Samakovlis, ChristosDeckmann, KlausGhoul, MarouaMarouaGhoul2025-07-142025-07-142025https://jlupub.ub.uni-giessen.de/handle/jlupub/20707https://doi.org/10.22029/jlupub-20057Airway basal cells are multipotent stem cell progenitors, which are crucial to maintaining the pseudostratified epithelium. Understanding the molecular mechanisms that regulate their differentiation is essential to unravel how normal and pathological airway epithelial regeneration occurs. With this aim, we performed single-cell RNA sequencing and pseudotime analysis of primary human bronchial epithelial cells (pHBECs) differentiated on air-liquid-interface (ALI) culture, revealing a selective upregulation of the transcription factor BHLHE40 in differentiating intermediate epithelial cells. Using our ALI cultures in combination with lentiviral-based gene transfer, we show that the overexpression of BHLHE40 in human basal cells increased their differentiation into club, goblet, and ciliated cells. Consistent with the overexpression results, the knockdown of BHLHE40 in the ALI cultures reduced basal epithelial cell differentiation. Interestingly, we demonstrate that BHLHE40-mediated loss of basal cell fate through increased differentiation was preceded earlier in the culture by an increase in Notch signaling on day 5. Previous studies have shown that Notch1 signaling activation in basal cells of the murine airway epithelium is associated with the downregulation of basal cell genes and upregulation of luminal differentiation markers. Furthermore, N1ICD overexpression in primary epithelial cells from the human mammary epithelium reduced TP63 expression. Taken together, we propose that BHLHE40-induced loss of basal cells and transition to an intermediate state involves Notch signaling. To investigate the molecular mechanisms of BHLHE40-induced basal-to-intermediate cell transition and identify its direct transcriptional targets involved in Notch signaling activation driving this transition, we did a 3-step selection by performing a comparative analysis of three datasets: the data from the published BHLHE40 A549 ChIP-sequencing, our differentially expressed transcription factors in the differentiation trajectory of the Submucosal glands (SMGs)-like basal cells toward secretory cells, and our Real-Time qPCR analysis of transduced A549 cells overexpressing GFP or BHLHE40-GFP. We ended up with 8 potential BHLHE40 targets: FOS, HIF1a, MYC, HMGA2, BNC1, ZFP36L1, FOSL1, and SOX9. To confirm that they are transcriptional targets of BHLHE40 in our pHBECs-derived ALI culture, we used CUT&RUN DNA enrichment combined with gene expression analysis of transduced cells overexpressing BHLHE40-GFP from ALI day 14. We show that, although BHLHE40 has bound to HIF1a, BNC1, FOSL1, MYC, HMGA2, and ZFP36L1 genomic regions, it only repressed BNC1 gene expression without affecting the other targets. Consistently, we show that BHLHE40 overexpression reduced the number of BNC1- expressing cells in the basal layer and that BNC1 expression was exclusive to basal cells, further confirming BHLHE40-mediated transcriptional repression of BNC1 in basal cells. Previous studies showed that BNC1 has a selectively high expression in basal cells of the skin epithelium, which goes down in the Suprabasal and differentiated layers. Consistently, its reduction was associated with the appearance of keratinocytes differentiation marker, suggesting its role in maintaining the undifferentiated basal cell state. In line with our results, we show a correlation between the downregulation of BNC1, the reduction of basal cell markers, and the increase in epithelial cell differentiation following BHLHE40 overexpression on day 14. Taken together, our data indicate that BHLHE40 transcriptionally represses BNC1 expression, which in turn induces the loss of basal cells and their differentiation. We hypothesized that BNC1 is the solely transcriptional target of BHLHE40 that is needed to maintain the undifferentiated basal cell state by negatively regulating Notch signaling in basal cells (e.g., by activating Notch inhibitor). Bulk RNA sequencing of sorted BHLHE40-GFP cells from ALI day 14 identified LFNG, a basal cell-specific Notch inhibitor, as a potential downstream target of BNC1, as its expression decreased following BHLHE40 overexpression. However, it remains unclear whether LFNG or other mediators contribute to the BHLHE40-BNC1-induced transition from basal to intermediate cells. Additionally, since TP63 expression is regulated by multiple signaling pathways in addition to Notch signaling, further investigation is needed to deepen our understanding of the molecular mechanisms driving BHLHE40-BNC1-mediated loss of TP63+ basal cells, focusing on identifying BNC1 downstream targets, which our findings suggest as a promising starting point for future studies. This will determine how BNC1 maintains TP63 expression in the human airway epithelium and whether Notch signaling is solely involved in BHLHE40-mediated regulation of basal cell differentiation. Considering our pHBECs-derived ALI culture as a re-generation model, our data suggest a potential of BHLHE40 to enhance the regeneration of the human airway epithelium upon airway injury through increasing differentiation. Also, our single-cell RNA sequencing and pseudotime analysis of differentiating ALI culture suggested the involvement of BHLHE40 in the SMGs-like basal cell differentiation trajectory towards secretory cells. Since basal cells of the SMGs have been shown to contribute to the surface airway epithelium (SAE) regeneration upon injury, we propose a potential role of BHLHE40 in enhancing regeneration via increasing differentiation of basal progenitor cells, both in the SAE and the SMGs. Collectively, we propose that BHLHE40 could serve as a target for therapeutic strategies aimed at enhancing tissue repair and regeneration in the airway epithelium.enAttribution-NonCommercial 4.0 Internationalcell differentiationbasal stem cellsmolecular mechanismshuman airway epitheliumddc:610