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（由适用提供）：核产品组蛋白一致的翻译后修改能够翻译局部染色质状态的变化，这些变化控制着基本DNA的可及性，从转录激活到基因沉默的调节过程。然而，借助当前的技术，不可能以当地特定方式测量组蛋白修饰的绝对密度。尽管是表观遗传学研究中的主要实验技术，但染色质免疫沉淀与深层测序（CHIP-SEQ）相连，但它具有许多严重的缺点：1。1.），这是一个相对测量，以任何外部尺度未切换到任何外部尺度，以相比进行比较。 2.）它采用具有区分特异性和对表位的亲和力的抗体试剂，而这些试剂反过来又是抽象变量的，但是在当前分析中，这些因素均未考虑到这些因素。因此，似乎与某些基因组基因座重叠的不同组蛋白修饰的峰不能有意义地比较。为了解决这些重大问题，我提出了一种新的方法，使用源自重组和半合成源作为内部标准品（Calchip-Seq）的核小体校准芯片序列数据。为此，带有给定标记的核小体将与由恒定强的核小体定位序列组成的DNA库重组，该核心体位于恒定的强核小体定位序列，该序列侧面是代表每个成员摩尔浓度的可变“条形码”，然后将其刺激到天然ChIP-Seq-seq实验的输入中。在具有修饰特异性抗体然后进行测序的免疫沉淀后，由外源性半同步核小体DNA系列产生的TAG计数将用作内部标准校准曲线，以绝对量化标记的标记密度，并在基因组全基因组数据集中的Chip-Seq的位置准确性。该基本方案将采用多种变化来以概念形式的证明进行校准，并严格检查芯片测量中实验错误的几个麻烦来源。该提案集中在开发Calchip-seq技术上，尽管许多潜在的应用可能有助于了解表观基因组如何介绍基因组信息的控制。 Calchip-seq在绝对尺度上比较组蛋白修饰密度的能力将是我们对染色质状态的理解，并首次启用一种与另一种细胞类型的一种修饰，从患者到另一种细胞类型之间的关键比较。我处于一个独特的位置，可以实现这一激进而迫切需要改进我们领域的最重要技术，因为我既有在制造半合成染色质的专业知识，又具有芯片序列实验的经验。