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

描述(申请人提供)：对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，这些变化支配着潜在DNA的可及性，调节从转录激活到基因沉默的过程。然而，在目前的技术下，不可能以特定于基因座的方式测量组蛋白修饰的绝对密度。尽管染色质免疫沉淀与深度测序(CHIP-SEQ)相结合是表观遗传学研究的核心实验技术，但它仍存在一些严重的缺陷。它是一种与任何外部尺度无关的相对测量，避免了实验之间的比较；它使用的抗体试剂对表位具有不同的特异性和亲和力，而表位的丰度又是可变的，但在目前的分析中没有考虑这些因素。因此，不同的组蛋白修饰的峰似乎重叠在特定的基因组座位上，不能进行有意义的比较。为了解决这些实质性的问题，我提出了一种新的方法来校准芯片序列数据，使用来自重组和半合成来源的一组核小体作为内部标准(calChIP-seq)。为此，带有给定标记的核小体将被重组为一个DNA文库，该文库由一个恒定的强核小体定位序列组成，两侧是代表每个成员S摩尔浓度的可变“条形码”，然后被插入到本地CHIP-SEQ实验的输入中。在用修饰特异性抗体进行免疫沉淀和测序后，外源半合成核小体DNA系列产生的标签计数将作为内部标准校准曲线，用于绝对量化标记密度，并在全基因组数据集中保持CHIP-SEQ的位置准确性。这一基本方案将用于多种变型中，以概念证明的形式校准芯片序列，并严格检查芯片测量中几个令人烦恼的实验误差来源。这项建议的中心是开发calChIP-seq技术，尽管提出了一些潜在的应用，这些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，并首次能够在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与另一种细胞类型之间进行关键比较。我处于一个独特的位置来完成这一对我们领域的根本性和迫切需要的改进？S最重要的技术，因为我既有制造半合成染色质的专业知识，也有芯片测序实验的经验。

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.