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（由申请人证明）：在这里，对核小体的组蛋白征服进行了翻译，这是染色质的，这些染色质是控制国际辅助性的。组蛋白以特异性方式进行了IQUE II。在实验中的比较。为了添加实质性的项目，我提出了一种新颖的方法，以校准芯片序列，使用一组核小体从辅助和半合成来源作为内部支架（calchip-seq），核体将带有一个给定的标记。由由核小体定位隔离的DNA组成的，该核小体定位隔离了，其侧面是一个可变的“条形码”，该核能抑制了每个成员S摩尔浓度，然后是跨跨天然CHIP-seq实验。在整个基因组数据集中的芯片序列。表观基因组有助于控制基因组信息。，从病人到另一个。这是最重要的技术，因为我俩都有在制作半合成染色质和芯片序列实验的经验方面具有专业知识。