Differential chromatin binding of the lung lineage transcription factor NKX2-1 resolves opposing murine alveolar cell fates in vivo [scRNA-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)对相同DNA序列的差异化利用，可催生不同的组织谱系，进而在单一代系内生成多种细胞类型；这一表观遗传（epigenetic）过程是祖细胞与干细胞生物学的核心议题。与之相关的基因组变化，尤其是原生体内组织中的此类变化，目前仍未得到充分阐释，本研究以小鼠肺为模型就此展开研究。在该模型中，同一谱系转录因子（transcription factor）NKX2-1可驱动两种截然相反的细胞命运，分别促成肺泡1型（Alveolar Type 1, AT1）与肺泡2型（Alveolar Type 2, AT2）细胞的分化。本研究证实，NKX2-1的细胞类型特异性功能，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（chromatin binding）特性。YAP/TAZ的缺失会将NKX2-1的结合位点从AT1特异性位点重定向至AT2特异性位点：若在祖细胞阶段敲除YAP/TAZ，会生成转录特征过度活化的AT2细胞；若在细胞命运定型后敲除，则会引发AT1细胞向AT2细胞的转分化。Nkx2-1突变的AT1与AT2细胞会获得独特的染色质开放位点，其中包含对应相反细胞命运的位点，同时这些细胞会转向胃肠道细胞命运，这表明表观遗传可塑性的范围远超转录可塑性。本研究结合单细胞或纯化细胞的基因组分析与精准遗传学手段，绘制了肺泡细胞命运与潜能的表观遗传调控图谱，并为解析谱系转录因子的体内功能提供了实验基准。本研究通过10X Genomics的Chromium单细胞基因表达解决方案平台，对经荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）纯化的肺上皮细胞、免疫细胞、内皮细胞与间充质细胞完成了测序处理。