Kim Samuel H, Marinov Georgi K, Greenleaf William J
Cancer Biology Programs, School of Medicine, Stanford University, Stanford, California 94305, USA.
Department of Genetics, School of Medicine, Stanford University, Stanford, California 94305, USA;
Genome Res. 2025 Jan 22;35(1):124-134. doi: 10.1101/gr.279621.124.
Gene regulation in most eukaryotes involves two fundamental processes: alterations in genome packaging by nucleosomes, with active -regulatory elements (CREs) generally characterized by open-chromatin configuration, and transcriptional activation. Mapping these physical properties and biochemical activities, through profiling chromatin accessibility and active transcription, is a key tool for understanding the logic and mechanisms of transcription and its regulation. However, the relationship between these two states has not been accessible to simultaneous measurement. To this end, we developed KAS-ATAC, a combination of the kethoxal-assisted ssDNA sequencing (KAS-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) methods for mapping single-stranded DNA (and thus active transcription) and chromatin accessibility, respectively, enabling the genome-wide identification of DNA fragments that are simultaneously accessible and contain ssDNA. We use KAS-ATAC to evaluate levels of active transcription over different CRE classes, to estimate absolute levels of transcribed accessible DNA over CREs, to map nucleosomal configurations associated with RNA polymerase activities, and to assess transcription factor association with transcribed DNA through transcription factor binding site (TFBS) footprinting. We observe lower levels of transcription over distal enhancers compared with promoters and distinct nucleosomal configurations around transcription initiation sites associated with active transcription. We find that most TFs associate equally with transcribed and nontranscribed DNA, but a few factors specifically do not exhibit footprints over ssDNA-containing fragments. We anticipate KAS-ATAC to continue to derive useful insights into chromatin organization and transcriptional regulation in other contexts in the future.
核小体对基因组包装的改变，其中活性调控元件（CRE）通常以开放染色质构型为特征，以及转录激活。通过分析染色质可及性和活性转录来绘制这些物理特性和生化活性图谱，是理解转录及其调控的逻辑和机制的关键工具。然而，这两种状态之间的关系一直无法同时测量。为此，我们开发了KAS-ATAC，它结合了乙二醛辅助单链DNA测序（KAS-seq）和转座酶可及染色质测序分析（ATAC-seq）方法，分别用于绘制单链DNA（从而活性转录）和染色质可及性图谱，能够在全基因组范围内鉴定同时可及且包含单链DNA的DNA片段。我们使用KAS-ATAC来评估不同CRE类别上的活性转录水平，估计CRE上转录可及DNA的绝对水平，绘制与RNA聚合酶活性相关的核小体构型图谱，并通过转录因子结合位点（TFBS）足迹分析来评估转录因子与转录DNA的关联。我们观察到，与启动子相比，远端增强子上的转录水平较低，并且在与活性转录相关的转录起始位点周围存在不同的核小体构型。我们发现，大多数转录因子与转录和未转录的DNA结合程度相同，但有一些因子在含单链DNA的片段上没有特异性足迹。我们预计KAS-ATAC未来将继续在其他背景下对染色质组织和转录调控产生有用的见解。
