JMJD5 couples with CDK9 to release the paused RNA polymerase II.

More than 30% of genes in higher eukaryotes are regulated by RNA polymerase II (Pol II) promoter proximal pausing. Pausing is released by the positive transcription elongation factor complex (P-TEFb). However, the exact mechanism by which this occurs and whether phosphorylation of the carboxyl-terminal domain of Pol II is involved in the process remains unknown. We previously reported that JMJD5 could generate tailless nucleosomes at position +1 from transcription start sites (TSS), thus perhaps enable progression of Pol II. Here we find that knockout of JMJD5 leads to accumulation of nucleosomes at position +1. Absence of JMJD5 also results in loss of or lowered transcription of a large number of genes. Interestingly, we found that phosphorylation, by CDK9, of Ser2 within two neighboring heptad repeats in the carboxyl-terminal domain of Pol II, together with phosphorylation of Ser5 within the second repeat, HR-Ser2p (1, 2)-Ser5p (2) for short, allows Pol II to bind JMJD5 via engagement of the N-terminal domain of JMJD5. We suggest that these events bring JMJD5 near the nucleosome at position +1, thus allowing JMJD5 to clip histones on this nucleosome, a phenomenon that may contribute to release of Pol II pausing.

Airway Progenitor Clone Formation Is Enhanced by Y-27632-Dependent Changes in the Transcriptome.

The application of conditional reprogramming culture (CRC) methods to nasal airway epithelial cells would allow more wide-spread incorporation of primary airway epithelial culture models into complex lung disease research. In this study, we adapted the CRC method to nasal airway epithelial cells, investigated the growth advantages afforded by this technique over standard culture methods, and determined the cellular and molecular basis of CRC cell culture effects. We found that the CRC method allowed the production of 7.1 × 10(10) cells after 4 passages, approximately 379 times more cells than were generated by the standard bronchial epithelial growth media (BEGM) method. These nasal airway epithelial cells expressed normal basal cell markers and could be induced to form a mucociliary epithelium. Progenitor cell frequency was significantly higher using the CRC method in comparison to the standard culture method, and progenitor cell maintenance was dependent on addition of the Rho-kinase inhibitor Y-27632. Whole-transcriptome sequencing analysis demonstrated widespread gene expression changes in Y-27632-treated basal cells. We found that Y-27632 treatment altered expression of genes fundamental to the formation of the basal cell cytoskeleton, cell-cell junctions, and cell-extracellular matrix (ECM) interactions. Importantly, we found that Y-27632 treatment up-regulated expression of unique basal cell intermediate filament and desmosomal genes. Conversely, Y-27632 down-regulated multiple families of protease/antiprotease genes involved in ECM remodeling. We conclude that Y-27632 fundamentally alters cell-cell and cell-ECM interactions, which preserves basal progenitor cells and allows greater cell amplification.

Impact of azithromycin on the clinical and antimicrobial effectiveness of tobramycin in the treatment of cystic fibrosis.

BACKGROUND: Concomitant use of oral azithromycin and inhaled tobramycin occurs in approximately half of US cystic fibrosis (CF) patients. Recent data suggest that this combination may be antagonistic. METHODS: Test the hypothesis that azithromycin reduces the clinical benefits of tobramycin by analyses of clinical trial data, in vitro modeling of P. aeruginosa antibiotic killing, and regulation of the MexXY efflux pump. RESULTS: Ongoing administration of azithromycin associates with reduced ability of inhaled tobramycin, as compared with aztreonam, to improve lung function and quality of life in a completed clinical trial. In users of azithromycin FEV1 (L) increased 0.8% during a 4-week period of inhaled tobramycin and an additional 6.4% during a subsequent 4-week period of inhaled aztreonam (P<0.005). CFQ-R respiratory symptom score decreased 1.8 points during inhaled tobramycin and increased 8.3 points during subsequent inhaled aztreonam (P<0.001). A smaller number of trial participants not using azithromycin had similar improvement in lung function and quality of life scores during inhaled tobramycin and inhaled aztreonam. In vitro, azithromycin selectively reduced the bactericidal effects tobramycin in cultures of clinical strains of P. aeruginosa, while up regulating antibiotic resistance through MexXY efflux. CONCLUSIONS: Azithromycin appears capable of reducing the antimicrobial benefits of tobramycin by inducing adaptive bacterial stress responses in P. aeruginosa, suggesting that these medications together may not be optimal chronic therapy for at least some patients.

Therapeutic Effects of α1-Antitrypsin on Psedumonas aeruginosa Infection in ENaC Transgenic Mice.

Cystic fibrosis (CF) is a genetic disease with many airway pathological features, including aberrant epithelial sodium channel (ENaC) function, persistent Pseudomonas aeruginosa (PA) infection and neutrophil-dominant inflammation. PA infection in CF airways is difficult to treat due to antibiotic resistance and other factors. Recently, α1-antitrypsin (A1AT) have been shown to be effective to reduce CF airway PA infection. However, there is a dearth of studies about the mechanisms underlying A1AT's therapeutic effects. The goal of our study is to provide an animal model of A1AT therapy in CF lungs. ENaC transgenic mice with PA infection were used as a CF-like model. Mice were intratracheally treated with PA or saline (control) in a fibrin plug. Two hours after PA infection, aerosolized A1AT were delivered to mouse lungs once daily. At day 1 and day 3 post PA infection, lung inflammation, PA load as well as host defence protein short palate, lung, and nasal epithelium clone 1 (SPLUNC1) were measured. At day 1 post PA infection when A1AT was delivered once to ENaC transgenic mouse lungs, A1AT did not reduce lung inflammation (e.g., neutrophils) and PA load. However, at day 3 post PA infection when ENaC transgenic mice received three repeated A1AT treatments, a significant decrease in airspace inflammation and PA load was observed. Although A1AT prevented the loss of SPLUNC1 in bronchoalveolar lavage fluid of PA-infected wild-type mice, it did not restore SPLUNC1 levels in ENaC transgenic mice. Our current study has provided a valid and quick A1AT therapeutic model in CF-like lungs that may serve as a platform for future mechanistic studies about how A1AT exerts beneficial effects in human CF patients.

Mycobacterium abscessus morphotype comparison in a murine model.

Pulmonary infections with Mycobacterium abscessus (M. abscessus) are increasingly prevalent in patients with lung diseases such as cystic fibrosis. M. abscessus exists in two morphotypes, smooth and rough, but the impact of morphotype on virulence is unclear. We developed an immune competent mouse model of pulmonary M. abscessus infection and tested the differences in host inflammatory response between the morphotypes of M. abscessus. Smooth and rough morphotypes of M. abscessus were isolated from the same American Type Culture Collection strain. Wild type and cystic fibrosis mice were intratracheally inoculated with known quantities of M. abscessus suspended in fibrin plugs. At the time of sacrifice lung and splenic tissues and bronchoalveolar lavage fluid were collected and cultured. Bronchoalveolar lavage fluid was analyzed for leukocyte count, differential and cytokine expression. Pulmonary infection with M. abscessus was present at both 3 days and 14 days post-inoculation in all groups at greater levels than systemic infection. Inoculation with M. abscessus rough morphotype resulted in more bronchoalveolar lavage fluid neutrophils compared to smooth morphotype at 14 days post-inoculation in both wild type (p = 0.01) and cystic fibrosis (p<0.01) mice. Spontaneous in vivo conversion from smooth to rough morphotype occurred in 12/57 (21%) of mice. These mice trended towards greater weight loss than mice in which morphotype conversion did not occur. In the described fibrin plug model of M. abscessus infection, pulmonary infection with minimal systemic dissemination is achieved with both smooth and rough morphotypes. In this model M. abscessus rough morphotype causes a greater host inflammatory response than the smooth based on bronchoalveolar lavage fluid neutrophil levels.