Fig 1: NKX2-1 binds chromatin in a cell-type-specific manner.a Genetic labeling and FACS purification of AT1 and AT2 nuclei using Wnt3aCre and SftpcCreER, respectively. Confocal images of immunostained lungs showing that both AT1 and AT2 cells express NKX2-1 while AT2 cells are distinguishable by LAMP3 and cuboidal E-Cadherin (ECAD). RosaSun1GFP marks the nuclear envelop (arrowhead; green in the diagram). All nuclei are Sytox Blue positive. 10-wk, 10-week-old. For SftpcCreER, 3 mg tamoxifen was administrated 3 days before tissue harvest. Scale: 10 µm. The color scheme (AT1: green; AT2: purple) applies to subsequent figures. b Validation of cell-type-specific ChIP-seq using an active promoter marker H3K4me3. Compared to the whole lung, the site (asterisk; same in subsequent figures) near an AT1 gene Spock2 shows enrichment in purified AT1 nuclei but depletion in purified AT2 nuclei, whereas the site near an AT2 gene Lamp3 shows the opposite pattern. A pan-epithelial gene Cdh1 (also known as E-Cadherin) is enriched in both AT1 and AT2 nuclei, whereas a mesenchymal gene Pdgfra is depleted in both. P, postnatal day. For SftpcCreER, 250 µg tamoxifen was administrated 3 days before tissue harvest. c ChIP-seq heatmaps (2.5 kb flanking the peak center; same in subsequent figures) of purified AT1 and AT2 nuclei for NKX2-1, H3K4me3 (active promoter), H3K27ac (putative active enhancer; note its bimodal pattern surrounding the center), and H3K4me1 (putative enhancer and active gene body), grouped by differential NKX2-1 binding into AT1-specific, AT2-specific, and common peaksets and sorted by the H3K4me3 signal. The common peakset is divided into lineage (epithelial) and housekeeping sets, based on scATAC-seq (Supplementary Fig. 2). Compared to the cell-type-specific and lineage sets, the housekeeping set has abundant H3K4me3, which also corresponds to depletion of H3K4me1 (top portion), whereas the bottom portion (curly bracket) is depleted of H3K4me3 and is enriched for the insulator CTCF motif. d Example NKX2-1 binding sites of the AT1-specific (Spock2), AT2-specific (Lamp3), lineage (Cdh1), and housekeeping (Gapdh) sets. The Y-axes are scaled via foreground normalization so that peak heights can be directly compared across samples; quantification and statistics are reported in the corresponding Supplementary Data files with marked peaks highlighted (same in subsequent figures).
Fig 2: NKX2-1-AS1 overexpression reduces CD274 expression levels in A549 cells in part by impairing NKX2-1 protein binding to the CD274 promoter. ChIP-qPCR analysis of NKX2-1 protein binding to (A) CD274 promoter (n = 4; p = 0.005), (B) CLDN1 promoter (n = 5), and (C) PTPN1 promoter (n = 5) in H441 cells transfected with NKX2-1-AS1 siRNAs or non-silencing control. (D) NKX2-1 co-transfection with the -1kbCD274-Luc vector results in higher luciferase activity (3-fold in the absence of NKX2-1-AS1, 0ug). NKX2-1-AS1 overexpression reduces the activity of the -1kbCD274 promoter in a dose-dependent manner both in the absence (E) (n = 3-4; ANOVA p = 0.003) or presence (F) (n = 3-4; ANOVA p = 0.0001) of NKX2-1 overexpression. NKX2-1-AS1 overexpression reduces the expression of the endogenous CD274 gene in a dose-dependent manner both in the absence (G) (n = 3-4; ANOVA p = 0.001) or presence (H) (n = 3-4; ANOVA p = 0.05) of NKX2-1 overexpression. (I) RIP-qPCR analysis of NKX2-1-AS1 pull down by NKX2-1 antibody compared to IgG control (n = 6; p = 0.05).
Fig 3: NKX2-1-AS1 follows tissue-specific patterns of expression similar to NKX2-1 in human cells. (A) Expression of NKX2-1-AS1 and NKX2-1 determined by RT-PCR in lung cell lines. The NKX2-1-AS1 PCR fragments were sequenced to confirm the identity of the sequence. (B) Relative expression patterns of NKX2-1-AS1 and NKX2-1 in tissues and cell lines, including normal human adult lung and thyroid and H441 and H661 cell lines, as determined by qPCR (n = 3; *p < 0.05; **p < 0.01). (C) 5'-RACE analysis of NKX2-1-AS1 in human thyroid identified multiple transcription initiation sites within the 500 bp 5' of the transcription initiation site reported in Ensembl. Thyroid total RNA was used as it has higher levels of NKX2-1-AS1 expression than the lung. The sensitivity of the method did not allow us to analyze lung RNA. Thirty clones, generated in three independent experiments, were sequenced. The black bar indicates the region recently identified in a genome wide analysis of accurate lncRNA transcription initiation sites13. (D) Time course analysis of NKX2-1-AS1 and NKX2-1 transcript stability in H441 cells by qPCR after inhibition of transcription by actinomycin D treatment (n = 3; *p < 0.05).
Fig 4: YAP/TAZ maintain AT1-specific NKX2-1 binding and cell fate, and prevent AT1-to-AT2 conversion.a Confocal images of immunostained lungs showing loss of YAP/TAZ in recombined AT1 cells (filled versus open arrowhead) in the Y/TWnt3a mutant. Scale: 10 µm. b NKX2-1 ChIP-seq heatmaps of purified AT1 nuclei from P15 Y/TWnt3a mutant and littermate control lungs, sorted by fold change and cross-referenced with NKX2-1 binding from Fig. 1. c Examples of NKX2-1 peaksets in b. d ScRNA-seq UMAP comparison of Y/TWnt3a mutant and littermate control epithelial cells with cell typing supported by the dot plot. AT1 cells in the mutant are higher in number, likely resulting from more efficient cell dissociation due to the phenotype in cell morphology (h). e Volcano plots (MAST differential expression) comparing AT1 (left) and AT2 (right) cells in d. f Monocle trajectory analysis of AT1 and AT2 cells in d, showing a linear transcriptomic shift from AT1 to AT2 cells in the control but via intermediate cells (bracket) in the mutant, which express a subset of AT1 (Spock2) and AT2 (Sftpc) genes as well as Sfn. g Monocle gene clusters with similar dynamics along the transcriptomic shift in f. h Confocal images of immunostained lungs showing AT2 markers (LAMP3 and SFTPC) in recombined cells in the Y/TWnt3a mutant (filled versus open arrowhead). Some SFTPC-expressing mutant cells still have extended morphology at P15 (open arrow), and become mostly cuboidal at 10 weeks. Scale: 10 µm. i Seurat module scores of gene sets associated with the 20% most decreased (left) or increased (right) NKX2-1 binding sites in b, plotted along the Monocle trajectory in f, showing concordant changes in NKX2-1 binding and gene expression. j Unsupervised principal component analysis of NKX2-1 binding across indicated color-coded samples. The E18.5 Y/TSox9 mutant is right-shifted as far as mature AT2 cells, whereas the P15 Y/TWnt3a mutant is between AT1 and AT2 cells, reflecting the exaggerated AT2 cells and intermediate cells in the respective mutants. k A diagram, reminiscent of and color-coded as in j, depicting the normal differentiation of progenitors toward AT1 and AT2 cells, as well as the drift in both NKX2-1 binding and transcriptome observed in Y/TSox9 and Y/TWnt3a mutants.
Fig 5: AT1-specific partner factors YAP/TAZ establish AT1-specific NKX2-1 binding and cell fate, and antagonize those of AT2 cells.a Top enriched HOMER de novo motifs (binomial test) for AT1-specific (TEAD), common (FOXA and CTCF), AT2-specific (CEBP) NKX2-1 binding sites. b Nuclear YAP/TAZ and CEBPA are specifically detected (arrowhead) in AT1 and AT2 cells, respectively. ShhCre genetically labels AT1 and AT2 nuclei, distinguishable by AT2 cell-specific LAMP3 and cuboidal ECAD. Scale: 10 µm. c Confocal images of immunostained E18.5 Y/TSox9 mutant and littermate control lungs received 3 mg tamoxifen (Tam) at E15.5, showing loss of nuclear YAP/TAZ from AT1 cells, as outlined by ECAD (arrowhead). Scale: 10 µm. d NKX2-1 ChIP-seq heatmaps of E18.5 Y/TSox9 mutant and littermate control (C) whole lungs, sorted by fold change and cross-referenced with NKX2-1 binding from Fig. 1. e Examples of NKX2-1 peaksets in d. Lamp3 peaks do not reach statistical significance. NKX2-1 binding for Spock2 and Lamp3 in 10-week cells are shown in Fig. 1 (same in Fig. 6c). f ScRNA-seq UMAP comparison of Y/TSox9 mutant and littermate control epithelial cells with cell typing supported by the dot plot. The mutant has a decrease in the number of AT1 cells accompanied by an increase in the number of AT2 cells. g Volcano plots (MAST differential expression) comparing AT1 (left) and AT2 (right) cells in f. Differentially expressed, curated lists of AT1 and AT2-specific genes (Supplementary Data 6) are labeled (same in subsequent figures). h Monocle trajectory analysis of AT1 and AT2 cells in f, showing a linear transcriptomic shift from AT1 to AT2 cells in the control but further extending to transcriptionally exaggerated AT2 cells (bracket) in the Y/TSox9 mutant. The progenitor marker Sox9 is present at a low level but normally higher in AT2 cells. i Monocle gene clusters with similar dynamics along the transcriptomic shift in h. j Seurat module scores of gene sets associated with the 20% most decreased (top) or increased (bottom) NKX2-1 binding sites in d, plotted along the Monocle trajectory in h, showing concordant changes in NKX2-1 binding and gene expression.
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