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

基本信息

批准号：
8724544
负责人：
Alexander Jackson Ruthenburg
金额：
$ 19.75万
依托单位：
UNIVERSITY OF CHICAGO
依托单位国家：
美国
项目类别：
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/8724544
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

项目摘要

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，从而控制底层 DNA 的可及性，调节从转录激活到基因沉默的过程。尽管染色质免疫沉淀是表观遗传学研究的核心实验技术，但它无法以位点特异性方式测量组蛋白修饰的绝对密度。测序 (ChIP-seq) 存在许多严重的缺点：1.) 它是一种不受任何外部尺度影响的相对测量，从而避免了比较实验；2.) 它使用的抗体试剂具有不同的特异性和亲和力。表位的丰度又存在差异，但在目前的分析中没有考虑到这些因素，因此无法对某些基因组位点上重叠的不同组蛋白修饰的峰值进行有意义的比较。针对这些重大问题，我提出了一种使用重组和半合成来源的一组核小体作为内标来校准 ChIP-seq 数据的新方法 (calChIP-seq) 为此，将使用文库重建带有给定标记的核小体。 DNA由恒定的强核小体定位序列组成，其两侧是代表每个成员的可变“条形码”？ s 摩尔浓度，然后加入天然 ChIP-seq 实验的输入中，然后用修饰特异性抗体进行免疫沉淀，然后进行测序，由外源半合成核小体 DNA 系列产生的标签计数将作为绝对值的内标校准曲线。在全基因组数据集中利用 ChIP-seq 的位置精度对标记密度进行量化该基本方案将用于许多变体中，以在概念验证中校准 ChIP-seq。尽管提出了许多可能有助于理解表观基因组如何有助于基因组信息控制的潜在应用，但该提案的重点是开发 calChIP-seq 技术。通过 calChIP-seq 在绝对范围内比较组蛋白修饰密度的能力将彻底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、一种细胞类型与另一种细胞类型以及患者之间的关键比较。对于另一个人来说，我处于一个独特的位置来完成我们领域急需的彻底改进。这是最重要的技术，因为我既有制作半合成染色质的专业知识，又有 ChIP 测序实验的经验。