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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE158201 | GEO | 2021/03/08

REPOSITORIES: GEO

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Project description:Alveoli are thin-walled sacs that serve as the gas exchange units of the lung. They are affected in devastating lung diseases including COPD, Idiopathic Pulmonary Fibrosis, and the major form (adenocarcinoma) of lung cancer, the leading cause of cancer deaths. The alveolar epithelium is composed of two morphologically distinct cell types: alveolar type (AT) 1 cells, exquisitely thin cells across which oxygen diffuses to reach the blood, and AT2 cells, specialized surfactant-secreting cells. Classical studies suggested that AT1 cells arise from AT2 cells during development and following injury, but more recent studies suggest other sources. Here we use histological and marker analysis, lineage tracing, and clonal analysis in mice to identify alveolar progenitor and stem cells and map their locations and potential in vivo. The results show that AT1 and AT2 cells arise independently during development from a bipotential progenitor. After birth, new AT1 cells derive from rare, long-lived, self-renewing AT2 cells, each producing a slowly expanding clonal focus of regenerated alveoli contiguous with the founder AT2 cell. This stem cell function of AT2 cells is broadly activated by diffuse AT1 cell injury, and AT2 self-renewal can be induced in vitro by EGF ligands and permanently activated in vivo by AT2 cell-specific targeting of the oncogenic KrasG12D allele, efficiently transforming AT2 cells into monoclonal adenomatous tumors that rapidly enlarge and prove fatal. Thus, there is a developmental switch in alveolar progenitor cells after birth, when mature AT2 cells function as facultative stem cells that contribute to local alveolar renewal, repair, and cancer. We propose that short-range signals from dying AT1 cells regulate AT2 stem cell activity: a signal transduced by EGFR-KRAS controls AT2 self-renewal and is hijacked during oncogenic transformation, and a separate signal controls reprogramming to AT1 cell fate. To compare expression between ATII and E18 BP populations, RNA was isolated from either population purified by FACS. Two populations are analyzed with 3 biological replicates per population.

Project description:Lung epithelial regeneration after acute injury requires coordination of complex cellular and molecular processes required for progenitor proliferation and differentiation of specialized alveolar cells to pattern the morphologically complex alveolar gas exchange surface. During regeneration, specialized Wnt-responsive alveolar epithelial progenitor (AEP) subset of alveolar type 2 (AT2) cells transition to alveolar type 1 (AT1) cells through specialized progenitor states, though the precise molecular and epigenetic determinants of these processes remain unclear. Here, we report a refined primary murine alveolar organoid assay which recapitulates critical aspects of in vivo regeneration, providing a tractable model to dissect these key regenerative processes. Clonal expansion of single AEPs generated complex alveolar organoids with extensive structural maturation and organization. These organoids contain properly patterned AT1 and AT2 cells surrounding numerous alveolar-like cavities with minimal structural contribution from mesenchymal cells, implying extensive cell autonomous regenerative function encoded in adult AEPs. Leveraging a time series of paired scRNAseq and scATACseq analysis, we identified the AEP state at single cell resolution and defined two distinct AEP to AT1 intermediate states: a widely reported transitional state defined by cell stress markers and a second state defined by differential activation of signaling pathways mediating AT1 cell differentiation. Transcriptional regulatory network (TRN) analysis demonstrates that these AT1 transition states are driven by distinct regulatory networks controlled in part by differential activity of the lung master regulatory factor Nkx2-1. Genetic ablation of Nkx2-1 in AEP-derived organoids is sufficient to cause irreversible transition to a proliferative stressed transitional state characterized by disorganized, uncontrolled growth. Finally, AEP-specific deletion of Nkx2-1 in adult mice leads to rapid loss of AEP state, clonal expansion, and disorganization of alveolar structure, implying a continuous requirement for Nkx2-1 in maintenance and function of adult lung progenitors. Together, these data provide new insight into lineage hierarchies in lung regeneration and implicate dynamic epigenetic maintenance via lineage transcription factors as central to control of facultative progenitor activity in AEPs.

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

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