A transcription factor network represses CFTR gene expression in airway epithelial cells.

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.

Regulatory dynamics of 11p13 suggest a role for EHF in modifying CF lung disease severity.

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.

Ets homologous factor (EHF) has critical roles in epithelial dysfunction in airway disease.

The airway epithelium forms a barrier between the internal and external environments. Epithelial dysfunction is critical in the pathology of many respiratory diseases, including cystic fibrosis. Ets homologous factor (EHF) is a key member of the transcription factor network that regulates gene expression in the airway epithelium in response to endogenous and exogenous stimuli. EHF, which has altered expression in inflammatory states, maps to the 5' end of an intergenic region on Chr11p13 that is implicated as a modifier of cystic fibrosis airway disease. Here we determine the functions of EHF in primary human bronchial epithelial (HBE) cells and relevant airway cell lines. Using EHF ChIP followed by deep sequencing (ChIP-seq) and RNA sequencing after EHF depletion, we show that EHF targets in HBE cells are enriched for genes involved in inflammation and wound repair. Furthermore, changes in gene expression impact cell phenotype because EHF depletion alters epithelial secretion of a neutrophil chemokine and slows wound closure in HBE cells. EHF activates expression of the SAM pointed domain-containing ETS transcription factor, which contributes to goblet cell hyperplasia. Our data reveal a critical role for EHF in regulating epithelial function in lung disease.

Ets homologous factor regulates pathways controlling response to injury in airway epithelial cells.

Ets homologous factor (EHF) is an Ets family transcription factor expressed in many epithelial cell types including those lining the respiratory system. Disruption of the airway epithelium is central to many lung diseases, and a network of transcription factors coordinates its normal function. EHF can act as a transcriptional activator or a repressor, though its targets in lung epithelial cells are largely uncharacterized. Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq), showed that the majority of EHF binding sites in lung epithelial cells are intergenic or intronic and coincide with putative enhancers, marked by specific histone modifications. EHF occupies many genomic sites that are close to genes involved in intercellular and cell-matrix adhesion. RNA-seq after EHF depletion or overexpression showed significant alterations in the expression of genes involved in response to wounding. EHF knockdown also targeted genes in pathways of epithelial development and differentiation and locomotory behavior. These changes in gene expression coincided with alterations in cellular phenotype including slowed wound closure and increased transepithelial resistance. Our data suggest that EHF regulates gene pathways critical for epithelial response to injury, including those involved in maintenance of barrier function, inflammation and efficient wound repair.

The inhibitor of phagocytosis, O-phospho-L-serine, suppresses Müller glia proliferation and cone cell regeneration in the light-damaged zebrafish retina.

The damaged zebrafish retina replaces lost neurons through a regenerative response that initiates with the asymmetric cell division of Müller glia to produce neuronal progenitor cells that proliferate and migrate to the damaged retinal layer, where they differentiate into the lost neuronal cell types. Because Müller glia are known to phagocytose apoptotic retinal cells during development, we tested if Müller glia engulfed apoptotic rod cell bodies in light-damaged retinas. After 24h of constant intense light, damaged retinas revealed both a strong nuclear TUNEL signal in photoreceptors and a weak cytoplasmic TUNEL signal in Müller glia, although Müller glial apoptosis is not observed in the light-damaged retina. Light damage of a rod-specific transgenic reporter line, Tg(XlRho:EGFP)(fl1), resulted in some Müller glia containing both TUNEL signal and EGFP, which indicated that this subset of Müller glia engulfed apoptotic photoreceptor cell bodies. To determine if phagocytosis induced the Müller glial proliferative response in the light-damaged retina, we utilized O-phospho-l-serine (L-SOP), a molecule that mimics the phosphatidylserine head group and partially blocks microglial phagocytosis of apoptotic cells. Intravitreal injection of L-SOP immediately prior to beginning constant intense light treatment: i) did not significantly reduce light-induced photoreceptor cell death, ii) significantly reduced the number of PCNA-positive Müller glia, and iii) significantly reduced the number of cone photoreceptors in the regenerated retina relative to control retinas. Because L-SOP is also a specific group III metabotropic glutamate receptor (mGluR) agonist, we also tested if the more potent specific group III agonist, L-2-amino-4-phosphonobutyrate (L-AP4), the specific group III antagonist (RS)-α-Methylserine-O-phosphate (MSOP) or the specific group I antagonist, L-2-amino-3-phophonopropanoic acid (L-AP3) affected Müller glial proliferation. We found no changes with any of these factors compared to control retinas, revealing that metabotropic glutamate receptors were not necessary in the Müller glia proliferative response. Furthermore, ascl1a and stat3 expression were unaffected in either the L-SOP or MSOP-injected retinas relative to controls, suggesting L-SOP disrupts Müller glia proliferation subsequent to or in parallel with ascl1a and stat3 activation. This implies that at least one signaling mechanism, in addition to the process disrupted by L-SOP, is required to activate Müller glia proliferation in the light-damaged retina.

Switching from an esterase to a hydroxynitrile lyase mechanism requires only two amino acid substitutions.

The alpha/beta hydrolase superfamily contains mainly esterases, which catalyze hydrolysis, but also includes hydroxynitrile lyases, which catalyze addition of cyanide to aldehydes, a carbon-carbon bond formation. Here, we convert a plant esterase, SABP2, into a hydroxynitrile lyase using just two amino acid substitutions. Variant SABP2-G12T-M239K lost the ability to catalyze ester hydrolysis (<0.9 mU/mg) and gained the ability to catalyze the release of cyanide from mandelonitrile (20 mU/mg, k(cat)/K(M) = 70 min(-1)M(-1)). This variant also catalyzed the reverse reaction, formation of mandelonitrile with low enantioselectivity: 20% ee (S), E = 1.5. The specificity constant for the lysis of mandelontrile is 13,000-fold faster than the uncatalyzed reaction and only 1300-fold less efficient (k(cat/)K(M)) than hydroxynitrile lyase from rubber tree.