Absolute nucleosome occupancy map for the Saccharomyces cerevisiae genome [ODM-nanopore-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）是真核生物中DNA包装的基本单位，其全基因组定位图谱是解析基因组调控机制的核心基础——核小体通过在基因组上的位置排布调控DNA的可及性。细胞群体的核小体图谱需要两类核心观测指标：DNA链上的核小体位置（“定位何处？”）以及群体水平的核小体占据率（“在多少细胞中存在？”）。当前所有已报道的全基因组核小体定位技术均属于产率型方法：这类技术仅能检测核小体结合的DNA（如MNase-seq、化学切割测序（chemical cleavage-seq））或非核小体结合的DNA（如ATAC-seq），却无法获取每个基因组区域内的总DNA总量。因此，这类方法仅能提供核小体位置信息，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——即特定基因组位置与时间点下，被核小体占据的DNA分子占该区域总DNA分子的比例。本研究建立了两种正交且可相互验证的实验方法，通过限制性内切酶与DNA甲基转移酶，实现了酿酒酵母（Saccharomyces cerevisiae）全基因组范围内的绝对核小体占据率测定。所获得的高分辨率（9 bp）图谱显示出均一的绝对占据率特征。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）鉴定的全部核小体中，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差异，每个酵母细胞中约存在57000~60000个核小体。少数绝对占据率较低的核小体并不与高转录基因体存在关联，而是与该处驱逐核小体的RSC染色质重塑复合物（RSC chromatin remodeling complex）的丰度升高显著相关，且在高转录或受调控基因的上游区域富集。本研究提供了一种定量检测方法与绝对维度的参考框架，可为未来染色质相关研究提供重要支撑。实验整体设计：通过ODM-seq（基于DNA甲基化的占据率测定，Occupancy via DNA methylation）与ORE-seq（基于限制性内切酶的占据率测定，Occupancy via Restriction Enzymes）对多组酿酒酵母染色质重复样本进行染色质占据率测定。