In mammals, the canonical histone H3 and the variant H3.3 are assembled into chromatin through replication-coupled and replication-independent (RI) histone deposition pathways, respectively, to play distinct roles in chromatin function. H3.3 is largely associated with transcriptionally active regions via the activity of RI histone chaperone, HIRA. However, the precise role of the RI pathway and HIRA in active transcription and the mechanisms by which H3.3 affects gene activity are not known. In this study, we show that HIRA is an essential factor for muscle development by establishing MyoD activation in myotubes. HIRA and Asf1a, but not CHD1 or Asf1b, mediate H3.3 incorporation in the promoter and the critical upstream regulatory regions of the MyoD gene. HIRA and H3.3 are required for epigenetic transition into the more permissive chromatin structure for polymerase II recruitment to the promoter, regardless of transcription-associated covalent modification of histones. Our results suggest distinct epigenetic management of the master regulator with RI pathway components for cellular differentiation.

Parafibromin, a component of the RNA polymerase II-associated PAF1 complex, is a tumor suppressor linked to hyperparathyroidism-jaw tumor syndrome and sporadic parathyroid carcinoma. Parafibromin induces cell cycle arrest by repressing cyclin D1 via an unknown mechanism. Here, we show that parafibromin interacts with the histone methyltransferase, SUV39H1, and functions as a transcriptional repressor. The central region (128-227 amino acids) of parafibromin is important for both the interaction with SUV39H1 and transcriptional repression. Parafibromin associated with the promoter and coding regions of cyclin D1 and was required for the recruitment of SUV39H1 and the induction of H3 K9 methylation but not H3 K4 methylation. RNA interference analysis showed that SUV39H1 was critical for cyclin D1 repression. These data suggest that parafibromin plays an unexpected role as a repressor in addition to its widely known activity associated with transcriptional activation. Parafibromin as a part of the PAF1 complex might downregulate cyclin D1 expression by integrating repressive H3 K9 methylation during transcription.

Menin, encoded by the multiple endocrine neoplasia type 1 ( MEN1 ) gene, is a tumor suppressor that leads to multiple endocrine tumors upon loss of its function. Menin functions as a transcriptional activator by tethering MLL complex to mediate histone H3 K4 methylation. It also functions as a repressor. However, the molecular mechanism of how menin contributes to the opposite outcome in gene expression is largely unknown. Here, we investigated the role of menin in the epigenetic regulation of transcription mediated by histone covalent modification. We show that the global methylation level of histone H3 K9, as well as H3 K4, was decreased in Men1 ^−/− MEF cells. Consistently, menin was able to interact with the suppressor of variegation 3–9 homolog family protein, SUV39H1, to mediate H3 K9 methylation. This interaction decreased when patient-derived MEN1 mutation was introduced into the SUV39H1-interaction domain. We show that menin mediated different chromatin changes depending on target genes. Chromatin immunoprecipitation studies showed that menin directly associated with the GBX2 promoter and menin-dependent recruitment of SUV39H1 was essential for chromatin remodeling and transcriptional regulation. These results provide a molecular basis of how menin functions as a transcriptional repressor and suggest that menin-dependent integration of H3 K9 methylation might play an important role in preventing tumors.

Mycoplasma pneumoniae, a major etiological agent of community-acquired pneumonia, exhibits distinct cyclic epidemic patterns recurring every three to five years. Several cases of co-infection with severe acute respiratory syndrome coronavirus 2 have been reported globally, resulting in unfavorable clinical manifestations. This study investigated the epidemiological features of the recent M. pneumoniae outbreak (May 2019–April 2020) using retrospective data from the last five years. Molecular test data for macrolide resistance and co-infection were obtained from the Seegene Medical Foundation. National medical expenditure and hospitalization rates were analyzed using data from The Health Insurance Review and Assessment Service of Korea. The macrolide resistance rate was 69.67%, peaking at 71.30% during the epidemic period, which was considerably higher than the 60.89% rate during non-epidemic periods. The co-infection rate with other respiratory pathogens was 88.49%; macrolide-resistant M. pneumoniae strains showed a 2.33% higher co-infection rate than the susceptible strains. The epidemic period had 15.43% higher hospitalization and 78.27% higher medical budget expenditure per patient than non-epidemic periods. The increased rates of macrolide resistance and co-infection observed in macrolide-resistant M. pneumoniae during the epidemic period highlight the importance of monitoring future outbreaks, especially considering macrolide resistance and the risk of co-infection with other pathogens.

Classification of clinical symptoms and diagnostic microbiology are essential to effectively employ antimicrobial therapy for lower respiratory tract infections (LRTIs) in a timely manner. Empirical antibiotic treatment without microbial identification hinders the selective use of narrow-spectrum antibiotics and effective patient treatment. Thus, the development of rapid and accurate diagnostic procedures that can be readily adopted by the clinic is necessary to minimize non-essential or excessive use of antibiotics and accelerate patient recovery from LRTI-induced damage. We developed and validated a multiplex real-time polymerase chain reaction (mRT-PCR) assay with good analytical performance and high specificity to simultaneously detect four bacterial pathogens causing pneumonia: Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and Moraxella catarrhalis. The analytical performance of mRT-PCR against target pathogens was evaluated by the limit of detection (LOD), specificity, and repeatability. Two hundred and ten clinical specimens from pneumonia patients were processed using an automatic nucleic acid extraction system for the “respiratory bacteria four” (RB4) mRT-PCR assay, and the results were directly compared to references from bacterial culture and/or Sanger sequencing. The RB4 mRT-PCR assay detected all target pathogens from sputum specimens with a coefficient of variation ranging from 0.29 to 1.71 and conservative LOD of DNA corresponding to 5 × 10² copies/reaction. The concordance of the assay with reference-positive specimens was 100%, and additional bacterial infections were detected from reference-negative specimens. Overall, the RB4 mRT-PCR assay showed a more rapid turnaround time and higher performance that those of reference assays. The RB4 mRT-PCR assay is a high-throughput and reliable tool that assists decision-making assessment and outperforms other standard methods. This tool supports patient management by considerably reducing the inappropriate use of antibiotics.

The detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in upper and lower respiratory specimens and coinfection with other respiratory pathogens in patients with coronavirus disease 2019 (COVID-19) was investigated. Study subjects (N=342) were retrospectively enrolled after being confirmed as SARS-CoV-2 positive, and their nasopharyngeal swab (NPS), oropharyngeal swab (OPS), and sputum specimens were restored for SARS-CoV-2 retesting and respiratory pathogen detection. The majority of the subjects (96.5, N=330) were confirmed as SARS-CoV-2 positive using NPS/OPS specimens. Among the COVID-19 patients (N=342), 7.9 (N=27) and 0.9 (N=3) were coinfected with respiratory viruses and Mycoplasma pneumoniae, respectively, yielding an 8.8 (N=30) overall respiratory pathogen coinfection rate. Of the respiratory virus coinfection cases (N=27), 92.6 (N=25) were coinfected with a single respiratory virus and 7.4 (N=2) with two viruses (metapneumovirus/adenovirus and rhinovirus/bocavirus). No triple coinfections of other respiratory viruses or bacteria with SARS-CoV-2 were detected. Respiratory viruses coinfected in the patients with COVID-19 were as follows: rhinovirus (N=7, 2.1), respiratory syncytial virus A and B (N=6, 1.8), non-SARS-CoV-2 coronaviruses (229E, NL63, and OC43, N=5, 1.5), metapneumovirus (N=4, 1.2), influenza A (N=3, 0.9), adenovirus (N=3, 0.9), and bocavirus (N=1, 0.3). In conclusion, the diagnostic value of utilizing NPS/OPS specimens is excellent, and, as the first report in Korea, coinfection with respiratory pathogens was detected at a rate of 8.8 in patients with COVID-19.