Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is commonly diagnosed by reverse transcription polymerase chain reaction (RT-PCR) to ...detect viral RNA in patient samples, but RNA extraction constitutes a major bottleneck in current testing. Methodological simplification could increase diagnostic availability and efficiency, benefitting patient care and infection control. Here, we describe methods circumventing RNA extraction in COVID-19 testing by performing RT-PCR directly on heat-inactivated or lysed samples. Our data, including benchmarking using 597 clinical patient samples and a standardised diagnostic system, demonstrate that direct RT-PCR is viable option to extraction-based tests. Using controlled amounts of active SARS-CoV-2, we confirm effectiveness of heat inactivation by plaque assay and evaluate various generic buffers as transport medium for direct RT-PCR. Significant savings in time and cost are achieved through RNA-extraction-free protocols that are directly compatible with established PCR-based testing pipelines. This could aid expansion of COVID-19 testing.
Understanding innate immune responses in COVID-19 is important to decipher mechanisms of host responses and interpret disease pathogenesis. Natural killer (NK) cells are innate effector lymphocytes ...that respond to acute viral infections but might also contribute to immunopathology. Using 28-color flow cytometry, we here reveal strong NK cell activation across distinct subsets in peripheral blood of COVID-19 patients. This pattern was mirrored in scRNA-seq signatures of NK cells in bronchoalveolar lavage from COVID-19 patients. Unsupervised high-dimensional analysis of peripheral blood NK cells furthermore identified distinct NK cell immunotypes that were linked to disease severity. Hallmarks of these immunotypes were high expression of perforin, NKG2C, and Ksp37, reflecting increased presence of adaptive NK cells in circulation of patients with severe disease. Finally, arming of CD56
NK cells was observed across COVID-19 disease states, driven by a defined protein-protein interaction network of inflammatory soluble factors. This study provides a detailed map of the NK cell activation landscape in COVID-19 disease.
X-chromosome inactivation and X-upregulation are the fundamental modes of chromosome-wide gene regulation that collectively achieve dosage compensation in mammals, but the regulatory link between the ...two remains elusive and the X-upregulation dynamics are unknown. Here, we use allele-resolved single-cell RNA-seq combined with chromatin accessibility profiling and finely dissect their separate effects on RNA levels during mouse development. Surprisingly, we uncover that X-upregulation elastically tunes expression dosage in a sex- and lineage-specific manner, and moreover along varying degrees of X-inactivation progression. Male blastomeres achieve X-upregulation upon zygotic genome activation while females experience two distinct waves of upregulation, upon imprinted and random X-inactivation; and ablation of Xist impedes female X-upregulation. Female cells carrying two active X chromosomes lack upregulation, yet their collective RNA output exceeds that of a single hyperactive allele. Importantly, this conflicts the conventional dosage compensation model in which naïve female cells are initially subject to biallelic X-upregulation followed by X-inactivation of one allele to correct the X dosage. Together, our study provides key insights to the chain of events of dosage compensation, explaining how transcript copy numbers can remain remarkably stable across developmental windows wherein severe dose imbalance would otherwise be experienced by the cell.
The Notch signaling pathway has fundamental roles in embryonic development and in the nervous system. The current model of receptor activation involves initiation via a force-induced conformational ...change. Here, we define conditions that reveal pulling force-independent Notch activation using soluble multivalent constructs. We treat neuroepithelial stem-like cells with molecularly precise ligand nanopatterns displayed from solution using DNA origami. Notch signaling follows with clusters of Jag1, and with chimeric structures where most Jag1 proteins are replaced by other binders not targeting Notch. Our data rule out several confounding factors and suggest a model where Jag1 activates Notch upon prolonged binding without appearing to need a pulling force. These findings reveal a distinct mode of activation of Notch and lay the foundation for the development of soluble agonists.
Altered DNA methylation patterns in CD4(+) T-cells indicate the importance of epigenetic mechanisms in inflammatory diseases. However, the identification of these alterations is complicated by the ...heterogeneity of most inflammatory diseases. Seasonal allergic rhinitis (SAR) is an optimal disease model for the study of DNA methylation because of its well-defined phenotype and etiology. We generated genome-wide DNA methylation (N(patients) = 8, N(controls) = 8) and gene expression (N(patients) = 9, Ncontrols = 10) profiles of CD4(+) T-cells from SAR patients and healthy controls using Illumina's HumanMethylation450 and HT-12 microarrays, respectively. DNA methylation profiles clearly and robustly distinguished SAR patients from controls, during and outside the pollen season. In agreement with previously published studies, gene expression profiles of the same samples failed to separate patients and controls. Separation by methylation (N(patients) = 12, N(controls) = 12), but not by gene expression (N(patients) = 21, N(controls) = 21) was also observed in an in vitro model system in which purified PBMCs from patients and healthy controls were challenged with allergen. We observed changes in the proportions of memory T-cell populations between patients (N(patients) = 35) and controls (N(controls) = 12), which could explain the observed difference in DNA methylation. Our data highlight the potential of epigenomics in the stratification of immune disease and represents the first successful molecular classification of SAR using CD4(+) T cells.
Birth weight is determined by the interplay between infant genetics and the intrauterine environment and is associated with several health outcomes in later life. Many studies have reported an ...association between birth weight and DNA methylation in infants and suggest that altered epigenetics may underlie birthweight-associated health outcomes. However, birth weight is a relatively nonspecific measure of fetal growth and consists of fat mass and fat-free mass which may have different effects on health outcomes which motivates studies of infant body composition and DNA methylation. Here, we combined genome-wide DNA methylation profiling of buccal cells from 47 full-term one-week old infants with accurate measurements of infant fat mass and fat-free mass using air-displacement plethysmography.
No significant association was found between DNA methylation in infant buccal cells and infant body composition. Moreover, no association between infant DNA methylation and parental body composition or indicators of maternal glucose metabolism were found.
Despite accurate measures of body composition, we did not identify any associations between infant body fatness and DNA methylation. These results are consistent with recent studies that generally have identified only weak associations between DNA methylation and birthweight. Although our results should be confirmed by additional larger studies, our findings may suggest that differences in DNA methylation between individuals with low and high body fatness may be established later in childhood.
5-methylcytosine (5mC) is converted to 5-hydroxymethylcytosine (5hmC) by the TET family of enzymes as part of a recently discovered active DNA de-methylation pathway. 5hmC plays important roles in ...regulation of gene expression and differentiation and has been implicated in T cell malignancies and autoimmunity. Here, we report early and widespread 5mC/5hmC remodeling during human CD4+ T cell differentiation ex vivo at genes and cell-specific enhancers with known T cell function. We observe similar DNA de-methylation in CD4+ memory T cells in vivo, indicating that early remodeling events persist long term in differentiated cells. Underscoring their important function, 5hmC loci were highly enriched for genetic variants associated with T cell diseases and T-cell-specific chromosomal interactions. Extensive functional validation of 22 risk variants revealed potentially pathogenic mechanisms in diabetes and multiple sclerosis. Our results support 5hmC-mediated DNA de-methylation as a key component of CD4+ T cell biology in humans, with important implications for gene regulation and lineage commitment.
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•5hmC remodeling is widespread during human CD4+ T cell differentiation•Early 5hmC gains predict loss of DNA methylation in differentiated cells•Early 5hmC remodeling in vitro predicts loss of DNA methylation in vivo•5hmC loci are enriched for functional T cell disease-associated genetic variants
Nestor et al. reveal widespread 5hmC-mediated DNA de-methylation during in vitro differentiation of human CD4+ T cells. They find that regions undergoing 5hmC remodeling are enriched for disease-associated regulatory regions.
Lentini and Reinius address issues in interpreting non-allelic gene expression measurements of dosage compensation during murine embryonic development.
During canonical Wnt signalling, the activity of nuclear β‐catenin is largely mediated by the TCF/LEF family of transcription factors. To challenge this view, we used the CRISPR/Cas9 genome editing ...approach to generate HEK 293T cell clones lacking all four TCF/LEF genes. By performing unbiased whole transcriptome sequencing analysis, we found that a subset of β‐catenin transcriptional targets did not require TCF/LEF factors for their regulation. Consistent with this finding, we observed in a genome‐wide analysis that β‐catenin occupied specific genomic regions in the absence of TCF/LEF. Finally, we revealed the existence of a transcriptional activity of β‐catenin that specifically appears when TCF/LEF factors are absent, and refer to this as β‐catenin‐GHOST response. Collectively, this study uncovers a previously neglected modus operandi of β‐catenin that bypasses the TCF/LEF transcription factors.
Synopsis
TCF/LEF‐deleted cells are used to challenge the assumption that Wnt/β‐catenin transcription is exclusively mediated by this transcription factor family. Genome‐wide gene expression and β‐catenin DNA binding analyses suggest that alternative transcription factors control a subset of β‐catenin target genes.
TCF/LEF quadruple knockout (d4TCF) and β‐catenin knockout (dBcat) HEK 293T cells were generated.
d4TCF cells display β‐catenin‐dependent gene regulation upon Wnt pathway activation.
β‐catenin binds to specific genomic regions in the absence of TCF/LEF.
The transcription factor FOXO4 is a candidate for TCF/LEF‐independent β‐catenin activity.
Genome‐wide gene expression and β‐catenin DNA binding analyses in TCF/LEF knockout cells reveal a subset of Wnt/β‐catenin target genes that do not depend on these transcription factors.
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