Institute of Epigenetics and Stem Cells, Helmholtz Zentrum München, Munich, Germany.
Institute of Functional Epigenetics, Helmholtz Zentrum München, Neuherberg, Germany.
Nucleic Acids Res. 2023 Dec 11;51(22):12303-12324. doi: 10.1093/nar/gkad1022.
Stochastic origin activation gives rise to significant cell-to-cell variability in the pattern of genome replication. The molecular basis for heterogeneity in efficiency and timing of individual origins is a long-standing question. Here, we developed Methylation Accessibility of TArgeted Chromatin domain Sequencing (MATAC-Seq) to determine single-molecule chromatin accessibility of four specific genomic loci. MATAC-Seq relies on preferential modification of accessible DNA by methyltransferases combined with Nanopore-Sequencing for direct readout of methylated DNA-bases. Applying MATAC-Seq to selected early-efficient and late-inefficient yeast replication origins revealed large heterogeneity of chromatin states. Disruption of INO80 or ISW2 chromatin remodeling complexes leads to changes at individual nucleosomal positions that correlate with changes in their replication efficiency. We found a chromatin state with an accessible nucleosome-free region in combination with well-positioned +1 and +2 nucleosomes as a strong predictor for efficient origin activation. Thus, MATAC-Seq identifies the large spectrum of alternative chromatin states that co-exist on a given locus previously masked in population-based experiments and provides a mechanistic basis for origin activation heterogeneity during eukaryotic DNA replication. Consequently, our single-molecule chromatin accessibility assay will be ideal to define single-molecule heterogeneity across many fundamental biological processes such as transcription, replication, or DNA repair in vitro and ex vivo.
随机起始激活导致基因组复制模式在细胞间产生显著的变异性。个体起始效率和时间异质性的分子基础是一个长期存在的问题。在这里，我们开发了靶向染色质结构域测序的甲基化可及性（MATAC-Seq）方法，以确定四个特定基因组位点的单分子染色质可及性。MATAC-Seq 依赖于甲基转移酶对可及 DNA 的优先修饰，结合纳米孔测序，直接读取甲基化的 DNA 碱基。将 MATAC-Seq 应用于选定的早期高效和晚期低效酵母复制起始点，揭示了染色质状态的巨大异质性。INO80 或 ISW2 染色质重塑复合物的破坏导致个别核小体位置发生变化，这与它们的复制效率变化相关。我们发现了一种具有可及核小体自由区域的染色质状态，同时具有定位良好的+1 和+2 核小体，这是有效起始激活的强有力预测因子。因此，MATAC-Seq 确定了在给定基因座上存在的大量替代染色质状态的范围，这些状态在基于群体的实验中以前被掩盖了，为真核 DNA 复制过程中的起始激活异质性提供了机制基础。因此，我们的单分子染色质可及性测定法将非常适合定义许多基本生物学过程（如体外和体内的转录、复制或 DNA 修复）中的单分子异质性。
