Heterogeneous single-molecule packaging of human mitochondrial nucleoids

During eukaryotic transcription, RNA polymerases must initiate and pause within a crowded, complex environment, surrounded by nucleosomes and other transcriptional activity. This environment creates a spatial arrangement along individual chromatin fibers ripe for both competition and coordination, yet these relationships remain largely unknown owing to the inherent limitations of traditional structural and sequencing methodologies. To address these limitations, we employed long-read chromatin fiber sequencing (Fiber-seq) to visualize RNA polymerases within their native chromatin context at single-molecule and near single-nucleotide resolution along up to 30 kb fibers. We demonstrate that Fiber-seq enables the identification of single-molecule RNA Polymerase (Pol) II and III transcription associated footprints, which, in aggregate, mirror bulk short-read sequencing-based measurements of transcription. We show that Pol II pausing destabilizes downstream nucleosomes, with frequently paused genes maintaining a short-term memory of these destabilized nucleosomes. Furthermore, we demonstrate pervasive direct coordination and anti-coordination between nearby Pol II genes, Pol III genes, transcribed enhancers, and insulator elements. This coordination is largely limited to spatially organized elements within 5 kb of each other, implicating short-range chromatin environments as a predominant determinant of coordinated polymerase initiation. Overall, transcription initiation reshapes surrounding nucleosome architecture and coordinates nearby transcriptional machinery along individual chromatin fibers. Overall design: PacBio sequencing of exogenously methylated genomic DNA from Drosophila S2 cells

在真核生物转录过程中，RNA聚合酶需在拥挤且复杂的染色质环境中起始转录并发生暂停，其周围环绕着核小体与其他转录活性复合物。该环境沿单条染色质纤维形成了兼具竞争与协同潜力的空间排布，但受传统结构生物学与测序方法的固有局限性限制，这类相互关系目前仍在很大程度上未被探明。为克服上述局限，本研究采用长读长染色质纤维测序（Fiber-seq）技术，在单分子及近单核苷酸分辨率下，对长达30 kb的染色质纤维进行成像，以观测天然染色质环境中的RNA聚合酶。本研究证实，Fiber-seq可识别单分子水平下与RNA聚合酶II（Pol II）、III（Pol III）转录相关的足迹信号，整体而言，该信号与基于批量短读长测序的转录水平检测结果高度相符。研究发现，Pol II的暂停会使下游核小体稳定性降低，而频繁发生暂停的基因会对这类稳定性降低的核小体保留“短期记忆”。此外，本研究还证实，邻近的Pol II基因、Pol III基因、转录增强子与绝缘子元件之间普遍存在直接的协同与拮抗调控关系。这类调控关系大多局限于彼此间距在5 kb以内的空间排布元件，表明短程染色质环境是调控聚合酶起始转录的主要决定因素。综上，转录起始会重塑周边核小体结构，并沿单条染色质纤维协调邻近的转录复合物。实验整体设计：对果蝇S2细胞的外源性甲基化基因组DNA进行PacBio测序。