Absolute nucleosome occupancy map for the Saccharomyces cerevisiae genome [ODM-seq]

Mapping of nucleosomes, the basic DNA packaging unit in eukaryotes, is fundamental for understanding genome regulation as nucleosomes modulate DNA access by their positioning along the genome. A cell population nucleosome map requires two observables: nucleosome positions along the DNA (“Where?”) and nucleosome occupancies across the population (“In how many cells?”). All available genome-wide nucleosome mapping techniques are yield methods as they score either nucleosomal (e.g., MNase-seq, chemical cleavage-seq) or non-nucleosomal (e.g., ATAC-seq) DNA but lose track of the total DNA population for each genomic region. Therefore, they only provide nucleosome positions and maybe compare relative occupancies between positions but cannot measure absolute nucleosome occupancy, which is the fraction of all DNA molecules occupied at a given position and time by a nucleosome. Here, we established two orthogonal and thereby crossvalidating approaches to measure absolute nucleosome occupancy across the Saccharomyces cerevisiae genome via restriction enzymes and DNA methyltransferases. The resulting high-resolution (9 bp) map shows uniform absolute occupancies. Most nucleosome positions are occupied in most cells: 97% of all nucleosomes called by chemical cleavage-seq have a mean absolute occupancy of 90 ± 6% (± SD). Depending on nucleosome position calling procedures, there are 57-60,000 nucleosomes per yeast cell. The few low absolute occupancy nucleosomes do not correlate with highly transcribed gene bodies, but with increased presence of the nucleosome-evicting RSC chromatin remodeling complex there and are enriched upstream of highly transcribed or regulated genes. Our work provides a quantitative method and reference frame in absolute terms for future chromatin studies. Overall design: Chromatin occupancy measurements by ODM-seq (Occupancy via DNA methylation) and ORE-seq (Occupancy via Restriction Enzymes) of several S. cerevisiae chromatin replicates

核小体（nucleosome）是真核生物（eukaryotes）中DNA的基本包装单元，其基因组定位图谱是解析基因组调控机制的核心基础——核小体通过沿基因组的位置排布，调控DNA的可及性。细胞群体水平的核小体图谱需要两类观测指标：其一为核小体在DNA链上的位置（即“定位何处”），其二为群体中的核小体占据率（即“在多少细胞中存在”）。

目前所有全基因组范围的核小体定位技术均属于产率型检测方法：这类技术仅能定量核小体结合的DNA（如微球菌核酸酶测序（MNase-seq）、化学切割测序（chemical cleavage-seq））或非核小体结合的DNA（如转座酶可及性测序（ATAC-seq）），但无法追踪每个基因组区域的总DNA群体总量。因此，这类技术仅能获取核小体位置信息，或可比较不同位置间的相对占据率，但无法测定绝对核小体占据率——绝对占据率指的是在特定位置与时间点上，被核小体占据的DNA分子占总DNA分子的比例。

本研究建立了两种正交且可相互验证的方法，通过限制性内切酶与DNA甲基转移酶，实现对酿酒酵母（Saccharomyces cerevisiae）全基因组范围内绝对核小体占据率的定量测定。所得的高分辨率（9 bp）图谱显示绝对占据率整体分布均一。绝大多数核小体位置在多数细胞中均被占据：通过化学切割测序（chemical cleavage-seq）鉴定的所有核小体中，97%的平均绝对占据率为90% ± 6%（±标准差，SD）。根据核小体位置鉴定流程的不同，每个酿酒酵母细胞中约存在57000~60000个核小体。

少数绝对占据率较低的核小体，并未与高转录活性的基因本体区域相关联，反而与该处核小体驱逐型染色质重塑复合物RSC的丰度升高相关，且在高转录活性或受调控基因的上游区域富集。本研究为未来的染色质研究提供了一套定量方法与绝对定量参考框架。

实验整体设计：对多组酿酒酵母染色质重复样本，分别通过ODM-seq（通过DNA甲基化测定占据率，Occupancy via DNA methylation）与ORE-seq（通过限制性内切酶测定占据率，Occupancy via Restriction Enzymes）开展染色质占据率检测。