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Biology subjects

Jurkowski, T. P.

Publications and source records attributed to Jurkowski, T. P..

5 recordsLinked to original sources

Epigenetic deregulation of IFN and WNT pathways in AT2 cells impairs alveolar regeneration (in COPD)

Chronic lung diseases, including chronic obstructive pulmonary disease (COPD), affect over 500 million people and are a leading cause of death worldwide. A common feature of both chronic and acute lung diseases is altered respiratory barrier integrity and impaired lung regeneration. We hypothesized that alveolar type 2 (AT2) cells, as alveolar epithelial progenitors, will carry molecular alterations that compromise alveolar regeneration in COPD. Sorted AT2 cells from ex-smokers with and without COPD at different disease stages were subjected to RNA sequencing and whole-genome bisulfite sequencing to generate unbiased transcriptome and DNA methylation maps of alveolar progenitors in the lung. Our analysis revealed genome-wide epigenetic changes in AT2 cells during COPD that were associated with global gene expression changes. Integrative data analysis uncovered a strong anti-correlation between gene expression and promoter methylation, suggesting that dysregulation of COPD-associated pathways in AT2 cells may be regulated by DNA methylation. Interferon (IFN) signaling was the top-upregulated pathway associated with the concomitant loss of promoter DNA methylation. Epigenetic regulation of the IFN pathway was validated in both global and targeted DNA demethylation assays in A549 cells. Notably, targeted DNA demethylation of IRF9 triggered upregulation of IFN signaling, mimicking the effects observed in COPD AT2 cells in the profiling data. Our findings suggest that COPD-triggered epigenetic alterations in AT2 cells may impair internal regeneration programs in human lung parenchyma.

cell biology↗

High-resolution epigenetic profiling identifies novel regulators of COPD in human lung fibroblasts

Patients with chronic obstructive pulmonary disease (COPD) are still waiting for curative treatments. Considering the environmental cause of COPD (e.g., cigarette smoke) and disease phenotypes, including stem-cell senescence and impaired differentiation, we hypothesized that COPD will be associated with altered epigenetic signaling in lung cells. We generated genome-wide DNA methylation maps at single CpG resolution of primary human lung fibroblasts (HLFs) isolated from distal parenchyma of ex-smoker controls and COPD patients, with both mild and severe disease. The epigenetic landscape is markedly changed in lung fibroblasts across COPD stages, with DNA methylation changes occurring predominantly in regulatory regions, including promoters and enhancers. RNA sequencing of matched fibroblasts demonstrated dysregulation of genes involved in proliferation, DNA repair, and extracellular matrix organization. Notably, we identified epigenetic and transcriptional dysregulation already in mild COPD patients, providing unique insights into early disease. Integration of profiling data identified 110 candidate regulators of disease phenotypes, including epigenetic factors. Using phenotypic screens, we verified the regulator capacity of multiple candidates and linked them to repair processes in the human lung. Our study provides first integrative high-resolution epigenetic and transcriptomic maps of human lung fibroblasts across stages of COPD. We reveal novel transcriptomic and epigenetic signatures associated with COPD onset and progression and identify new candidate regulators involved in the pathogenesis of chronic respiratory diseases. The presence of various epigenetic factors among the candidates demonstrates that epigenetic regulation in COPD is an exciting research field that holds promise for novel therapeutic avenues for patients.

cell biology↗

Pronounced sequence specificity of the TET enzyme catalytic domain guides its cellular function

TET (ten-eleven translocation) enzymes catalyze the oxidation of 5-methylcytosine bases in DNA, thus driving active and passive DNA demethylation. Here, we report that the catalytic cores of mammalian TET enzymes favor CpGs embedded within bHLH and bZIP transcription factor binding sites, with 250-fold preference in vitro. Crystal structures and molecular dynamics calculations show that sequence preference is caused by intra-substrate interactions and CpG flanking sequence indirectly affecting enzyme conformation. TET sequence preferences are physiologically relevant as they explain the rates of DNA demethylation in TET-rescue experiments in culture and in vivo within the zygote and germline. Most and least favorable TET motifs represent DNA sites that are bound by methylation-sensitive immediate-early transcription factors and OCT4, respectively, illuminating TET function in transcriptional responses and pluripotency support. One-Sentence SummaryThe catalytic domains of the enzymes that facilitate passive and drive active DNA demethylation have intrinsic sequence preferences that target DNA demethylation to bHLH and bZIP transcription factor binding sites.

molecular biology↗

CONTAIN: An open-source shipping container laboratory optimisedfor automated COVID-19 diagnostics

The COVID-19 pandemic has challenged diagnostic systems globally. Expanding testing capabilities to conduct population-wide screening for COVID-19 requires innovation in diagnostic services at both the molecular and industrial scale. No report to-date has considered the complexity of laboratory infrastructure in conjunction with the available molecular assays to offer a standardised solution to testing. Here we present CONTAIN. A modular biosafety level 2+ laboratory optimised for automated RT-qPCR COVID-19 testing based on a standard 40ft shipping container. Using open-source liquid-handling robots and RNA extraction reagents we demonstrate a reproducible workflow for RT-qPCR COVID-19 testing. With five OT2 liquid handlers, a single CONTAIN unit reaches a maximum daily testing capacity of 2400 tests/day. We validate this workflow for automated RT-qPCR testing, using both synthetic SARS-CoV-2 samples and patient samples from a local NHS hospital. Finally, we discuss the suitability of CONTAIN and its flexibility in a range of diagnostic testing scenarios including high-density urban environments and mobile response units. Visual abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/106625v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@18acad6org.highwire.dtl.DTLVardef@10ae5f1org.highwire.dtl.DTLVardef@7e34d3org.highwire.dtl.DTLVardef@1be3815_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

Bio-On-Magnetic-Beads (BOMB): Open platform for high-throughput nucleic acid extraction and manipulation

Current molecular biology laboratories rely heavily on the purification and manipulation of nucleic acids. Yet, commonly used centrifuge-and column-based protocols require specialised equipment, often use toxic reagents and are not economically scalable or practical to use in a high-throughput manner. Although it has been known for some time that magnetic beads can provide an elegant answer to these issues, the development of open-source protocols based on beads has been limited. In this article, we provide step-by-step instructions for an easy synthesis of functionalised magnetic beads, and detailed protocols for their use in the high-throughput purification of plasmids, genomic DNA and total RNA from different sources, as well as environmental TNA and PCR amplicons. We also provide a bead-based protocol for bisulfite conversion, and size selection of DNA and RNA fragments. Comparison to other methods highlights the capability, versatility and extreme cost-effectiveness of using magnetic beads. These open source protocols and the associated webpage (https://bomb.bio) can serve as a platform for further protocol customisation and community engagement.

molecular biology↗