Differential chromatin binding of the lung lineage transcription factor NKX2-1 resolves opposing murine alveolar cell fates in vivo [scATAC-Seq]

Differential use of identical DNA sequences leads to distinct tissue lineages and then multiple cell types within a lineage, an epigenetic process central to progenitor and stem cell biology. The associated genomic changes, especially in native tissues, remain insufficiently understood, and are hereby addressed in the mouse lung, where the same lineage transcription factor NKX2-1 promotes the diametrically opposed alveolar type 1 (AT1) versus AT2 cell fate. We show that the cell-type-specific function of NKX2-1 is attributed to its differential chromatin binding that is acquired or retained during development in coordination with partner transcriptional factors. Loss of YAP/TAZ redirects NKX2-1 from its AT1-specific to AT2-specific binding sites, leading to transcriptionally exaggerated AT2 cells when deleted in progenitors or AT1-to-AT2 conversion when deleted after fate commitment. Nkx2-1 mutant AT1 and AT2 cells gain distinct accessible sites including those of the opposite fate while adopting the gastrointestinal fate, suggesting an epigenetic plasticity larger than a transcriptional one. Our genomic analysis of single or purified cells, coupled with precision genetics, provides an epigenetic roadmap of alveolar cell fate and potential, and introduces an experimental benchmark for unraveling the in vivo function of lineage transcription factors. FACS-purified lung epithelial, immune, endothelial, and mesenchymal cells were processed through the Chromium Single Cell Gene Expression Solution Platform (10X Genomics)

相同DNA序列的差异化使用可催生不同的组织谱系，并在单一谱系内进一步生成多种细胞类型，这一表观遗传过程是祖细胞与干细胞生物学的核心内容。目前针对该过程的相关基因组变化（尤其是在原生组织中）的认知仍存在不足，本研究以小鼠肺脏为模型展开针对性探讨。小鼠肺脏中，同一谱系的转录因子NKX2-1可调控两种截然相反的细胞命运：肺泡1型（alveolar type 1, AT1）与肺泡2型（alveolar type 2, AT2）细胞命运。本研究证实，NKX2-1的细胞类型特异性功能，来源于其在发育过程中与协同转录因子协同作用下获得或保留的差异化染色质结合特性。YAP/TAZ的缺失会将NKX2-1的结合位点从AT1特异性结合位点重定向至AT2特异性结合位点；若在祖细胞阶段敲除YAP/TAZ，会导致转录层面过度活化的AT2细胞；若在细胞命运确定后敲除，则会引发AT1向AT2的细胞转化。Nkx2-1突变的AT1与AT2细胞会获得与自身命运相反的染色质可及位点，同时转向胃肠道细胞命运，这表明表观遗传可塑性远超转录可塑性。本研究通过对单细胞或纯化细胞的基因组分析，结合精准遗传学手段，绘制了肺泡细胞命运与潜能的表观遗传图谱，并为解析谱系特异性转录因子的体内功能提供了实验基准。本研究对经荧光激活细胞分选术（Fluorescence-Activated Cell Sorting, FACS）纯化的肺上皮细胞、免疫细胞、内皮细胞及间充质细胞，采用10X Genomics公司的Chromium单细胞基因表达解决方案平台完成测序处理。