Nucleosome occupancy reveals regulatory elements of the CFTR promoter

Access to regulatory elements of the genome can be inhibited by nucleosome core particles arranged along the DNA strand. Hence, sites that are accessible by transcription factors may be located by using nuclease digestion to identify the relative nucleosome occupancy of a genomic region. In order to define novel cis regulatory elements in the ∼2.7-kb promoter region of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, we define its nucleosome occupancy. This profile reveals the precise positions of nucleosome-free regions (NFRs), both cell-type specific and others apparently unrelated to CFTR-expression level and offer the first high-resolution map of the chromatin structure of the entire CFTR promoter in relevant cell types. Several of these NFRs are strongly bound by nuclear factors in a sequence-specific manner, and directly influence CFTR promoter activity. Sequences within the NFR1 and NFR4 elements are highly conserved in many human gene promoters. Moreover, NFR1 contributes to promoter activity of another gene, angiopoietin-like 3 (ANGPTL3), while NFR4 is constitutively nucleosome-free in promoters genome wide. Conserved motifs within NFRs of the CFTR promoter also show a high level of protection from DNase I digestion genome-wide, and likely have important roles in the positioning of nucleosome core particles more generally

An insulator element 3′ to the CFTR gene binds CTCF and reveals an active chromatin hub in primary cells

Regulation of expression of the CFTR gene is poorly understood. Elements within the basal promoter of the gene do not fully explain CFTR expression patterns, suggesting that cis-regulatory elements are located elsewhere, either within the locus or in adjacent chromatin. We previously mapped DNase I hypersensitive sites (DHS) in 400 kb spanning the CFTR locus including a cluster of sites close to the 3′-end of the gene. Here we focus on a DHS at +6.8 kb from the CFTR translation end-point to evaluate its potential role in regulating expression of the gene. This DHS, which encompasses a consensus CTCF-binding site, was evident in primary human epididymis cells that express abundant CFTR mRNA. We show by DNase I footprinting and electophoretic mobility shift assays that the cis-regulatory element within this DHS binds CTCF in vitro. We further demonstrate that the element functions as an enhancer blocker in a well-established in vivo assay, and by using chromatin immunoprecipitation that it recruits CTCF in vivo. Moreover, we reveal that in primary epididymis cells, the +6.8 kb DHS interacts closely with the CFTR promoter, suggesting that the CFTR locus exists in a looped conformation, characteristic of an active chromatin hub

The Hellenic type of nondeletional hereditary persistence of fetal hemoglobin results from a novel mutation (g.-109G>T) in the HBG2 gene promoter

Nondeletional hereditary persistence of fetal hemoglobin (nd-HPFH), a rare hereditary condition resulting in elevated levels of fetal hemoglobin (Hb F) in adults, is associated with promoter mutations in the human fetal globin (HBG1 and HBG2) genes. In this paper, we report a novel type of nd-HPFH due to a HBG2 gene promoter mutation (HBG2:g.-109G>T). This mutation, located at the 3′ end of the HBG2 distal CCAAT box, was initially identified in an adult female subject of Central Greek origin and results in elevated Hb F levels (4.1%) and significantly increased Gγ-globin chain production (79.2%). Family studies and DNA analysis revealed that the HBG2:g.-109G>T mutation is also found in the family members in compound heterozygosity with the HBG2:g.-158C>T single nucleotide polymorphism or the silent HBB:g.-101C>T β-thalassemia mutation, resulting in the latter case in significantly elevated Hb F levels (14.3%). Electrophoretic mobility shift analysis revealed that the HBG2:g.-109G>T mutation abolishes a transcription factor binding site, consistent with previous observations using DNA footprinting analysis, suggesting that guanine at position HBG2/1:g.-109 is critical for NF-E3 binding. These data suggest that the HBG2:g-109G>T mutation has a functional role in increasing HBG2 transcription and is responsible for the HPFH phenotype observed in our index cases

Corrigendum to "European contribution to the study of ROS:A summary of the findings and prospects for the future from the COST action BM1203 (EU-ROS)" [Redox Biol. 13 (2017) 94-162]

The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed

European contribution to the study of ROS: A summary of the findings and prospects for the future from the COST action BM1203 (EU-ROS).

The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed.The EU-ROS consortium (COST Action BM1203) was supported by the European Cooperation in Science and Technology (COST). The present overview represents the final Action dissemination summarizing the major achievements of COST Action BM1203 (EU-ROS) as well as research news and personal views of its members. Some authors were also supported by COST Actions BM1005 (ENOG) and BM1307 (PROTEOSTASIS), as well as funding from the European Commission FP7 and H2020 programmes, and several national funding agencies

General anaesthetic and airway management practice for obstetric surgery in England: a prospective, multi-centre observational study

There are no current descriptions of general anaesthesia characteristics for obstetric surgery, despite recent changes to patient baseline characteristics and airway management guidelines. This analysis of data from the direct reporting of awareness in maternity patients' (DREAMY) study of accidental awareness during obstetric anaesthesia aimed to describe practice for obstetric general anaesthesia in England and compare with earlier surveys and best-practice recommendations. Consenting patients who received general anaesthesia for obstetric surgery in 72 hospitals from May 2017 to August 2018 were included. Baseline characteristics, airway management, anaesthetic techniques and major complications were collected. Descriptive analysis, binary logistic regression modelling and comparisons with earlier data were conducted. Data were collected from 3117 procedures, including 2554 (81.9%) caesarean deliveries. Thiopental was the induction drug in 1649 (52.9%) patients, compared with propofol in 1419 (45.5%). Suxamethonium was the neuromuscular blocking drug for tracheal intubation in 2631 (86.1%), compared with rocuronium in 367 (11.8%). Difficult tracheal intubation was reported in 1 in 19 (95%CI 1 in 16-22) and failed intubation in 1 in 312 (95%CI 1 in 169-667). Obese patients were over-represented compared with national baselines and associated with difficult, but not failed intubation. There was more evidence of change in practice for induction drugs (increased use of propofol) than neuromuscular blocking drugs (suxamethonium remains the most popular). There was evidence of improvement in practice, with increased monitoring and reversal of neuromuscular blockade (although this remains suboptimal). Despite a high risk of difficult intubation in this population, videolaryngoscopy was rarely used (1.9%)