OTX2 regulates CFTR expression during endoderm differentiation and occupies 3′ cis‐regulatory elements

Abstract

Background: Cell-specific and developmental mechanisms contribute to expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene; however, its developmental regulation is poorly understood. Here we use human induced pluripotent stem cells differentiated into pseudostratified airway epithelial cells to study these mechanisms. Results: Changes in gene expression and open chromatin profiles were investigated by RNA-seq and ATAC-seq, and revealed that alterations in CFTR expression are associated with differences in stage-specific open chromatin. Additionally, two novel open chromatin regions, at +19.6 kb and +22.6 kb 3 0 to the CFTR translational stop signal, were observed in definitive endoderm (DE) cells, prior to an increase in CFTR expression in anterior foregut endoderm (AFE) cells. Chromatin studies in DE and AFE cells revealed enrichment of active enhancer marks and occupancy of OTX2 at these sites in DE cells. Loss of OTX2 in DE cells alters histone modifications across the CFTR locus and results in a 2.5-fold to 5-fold increase in CFTR expression. However, deletion of the +22.6 kb site alone does not affect CFTR expression in DE or AFE cells.Conclusions: These results suggest that a network of interacting cis-regulatory elements recruit OTX2 to the locus to impact CFTR expression during early endoderm differentiation.

Cellular heterogeneity in the 16HBE14o− airway epithelial line impacts biological readouts

“…The −33 kb element is adjacent to the strong airway‐selective enhancer at −35 kb, and like this CRE and another enhancer at −44 kb, has weak RNAPII enrichment in 16HBE14o − cells (Figure S2 ). In contrast, this site is not accessible in ATAC‐seq nor is it enriched for RNAPII in Calu3, a lung carcinoma cell line that also expresses CFTR (Figure S2 ), nor primary bronchial epithelial cells (Kerschner et al, 2021 ). Conversely, another CRE of unknown function, located in intron 10 (i10c) is of higher intensity in CFTR low cells compare to 16HBE14o − or CFTR high cells (Figures 2 and S2 , dark blue arrow).…”

“…CFTR expression is controlled by distal and intronic cis ‐regulatory elements (CREs) and adopts cell‐type specific 3D conformations, which are required for normal CRE‐promoter interactions. We showed previously that deletion of CREs or depletion of activating transcription factors (TFs) disrupts these higher order chromatin interactions and alters CFTR expression levels (Kerschner et al, 2021 ; NandyMazumdar et al, 2020 , 2021 ; Paranjapye et al, 2021 , 2022 ; Yang et al, 2016 ; Yin et al, 2022 ). Hence, we next investigated whether the CFTR locus adopts different chromatin conformations and 3D interactions in the CFTR high and CFTR low clonal cells.…”

“…Focusing first on the CFTR locus and its CREs, open chromatin profiling revealed accessibility differences at −33 kb upstream of the CFTR promoter and at a site within intron 10 (legacy, Refseq intron 11iii) of the gene (Figure 2 ). The −33 kb CRE, which to date is functionally uncharacterized, is only observed in 16HBE14o − cells and not in the CFTR ‐expressing lung adenocarcinoma cell line Calu3 (Figure S2 ) or primary human bronchial epithelial cells (Kerschner et al, 2021 ; Suzuki et al, 2020 ). This site is more open in CFTR high cells compared to parental 16HBE14o − or CFTR low cells and it is enriched for RNAPII and H3K27ac in bulk 16HBE14o − cells (NandyMazumdar et al, 2020 ).…”

“…The i10c CRE chromatin is also open in many cell types profiled in the ENCODE database which do not express CFTR . In addition to being a site of open chromatin in 16HBE14o − cells (NandyMazumdar et al, 2020 ; Zhang et al, 2013 ), i10c interacts with the transcription factor orthodenticle homeobox 2 (OTX2) where it represses CFTR in definitive endoderm cells (Kerschner et al, 2021 ). Consistent with the earlier data, the accessibility of this element is inversely correlated to CFTR expression, again suggesting a repressive role in these cells.…”

“…We identified and characterized two strong airway‐selective enhancers at −35 kb and −44 kb upstream of the CFTR promoter (Kerschner et al, 2022 ; NandyMazumdar et al, 2020 ; NandyMazumdar et al, 2021 ; Paranjapye et al, 2022 ; Zhang et al, 2012 , 2013 , 2015 ). These enhancers, and many of the transcription factors and mechanisms driving their activity, are conserved in other airway cells including the lung carcinoma cell line Calu3, primary human bronchial epithelial cells, iPSC‐derived airway epithelial cells (Kerschner et al, 2021 ; Mutolo et al, 2018 ; NandyMazumdar et al, 2020 , 2021 ; Paranjapye et al, 2022 ) and adult and fetal human tissues (Bergougnoux et al, 2014 ). DNA sequence analysis of 16HBE14o − cells indicates that at least one copy of the pSV40 (ori‐) plasmid has integrated into intron 6 of the CFTR locus, and it has been suggested that this results in mono‐allelic expression of CFTR in these cells (Valley et al, 2019 ).…”