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Cellular heterogeneity in the 16HBE14o- airway epithelial line impacts functional readouts [ATAC-seq]

ABSTRACT: The airway epithelial cell line, 16HBE14o-, is an important cell model for studying airway disease. 16HBE14o- cells were originally generated from primary human bronchial epithelial cells by SV40-mediated immortalization, a process that is associated with genomic instability through long-term culture. Here we explore the heterogeneity of these cells, with respect to expression of the cystic fibrosis transmembrane conductance regulator (CFTR) transcript and protein. We isolate clones of 16HBE14o- with stably higher and lower levels of CFTR in comparison to bulk 16HBE14o-, designated CFTRhigh and CFTRlow. Detailed characterization of the CFTR locus in these clones by ATAC-seq and 4C-seq showed open chromatin profiles and higher order chromatin structure that correlate with CFTR expression levels. . Transcriptomic profiling of CFTRhigh and CFTRlow cells showed that the CFTRhigh cells had an elevated inflammatory/ innate immune response phenotype. These results encourage caution in interpreting functional data from clonal lines of 16HBE14o- cells, generated after genomic or other manipulations.

ORGANISM(S): Homo sapiens

PROVIDER: GSE220853 | GEO | 2023/06/07

REPOSITORIES: GEO

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Cellular heterogeneity in the 16HBE14o- airway epithelial line impacts functional readouts [RNA-seq]

Project description:The airway epithelial cell line, 16HBE14o-, is an important cell model for studying airway disease. 16HBE14o- cells were originally generated from primary human bronchial epithelial cells by SV40-mediated immortalization, a process that is associated with genomic instability through long-term culture. Here we explore the heterogeneity of these cells, with respect to expression of the cystic fibrosis transmembrane conductance regulator (CFTR) transcript and protein. We isolate clones of 16HBE14o- with stably higher and lower levels of CFTR in comparison to bulk 16HBE14o-, designated CFTRhigh and CFTRlow. Detailed characterization of the CFTR locus in these clones by ATAC-seq and 4C-seq showed open chromatin profiles and higher order chromatin structure that correlate with CFTR expression levels. . Transcriptomic profiling of CFTRhigh and CFTRlow cells showed that the CFTRhigh cells had an elevated inflammatory/ innate immune response phenotype. These results encourage caution in interpreting functional data from clonal lines of 16HBE14o- cells, generated after genomic or other manipulations.

2023-06-07 | GSE220854 | GEO

Cellular heterogeneity in the 16HBE14o- airway epithelial line impacts functional readouts [4C-seq]

2023-06-07 | GSE220852 | GEO

The impact of genomic distance on enhancer-promoter interactions at the CFTR locus.

Project description:We identified and characterized multiple cell-type selective enhancers of the CFTR gene promoter in previous work and demonstrated active looping of these elements to the promoter. Here we address the impact of genomic spacing on these enhancer:promoter interactions and on CFTR gene expression. Using CRISPR/Cas9, we generated clonal cell lines with deletions between the -35kb airway enhancer and the CFTR promoter in the 16HBE14o- airway cell line, or between the intron 1 (185 + 10kb) intestinal enhancer and the promoter in the Caco2 intestinal cell line. The effect of these deletions on CFTR transcript abundance, as well as the 3D looping structure of the locus was investigated in triplicate clones of each modification. Our results indicate that both small and larger deletions upstream of the promoter can perturb CFTR expression and -35kb enhancer:promoter interactions in the airway cells, though the larger deletions are more impactful. In contrast, the small intronic deletions have no effect on CFTR expression and intron 1 enhancer:promoter interactions in the intestinal cells, whereas larger deletions do. Clonal variation following a specific CFTR modification is a confounding factor particularly in 16HBE14o- cells.

2024-03-07 | GSE239930 | GEO

Human bronchial epithelial cell lines: 16HBE14o- vs. beta/gammaENaC-16HBE14o-, Primary cells NHBE vs. DHBE-CF

Project description:Transcriptional profiling of human bronchial epithelial cell lines and primary cells. Parental normal 16HBE14o- cells were stably transfected with beta/gammaENaC to generate beta/gammaENaC-16HBE14o- cells. NHBE and DHBE-CF were obtained from TaKaRa.

2018-01-03 | GSE101829 | GEO

An ectopic enhancer restores CFTR expression through de novo chromatin looping [4C-seq]

Project description:Transcription of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is regulated by a set of ubiquitous and cell-type selective cis-regulatory elements (CREs). These elements include extragenic and intronic enhancers that bind cell-type specific transcription factors, as well as architectural protein bound structural elements. We previously reported that loss of the airway-selective -35kb enhancer in 16HBE14o- airway epithelial cells disrupts normal enhancer-promoter looping at the CFTR locus and nearly abolishes its expression. Here we expand on previous work, and explore the effect of relocation of the 500bp -35kb enhancer core to an ectopic site in intron 1 of CFTR on gene expression and chromatin dynamics. We find that although genomic relocation of this element is not able to fully restore CFTR expression to wild-type levels, the -35kb sequence is able establish a de novo enhancer signature at chromatin at the site of insertion. Additionally, the relocated -35kb enhancer is able to form chromatin contacts with known CFTR CREs. These studies reveal important direct chromatin effects, as well as limitations, of CFTR enhancers, and provide important considerations when targeting the CFTR locus for novel therapeutic gene editing.

2022-12-13 | GSE203559 | GEO

An ectopic enhancer restores CFTR expression through de novo chromatin looping [ATAC-seq]

2022-12-13 | GSE203558 | GEO

Gene expression data from human bronchial epithelial cell lines: comparison between normal and CFTR-deficient cells

Project description:Whole transcriptional analysis of normal 16HBE14o- cells, cystic fibrosis (CF) patients-derived CFBE41o- cells, and WT-CFTR-rescued CFBE41o- cells. We utilized the gene expression data to understand the transcriptional regulation underlying the loss of CFTR function.

2019-01-31 | GSE110289 | GEO

Phosphoproteome of airway epithelial cell in response to infection with Staphylococcus aureus

Project description:Here we used an unbiased proteomic and phosphoproteomic approach to characterize the response of 16HBE14o- airway epithelial cells following infection with S. aureus.

2016-12-14 | PXD002776 | Pride

Secretory Cells Dominate Airway CFTR Expression and Function in Human Airway Superficial Epithelia [single-cell RNA-seq]

Project description:Purpose: To identify the cell types that contribute to CFTR expression and function within the proximal-distal axis of the normal human lung. Methods: Single cell RNA-seq (scRNA-seq) was performed on freshly isolated human large and small airway epithelial cells. Single cell-based RNA in situ hybridization (scRNA-ISH) and quantitative RT-PCR (scqRT-PCR) were performed for validation. In vitro culture systems correlated CFTR function to cell types. Lentiviruses were used for cell-type specific transduction of wild-type CFTR in CF cells. Results: scRNA-seq identified secretory cells as dominating CFTR expression in normal human large and particularly small airways, followed by basal cells. Ionocytes expressed the highest CFTR levels but were rare, while expression in ciliated cells was infrequent and low. scRNA-ISH, and scqRT-PCR confirmed the scRNA-seq findings. CF lungs exhibited distributions of CFTR and ionocytes similar to normal controls. CFTR-mediated Cl- secretion tracked secretory cell, not ionocyte, densities. Further, the nucleotide-purinergic regulatory system that controls CFTR-mediated hydration was associated with secretory cells. Lentiviral transduction of wild-type CFTR produced CFTR-mediated Cl- secretion in CF airway secretory cells but not ciliated cells. Conclusions: Secretory cells dominate CFTR expression and function in human airway superficial epithelia. CFTR therapies may need to restore CFTR function to multiple cell types, with a focus on secretory cells.

2020-12-16 | GSE160673 | GEO

Secretory Cells Dominate Airway CFTR Expression and Function in Human Airway Superficial Epithelia [bulk RNA]

2020-12-16 | GSE160674 | GEO

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