Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause the inherited disorder cystic fibrosis (CF). Lung disease is the major cause of CF morbidity, though CFTR expression levels are substantially lower in the airway epithelium than in pancreatic duct and intestinal epithelia, which also show compromised function in CF. Recently developed small molecule therapeutics for CF are highly successful for one specific CFTR mutation and have a positive impact on others. However, the low abundance of CFTR transcripts in the airway limits the opportunity for drugs to correct the defective substrate. Elucidation of the transcriptional mechanisms for the CFTR locus has largely focused on intragenic and intergenic tissue-specific enhancers and their activating trans-factors. Here, we investigate whether the low CFTR levels in the airway epithelium result from the recruitment of repressive proteins directly to the locus. Using an siRNA screen to deplete ∼1500 transcription factors (TFs) and associated regulatory proteins in Calu-3 lung epithelial cells, we identified nearly 40 factors that upon depletion elevated CFTR mRNA levels more than 2-fold. A subset of these TFs was validated in primary human bronchial epithelial cells. Among the strongest repressors of airway expression of CFTR were Krüppel-like factor 5 and Ets homologous factor, both of which have pivotal roles in the airway epithelium. Depletion of these factors, which are both recruited to an airway-selective cis-regulatory element at −35 kb from the CFTR promoter, improved CFTR production and function, thus defining novel therapeutic targets for enhancement of CFTR.

Introduction

Tissue-specific regulation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which when mutated causes the common inherited disorder cystic fibrosis (CF), has been a topic of intensive investigation (reviewed in refs [13]). The results revealed key aspects of locus architecture, intergenic and intronic regulatory elements with cell-specific signatures, and a transcription factor (TF) network recruited to these elements reflecting the differentiated functions of the host cell. These features of CFTR are a paradigm for other genes with complex control mechanisms, which involve cis-regulatory elements outside the gene promoter. CFTR lies within a topologically associated domain (TAD) that is limited by regions occupied by the architectural proteins CCCTC-binding factor (CTCF) and cohesin complex [4,5]. Within the TAD, which extends from −80.1 kb 5′ to the CFTR promoter to +48.9 kb 3′ to the last coding base, cell-selective cis-regulatory elements are recruited to the gene promoter to drive gene expression (Figure 1). These elements are located both in introns and in intergenic regions, and several of them encompass tissue-specific enhancers. In the intestinal epithelium, the pioneer TF Forkhead box A2 (FOXA2) has a pivotal role in driving gene expression through intronic enhancers and in establishing the 3D structure of the active locus [4,6,7]. It may also have an important role in the recruitment of other TFs including hepatocyte nuclear factor 1 (HNF1) [8,9] and Caudal type homeobox 2 (CDX2) to the cis-regulatory elements. In the airway epithelium, two cis-elements outside the gene at −44 and −35 kb upstream of the gene promoter are important in conferring tissue-specific expression [1012]. Both the sites encompass airway-selective enhancers, which appear to be functionally linked, though they recruit different TFs. The −44 kb site is associated with an antioxidant response element (ARE) that may serve as an environmental/stress sensor. Under normal conditions, this site is occupied by repressor BTB and CNC homology 1, basic leucine zipper TF (Bach1), and v-Maf avian musculoaponeurotic fibrosarcoma oncogene homolog K (MafK) heterodimers. After natural antioxidant treatment (sulforaphane, SFN), nuclear factor, erythroid 2-like 2 (Nrf2) translocates into the nucleus and displaces these repressive factors and activates CFTR expression. Site-directed mutagenesis shows that both the ARE and an adjacent NF-κB-binding site are required for activation. The −35 kb enhancer is responsive to the immune-mediators interferon regulatory factor 1 and 2 (IRF1 and IRF2), IRF1 activating and IRF2 repressing the enhancer function. However, the functional complexity of this site and the requirement of other factors, including nuclear factor Y (NF-Y), to maintain active histone marks at the site implicate additional mechanisms underlying its function. Our goal was to reveal other key pathways and TFs that contribute to the control of CFTR expression in airway epithelial cells.

Histone modifications across the CFTR locus in airway epithelial cells.

Figure 1.
Histone modifications across the CFTR locus in airway epithelial cells.

UCSC genome browser graphic showing occupancy of repressive (H3K9me2, H3K9me3 and H3K27me3) and active (H3K4me3 and H3K27ac) histone modifications by ChIP-seq in Calu-3 and primary HBE cells. Blue triangles denote the TAD boundaries. The CFTR promoter is marked by a black arrowhead. Red and gray arrows denote repressive histone marks at cis-regulatory elements. Of note the promoter carries active marks (turquoise arrows) in Calu-3 and HBE cells but also repressive marks (purple arrow) in HBE cells. Cis-regulatory elements/DHS as reviewed in ref. [3].

Figure 1.
Histone modifications across the CFTR locus in airway epithelial cells.

UCSC genome browser graphic showing occupancy of repressive (H3K9me2, H3K9me3 and H3K27me3) and active (H3K4me3 and H3K27ac) histone modifications by ChIP-seq in Calu-3 and primary HBE cells. Blue triangles denote the TAD boundaries. The CFTR promoter is marked by a black arrowhead. Red and gray arrows denote repressive histone marks at cis-regulatory elements. Of note the promoter carries active marks (turquoise arrows) in Calu-3 and HBE cells but also repressive marks (purple arrow) in HBE cells. Cis-regulatory elements/DHS as reviewed in ref. [3].

Of direct relevance to this objective is one aspect of CFTR regulation that is not well understood, specifically how very low CFTR expression is maintained in the airway epithelium in comparison with the much higher levels evident in pancreatic duct, intestinal epithelium, and male genital ducts [1317]. Moreover, the developmental profile of CFTR suggests that the locus may be subject to repression, since transcript abundance is highest in the early to mid-trimester of human and sheep gestation and then appears to fall gradually to reach the low levels characteristic of the lung epithelium at birth [18]. To test the hypothesis that repressive TFs or other chromatin-associated factors (CAFs) were responsible for this phenomenon, we screened a library of siRNAs designed to target ∼1500 TFs and CAFs in Calu-3 cells. We identified ∼50 siRNAs that reproducibly increased CFTR expression levels more than 2-fold in replica screens. Factors with documented global roles in gene activation or repression, together with TFs with very low expression levels in Calu-3 and human bronchial epithelial (HBE) cells, according to our RNA-seq data (FPKM <1), were not evaluated further. Here, we describe 37 TFs, which upon depletion increase CFTR transcript levels and were not previously associated with regulation of CFTR expression. We show that several of these TFs also repress CFTR protein expression and directly regulate CFTR by recruitment to cis-regulatory elements at the locus, most notably the upstream airway-selective enhancers or the promoter. These data generate new targets and potential avenues to augment CFTR transcript and protein expression for therapeutic benefit.

Experimental

Cell culture

Calu-3 cells were obtained from ATCC and cultured by standard methods. Passage 1 (P1) primary bronchial epithelial cells were obtained under protocol #03-1396 approved by the University of North Carolina at Chapel Hill Biomedical Institutional Review Board. Informed consent was obtained from authorized representatives of all organ donors. Cells were grown on collagen-coated plastic in bronchial epithelial growth medium [19]. Passage 2 or 3 primary cells were used for the study.

TF siRNA screen and validation

The Dharmacon siGENOME SMARTpool siRNA Library — Human Transcription Factor (G-005805 Lot Number 13107) was used (GE Healthcare). This library comprises twenty 96-well plates with each well containing a pool of four siRNAs that target a single human TF. In total, 1528 TFs were targeted. Either 6 pMol of an siRNA pool or non-targeting negative control (D-001210-02-05) was reverse-transfected with Lipofectamine RNAiMax (ThermoFisher) into 25 000 Calu-3 cells in a 96-well plate in duplicate. After 72 h, Calu-3 cells were lysed for RNA extraction, DNase I-treated and cDNA-synthesized using the Cell-to-Ct Kit (Ambion). Untransfected Calu-3 cells were used as controls to ensure similar CFTR expression levels across all plates used in the screen. CFTR and β2 microglobulin (β2M) transcript levels were determined in duplicate using 4μl of cDNA in a multiplexed Taqman assay (CFTR probe 5′-FAM; β2M probe 5′-JOE) on a QuantStudio 6 Flex Real-Time PCR System (Applied BioSystems). CFTR transcript values were normalized to β2M and compared with the non-targeting negative controls to determine fold changes in CFTR expression. Select factors that showed an increase in CFTR/β2M >2-fold were repeated. For validation experiments, CFTR mRNA expression was then measured relative to the geometric mean of three normalizers β2M, phosphoglycerate kinase (PGK1) and β-actin (ACTB) Taqman primer/probe sets (Supplementary Table S1A) in optimized multiplex RT-qPCRs. All factors were validated with Dharmacon siRNA reagents and for the factors of most immediate interest Krüppel-like factor 5 (KLF5), Ets homologous factor (EHF) and Bromodomain containing protein 8 (BRD8), siRNAs from Ambion (LT) were also used (Figures 2A and 4A). SYBR green RT-qPCR assays were used to confirm siRNA-mediated depletion of each factor (Supplementary Figure S1 and Table S1C). The negative control siRNA value is taken as 1 and specific siRNA levels for each factor compared with that.

Depletion of multiple TFs enhances CFTR transcript levels in Calu-3 cells.

Figure 2.
Depletion of multiple TFs enhances CFTR transcript levels in Calu-3 cells.

siRNA-mediated depletion (KD) of (A) BRD8, EHF, KLF5 and (B) ING2, NR2F2, enhances CFTR mRNA abundance. RT-qPCR measurements of CFTR normalized to the geometric mean of three HKG controls, β2M, PGK and ACTB, and shown relative to NC. Bar shows average of three experiments with standard error of the mean. Key: ***P ≤ 0.001, ****P ≤ 0.0001 by an unpaired two-tailed Student's t-test.

Figure 2.
Depletion of multiple TFs enhances CFTR transcript levels in Calu-3 cells.

siRNA-mediated depletion (KD) of (A) BRD8, EHF, KLF5 and (B) ING2, NR2F2, enhances CFTR mRNA abundance. RT-qPCR measurements of CFTR normalized to the geometric mean of three HKG controls, β2M, PGK and ACTB, and shown relative to NC. Bar shows average of three experiments with standard error of the mean. Key: ***P ≤ 0.001, ****P ≤ 0.0001 by an unpaired two-tailed Student's t-test.

CFTR protein detection by western blot

To assay the effect of siRNA depletion TFs on CFTR protein expression, transfections were done as above, and at 72 h post-transfection, whole cell lysates were collected using NET buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, and 1 mM Na2EDTA) supplemented with Triton X-100 and protease inhibitors (Sigma). Protein concentration was determined by the Bradford assay. SDS sample loading buffer with β-mercaptoethanol was added and samples were heated at 55°C for 15 min. These samples were resolved by SDS–PAGE, transferred to Immobilon membrane, and western blots were probed with anti-CFTR 596 (Cystic Fibrosis Foundation) and β-tubulin (T4026, Sigma–Aldrich) with ECL detection. Each western blot was probed only once. Films were scanned by densitometry, and the intensities of CFTR and β-tubulin were compared using Photoshop CS6 Extended and/or ImageJ.

ChIP-seq and ChIP-qPCR

Antibodies used in ChIP-seq and ChIP-qPCR were against EHF (Clone 5A.5) [20] and KLF5 (Active motif 61100; Santa Cruz sc-398470). ChIP-seq was performed as described previously [21,22]. For primers, see Supplementary Table S1B. EHF ChIP-seq data are deposited at GEO (GSE:85403).

Chloride conductance assay

Assays were performed as described previously [23], and calibration of intracellular MQAE [N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide] was performed as in ref. [24]. Briefly, once confluent, Calu-3 cells growing on 96-well tissue culture plates were loaded with 1 mM MQAE in DMEM low-glucose culture medium with 10% FCS in 5% CO2, overnight at 37°C. Cells were washed and then equilibrated in a high chloride, loading buffer (137 mM NaCl, 2.7 mM KCl, 0.7 mM CaCl2, 1.5 mM KH2PO4, and 8.1 mM Na2HPO4, pH 7.4) for 1 h. This buffer was then replaced with a high nitrate, efflux buffer (137 mM NaNO3, 2.7 mM KNO3, 0.7 mM Ca(NO3)2, 1.5 mM KH2PO4, and 8.1 mM Na2HPO4, pH 7.4), with or without 5 μM the CFTR-activator Forskolin. MQAE fluorescence readings at 360 nm excitation/460 nm emission were immediately recorded every minute for 10 min in a Spectramax M5 fluorescence plate reader (Molecular Devices).

Results

Repressive histone marks are evident at cis-regulatory elements in the CFTR locus in airway epithelial cells

To test the hypothesis that the low abundance of CFTR transcripts in the airway epithelium was a consequence of the recruitment of transcriptional repressors, we first sought evidence for repressive histone modifications at the locus. We inspected our genome-wide ChIP-seq data for active (H3K27ac and H3K4me3) and repressive (H3K9me2, H3K9me3 and H3K27me3) histone marks in Calu-3 lung adenocarcinoma cells and primary HBE cells [21,22,25]. These data revealed repressive marks at many known cis-regulatory elements in addition to other sites within the CFTR locus (red and gray arrows in Figure 1).

siRNA screen for factors that repress CFTR expression

Next, to identify factors that might recruit negative histone marks to the locus, we performed an siRNA screen for repressive TFs in Calu-3 cells. The Dharmacon siRNA Library targeting human TFs includes TFs, some CAFs and certain other factors associated with transcriptional regulation. Here, we consider all factors together and do not subdivide them according to specific functions (activating or repressing) since these are often cell-context dependent. In the first replicated screen of siRNAs against 1528 TFs, we applied a filter to the data analysis to remove factors with low expression levels (FPKM <1) in Calu-3 cells, which reduced the total factors analyzed further to 1058. CFTR expression after specific factor depletion was normalized to β2-microglobulin transcript levels and compared with replicated (n = 4) negative control (NC) siRNA values. Fifty-four factors were identified that upon siRNA-mediated depletion increased the normalized (CFTR/β2M compared with NC2) ratio of CFTR expression more than 2-fold. For each siRNA plate, positive controls for transfection efficiency were provided by siRNAs specific for FOXA1 and FOXA2, which we showed previously to be activating TFs at the CFTR locus [6,7]. An additional replica experiment was performed on the 54 factors with two independent transfections to generate n = 4 assays on each factor. The ratio of CFTR to β2 microglobulin compared with NC was then averaged across experiments and siRNAs against TFs that no longer caused a CFTR change of >2-fold were excluded from further analysis, reducing the number to 50. Thirty-seven of these are shown in Table 1. Also shown in Table 1 are the expression levels (FPKM) of each factor in RNA-seq data (average from n = 3) from Calu-3 and HBE cells. In all further experiments on a smaller number of candidate factors that repressed CFTR expression, siRNA-mediated depletion of each factor was confirmed by RT-qPCR (Supplementary Figure S1).

Table 1
TFs that when depleted increase CFTR mRNA levels ≥2-fold in Calu-3 cells
Gene CFTR/β2M change1 Calu-3 FPKM** HBE FPKM** 
ING2 3.00 7.38 10.10 
ANKFY1 2.83 8.01 11.52 
NR2F2 2.71 44.65 10.21 
BRD8 2.58 37.76 9.47 
KLF5 2.58 79.54 122.97 
ZNF148 2.52 13.44 6.93 
TCF7L2 2.45 22.52 4.15 
TGIF1 2.41 47.48 38.61 
VGLL1 2.39 80.18 9.71 
TLE1 2.36 30.09 10.32 
ANKRD54 2.34 16.48 24.21 
EHF 2.32 72.53 189.09 
SMARCC2 2.31 28.88 17.51 
MDM2 2.30 14.84 35.66 
BRCA1 2.29 8.00 2.89 
ANKZF1 2.29 22.62 23.95 
GTF2IRD1 2.28 11.93 24.06 
ATF5 2.25 34.38 59.74 
ECSIT 2.25 20.23 13.86 
SATB2 2.24 1.146 2.69 
SREBF2 2.23 131.37 67.47 
HDAC1 2.19 206.77 42.60 
SMARCE1 2.17 315.31 39.85 
BTG1 2.16 52.28 34.37 
PLRG1 2.14 36.62 21.04 
IRF2BP1 2.13 15.08 7.32 
RBAK 2.13 4.05 8.48 
CASKIN1 2.13 5.51 5.59 
FOXE1 2.11 1.21 5.26 
GATAD2A 2.10 40.31 14.27 
ZNF232 2.07 3.63 4.23 
CDKN2B 2.07 63.80 16.03 
TARBP1 2.05 9.47 11.10 
TEAD4 2.05 21.13 1.91 
NR1D1 2.04 23.90 22.81 
ZNF33A 2.02 6.19 5.81 
TAF4B 2.00 3.82 1.62 
Gene CFTR/β2M change1 Calu-3 FPKM** HBE FPKM** 
ING2 3.00 7.38 10.10 
ANKFY1 2.83 8.01 11.52 
NR2F2 2.71 44.65 10.21 
BRD8 2.58 37.76 9.47 
KLF5 2.58 79.54 122.97 
ZNF148 2.52 13.44 6.93 
TCF7L2 2.45 22.52 4.15 
TGIF1 2.41 47.48 38.61 
VGLL1 2.39 80.18 9.71 
TLE1 2.36 30.09 10.32 
ANKRD54 2.34 16.48 24.21 
EHF 2.32 72.53 189.09 
SMARCC2 2.31 28.88 17.51 
MDM2 2.30 14.84 35.66 
BRCA1 2.29 8.00 2.89 
ANKZF1 2.29 22.62 23.95 
GTF2IRD1 2.28 11.93 24.06 
ATF5 2.25 34.38 59.74 
ECSIT 2.25 20.23 13.86 
SATB2 2.24 1.146 2.69 
SREBF2 2.23 131.37 67.47 
HDAC1 2.19 206.77 42.60 
SMARCE1 2.17 315.31 39.85 
BTG1 2.16 52.28 34.37 
PLRG1 2.14 36.62 21.04 
IRF2BP1 2.13 15.08 7.32 
RBAK 2.13 4.05 8.48 
CASKIN1 2.13 5.51 5.59 
FOXE1 2.11 1.21 5.26 
GATAD2A 2.10 40.31 14.27 
ZNF232 2.07 3.63 4.23 
CDKN2B 2.07 63.80 16.03 
TARBP1 2.05 9.47 11.10 
TEAD4 2.05 21.13 1.91 
NR1D1 2.04 23.90 22.81 
ZNF33A 2.02 6.19 5.81 
TAF4B 2.00 3.82 1.62 
*

Denotes average of duplicate or ** triplicate assays. Factors highlighted in bold were studied further.

Validation of TFs that repress CFTR expression

Among the factors with the most repressive effect on CFTR transcript levels (Table 1) were several of particular interest: firstly, inhibitor of growth family member 2 (ING2), which associates with both histone acetyltransferases (HATs) and histone deacetylases (HDACs) to modulate gene expression. It is a component of the Sin3A deacetylase (repressive) complex, which interacts with HDAC1 and HDAC2 [26], and may repress CFTR transcription by several mechanisms [10,27]. Next, nuclear receptor subfamily 2 group F, member 2 (NR2F2) is a member of steroid hormone superfamily of nuclear receptors, which were implicated in CFTR regulation previously [28,29]. BRD8, also a strong repressor of CFTR transcription in the present study, interacts with the thyroid hormone receptor, probably as a co-activator, and amplifies thyroid hormone-dependent activation of genes [30,31]. KLF5 is involved in lung development and function [32] and is a candidate CFTR transcriptional regulator since we showed previously that its motif is overrepresented in open chromatin in human tracheal epithelial (HTE) cells [33]. Forkhead box E1 (FOXE1) is involved in thyroid morphogenesis/function and CFTR is expressed in the thyroid epithelium. Both TGF-β-induced factor homeobox 1 (TGIF1) and the cofactor transducin-like enhancer of split 1 (TLE1) are logical candidates for a role in CFTR expression, which may be regulated by immune and inflammatory response in the airway [2,10]. Moreover, the important role of EHF in the human lung epithelium was reported in our previous work [21,22]. Validation of the significant repressive effect of BRD8, EHF, KLF5, ING2 and NR2F2 on CFTR mRNA expression in Calu-3 cells was provided by three independent siRNA transfection experiments (Figure 2). CFTR expression was normalized to the geometric means of three housekeeping gene (HKG) control transcripts, β2M, PGK1 and ACTB, to ensure that results were not influenced by siRNAs also altering the expression of the normalizer. As before, siRNAs specific for FOXA1/FOXA2 provided the transfection control, reducing CFTR mRNA expression by more than 50%. Of note for BRD8, EHF and KLF5, siRNA from a different supplier (Ambion) with the relevant negative control was used to validate the earlier results obtained using the Dharmacon siRNAs (Figure 2A). The most repressive factors were KLF5 (6-fold), EHF and BRD8 (both ∼3-fold).

Impact of TF depletion on CFTR protein abundance in Calu-3 cells

Little is known about the correlation between CFTR mRNA levels and the abundance of CFTR protein, so we could not assume that an increase in CFTR mRNA abundance would be therapeutically useful. To determine the impact on CFTR protein abundance of depletion of five factors that repress CFTR mRNA levels (BRD8, EHF, KLF5, ING2 and NR2F2), western blots were performed. Protein was extracted from Calu-3 cells 72 h after TF siRNA delivery, and CFTR was detected with antibody 596 (CFF) and normalized to β-tubulin levels on the same membrane. Figure 3A shows representative western blots, where depletion of BRD8, EHF, KLF5, ING2 and NR2F2 augments CFTR protein expression in three replicates of Calu-3 cells in comparison with non-targeting control siRNAs. Again the impact on CFTR protein of repressing KLF5 was the greatest effect. Quantification of data from ≥3 western blots from independent experiments is shown in Figure 3B, where CFTR abundance relative to β-tubulin is compared with the relevant negative control (Ambion or Dharmacon) for each siRNA, though only the Ambion NC is shown.

Depletion of KLF5, EHF, ING2, NR2F2 and BRD8 enhances CFTR protein abundance in Calu-3 cells.

Figure 3.
Depletion of KLF5, EHF, ING2, NR2F2 and BRD8 enhances CFTR protein abundance in Calu-3 cells.

siRNA-mediated depletion of BRD8, EHF, KLF5, compared with NCA and ING2, NR2F2, FOXA1/A2 compared with NCD. (A) Western blot probed with CFTR antibody 596 (CFF) or β-tubulin as a loading control. FOXA1/A2 are known activators of CFTR expression (NCA = Ambion, NCD = Dharmacon negative controls). (B) Quantification of densitometry from ≥3 western blots shows pooled data on the impact of depletion of each factor on CFTR protein abundance. Bar shows average with standard error of the mean. Key: ns: not significant. P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001 by an unpaired two-tailed Student's t-test.

Figure 3.
Depletion of KLF5, EHF, ING2, NR2F2 and BRD8 enhances CFTR protein abundance in Calu-3 cells.

siRNA-mediated depletion of BRD8, EHF, KLF5, compared with NCA and ING2, NR2F2, FOXA1/A2 compared with NCD. (A) Western blot probed with CFTR antibody 596 (CFF) or β-tubulin as a loading control. FOXA1/A2 are known activators of CFTR expression (NCA = Ambion, NCD = Dharmacon negative controls). (B) Quantification of densitometry from ≥3 western blots shows pooled data on the impact of depletion of each factor on CFTR protein abundance. Bar shows average with standard error of the mean. Key: ns: not significant. P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001 by an unpaired two-tailed Student's t-test.

Depletion of KLF5, BRD8 and ING2 enhances CFTR transcript levels in HBE cells.

Figure 4.
Depletion of KLF5, BRD8 and ING2 enhances CFTR transcript levels in HBE cells.

siRNA-mediated depletion (KD) of (A) BRD8, EHF, KLF5 and (B) ING2, NR2F2 enhances CFTR mRNA abundance. RT-qPCR measurements of CFTR normalized to the geometric mean of three HKG controls, β2M, PGK, and ACTB and shown relative to NC. Bar shows standard error of the mean. Key: ns: not significant. P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001 by an unpaired two-tailed Student's t-test.

Figure 4.
Depletion of KLF5, BRD8 and ING2 enhances CFTR transcript levels in HBE cells.

siRNA-mediated depletion (KD) of (A) BRD8, EHF, KLF5 and (B) ING2, NR2F2 enhances CFTR mRNA abundance. RT-qPCR measurements of CFTR normalized to the geometric mean of three HKG controls, β2M, PGK, and ACTB and shown relative to NC. Bar shows standard error of the mean. Key: ns: not significant. P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001 by an unpaired two-tailed Student's t-test.

Impact of TF depletion in primary HBE cells

Among critical cell types that will be a target of new CF therapeutics are the epithelial cells lining the human lung. Hence, we next investigated whether the TFs that were the most effective repressors of CFTR mRNA and protein expression in Calu-3 cells had similar properties in HBE cells. siRNAs specific for BRD8, EHF, KLF5, ING2, NR2F2 and relevant NCs were transfected into p2 primary HBE cells and RNA extracted after 96 h. Consistent with the results in Calu-3 cells, depletion of BRD8, KLF5 and ING2 caused significant enhancement of the CFTR to normalizer ratio (geometric mean of three control transcripts, β2M, PGK1 and ACTB) in HBE cells (n = 3 donors) (Figure 4). Depletion of EHF and NR2F2 increased CFTR expression somewhat though values did not reach statistical significance. CFTR expression levels in HBE cells are too low to reliably assay the effect of siRNA depletion on CFTR protein abundance by western blot.

EHF and KLF5 regulate CFTR expression through an upstream cis-regulatory element

Though it is likely that many of the factors that activate or repress CFTR expression do so indirectly, through complex networks of TFs, we were particularly interested in those that targeted the CFTR locus through direct occupancy of its cis-regulatory elements. We recently revealed the role of EHF in modulating pathways that contribute to lung disease severity in CF patients [21,22] and in that context we generated genome-wide occupancy data for EHF by ChIP-seq. Inspection of these data suggests that EHF may directly regulate CFTR in HBE cells. Peaks of EHF occupancy are seen at previously characterized regulatory elements for CFTR, in DNase I hypersensitive sites (DHS) −35 kb from the transcription start site [10], within intron 23 (legacy nomenclature) [12,16] and at 48.9 kb 3′ to the locus [4] (Figure 5A). Though the impact of EHF depletion on CFTR expression was not significant in HBE cells, this may be due to the substantial donor-to-donor variation in EHF levels that was observed in the primary cells ([22], data not shown). Since, CFTR protein is at very low abundance in HBE cells grown on plastic, below the levels that can be reliably detected by western blot, the effect of EHF on its expression was again investigated in Calu-3 cells. EHF was depleted by siRNA in Calu-3 cells, and CFTR mRNA and protein expression were measured by RT-qPCR and western blot, respectively (Figure 5B,C). Confirming our initial screening data that showed EHF as a repressor of the CFTR gene, EHF depletion significantly increased CFTR mRNA abundance, and this correlated with increased CFTR protein.

Repression of CFTR expression by EHF.

Figure 5.
Repression of CFTR expression by EHF.

(A) UCSC genome browser graphic showing EHF ChIP-seq peaks (yellow) at the CFTR locus. Legacy nomenclature for CFTR DHS is shown. HBE DHS track from ref. [4]. (B) CFTR mRNA expression relative to β2M control in NC and EHF-depleted (siRNA) Calu-3 cells. (C) CFTR protein (antibody 596) relative to β-tubulin, assayed by scanning western blots of NC and EHF-depleted (siRNA) Calu-3 cells. (B and C) Bars show the average of experiments (B, n = 3; C, n = 4) with SEM. *P < 0.05; ***P < 0.001 by an unpaired two-tailed Student's t-test.

Figure 5.
Repression of CFTR expression by EHF.

(A) UCSC genome browser graphic showing EHF ChIP-seq peaks (yellow) at the CFTR locus. Legacy nomenclature for CFTR DHS is shown. HBE DHS track from ref. [4]. (B) CFTR mRNA expression relative to β2M control in NC and EHF-depleted (siRNA) Calu-3 cells. (C) CFTR protein (antibody 596) relative to β-tubulin, assayed by scanning western blots of NC and EHF-depleted (siRNA) Calu-3 cells. (B and C) Bars show the average of experiments (B, n = 3; C, n = 4) with SEM. *P < 0.05; ***P < 0.001 by an unpaired two-tailed Student's t-test.

Next, we examined ChIP-seq data for KLF5 in Calu-3 cells and also observed a peak of occupancy for this factor at the −35 kb DHS in replicate experiments (Figure 6A), though not at the intron 23 and +48.9 kb cis-elements which bound EHF. These data suggest that the −35 kb site may encompass an element with a key role in fine-tuning airway expression of CFTR.

Depletion of KLF5 enhances CFTR chloride channel activity in Calu-3 cells.

Figure 6.
Depletion of KLF5 enhances CFTR chloride channel activity in Calu-3 cells.

(A) UCSC genome browser graphic showing KL5 ChIP-seq peak at the −35 kb airway-selective DHS at the CFTR locus (blue arrow), with the CFTR gene promoter highlighted (gray arrow). (B) Western blot probed with CFTR antibody 596 and β-tubulin as a loading control shows the impact of siRNA-mediated depletion of KLF5 compared with NC on CFTR abundance in representative samples used for MQAE assays shown in (C). (C) MQAE assay shows change in fluorescence with time, with (cAMP) or without (V) the cAMP agonist forskolin. NC siRNA (V) blue, (cAMP) red; KLF5 siRNA (V) gray, (cAMP) teal.

Figure 6.
Depletion of KLF5 enhances CFTR chloride channel activity in Calu-3 cells.

(A) UCSC genome browser graphic showing KL5 ChIP-seq peak at the −35 kb airway-selective DHS at the CFTR locus (blue arrow), with the CFTR gene promoter highlighted (gray arrow). (B) Western blot probed with CFTR antibody 596 and β-tubulin as a loading control shows the impact of siRNA-mediated depletion of KLF5 compared with NC on CFTR abundance in representative samples used for MQAE assays shown in (C). (C) MQAE assay shows change in fluorescence with time, with (cAMP) or without (V) the cAMP agonist forskolin. NC siRNA (V) blue, (cAMP) red; KLF5 siRNA (V) gray, (cAMP) teal.

KLF5 depletion enhances CFTR channel activity

To be of therapeutic use, the inhibition of a repressive TF that increased CFTR mRNA and protein abundance would also need to enhance activity of the CFTR chloride channel activity in relevant epithelial cells. Since KLF5 was apparently the TF that most robustly repressed CFTR levels, we tested the effect of its depletion on CFTR channel activity by fluorescent MQAE assays in Calu-3 cells. Enhanced CFTR protein levels after siRNA-mediated depletion of KLF5 (KLF5si) are shown by western blot (Figure 6B) in one of the samples used in the MQAE assays shown in Figure 6C. In replicate experiments, we observed that not only did siRNA-mediated depletion of KLF5 increase basal chloride channel activity in Calu-3 cells (gray compared with blue traces), it also substantially elevated forskolin-activated CFTR conductance (teal compared with red) (Figure 6C). The specificity of the forskolin response was confirmed by CFTR inhibitor inh172 (Supplementary Figure S2).

Discussion

The CFTR locus is among the best-studied single gene loci in the human genome, in part, due to its association with a common genetic disease (reviewed in refs [13]). Its control mechanisms provide many insights into how cis-regulatory elements in non-coding regions of the genome organize chromatin structure and co-ordinate tissue-specific expression. Most studies to date focused on pathways that activate CFTR gene expression and the associated TF repertoire. Here, we identify a network of TFs that repress CFTR transcription in airway epithelial cells, which maintain low levels of expression in comparison with epithelial cells in the digestive tract. These repressive TFs function both directly through cis-elements at the locus and indirectly by other mechanism including through other TFs and possibly microRNAs. The data reveal an important component of the complex fine-tuning of CFTR expression, which is directly relevant to the development of novel therapeutics for CF.

Repression of ∼100 factors increased CFTR levels between 1.6- and 2-fold, and of ∼50 factors >2-fold in the initial replicated screen. However, for practical reasons, we chose the arbitrary >2-fold cut-off for further analysis, though it is possible that factors with an important role in CFTR regulation are in the 1.6–2-fold group. Among the 37 factors that were validated to repress CFTR transcription more than 2-fold, a smaller group is of particular interest due either to their relevance to CF biology and pathology or their status as therapeutic targets in other contexts. For example, NR2F2 is an orphan (no endogenous ligand identified) nuclear receptor in the steroid thyroid hormone superfamily. Several members of this superfamily are already targets for pharmacological intervention due to their potential role in cancer [34]. Another CFTR repressor is BRD8, which interacts with the thyroid hormone receptor and may function as a co-activator of hormone-activated nuclear receptors. BRD8 contains two bromodomains, protein modules that specifically recognize acetylated lysine residues on histone tails and other CAFs. The distinct hydrophobic cleft within bromodomains makes them attractive targets for synthetic small molecule drugs [3537]. Indeed, several clinical studies using small molecule inhibitors of bromodomains are currently underway, and another orphan bromodomain protein BRD9 was recently effectively targeted by this approach [38].

Human KLF5, originally cloned from placenta as BTEB2 (basic transcription element binding protein 2 [39]), is a zinc finger (GC-box) TF with key roles in multiple tissues. It is a member of the Krüppel-like family of TFs and its mouse homolog was initially named IKlf5 due to its abundance in the digestive tract. It showed developmental regulation, being enriched in early endoderm [40], though its expression was restricted to the intestinal epithelium with highest levels in intestinal crypt cells [41]. Later studies also found Klf5 to be developmentally regulated in the mouse bronchi and trachea [42] and to be involved in β-catenin-independent Wnt-1 signaling [43]. Most relevant to our observations on the role of KLF5 in repression of CFTR in lung epithelial cells are data showing that Klf5 is required for morphogenesis and function of the perinatal mouse lung [32]. Conditional loss of Klf5 from the respiratory epithelium caused mice to die immediately after birth due to respiratory distress, in part caused by immaturity of the respiratory epithelium and decreased surfactant levels. Loss of KLF5 from bronchial epithelial cells was associated with reduced levels of the CAAT/enhancer-binding protein alpha (CEBPα) and secretoglobulin family 1A member 1 (SCGB1A1/CCSP) TFs, though probably through an indirect mechanism [32]. The impact of loss on Klf5 on other activating TFs was not investigated in detail. MatInspector analysis of the airway-selective −35 kb cis-element in the CFTR locus shows three predicted KLF-binding motifs (EKLF or GKLF) though none specifically for KLF5, despite the observed peak of occupancy of this factor in Calu-3 ChIP-seq data. This raises the possibility that KLF5 is recruited to this site by another DNA-binding protein, through which it exerts its repressive action. Most studies on KLF5 focus on its role as a transcriptional activator and it is known to be a core regulator of intestinal oncogenesis [44]. KLF5 is down-regulated during epithelial–mesenchymal transition (EMT) and its loss induces EMT in the absence of EGF/TGF-β activation of EMT. In this context, KLF5 maintains epithelial characteristics by transcriptionally activating miR-200 family member in epithelial cells [45]. Interestingly, miR-200b was recently shown to down-regulate CFTR expression during hypoxia [46], suggesting an additional pathway for repression of CFTR by KLF5.

EHF was originally cloned from mouse pituitary somatotroph tumors [47]. In parallel with the observations of KLF5 as a repressive factor acting through miRNAs, EHF represses miR-424 in normal prostate epithelial cells. However, miR-424 is up-regulated in prostate tumors and is associated with the aggressive features of the tumor, since it impairs the E3 ubiquitin ligase COP1 and in turn activates STAT3 [48]. Prostate tumors show an inverse correlation between EHF levels and miR-424. Though EHF is quite well studied in epithelial cancers, its role in normal epithelial cells has received less attention. It is involved in the wound response in the mouse cornea [49] and in differentiation of the skin [50], and it regulates important functions of the airway epithelium including wound repair, inflammation and barrier function [21,22]. We also showed by ChIP-seq for EHF and RNA-seq after EHF depletion in primary HBE cells that EHF directly regulates many other TFs with a key role in the airway epithelium [22]. Among genes with nearby peaks of EHF occupancy in replicated EHF ChIP-seq datasets and/or differentially expressed genes (DEG) after EHF depletion in primary HBE cells were GATA-binding protein 6 (GATA6), HOP Homeobox (HOPX), KLF5, SAM-pointed domain containing ETS TF (SPDEF), retinoic acid receptor beta (RARB), FOXA1 and FOXA2. HOPX, KLF5 and RARB abundance significantly increased after EHF depletion and SPDEF levels significantly decreased. EHF occupancy at intergenic sites 5′ of the HOPX, RARB and FOXA1 genes and at the promoter of SPDEF was confirmed by ChIP-qPCR in primary HBE cells. We also showed that FOXA1 co-regulates gene expression with EHF [22].

Our observation that EHF represses CFTR expression may also be pertinent to understanding the mechanisms underlying the association of modifier genes with CF lung disease severity [51,52]. In these genome-wide association studies among the highest P-value single-nucleotide polymorphisms (SNPs) correlating with CF lung phenotype were several within a non-coding (intergenic) region of chromosome 11p13. Though the relationship of these SNPs to expression of nearby loci has yet to be determined, they appear to lie within the same TAD or sub-TAD as the EHF locus [25], suggesting a connection with cis-regulatory elements for this gene.

This remarkable complexity of interactions in the TF network in airway epithelial cells makes direct correlation between the role of a single TF in the regulation of expression of CFTR, or indeed any locus, a major challenge. However, since a major therapeutic goal is to identify TFs that repress CFTR expression, in order to compromise their activity, whether they do so directly or indirectly or both, may not be important. Of more concern is the involvement of KLF5 and EHF, together with several other CFTR repressive TFs identified in the present study, in epithelial cancers. This will necessitate a highly focused approach to inhibiting recruitment of the repressive factors to specific regulatory elements at the CFTR locus. For example, targeting the airway cis-regulatory element for CFTR at −35 kb with specific guide RNAs, Cas9 and a transcriptional activator (reviewed in refs [53,54]) could be used to nudge this element toward enhancer function and prevent the recruitment of repressive factors.

Abbreviations

     
  • ACTB

    β-actin

  •  
  • ARE

    antioxidant response element

  •  
  • BRD8

    Bromodomain containing protein 8

  •  
  • CAFs

    chromatin-associated factors

  •  
  • CF

    cystic fibrosis

  •  
  • CFTR

    cystic fibrosis transmembrane conductance regulator

  •  
  • DHS

    DNase I hypersensitive site

  •  
  • EHF

    Ets homologous factor

  •  
  • EMT

    epithelial–mesenchymal transition

  •  
  • FOXA2

    Forkhead box A2

  •  
  • FOXE1

    Forkhead box E1

  •  
  • FPKM

    fragments per kilobase of transcript per million mapped reads

  •  
  • HBE

    human bronchial epithelial

  •  
  • HDACs

    histone deacetylases

  •  
  • HKG

    housekeeping gene

  •  
  • ING2

    inhibitor of growth family member 2

  •  
  • IRF1 and IRF2

    interferon regulatory factors 1 and 2

  •  
  • KLF5

    Krüppel-like factor 5

  •  
  • MQAE

    N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide

  •  
  • NC

    negative control

  •  
  • NR2F2

    nuclear receptor subfamily 2 group F, member 2

  •  
  • PGK

    phosphoglycerate kinase

  •  
  • RARB

    retinoic acid receptor beta

  •  
  • SNPs

    single-nucleotide polymorphisms

  •  
  • SPDEF

    SAM-pointed domain containing ETS TF

  •  
  • TAD

    topologically associated domain

  •  
  • TFs

    transcription factors

  •  
  • TGIF1

    TGFβ-induced factor homeobox 1

  •  
  • TLE1

    transducin-like enhancer of split 1

  •  
  • β2M

    β2 microglobulin

Author Contribution

M.J.M., S-H.L., S.L.F., and J.A.B. performed experiments and analyzed data. A.H. analyzed data and wrote the manuscript. All authors edited and approved the manuscript.

Funding

This work was funded by the Cystic Fibrosis Foundation [Harris14G0, Harris 16G0 and 15XX0]; National Institutes of Health [R01HL094585, R01HD068901 and R01HL117843 (PI: A.H.); F30HL124925 (S.F.)].

Acknowledgments

The authors thank Drs S. Ghosh and A. Hoffman for generating ChIP-seq data for negative histone marks in Calu-3 and HBE cells; Drs C.J. Ott and J.L. Kerschner for helpful discussions; also Dr S. Randell and colleagues for HBE cells, which were provided with support from NIH [P30DK065988]; Cystic Fibrosis Foundation [BOUCHE15R0], and for contributions.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

References

References
1
Ott
,
C.J.
,
Blackledge
,
N.P.
,
Leir
,
S.-H.
and
Harris
,
A.
(
2009
)
Novel regulatory mechanisms for the CFTR gene
.
Biochem. Soc. Trans.
37
,
843
848
2
Gillen
,
A.E.
and
Harris
,
A
. (
2012
)
Transcriptional regulation of CFTR gene expression
.
Front. Biosci.
4
,
587
592
3
Gosalia
,
N.
and
Harris
,
A
. (
2015
)
Chromatin dynamics in the regulation of CFTR expression
.
Genes
6
,
543
558
4
Yang
,
R.
,
Kerschner
,
J.L.
,
Gosalia
,
N.
,
Neems
,
D.
,
Gorsic
,
L.K.
,
Safi
,
A.
et al. 
(
2016
)
Differential contribution of cis-regulatory elements to higher order chromatin structure and expression of the CFTR locus
.
Nucleic Acids Res.
44
,
3082
3094
5
Smith
,
E.M.
,
Lajoie
,
B.R.
,
Jain
,
G.
and
Dekker
,
J.
(
2016
)
Invariant TAD boundaries constrain cell-type-specific looping interactions between promoters and distal elements around the CFTR locus
.
Am. J. Hum. Genet.
98
,
185
201
6
Kerschner
,
J.L.
and
Harris
,
A.
(
2012
)
Transcriptional networks driving enhancer function in the CFTR gene
.
Biochem. J.
446
,
203
212
7
Kerschner
,
J.L.
,
Gosalia
,
N.
,
Leir
,
S.-H.
and
Harris
,
A.
(
2014
)
Chromatin remodeling mediated by the FOXA1/A2 transcription factors activates CFTR expression in intestinal epithelial cells
.
Epigenetics
9
,
557
565
8
Mouchel
,
N.
,
Henstra
,
S.A.
,
McCarthy
,
V.A.
,
Williams
,
S.H.
,
Phylactides
,
M.
and
Harris
,
A.
(
2004
)
HNF1alpha is involved in tissue-specific regulation of CFTR gene expression
.
Biochem. J.
378
,
909
918
9
Ott
,
C.J.
,
Suszko
,
M.
,
Blackledge
,
N.P.
,
Wright
,
J.E.
,
Crawford
,
G.E.
and
Harris
,
A.
(
2009
)
A complex intronic enhancer regulates expression of the CFTR gene by direct interaction with the promoter
.
J. Cell Mol. Med.
13
,
680
692
10
Zhang
,
Z.
,
Leir
,
S.-H.
and
Harris
,
A.
(
2013
)
Immune mediators regulate CFTR expression through a bifunctional airway-selective enhancer
.
Mol. Cell. Biol.
33
,
2843
2853
11
Zhang
,
Z.
,
Leir
,
S.H.
and
Harris
,
A.
(
2015
)
Oxidative stress regulates CFTR gene expression in human airway epithelial cells through a distal antioxidant response element
.
Am. J. Respir. Cell Mol. Biol.
52
,
387
396
12
Zhang
,
Z.
,
Ott
,
C.J.
,
Lewandowska
,
M.A.
,
Leir
,
S.H.
and
Harris
,
A.
(
2012
)
Molecular mechanisms controlling CFTR gene expression in the airway
.
J. Cell. Mol. Med.
16
,
1321
1330
13
Harris
,
A.
,
Chalkley
,
G.
,
Goodman
,
S.
and
Coleman
,
L.
(
1991
)
Expression of the cystic fibrosis gene in human development
.
Development
113
,
305
310
PMID:
[PubMed]
14
Crawford
,
I.
,
Maloney
,
P.C.
,
Zeitlin
,
P.L.
,
Guggino
,
W.B.
,
Hyde
,
S.C.
,
Turley
,
H.
et al. 
(
1991
)
Immunocytochemical localization of the cystic fibrosis gene product CFTR
.
Proc. Natl Acad. Sci. U.S.A.
88
,
9262
9266
15
Hyde
,
K.
,
Reid
,
C.J.
,
Tebbutt
,
S.J.
,
Weide
,
L.
,
Hollingsworth
,
M.A.
and
Harris
,
A.
(
1997
)
The cystic fibrosis transmembrane conductance regulator as a marker of human pancreatic duct development
.
Gastroenterology
113
,
914
919
16
Ott
,
C.J.
,
Blackledge
,
N.P.
,
Kerschner
,
J.L.
,
Leir
,
S.-H.
,
Crawford
,
G.E.
,
Cotton
,
C.U.
et al. 
(
2009
)
Intronic enhancers coordinate epithelial-specific looping of the active CFTR locus
.
Proc. Natl Acad. Sci. U.S.A.
106
,
19934
19939
17
Leir
,
S.-H.
,
Browne
,
J.A.
,
Eggener
,
S.E.
and
Harris
,
A.
(
2015
)
Characterization of primary cultures of adult human epididymis epithelial cells
.
Fertil. Steril.
103
,
647
654.e1
18
Broackes-Carter
,
F.C.
,
Mouchel
,
N.
,
Gill
,
D.
,
Hyde
,
S.
,
Bassett
,
J.
and
Harris
,
A.
(
2002
)
Temporal regulation of CFTR expression during ovine lung development: implications for CF gene therapy
.
Hum. Mol. Genet.
11
,
125
131
19
Fulcher
,
M.L.
,
Gabriel
,
S.
,
Burns
,
K.A.
,
Yankaskas
,
J.R.
and
Randell
,
S.H.
(
2005
)
Well-differentiated human airway epithelial cell cultures
.
Methods Mol. Med.
107
,
183
206
PMID:
[PubMed]
20
Tugores
,
A.
,
Le
,
J.
,
Sorokina
,
I.
,
Snijders
,
A.J.
,
Duyao
,
M.
,
Reddy
,
P.S.
et al. 
(
2001
)
The epithelium-specific ETS protein EHF/ESE-3 is a context-dependent transcriptional repressor downstream of MAPK signaling cascades
.
J. Biol. Chem.
276
,
20397
20406
21
Fossum
,
S.L.
,
Mutolo
,
M.J.
,
Yang
,
R.
,
Dang
,
H.
,
O'Neal
,
W.K.
,
Knowles
,
M.R.
et al. 
(
2014
)
Ets homologous factor regulates pathways controlling response to injury in airway epithelial cells
.
Nucleic Acids Res.
42
,
13588
13598
22
Fossum
,
S.L.
,
Mutolo
,
M.J.
,
Tugores
,
A.
,
Ghosh
,
S.
,
Randell
,
S.H.
,
Jones
,
L.C.
et al. 
(
2017
)
Ets homologous factor (EHF) has critical roles in epithelial dysfunction in airway disease
.
J. Biol. Chem.
292
,
10938
10949
23
West
,
M.R.
and
Molloy
,
C.R.
(
1996
)
A microplate assay measuring chloride ion channel activity
.
Anal. Biochem.
241
,
51
58
24
Amaral
,
M.D.
and
Kunzelmann
,
K.
(
2011
)
Cystic Fibrosis: Diagnosis and Protocols
.
Springer
25
Stolzenburg
,
L.R.
,
Yang
,
R.
,
Kerschner
,
J.L.
,
Fossum
,
S.
,
Xu
,
M.
,
Hoffmann
,
A.
et al. 
(
2017
)
Regulatory dynamics of 11p13 suggest a role for EHF in modifying CF lung disease severity
.
Nucleic Acids Res.
45
,
8773
8784
26
Laherty
,
C.D.
,
Yang
,
W.-M.
,
Sun
,
J.-M.
,
Davie
,
J.R.
,
Seto
,
E.
and
Eisenman
,
R.N.
(
1997
)
Histone deacetylases associated with the mSin3 corepressor mediate mad transcriptional repression
.
Cell
89
,
349
356
27
Ramachandran
,
S.
,
Karp
,
P.H.
,
Jiang
,
P.
,
Ostedgaard
,
L.S.
,
Walz
,
A.E.
,
Fisher
,
J.T.
Jr.
et al. 
(
2012
)
A microRNA network regulates expression and biosynthesis of wild-type and ΔF508 mutant cystic fibrosis transmembrane conductance regulator
.
Proc. Natl Acad. Sci. U.S.A.
109
,
13362
13367
28
Yigit
,
E.
,
Bischof
,
J.M.
,
Zhang
,
Z.
,
Ott
,
C.J.
,
Kerschner
,
J.L.
,
Leir
,
S.-H.
et al. 
(
2013
)
Nucleosome mapping across the CFTR locus identifies novel regulatory factors
.
Nucleic Acids Res.
41
,
2857
2868
29
Blackledge
,
N.P.
,
Ott
,
C.J.
,
Gillen
,
A.E.
and
Harris
,
A.
(
2009
)
An insulator element 3′ to the CFTR gene binds CTCF and reveals an active chromatin hub in primary cells
.
Nucleic Acids Res.
37
,
1086
1094
30
Monden
,
T.
,
Wondisford
,
F.E.
and
Hollenberg
,
A.N.
(
1997
)
Isolation and characterization of a novel ligand-dependent thyroid hormone receptor-coactivating protein
.
J. Biol. Chem.
272
,
29834
29841
31
Hollenberg
,
A.N.
,
Monden
,
T.
,
Madura
,
J.P.
,
Lee
,
K.
and
Wondisford
,
F.E.
(
1996
)
Function of nuclear co-repressor protein on thyroid hormone response elements is regulated by the receptor A/B domain
.
J. Biol. Chem.
271
,
28516
28520
32
Wan
,
H.
,
Luo
,
F.
,
Wert
,
S.E.
,
Zhang
,
L.
,
Xu
,
Y.
,
Ikegami
,
M.
et al. 
(
2008
)
Kruppel-like factor 5 is required for perinatal lung morphogenesis and function
.
Development
135
,
2563
2572
33
Bischof
,
J.M.
,
Ott
,
C.J.
,
Leir
,
S.-H.
,
Gosalia
,
N.
,
Song
,
L.
,
London
,
D.
et al. 
(
2012
)
A genome-wide analysis of open chromatin in human tracheal epithelial cells reveals novel candidate regulatory elements for lung function
.
Thorax
67
,
385
391
34
Safe
,
S.
,
Jin
,
U.-H.
,
Hedrick
,
E.
,
Reeder
,
A.
and
Lee
,
S.-O.
(
2014
)
Minireview: role of orphan nuclear receptors in cancer and potential as drug targets
.
Mol. Endocrinol.
28
,
157
172
35
Filippakopoulos
,
P.
and
Knapp
,
S.
(
2014
)
Targeting bromodomains: epigenetic readers of lysine acetylation
.
Nat. Rev. Drug Discov.
13
,
337
356
36
Filippakopoulos
,
P.
,
Picaud
,
S.
,
Mangos
,
M.
,
Keates
,
T.
,
Lambert
,
J.P.
,
Barsyte-Lovejoy
,
D.
et al. 
(
2012
)
Histone recognition and large-scale structural analysis of the human bromodomain family
.
Cell
149
,
214
231
37
Filippakopoulos
,
P.
,
Qi
,
J.
,
Picaud
,
S.
,
Shen
,
Y.
,
Smith
,
W.B.
,
Fedorov
,
O.
et al. 
(
2010
)
Selective inhibition of BET bromodomains
.
Nature
468
,
1067
1073
38
Hohmann
,
A.F.
,
Martin
,
L.J.
,
Minder
,
J.L.
,
Roe
,
J.S.
,
Shi
,
J.
,
Steurer
,
S.
et al. 
(
2016
)
Sensitivity and engineered resistance of myeloid leukemia cells to BRD9 inhibition
.
Nat. Chem. Biol.
12
,
672
679
39
Sogawa
,
K.
,
Imataka
,
H.
,
Yamasaki
,
Y.
,
Kusume
,
H.
,
Abe
,
H.
and
Fujii-Kuriyama
,
Y.
(
1993
)
cDNA cloning and transcriptional properties of a novel GC box-binding protein, BTEB2
.
Nucleic Acids Res.
21
,
1527
1532
40
Gu
,
G.
,
Wells
,
J.M.
,
Dombkowski
,
D.
,
Preffer
,
F.
,
Aronow
,
B.
and
Melton
,
D.A.
(
2004
)
Global expression analysis of gene regulatory pathways during endocrine pancreatic development
.
Development
131
,
165
179
41
Conkright
,
M.D.
,
Wani
,
M.A.
,
Anderson
,
K.P.
and
Lingrel
,
J.B.
(
1999
)
A gene encoding an intestinal-enriched member of the Krüppel-like factor family expressed in intestinal epithelial cells
.
Nucleic Acids Res.
27
,
1263
1270
42
Ohnishi
,
S.
,
Laub
,
F.
,
Matsumoto
,
N.
,
Asaka
,
M.
,
Ramirez
,
F.
,
Yoshida
,
T.
et al. 
(
2000
)
Developmental expression of the mouse gene coding for the Krüppel-like transcription factor KLF5
.
Dev. Dyn.
217
,
421
429
43
Ziemer
,
L.T.
,
Pennica
,
D.
and
Levine
,
A.J.
(
2001
)
Identification of a mouse homolog of the human BTEB2 transcription factor as a beta-catenin-independent Wnt-1-responsive gene
.
Mol. Cell Biol.
21
,
562
574
44
Nakaya
,
T.
,
Ogawa
,
S.
,
Manabe
,
I.
,
Tanaka
,
M.
,
Sanada
,
M.
,
Sato
,
T.
et al. 
(
2014
)
KLF5 regulates the integrity and oncogenicity of intestinal stem cells
.
Cancer Res.
74
,
2882
2891
45
Zhang
,
B.
,
Zhang
,
Z.
,
Xia
,
S.
,
Xing
,
C.
,
Ci
,
X.
,
Li
,
X.
et al. 
(
2013
)
KLF5 activates microRNA 200 transcription to maintain epithelial characteristics and prevent induced epithelial-mesenchymal transition in epithelial cells
.
Mol. Cell Biol.
33
,
4919
4935
46
Bartoszewska
,
S.
,
Kamysz
,
W.
,
Jakiela
,
B.
,
Sanak
,
M.
,
Kroliczewski
,
J.
,
Bebok
,
Z.
et al. 
(
2017
)
miR-200b downregulates CFTR during hypoxia in human lung epithelial cells
.
Cell Mol. Biol. Lett.
22
,
23
47
Bochert
,
M.A.
,
Kleinbaum
,
L.A.
,
Sun
,
L.-Y.
and
Burton
,
F.H.
(
1998
)
Molecular cloning and expression of Ehf, a new member of the ets transcription factor/oncoprotein gene family
.
Biochem. Biophys. Res. Commun.
246
,
176
181
48
Dallavalle
,
C.
,
Albino
,
D.
,
Civenni
,
G.
,
Merulla
,
J.
,
Ostano
,
P.
,
Mello-Grand
,
M.
et al. 
(
2016
)
MicroRNA-424 impairs ubiquitination to activate STAT3 and promote prostate tumor progression
.
J. Clin. Invest.
126
,
4585
4602
49
Stephens
,
D.N.
,
Klein
,
R.H.
,
Salmans
,
M.L.
,
Gordon
,
W.
,
Ho
,
H.
and
Andersen
,
B.
(
2013
)
The Ets transcription factor EHF as a regulator of cornea epithelial cell identity
.
J. Biol. Chem.
288
,
34304
34324
50
Rubin
,
A.J.
,
Barajas
,
B.C.
,
Furlan-Magaril
,
M.
,
Lopez-Pajares
,
V.
,
Mumbach
,
M.R.
,
Howard
,
I.
et al. 
(
2017
)
Lineage-specific dynamic and pre-established enhancer–promoter contacts cooperate in terminal differentiation
.
Nat. Genet.
49
,
1522
1528
51
Wright
,
F.A.
,
Strug
,
L.J.
,
Doshi
,
V.K.
,
Commander
,
C.W.
,
Blackman
,
S.M.
,
Sun
,
L.
et al. 
(
2011
)
Genome-wide association and linkage identify modifier loci of lung disease severity in cystic fibrosis at 11p13 and 20q13.2
.
Nat. Genet.
43
,
539
546
52
Corvol
,
H.
,
Blackman
,
S.M.
,
Boëlle
,
P.-Y.
,
Gallins
,
P.J.
,
Pace
,
R.G.
,
Stonebraker
,
J.R.
et al. 
(
2015
)
Genome-wide association meta-analysis identifies five modifier loci of lung disease severity in cystic fibrosis
.
Nat. Commun.
6
,
8382
53
La Russa
,
M.F.
and
Qi
,
L.S.
(
2015
)
The new state of the art: Cas9 for gene activation and repression
.
Mol. Cell Biol.
35
,
3800
3809
54
Vora
,
S.
,
Tuttle
,
M.
,
Cheng
,
J.
and
Church
,
G.
(
2016
)
Next stop for the CRISPR revolution: RNA-guided epigenetic regulators
.
FEBS J.
283
,
3181
3193