Calibrated ChIP-seq: determining local histone modification density genome-wide

校准 ChIP-seq：确定全基因组局部组蛋白修饰密度

• 批准号: 8571936
• 负责人: Alexander Jackson Ruthenburg
• 金额: $ 23.14万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-22 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8571936
• 关键词: Accounting; Address; Affinity; Antibodies; Biology; Calibration; Cancer Etiology; Cell physiology; ChIP-seq; Chemicals; Chromatin; Chromatin Structure; Coupled; DNA; DNA Library; Data; Data Set; Deposition; Development; Epigenetic Process; Epitopes; Gene Silencing; Generations; Genome; Genomics; Histones; Immunoprecipitation; Libraries; Ligation; Measurement; Measures; Methodology; Methods; Modification; Molecular; Nuclear; Nucleosome Core Particle; Nucleosomes; Patients; Pattern; Positioning Attribute; Post-Translational Protein Processing; Process; Proteins; Protocols documentation; Reagent; Recombinants; Relative (related person); Research; Resolution; Role; Sampling; Scheme; Series; Source; Specificity; Stem Cell Development; Stem cells; Techniques; Technology; Time; Transcriptional Activation; Variant; Work; cell type; chromatin immunoprecipitation; comparative; deep sequencing; density; diabetic; epigenome; experience; genome-wide; glucose tolerance; histone modification; insight; leukemia; member; new technology; novel; novel strategies; public health relevance; reconstitution; research study

DESCRIPTION (provided by applicant): Post-translational modifications on the histone constituents of nucleosomes are able to transduce changes in local chromatin states that govern the accessibility of underlying DNA, regulating processes that range from transcriptional activation to gene silencing. Yet with present technology, it is impossible to measure the absolute densities of histone modifications in a locus specific manner. Despite serving as the central experimental technique in epigenetics research, chromatin immunoprecipitation coupled to deep sequencing (ChIP-seq) suffers from a number of serious drawbacks: 1.) it is a relative measurement unthered to any external scale in a way that obviates comparison amongst experiments; and 2.) it employs antibody reagents that have differing specificity and affinity for epitopes, which are in turn variable in abundance, yet none of these factors are taken into account in present analysis. Consequently, the peaks of different histone modifications that seem to overlap on certain genomic loci cannot be meaningfully compared. To address these substantial problems, I propose a novel approach to calibrate ChIP-seq data using a panel of nucleosomes derived from recombinant and semisynthetic sources as internal standards (calChIP-seq). To that end, nucleosomes bearing a given mark will be reconstituted with a library of DNAs composed of a constant strong nucleosome positioning sequence that is flanked by a variable "barcode" that represents each member¿s molar concentration, then spiked into the input of a native ChIP-seq experiment. After immunoprecipitation with modification-specific antibodies followed by sequencing, the tag counts resulting from the exogenous semisynthetic nucleosome DNA series will serve as an internal-standard calibration curve for absolute quantification of mark density with the positional accuracy of ChIP-seq in a genome-wide data set. This basic scheme will be employed in a number of variations to calibrate ChIP-seq in a proof of concept form and critically examine several troublesome sources of experimental error in ChIP measurements. This proposal is centered on developing the calChIP-seq technology, although a number of potential applications that could substantially contribute to understanding how the epigenome contributes to the control of genomic information are presented. The ability to make comparisons of histone modification density on an absolute scale by calChIP-seq will be transformative for our understanding of chromatin states and enable for the first time crucial comparisons between one modification to another, one cell type to another, and from patient to another. I am in a unique position to accomplish this radical and desperately needed improvement to our field¿s most important technology in that I have both expertise in making semisynthetic chromatin and experience with ChIP-sequencing experiments.

描述（由申请人提供）：核小体组蛋白成分的翻译后修饰能够转导局部染色质状态的变化，从而控制潜在DNA的可及性，调节从转录激活到基因沉默的过程。然而，以目前的技术，不可能以特定位点的方式测量组蛋白修饰的绝对密度。尽管作为表观遗传学研究的中心实验技术，染色质免疫沉淀耦合深度测序（ChIP-seq）存在许多严重的缺点：1.它是一种相对的测量方法，不受任何外部尺度的影响，在某种程度上避免了实验之间的比较；2)它使用的抗体试剂对表位具有不同的特异性和亲和力，而表位的丰度又不同，但在本分析中没有考虑这些因素。因此，似乎重叠在某些基因组位点上的不同组蛋白修饰的峰值不能进行有意义的比较。为了解决这些实质性问题，我提出了一种新的方法来校准ChIP-seq数据，使用一组来自重组和半合成来源的核小体作为内部标准（calChIP-seq）。为此，带有给定标记的核小体将用一个由恒定的强核小体定位序列组成的dna文库进行重组，该序列的两侧是一个可变的“条形码”，代表每个成员的摩尔浓度，然后将其添加到本地ChIP-seq实验的输入中。经过修饰特异性抗体免疫沉淀和测序后，外源半合成核小体DNA序列产生的标记计数将作为内标校准曲线，用于标记密度的绝对定量，并具有ChIP-seq在全基因组数据集中的定位精度。这个基本方案将在许多变化中使用，以概念证明的形式校准ChIP-seq，并严格检查ChIP测量中几个麻烦的实验误差来源。本提案的核心是开发calChIP-seq技术，尽管许多潜在的应用可能有助于理解表观基因组如何有助于控制基因组信息。通过calChIP-seq在绝对尺度上比较组蛋白修饰密度的能力将改变我们对染色质状态的理解，并首次实现一种修饰与另一种修饰之间、一种细胞类型与另一种细胞类型之间以及患者与另一种细胞类型之间的关键比较。我处于一个独特的位置，以完成这一激进的和迫切需要的改进，我们的领域最重要的技术，因为我有专业知识，使半合成染色质和经验与芯片测序实验。