound to promoters and distal regulatory elements (enhancers), and finally the structural interplay of regulators, RNA polymerase II, and chromatin then lead to regulated gene expression. In recent years, a number of methods called chromatin (conformation) capture (CC) have been developed and used to capture dynamic and stable DNA contacts that constitute genome architecture and potential regulatory interactions. A major limitation of these methods is that they all depend on a single crosslinker, formaldehyde, which crosslinks DNA to proteins as well as proteins to other proteins. This complicates the interpretation of the observed 'DNA-DNA' contacts and it lacks distance information. Here we propose an orthogonal strategy, named Distance-Hi-C (D-Hi-C), where we design, test, and apply a battery of photo-activated crosslinkers, designed to directly measure distances between interacting sites genome-wide. These bivalent crosslinkers consist of two reactive groups separated by a linker. DNA intercalaters that can be crosslinked by photo-activation (i.e. Psoralen) will be used as the reactive groups. Linkers will be of varying precise lengths, and either flexible or rigid in nature so that the 3-dimensional distance between crosslinked loci can be inferred. Such DNA-specific bivalent crosslinking reagents, when substituted for formaldehyde in Hi-C protocol, produces space constraints revealing enhancer- promoter interactions and potentially allowing the inference of the 3D arrangement of the nuclear genome with unprecedented precision. Moreover, D-Hi-C is expected to lower backgrounds and allow examination of short and moderate range interactions, which are obscured by high backgrounds of current methods. Addition of groups such as digoxigenin to the bivalent crosslinkers, in addition to biotin incorporated to the ligation products, will enable better purification and thus deeper examination of genomic interactions. Finally, sampling DNA distances in time following gene activation provides a means of exploring the 4D architecture and setting critical limits in evaluating mechanisms of gene activation. The specific aims are to 1) synthesize a battery of bivalent crosslinkers and evaluate their ability to crosslink DNA in vitro; 2) test D-Hi-C crosslinkers relative to formaldehyde in the Drosophila nuclei model; 3) apply new crosslinkers to tier 1, ENCODE GM12878 and K562 cell lines to test their efficacy in human cells; and 4) test locus-specific photo-crosslinking that will allow a more focused and thorough examination of locus-specific interactions with the genome in a time course of gene activation. The development of these methodologies will be broadly useful in providing critical insights into gene regulatory mechanisms that are operative in normal animal development and homeostasis and that go awry in diseases like cancer.

 描述（由适用提供）：DNA的核组织很复杂，由多层组成。 At the lowest resolution, large sections of chromosomes are packed in territory, at a higher level chromosomes are organized in topologically-associated domains that provide a framework for interactions of transcription factors (TFs) bound to promoters and discal regulatory elements (enhancers), and finally the structural interplay of regulators, RNA polymerase II, and chromatin then lead to regulated gene expression.近年来，已经开发了许多称为染色质（构象）捕获（CC）的方法，并用于捕获构成基因组结构和潜在调节相互作用的动态和稳定的DNA接触。这些方法的主要局限性是它们都取决于单个交联甲醛，该交联甲醛将DNA交联至蛋白质以及蛋白质与其他蛋白质。这使观察到的“ DNA-DNA”接触的解释变得复杂，并且缺乏距离信息。在这里，我们提出了一种正交策略，称为Dance-Hi-C（D-Hi-C），在该策略中，我们设计，测试和应用一系列光激活的交联链，旨在直接测量整个基因组相互作用位点之间的距离。这些二价交联器由两个被接头分隔的反应组组成。可以通过光激活（即牛cor）交联的DNA插入量将用作反应性基团。链接器的精度长度不同，并且本质上的柔性或刚性，因此可以推断出交联局部之间的3维距离。这种DNA特异性的二价交联试剂在HI-C方案中取代甲醛时会产生空间约束，从而揭示了增强子 - 启动子相互作用，并有可能允许用前所未有的精度推断核基因组的3D排列。此外，D-HI-C有望降低背景，并允许检查短和中等范围的相互作用，这些相互作用被当前方法的高背景所掩盖。除了与结扎产物合并的生物素外，在二价交联中添加诸如二氧基素的组还可以更好地纯化，从而更深入地检查基因组相互作用。最后，在基因激活后的时间中对DNA距离进行采样提供了一种探索4D体系结构并在评估基因激活机制时设定关键限制的方法。具体目的是1）合成一组二价交联链，并评估其体外交联DNA的能力； 2）果蝇核模型中相对于甲醛的测试D-HI-C交联剂； 3）将新的交联器应用于第1层，编码GM12878和K562细胞系以测试其在人类细胞中的效率； 4）测试基因座特异性的照片连接，将在基因激活的时间过程中更加集中和彻底检查与基因组特异性相互作用。这些方法的开发将在对正常动物发育和稳态中运行的基因调节机制的关键见解方面广泛有用，并且在癌症等疾病中出现了问题。