Multi-scale chromatin footprinting reveals wide-spread alterations to the structure of DNA regulatory elements [SHARE-Seq]

Cis-regulatory elements (CREs) are bound by diverse chromatin-associated proteins, cooperate to establish gene expression, and change in structure over time to alter cell fate and function1–3. Here, we develop a footprinting framework that uses deep learning to correct Tn5 sequence bias, improving accuracy and reducing false positives. This framework additionally identifies the interaction of DNA with objects of various sizes. Using these ‘multi-scale footprints’ we find that transcription factor (TF) binding occurs at well-defined distances from nucleosomes. Further, we demonstrate that multi-scale footprints, which include the positions of nucleosomes, enable more accurate inference of TF binding. Using multi-scale footprinting with single-cell multi-omics, we discover wide-spread structural and functional changes of CREs across hematopoiesis, wherein nucleosomes slide, expose DNA for TF binding, and promote gene expression. Finally, we apply this single-cell and footprint approach to characterize age-associated structural changes to CREs within hematopoietic stem cells and identify a spectrum of cells harboring age-associated changes to the epigenome. Interestingly, we find extensive changes to the structure of CREs upon aging, with many age-associated changes altering CRE structure while preserving overall accessibility. Collectively, we reveal the dynamics and functional importance of CRE structure, highlighting changes to gene regulation at single-cell and single-base-pair resolution. SHARE-Seq of HepG2 cells

顺式调控元件（cis-regulatory elements, CREs）可与多种染色质相关蛋白结合，协同调控基因表达，并随时间改变自身结构以重塑细胞命运与功能[1-3]。本研究构建了一套基于深度学习的足迹分析框架，能够校正Tn5序列偏好性，提升分析精度并降低假阳性结果。该框架还可识别不同尺寸的分子与DNA的互作模式。利用这类“多尺度足迹”，我们发现转录因子（transcription factor, TF）的结合位点与核小体（nucleosomes）之间存在明确的间距规律。进一步研究证实，包含核小体位置信息的多尺度足迹可更精准地推断转录因子结合事件。将多尺度足迹分析与单细胞多组学技术联用，我们揭示了造血过程中顺式调控元件广泛存在的结构与功能变化：核小体发生滑动，暴露DNA区段以允许转录因子结合，进而促进基因表达。最后，我们将该单细胞足迹分析方法应用于造血干细胞内与衰老相关的顺式调控元件结构变化研究，鉴定出一系列携带表观基因组（epigenome）衰老相关改变的细胞亚群。值得注意的是，我们发现衰老过程中顺式调控元件的结构发生了广泛重塑，诸多与衰老相关的改变在维持整体染色质可及性的前提下，仅改变顺式调控元件的局部结构。综上，本研究揭示了顺式调控元件结构的动态变化与功能重要性，以单细胞及单碱基分辨率解析了基因调控的改变过程。针对HepG2细胞的SHARE-Seq数据集