Integrative Single-Cell Analysis of Gene Expression and Chromatin Accessibility Reveals CTCF as a Key Regulator of Stem Cell Differentiation and Lung Morphogenesis [multi-omics]

The lungs develop into an intricate tree-like structure through proximal-distal patterning and branching morphogenesis. However, the gene regulatory programs that dictate embryonic lung development remain largely unclear. Here, we profile gene expression and chromatin accessibility in mouse embryonic lungs to generate a multi-omics atlas at single-cell resolution. By integrative analyses, we dissect 10 cell type-specific gene expression signatures and characterize177,990 cis-regulatory elements (CREs), 36,114 differential accessible chromatin regions (DARs), 27,628 gene-to-peak linkages, 632 highly regulated genes (HRGs), and their binding transcription factors (TFs). We also explore candidate regulators driving lung progenitor developmental trajectory and discover the important role of the AP-1 complex in mesenchymal differentiation. Based on this multi-modal dataset, we delineate gene regulatory networks (GRNs) across various cell types. Finally, using the Ctcf conditional knockout mouse model combined with multiple approaches, we reveal CTCF as a crucial regulator of Sox2+ progenitor specification and lung branching morphogenesis by reprogramming transcriptome and chromatin accessibility. Therefore, this study provides multi-omics resources and mechanistic insights for transcription regulation of lung morphogenesis. To comprehensively study the heterogeneity and regulatory mechanisms of lung morphogenesis, we performed multi-omics profiling of nuclei extracted from embryonic day 12.5 (E12.5) lungs using 10x Genomics paired snRNA-seq and snATAC-seq techniques.肺通过近端-远端模式化（proximal-distal patterning）与分支形态发生（branching morphogenesis）过程发育为复杂的树状结构。然而，调控胚胎肺发育的基因调控程序目前仍未被完全阐明。本研究对小鼠胚胎肺的基因表达与染色质可及性进行分析，构建了单细胞分辨率下的多组学图谱。通过整合分析，本研究解析了10种细胞类型特异性基因表达特征，并鉴定出177990个顺式调控元件（cis-regulatory elements, CREs）、36114个差异开放染色质区域（differential accessible chromatin regions, DARs）、27628个基因-峰关联、632个高度调控基因（highly regulated genes, HRGs）及其结合的转录因子（transcription factors, TFs）。本研究还筛选了驱动肺祖细胞发育轨迹的候选调控因子，并揭示了AP-1复合物在间充质分化中的关键作用。基于该多模态数据集，本研究绘制了不同细胞类型中的基因调控网络（gene regulatory networks, GRNs）。最后，本研究利用Ctcf条件性敲除小鼠模型结合多种实验手段，发现CTCF通过重编程转录组与染色质可及性，作为关键调控因子参与祖细胞特化与肺分支形态发生过程。综上，本研究为肺形态发生的转录调控提供了多组学资源与机制解析视角。为全面解析肺形态发生的异质性与调控机制，本研究采用10x Genomics的单细胞核RNA测序（snRNA-seq）与单细胞核ATAC测序（snATAC-seq）双技术联用方案，对胚胎第12.5天（E12.5）的肺细胞核进行了多组学表征分析。