Ariel - Volume 10 Number 3

Executive Editors Madalyn Schaefgen David Reich Business Manager David Reich News Editors Medical College Edward Zurad CAHS John Guardiani World Mark Zwanger Features Editors Meg Trexler Jim O\u27Brien Editorials Editor Jeffrey Banyas Photography and Sports Editor Stuart Singer Commons Editor Brenda Peterso

Targeted epigenetic editing of SPDEF reduces mucus production in lung epithelial cells

Airway mucus hypersecretion contributes to the morbidity and mortality in patients with chronic inflammatory lung diseases. Reducing mucus production is crucial for improving patients' quality of life. The transcription factor SAM-pointed domain-containing Ets-like factor (SPDEF) plays a critical role in the regulation of mucus production and, therefore, represents a potential therapeutic target. This study aims to reduce lung epithelial mucus production by targeted silencing SPDEF using the novel strategy, epigenetic editing. Zinc fingers and CRISPR/dCas platforms were engineered to target repressors (KRAB, DNA methyltransferases, histone methyltransferases) to the SPDEF promoter. All constructs were able to effectively suppress both SPDEF mRNA and protein expression, which was accompanied by inhibition of downstream mucus-related genes [anterior gradient 2 (AGR2), mucin 5AC (MUC5AC)]. For the histone methyltransferase G9A, and not its mutant or other effectors, the obtained silencing was mitotically stable. These results indicate efficient SPDEF silencing and downregulation of mucus-related gene expression by epigenetic editing, in human lung epithelial cells. This opens avenues for epigenetic editing as a novel therapeutic strategy to induce long-lasting mucus inhibition

Regulation of DNA Methylation Patterns by CK2-Mediated Phosphorylation of Dnmt3a

DNA methylation is a central epigenetic modification that is established by de novo DNA methyltransferases. The mechanisms underlying the generation of genomic methylation patterns are still poorly understood. Using mass spectrometry and a phosphospecific Dnmt3a antibody, we demonstrate that CK2 phosphorylates endogenous Dnmt3a at two key residues located near its PWWP domain, thereby downregulating the ability of Dnmt3a to methylate DNA. Genome-wide DNA methylation analysis shows that CK2 primarily modulates CpG methylation of several repeats, most notably of Alu SINEs. This modulation can be directly attributed to CK2-mediated phosphorylation of Dnmt3a. We also find that CK2-mediated phosphorylation is required for localization of Dnmt3a to heterochromatin. By revealing phosphorylation as a mode of regulation of de novo DNA methyltransferase function and by uncovering a mechanism for the regulation of methylation at repetitive elements, our results shed light on the origin of DNA methylation patterns

VarLOCK - sequencing independent, rapid detection of SARS-CoV-2 variants of concern for point-of-care testing, qPCR pipelines and national wastewater surveillance

The COVID-19 pandemic continues to pose a threat to the general population. The ongoing vaccination programs provide protection to individuals and facilitate the opening of society and a return to normality. However, emergent and existing SARS-CoV-2 variants capable of evading the immune system endanger the efficacy of the vaccination strategy. To preserve the efficacy of SARS-CoV-2 vaccination globally, aggressive and effective surveillance for known and emerging SARS-CoV-2 Variants of Concern (VOC) is required. Rapid and specific molecular diagnostics can provide speed and coverage advantages compared to genomic sequencing alone, benefitting the public health response and facilitating VOC containment. In this work, we expand the recently developed SARS-CoV-2 CRISPR-Cas detection technology (SHERLOCK) to allow rapid and sensitive discrimination of VOCs, that can be used at point of care and/or implemented in the pipelines of small or large testing facilities, and even determine proportion of VOCs in pooled population-level wastewater samples. This technology aims to complement the ongoing sequencing efforts to allow facile and, crucially, rapid identification of individuals infected with VOCs to help break infection chains. Here, we show the optimisation of our VarLOCK assays (Variant-specific SHERLOCK) for multiple specific mutations in the S gene of SARS-CoV-2 and validation with samples from the Cardiff University Testing Service. We also show the applicability of VarLOCK to national wastewater surveillance of SARS-CoV-2 variants. In addition, we show the rapid adaptability of the technique for new and emerging VOCs such as Omicron

Lazarus1, a DUF300 Protein, Contributes to Programmed Cell Death Associated with Arabidopsis acd11 and the Hypersensitive Response

Programmed cell death (PCD) is a necessary part of the life of multi-cellular organisms. A type of plant PCD is the defensive hypersensitive response (HR) elicited via recognition of a pathogen by host resistance (R) proteins. The lethal, recessive accelerated cell death 11 (acd11) mutant exhibits HR-like accelerated cell death, and cell death execution in acd11 shares genetic requirements for HR execution triggered by one subclass of R proteins

Rewriting DNA Methylation Signatures at Will:The Curable Genome Within Reach?

DNA methyltransferases are important enzymes in a broad range of organisms. Dysfunction of DNA methyltransferases in humans leads to many severe diseases, including cancer. This book focuses on the biochemical properties of these enzymes, describing their structures and mechanisms in bacteria, humans and other species, including plants, and also explains the biological processes of reading of DNA methylation and DNA demethylation. It covers many emerging aspects of the biological roles of DNA methylation functioning as an essential epigenetic mark and describes the role of DNA methylation in diseases. Moreover, the book explains modern technologies, like targeted rewriting of DNA methylation by designed DNA methyltransferases, as well as technological applications of DNA methyltransferases in DNA labelling. Finally, the book summarizes recent methods for the analysis of DNA methylation in human DNA. Overall, this book represents a comprehensive state-of-the-art- work and is a must-have for advanced researchers in the field of DNA methylation and epigenetics

Structure Analysis of Entamoeba histolytica DNMT2 (EhMeth)

In eukaryotes, DNA methylation is an important epigenetic modification that is generally involved in gene regulation. Methyltransferases (MTases) of the DNMT2 family have been shown to have a dual substrate specificity acting on DNA as well as on three specific tRNAs (tRNAAsp, tRNAVal, tRNAGly). Entamoeba histolytica is a major human pathogen, and expresses a single DNA MTase (EhMeth) that belongs to the DNMT2 family and shows high homology to the human enzyme as well as to the bacterial DNA MTase M.HhaI. The molecular basis for the recognition of the substrate tRNAs and discrimination of non-cognate tRNAs is unknown. Here we present the crystal structure of the cytosine-5-methyltransferase EhMeth at a resolution of 2.15 Å, in complex with its reaction product S-adenosyl-L-homocysteine, revealing all parts of a DNMT2 MTase, including the active site loop. Mobility shift assays show that in vitro the full length tRNA is required for stable complex formation with EhMeth

DNA Methylation Analysis of Chromosome 21 Gene Promoters at Single Base Pair and Single Allele Resolution

Differential DNA methylation is an essential epigenetic signal for gene regulation, development, and disease processes. We mapped DNA methylation patterns of 190 gene promoter regions on chromosome 21 using bisulfite conversion and subclone sequencing in five human cell types. A total of 28,626 subclones were sequenced at high accuracy using (long-read) Sanger sequencing resulting in the measurement of the DNA methylation state of 580427 CpG sites. Our results show that average DNA methylation levels are distributed bimodally with enrichment of highly methylated and unmethylated sequences, both for amplicons and individual subclones, which represent single alleles from individual cells. Within CpG-rich sequences, DNA methylation was found to be anti-correlated with CpG dinucleotide density and GC content, and methylated CpGs are more likely to be flanked by AT-rich sequences. We observed over-representation of CpG sites in distances of 9, 18, and 27 bps in highly methylated amplicons. However, DNA sequence alone is not sufficient to predict an amplicon's DNA methylation status, since 43% of all amplicons are differentially methylated between the cell types studied here. DNA methylation in promoter regions is strongly correlated with the absence of gene expression and low levels of activating epigenetic marks like H3K4 methylation and H3K9 and K14 acetylation. Utilizing the single base pair and single allele resolution of our data, we found that i) amplicons from different parts of a CpG island frequently differ in their DNA methylation level, ii) methylation levels of individual cells in one tissue are very similar, and iii) methylation patterns follow a relaxed site-specific distribution. Furthermore, iv) we identified three cases of allele-specific DNA methylation on chromosome 21. Our data shed new light on the nature of methylation patterns in human cells, the sequence dependence of DNA methylation, and its function as epigenetic signal in gene regulation. Further, we illustrate genotype–epigenotype interactions by showing novel examples of allele-specific methylation