Airway injury induces alveolar epithelial and mesenchymal responses mediated by macrophages.

Acute injury in the airways or the lung activates local progenitors and stimulates changes in cell-cell interactions to restore homeostasis, but it is not appreciated how more distant niches are impacted. We utilized mouse models of airway-specific epithelial injury to examine secondary tissue-wide alveolar, immune, and mesenchymal responses. Single-cell transcriptomics and in vivo validation revealed transient, tissue-wide proliferation of alveolar type 2 (AT2) progenitor cells after club cell-specific ablation. The AT2 cell proliferative response was reliant on alveolar macrophages (AMs) via upregulation of Spp1 which encodes the secreted factor Osteopontin. A previously uncharacterized mesenchymal population we termed Mesenchymal Airway/Adventitial Niche Cell 2 (MANC2) also exhibited dynamic changes in abundance and a pro-fibrotic transcriptional signature after club cell ablation in an AM-dependent manner. Overall, these results demonstrate that acute airway damage can trigger distal lung responses including altered cell-cell interactions that may contribute to potential vulnerabilities for further dysregulation and disease.

Organoid modeling reveals the tumorigenic potential of the alveolar progenitor cell state.

Alveolar type 2 (AT2) cells, the epithelial progenitor cells of the distal lung, are known to be the prominent cell of origin for lung adenocarcinoma. The regulatory programs that control chromatin and gene expression in AT2 cells during the early stages of tumor initiation are not well understood. Here, we dissected the response of AT2 cells to Kras activation and p53 loss (KP) using combined single cell RNA and ATAC sequencing in an established tumor organoid system. Multi-omic analysis showed that KP tumor organoid cells exhibit two major cellular states: one more closely resembling AT2 cells (SPC-high) and another with loss of AT2 identity (hereafter, Hmga2-high). These cell states are characterized by unique transcription factor (TF) networks, with SPC-high states associated with TFs known to regulate AT2 cell fate during development and homeostasis, and distinct TFs associated with the Hmga2-high state. CD44 was identified as a marker of the Hmga2-high state, and was used to separate organoid cultures for functional comparison of these two cell states. Organoid assays and orthotopic transplantation studies indicated that SPC-high cells have higher tumorigenic capacity in the lung microenvironment compared to Hmga2-high cells. These findings highlight the utility of understanding chromatin regulation in the early oncogenic versions of epithelial cells, which may reveal more effective means to intervene the progression of Kras-driven lung cancer.

Binary outcomes of enhancer activity underlie stable random monoallelic expression.

Mitotically stable random monoallelic gene expression (RME) is documented for a small percentage of autosomal genes. We developed an in vivo genetic model to study the role of enhancers in RME using high-resolution single-cell analysis of natural killer (NK) cell receptor gene expression and enhancer deletions in the mouse germline. Enhancers of the RME NK receptor genes were accessible and enriched in H3K27ac on silent and active alleles alike in cells sorted according to allelic expression status, suggesting enhancer activation and gene expression status can be decoupled. In genes with multiple enhancers, enhancer deletion reduced gene expression frequency, in one instance converting the universally expressed gene encoding NKG2D into an RME gene, recapitulating all aspects of natural RME including mitotic stability of both the active and silent states. The results support the binary model of enhancer action, and suggest that RME is a consequence of general properties of gene regulation by enhancers rather than an RME-specific epigenetic program. Therefore, many and perhaps all genes may be subject to some degree of RME. Surprisingly, this was borne out by analysis of several genes that define different major hematopoietic lineages, that were previously thought to be universally expressed within those lineages: the genes encoding NKG2D, CD45, CD8α, and Thy-1. We propose that intrinsically probabilistic gene allele regulation is a general property of enhancer-controlled gene expression, with previously documented RME representing an extreme on a broad continuum.