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Cellular heterogeneity in the 16HBE14o- airway epithelial line impacts functional readouts [4C-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: GSE220852 | GEO | 2023/06/07

REPOSITORIES: GEO

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

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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.

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

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