Search

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 (hiPSCs) 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, 2 novel open chromatin regions, at +19.6kb and +22.6kb 3' 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 +22.6kb 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. This article is protected by copyright. All rights reserved.

The availability of robust protocols to differentiate induced pluripotent stem cells (iPSCs) into many human cell lineages has transformed research into the origins of human disease. The efficacy of differentiating iPSCs into specific cellular models is influenced by many factors including both intrinsic and extrinsic features. Among the most challenging models is the generation of human bronchial epithelium at air‐liquid interface (HBE‐ALI), which is the gold standard for many studies of respiratory diseases including cystic fibrosis. Here, we perform open chromatin mapping by ATAC‐seq and transcriptomics by RNA‐seq in parallel, to define the functional genomics of key stages of the iPSC to HBE‐ALI differentiation. Within open chromatin peaks, the overrepresented motifs include the architectural protein CTCF at all stages, while motifs for the FOXA pioneer and GATA factor families are seen more often at early stages, and those regulating key airway epithelial functions, such as EHF, are limited to later stages. The RNA‐seq data illustrate dynamic pathways during the iPSC to HBE‐ALI differentiation, and also the marked functional divergence of different iPSC lines at the ALI stages of differentiation. Moreover, a comparison of iPSC‐derived and lung donor‐derived HBE‐ALI cultures reveals substantial differences between these models.

E74‐like factor 5 (ELF5) and ETS‐homologous factor (EHF) are epithelial selective ETS family transcription factors (TFs) encoded by genes at chr11p13, a region associated with cystic fibrosis (CF) lung disease severity. EHF controls many key processes in lung epithelial function so its regulatory mechanisms are important. Using CRISPR/Cas9 technology, we removed three key cis‐regulatory elements (CREs) from the chr11p13 region and also activated multiple open chromatin sites with CRISPRa in airway epithelial cells. Deletion of the CREs caused subtle changes in chromatin architecture and site‐specific increases in EHF and ELF5. CRISPRa had most effect on ELF5 transcription. ELF5 levels are low in airway cells but higher in LNCaP (prostate) and T47D (breast) cancer cells. ATAC‐seq in these lines revealed novel peaks of open chromatin at the 5’ end of chr11p13 associated with an expressed ELF5 gene. Furthermore, 4C‐seq assays identified direct interactions between the active ELF5 promoter and sites within the EHF locus, suggesting coordinate regulation between these TFs. ChIP‐seq for ELF5 in T47D cells revealed ELF5 occupancy within EHF introns 1 and 6, and siRNA‐mediated depletion of ELF5 enhanced EHF expression. These results define a new role for ELF5 in lung epithelial biology.

It is estimated that up to 5% of cystic fibrosis transmembrane conductance regulator (CFTR) pathogenic alleles are unidentified. Some of these errors may lie in noncoding regions of the locus and affect gene expression. To identify regulatory element variants in the CFTR locus, SureSelect targeted enrichment of 460 kb encompassing the gene was optimized to deep sequence genomic DNA from 80 CF patients with an unequivocal clinical diagnosis but only one or no CFTR-coding region pathogenic variants. Bioinformatics tools were used to identify sequence variants and predict their impact, which were then assayed in transient reporter gene luciferase assays. The effect of five variants in the CFTR promoter and four in an intestinal enhancer of the gene were assayed in relevant cell lines. The initial analysis of sequence data revealed previously known CF-causing variants, validating the robustness of the SureSelect design, and showed that 85 of 160 CF alleles were undefined. Of a total 1737 variants revealed across the extended 460-kb CFTR locus, 51 map to known CFTR cis-regulatory elements, and many of these are predicted to alter transcription factor occupancy. Four promoter variants and all those in the intestinal enhancer significantly repress reporter gene activity. These data suggest that CFTR regulatory elements may harbor novel CF disease-causing variants that warrant further investigation, both for genetic screening protocols and functional assays.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), but are not good predictors of lung phenotype. Genome-wide association studies (GWAS) previously identified additional genomic sites associated with CF lung disease severity. One of these, at chromosome 11p13, is an intergenic region between Ets homologous factor (EHF) and Apaf-1 interacting protein (APIP). Our goal was to determine the functional significance of this region, which being intergenic is probably regulatory. To identify cis-acting elements, we used DNase-seq and H3K4me1 and H3K27Ac ChIP-seq to map open and active chromatin respectively, in lung epithelial cells. Two elements showed strong enhancer activity for the promoters of EHF and the 5′ adjacent gene E47 like ETS transcription factor 5 (ELF5) in reporter gene assays. No enhancers of the APIP promoter were found. Circular chromosome conformation capture (4C-seq) identified direct physical interactions of elements within 11p13. This confirmed the enhancer-promoter associations, identified additional interacting elements and defined topologically associating domain (TAD) boundaries, enriched for CCCTC-binding factor (CTCF). No strong interactions were observed with the APIP promoter, which lies outside the main TAD encompassing the GWAS signal. These results focus attention on the role of EHF in modifying CF lung disease severity.

Organoid cultures derived from primary human tissues facilitate the study of disease processes and the development of new therapeutics. Most men with cystic fibrosis (CF) are infertile due to defects in the epididymis and vas deferens; however, the causative mechanisms are still unclear. We used human epididymis epithelial cell (HEE) organoids and polarized HEE cell cultures to assay the CF transmembrane conductance regulator (CFTR) in the human epididymis. 3D HEE organoids and polarized 2D HEE cell cultures on membrane inserts were established from human caput epididymis. Single-cell RNA sequencing (scRNA-seq) was performed to map cell-type-specific gene expression in the organoids. Using forskolin (FSK) to activate CFTR and inhibitor CFTRinh(172) to block its activity, we assessed how CFTR contributes to organoid swelling and epithelial barrier function. The scRNA-seq data showed key caput epididymis cell types present in HEE organoid cultures. FSK at 10 μM induced HEE organoid swelling by 20% at 16 hrs, while 5 and 10 μM CFTRinh(172) treatment significantly reduced HEE organoid size. In transepithelial resistance (TER) measurements, FSK reduced TER, while inhibition of CFTR increased TER; also, depletion of CFTR with specific siRNAs significantly increased TER. FSK treatment significantly increased the flux of 4-kDa but not 70kDa dextran, suggesting activation of CFTR mainly enhances transcellular diffusion. We have demonstrated that CFTR contributes to the maintenance of HEE cell TER and that cultured HEE organoids are a useful model to investigate human epididymis function. This research facilitates progress in elucidating how CFTR-dependent cellular processes impair fertility in CF.

Higher order chromatin structure establishes domains that organize the genome and coordinate gene expression. However, the molecular mechanisms controlling transcription of individual loci within a topological domain (TAD) are not fully understood. The cystic fibrosis transmembrane conductance regulator (CFTR) gene provides a paradigm for investigating these mechanisms. CFTR occupies a TAD bordered by CTCF/cohesin binding sites within which are cell-type-selective cis-regulatory elements for the locus. We showed previously that intronic and extragenic enhancers, when occupied by specific transcription factors, are recruited to the CFTR promoter by a looping mechanism to drive gene expression. Here we use a combination of CRISPR/Cas9 editing of cis-regulatory elements and siRNA-mediated depletion of architectural proteins to determine the relative contribution of structural elements and enhancers to the higher order structure and expression of the CFTR locus. We found the boundaries of the CFTR TAD are conserved among diverse cell types and are dependent on CTCF and cohesin complex. Removal of an upstream CTCF-binding insulator alters the interaction profile, but has little effect on CFTR expression. Within the TAD, intronic enhancers recruit cell-type selective transcription factors and deletion of a pivotal enhancer element dramatically decreases CFTR expression, but has minor effect on its 3D structure.

Using affinity purification MS approaches, we have identified a novel role for casein kinase II (CKII) in the modification of the polymerase associated factor complex (PAF-C). Our data indicate that the facilitates chromatin transcription complex (FACT) interacts with CKII and may facilitate PAF complex phosphorylation. Posttranslational modification analysis of affinity-isolated PAF-C shows extensive CKII phosphorylation of all five subunits of PAF-C, although CKII subunits were not detected as interacting partners. Consistent with this, recombinant CKII or FACT-associated CKII isolated from cells can phosphorylate PAF-C in vitro, whereas no intrinsic kinase activity was detected in PAF-C samples. Significantly, PAF-C purifications combined with stable isotope labeling in cells (SILAC) quantitation for PAF-C phosphorylation from wild-type and CKII temperature-sensitive strains (cka1Δ cka2-8) showed that PAF-C phosphorylation at consensus CKII sites is significantly reduced in cka1Δ cka2-8 strains. Consistent with a role of CKII in FACT and PAF-C function, we show that decreased CKII function in vivo results in decreased levels of histone H2B lysine 123 monoubiquitylation, a modification dependent on FACT and PAF-C. Taken together, our results define a coordinated role of CKII and FACT in the regulation of RNA polymerase II transcription through chromatin via phosphorylation of PAF-C.

Mutations in hepatocyte nuclear factor 1 transcription factors (HNF1α/β) are associated with diabetes. These factors are well studied in the liver, pancreas and kidney, where they direct tissue-specific gene regulation. However, they also have an important role in the biology of many other tissues, including the intestine. We investigated the transcriptional network governed by HNF1 in an intestinal epithelial cell line (Caco2). We used chromatin immunoprecipitation followed by direct sequencing (ChIP-seq) to identify HNF1 binding sites genome-wide. Direct targets of HNF1 were validated using conventional ChIP assays and confirmed by siRNA-mediated depletion of HNF1, followed by RT-qPCR. Gene ontology process enrichment analysis of the HNF1 targets identified multiple processes with a role in intestinal epithelial cell function, including properties of the cell membrane, cellular response to hormones, and regulation of biosynthetic processes. Approximately 50% of HNF1 binding sites were also occupied by other members of the intestinal transcriptional network, including hepatocyte nuclear factor 4A (HNF4A), caudal type homeobox 2 (CDX2), and forkhead box A2 (FOXA2). Depletion of HNF1 in Caco2 cells increases FOXA2 abundance and decreases levels of CDX2, illustrating the coordinated activities of the network. These data suggest that HNF1 plays an important role in regulating intestinal epithelial cell function, both directly and through interactions with other intestinal transcription factors.

SUMMARY Spt6 is a histone chaperone that associates with RNA polymerase II and deposits nucleosomes in the wake of transcription. Although Spt6 has an essential function in nucleosome deposition, it is not known whether this function is influenced by post-translational modification. Here, we report that casein kinase II (CKII) phosphorylation of Spt6 is required for nucleosome occupancy at the 5′ ends of genes to prevent aberrant antisense transcription and enforce transcriptional directionality. Mechanistically, we show that CKII phosphorylation of Spt6 promotes the interaction of Spt6 with Spn1, a binding partner required for chromatin reassembly and full recruitment of Spt6 to genes. Our study defines a function for CKII phosphorylation in transcription and highlights the importance of post-translational modification in histone chaperone function., In Brief Dronamraju et al. show that the N terminus of Spt6 is phosphorylated by casein kinase II, which is required for proper Spt6-Spn1 interaction. CKII phosphorylation of Spt6 is pivotal to maintain nucleosome occupancy at the 5′ ends of genes, suppression of antisense transcription from the 5′ ends, and resistance to genotoxic agents.