Widespread genomic de novo DNA methylation occurs following CD8+ T cell activation and proliferation.

This research investigates the intricate dynamics of DNA methylation in the hours following CD8+ T cell activation, during a critical yet understudied temporal window. DNA methylation is an epigenetic modification central to regulation of gene expression and directing immune responses. Our investigation spanned 96-h post-activation and unveils a nuanced tapestry of global and site-specific methylation changes. We identified 15,626 significant differentially methylated CpGs spread across the genome, with the most significant changes occurring within the genes ADAM10, ICA1, and LAPTM5. While many changes had modest effect sizes, approximately 120 CpGs exhibited a log2FC above 1.5, with cell activation and proliferation pathways the most affected. Relatively few of the differentially methylated CpGs occurred along adjacent gene regions. The exceptions were seven differentially methylated gene regions, with the Human T cell Receptor Alpha Joining Genes demonstrating consistent methylation change over a 3kb window. We also investigated whether an inflammatory environment could alter DNA methylation during activation, with proliferating cells exposed to the oxidant glycine chloramine. No substantial differential methylation was observed in this context. The temporal perspective of early activation adds depth to the evolving field of epigenetic immunology, offering insights with implications for therapeutic innovation and expanding our understanding of epigenetic modulation in immune function.

Epigenetic changes associated with Bacillus Calmette-Guerin (BCG) treatment in bladder cancer.

Bacillus Calmette-Guérin (BCG) treatment for non-muscle invasive bladder cancer (NMIBC) is an established immunotherapeutic, however, a significant portion of patients do not respond to treatment. Despite extensive research into the therapeutic mechanism of BCG, gaps remain in our understanding. This review specifically focuses on the epigenomic contributions in the immune microenvironment, in the context of BCG treatment for NMIBC. We also summarise the current understanding of NMIBC epigenetic characteristics, and discuss how future targeted strategies for BCG therapy should incorporate epigenomic biomarkers in conjunction with genomic biomarkers.

Site-specific decreases in DNA methylation in replicating cells following exposure to oxidative stress.

Oxidative stress is a common feature of inflammation-driven cancers, and it promotes genomic instability and aggressive tumour phenotypes. It is known that oxidative stress transiently modulates gene expression through the oxidation of transcription factors and associated regulatory proteins. Neutrophils are our most abundant white blood cells and accumulate at sites of infection and inflammation. Activated neutrophils produce hypochlorous acid and chloramines, which can disrupt DNA methylation by oxidizing methionine. The goal of the current study was to determine whether chloramine exposure results in sequence-specific modifications in DNA methylation that enable long-term alterations in transcriptional output. Proliferating Jurkat T-lymphoma cells were exposed to sublethal doses of glycine chloramine and differential methylation patterns were compared using Illumina EPIC 850 K bead chip arrays. There was a substantial genome-wide decrease in methylation 4 h after exposure that correlated with altered RNA expression for 24 and 48 h, indicating sustained impacts on exposed cells. A large proportion of the most significant differentially methylated CpG sites were situated towards chromosomal ends, suggesting that these regions are most susceptible to inhibition of maintenance DNA methylation. This may contribute to epigenetic instability of chromosomal ends in rapidly dividing cells, with potential implications for the regulation of telomere length and cellular longevity.

Genome-wide impact of hydrogen peroxide on maintenance DNA methylation in replicating cells.

BACKGROUND: Environmental factors, such as oxidative stress, have the potential to modify the epigenetic landscape of cells. We have previously shown that DNA methyltransferase (DNMT) activity can be inhibited by sublethal doses of hydrogen peroxide (H2O2). However, site-specific changes in DNA methylation and the reversibility of any changes have not been explored. Using bead chip array technology, differential methylation was assessed in Jurkat T-lymphoma cells following exposure to H2O2. RESULTS: Sublethal H2O2 exposure was associated with an initial genome-wide decrease in DNA methylation in replicating cells, which was largely corrected 72 h later. However, some alterations were conserved through subsequent cycles of cell division. Significant changes to the variability of DNA methylation were also observed both globally and at the site-specific level. CONCLUSIONS: This research indicates that increased exposure to H2O2 can result in long-term alterations to DNA methylation patterns, providing a mechanism for environmental factors to have prolonged impact on gene expression.

Author Correction: Human gut microbiome changes during a 10 week Randomised Control Trial for micronutrient supplementation in children with attention deficit hyperactivity disorder.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The Driver of Extreme Human-Specific Olduvai Repeat Expansion Remains Highly Active in the Human Genome.

Sequences encoding Olduvai protein domains (formerly DUF1220) show the greatest human lineage-specific increase in copy number of any coding region in the genome and have been associated, in a dosage-dependent manner, with brain size, cognitive aptitude, autism, and schizophrenia. Tandem intragenic duplications of a three-domain block, termed the Olduvai triplet, in four NBPF genes in the chromosomal 1q21.1-0.2 region, are primarily responsible for the striking human-specific copy number increase. Interestingly, most of the Olduvai triplets are adjacent to, and transcriptionally coregulated with, three human-specific NOTCH2NL genes that have been shown to promote cortical neurogenesis. Until now, the underlying genomic events that drove the Olduvai hyperamplification in humans have remained unexplained. Here, we show that the presence or absence of an alternative first exon of the Olduvai triplet perfectly discriminates between amplified (58/58) and unamplified (0/12) triplets. We provide sequence and breakpoint analyses that suggest the alternative exon was produced by an nonallelic homologous recombination-based mechanism involving the duplicative transposition of an existing Olduvai exon found in the CON3 domain, which typically occurs at the C-terminal end of NBPF genes. We also provide suggestive in vitro evidence that the alternative exon may promote instability through a putative G-quadraplex (pG4)-based mechanism. Lastly, we use single-molecule optical mapping to characterize the intragenic structural variation observed in NBPF genes in 154 unrelated individuals and 52 related individuals from 16 families and show that the presence of pG4-containing Olduvai triplets is strongly correlated with high levels of Olduvai copy number variation. These results suggest that the same driver of genomic instability that allowed the evolutionarily recent, rapid, and extreme human-specific Olduvai expansion remains highly active in the human genome.

Human gut microbiome changes during a 10 week Randomised Control Trial for micronutrient supplementation in children with attention deficit hyperactivity disorder.

It has been widely hypothesized that both diet and the microbiome play a role in the regulation of attention-deficit/hyperactivity disorder (ADHD) behaviour. However, there has been very limited scientific investigation into the potential biological connection. We performed a 10-week pilot study investigating the effects of a broad spectrum micronutrient administration on faecal microbiome content, using 16S rRNA gene sequencing. The study consisted of 17 children (seven in the placebo and ten in the treatment group) between the ages of seven and 12 years, who were diagnosed with ADHD. We found that micronutrient treatment did not drive large-scale changes in composition or structure of the microbiome. However, observed OTUs significantly increased in the treatment group, and showed no mean change in the placebo group. The differential abundance and relative frequency of Actinobacteria significantly decreased post- micronutrient treatment, and this was largely attributed to species from the genus Bifidobacterium. This was compensated by an increase in the relative frequency of species from the genus Collinsella. Further research is required to establish the role that Bifidobacterium contribute towards neuropsychiatric disorders; however, these findings suggest that micronutrient administration could be used as a safe, therapeutic method to modulate Bifidobacterium abundance, which could have potential implications for modulating and regulating ADHD behaviour. Our pilot study provides an initial observation into this area of research, and highlights an interesting avenue for further investigation in a larger cohort. Furthermore, these novel results provide a basis for future research on the biological connection between ADHD, diet and the microbiome.

Can we predict treatment response in children with ADHD to a vitamin-mineral supplement? An investigation into pre-treatment nutrient serum levels, MTHFR status, clinical correlates and demographic variables.

BACKGROUND: Intent-to-treat analyses from a randomized controlled trial showed significant between-group differences favouring micronutrient treatment on the Clinical Global Impression-Improvement, but no group differences on clinician, parent and teacher ratings of overall ADHD symptoms. There was an advantage of micronutrients over placebo in improving overall function, emotional regulation, aggression, and reducing impairment as well as improving inattention based on clinician but not parent observation. No group differences were observed on hyperactive-impulsive symptoms. We investigated predictors of response defined by pre-treatment variables. METHOD: We conducted analyses of data from a clinical trial of children (7-12 years) with ADHD, whereby participants were randomized to receive micronutrients or placebo for 10 weeks followed by a 10 week open-label (OL) phase. We included only children who had been exposed to micronutrients for a full 10 week period and demonstrated satisfactory adherence, either in RCT phase (n = 40) or OL phase (those who received placebo during RCT phase; n = 31). Seven outcomes were examined: change in ADHD symptoms (clinician/parent), ADHD responder, overall responder, change in mood, change in functioning, and change in aggression. Demographic, developmental variables, current clinical and physical characteristics, MTHFR genotype at two common variants, and pre-treatment serum/plasma levels (vitamin D, B12, folate, zinc, copper, iron, ferritin, potassium, calcium, magnesium, and homocysteine) were all considered as putative predictors. RESULTS: Substantial nutrient deficiencies pre-treatment were observed only for vitamin D (13%) and copper (15%), otherwise most children entered the trial with nutrient levels falling within expected ranges. Regression analyses showed varying predictors across outcomes with no one predictor being consistently identified across different variables. Lower pre-treatment folate and B12 levels, being female, greater severity of symptoms and co-occurring disorders pre-treatment, more pregnancy complications and fewer birth problems were identified as possible predictors of greater improvement for some but not all outcome measures although predictive values were weak. Lower IQ and higher BMI predicted greater improvement in aggression. CONCLUSIONS: This study replicates Rucklidge et al. (2014b) showing the limited value of using serum nutrient levels to predict treatment response although we cannot rule out that other non-assayed nutrient levels may be more valuable. Additionally, no specific demographic or clinical characteristics, including MTHFR genetic status, were identified that would preclude children with ADHD from trying this treatment approach.

Methylomic changes in response to micronutrient supplementation and MTHFR genotype.

Exposure times and dosage required for dietary components to modify DNA methylation patterns are largely unknown. AIM: This exploratory research represents the first genome-wide analysis of DNA methylation changes during a randomized-controlled-trial (RCT) for dietary supplementation with broad spectrum vitamins, minerals and amino acids in humans. METHODS: Genome-wide changes in methylation from paired, peripheral blood samples were assessed using the Infinium Methylation EPIC 850 K array. RESULTS: Methylation increased at 84% of the most significant differentially methylated CpGs; however, none showed significance after adjustment for genome-wide testing. CONCLUSION: Micronutrient supplementation is unlikely to have a substantial biological effect on DNA methylation over 10 weeks; however, the trend toward hypermethylation that we observed is likely to become more marked with longer exposure periods.

Epigenetics, nutrition and mental health. Is there a relationship?

Many aspects of human development and disease are influenced by the interaction between genetic and environmental factors. Understanding how our genes respond to the environment is central to managing health and disease, and is one of the major contemporary challenges in human genetics. Various epigenetic processes affect chromosome structure and accessibility of deoxyribonucleic acid (DNA) to the enzymatic machinery that leads to expression of genes. One important epigenetic mechanism that appears to underlie the interaction between environmental factors, including diet, and our genome, is chemical modification of the DNA. The best understood of these modifications is methylation of cytosine residues in DNA. It is now recognized that the pattern of methylated cytosines throughout our genomes (the 'methylome') can change during development and in response to environmental cues, often with profound effects on gene expression. Many dietary constituents may indirectly influence genomic pathways that methylate DNA, and there is evidence for biochemical links between nutritional quality and mental health. Deficiency of both macro- and micronutrients has been associated with increased behavioural problems, and nutritional supplementation has proven efficacious in treatment of certain neuropsychiatric disorders. In this review we examine evidence from the fields of nutrition, developmental biology, and mental health that supports dietary impacts on epigenetic processes, particularly DNA methylation. We then consider whether such processes could underlie the demonstrated efficacy of dietary supplementation in treatment of mental disorders, and whether targeted manipulation of DNA methylation patterns using controlled dietary supplementation may be of wider clinical value.

Methylated Cytosine Maintains G-Quadruplex Structures during Polymerase Chain Reaction and Contributes to Allelic Dropout.

The promoter of the human imprinted gene MEST is differentially methylated with respect to the parent of origin and contains several non B-DNA motifs that are capable of forming G-quadruplexes. These factors can contribute to a consistent allelic dropout (ADO) of the maternally methylated DNA during polymerase chain reaction (PCR) analysis of such gene regions. Here, we directly investigate the cause of allelic dropout by applying fluorescent techniques to visualize polymerase amplification and arrest during PCR of differentially methylated DNA templates. We demonstrate that polymerase arrest corresponds to previously characterized G-quadruplex-forming motifs at the MEST promoter region and occurs at equivalent sites on both methylated and nonmethylated DNA templates. However, during PCR, polymerase arrest can be observed on the methylated template for several cycles longer than on the nonmethylated template, and this results in an amplification lag and a lower yield of full length amplicons. We demonstrate that this delay in amplification is sufficient to cause complete ADO during PCR, providing a mechanistic basis for the previously observed genotyping error at this locus.

Allelic Dropout During Polymerase Chain Reaction due to G-Quadruplex Structures and DNA Methylation Is Widespread at Imprinted Human Loci.

Loss of one allele during polymerase chain reaction (PCR) amplification of DNA, known as allelic dropout, can be caused by a variety of mechanisms. Allelic dropout during PCR may have profound implications for molecular diagnostic and research procedures that depend on PCR and assume biallelic amplification has occurred. Complete allelic dropout due to the combined effects of cytosine methylation and G-quadruplex formation was previously described for a differentially methylated region of the human imprinted gene, MEST We now demonstrate that this parent-of-origin specific allelic dropout can potentially occur at several other genomic regions that display genomic imprinting and have propensity for G-quadruplex formation, including AIM1, BLCAP, DNMT1, PLAGL1, KCNQ1, and GRB10 These findings demonstrate that systematic allelic dropout during PCR is a general phenomenon for regions of the genome where differential allelic methylation and G-quadruplex motifs coincide, and suggest that great care must be taken to ensure biallelic amplification is occurring in such situations.

Structural Analysis of G-Quadruplex Formation at the Human MEST Promoter.

The promoter region of the imprinted gene MEST contains several motifs capable of forming G-quadruplex (G4) structures, which appear to contribute to consistent allelic dropout during polymerase chain reaction (PCR) analysis of this region. Here, we extend our previous analysis of MEST G4 structures by applying fluorescent footprinting techniques to assess non B-DNA structure and topology in dsDNA from the full MEST promoter region, under conditions that mimic PCR. We demonstrate that the buffer used for PCR provides an extremely favourable milieu for G4 formation, and that cytosine methylation helps maintain G4 structures during PCR. Additionally, we demonstrate G4 formation at motifs not previously identified through bioinformatic analysis of the MEST promoter, and provide nucleotide level resolution for topological reconstruction of these structures. These observations increase our understanding of the mechanisms through which methylation and G4 contribute towards allelic drop-out during PCR.

[Letter to the Editor] Many commercial hot-start polymerases demonstrate activity prior to thermal activation.

Address correspondence to Martin A. Kennedy, Department of Pathology & Carney Centre for Pharmacogenomics, University of Otago, Christchurch, PO Box 4345, Christchurch, New Zealand. E-mail: martin.kennedy@otago.ac.nz.

Fluorescence Methods for Probing G-Quadruplex Structure in Single- and Double-Stranded DNA.

Interest in exploring G-quadruplex (G4) structures in nucleic acids is growing as it becomes more widely recognized that these structures have many interesting biological roles and chemical properties. Probing the G4-forming potential of DNA with dimethyl sulfate, polymerase stop assays, or nuclease digestion are three commonly used techniques that usually employ radio-isotopic labels for visualization. However, as fluorescent labeling methods have grown in popularity and versatility, many laboratories have moved away from the routine use of radio-isotopic methods. We have adapted traditional procedures for structural analysis of G4-forming DNA sequences by using fluorescent labels and capillary electrophoresis and demonstrate their application to well-studied G4 structures, including c-MYC PU27 G4. The three fluorescent assays described here allow interrogation of G4 structures in double- and single-stranded DNA substrates, using either chemical or enzymatic cleavage. When combined, these techniques can provide valuable information for the investigation of G4 topology and structure, as well as visualizing any structural effects caused by interaction of quadruplexes with the complementary C-rich DNA strand.

Circular replication-associated protein encoding DNA viruses identified in the faecal matter of various animals in New Zealand.

In recent years, innovations in molecular techniques and sequencing technologies have resulted in a rapid expansion in the number of known viral sequences, in particular those with circular replication-associated protein (Rep)-encoding single-stranded (CRESS) DNA genomes. CRESS DNA viruses are present in the virome of many ecosystems and are known to infect a wide range of organisms. A large number of the recently identified CRESS DNA viruses cannot be classified into any known viral families, indicating that the current view of CRESS DNA viral sequence space is greatly underestimated. Animal faecal matter has proven to be a particularly useful source for sampling CRESS DNA viruses in an ecosystem, as it is cost-effective and non-invasive. In this study a viral metagenomic approach was used to explore the diversity of CRESS DNA viruses present in the faeces of domesticated and wild animals in New Zealand. Thirty-eight complete CRESS DNA viral genomes and two circular molecules (that may be defective molecules or single components of multicomponent genomes) were identified from forty-nine individual animal faecal samples. Based on shared genome organisations and sequence similarities, eighteen of the isolates were classified as gemycircularviruses and twelve isolates were classified as smacoviruses. The remaining eight isolates lack significant sequence similarity with any members of known CRESS DNA virus groups. This research adds significantly to our knowledge of CRESS DNA viral diversity in New Zealand, emphasising the prevalence of CRESS DNA viruses in nature, and reinforcing the suggestion that a large proportion of CRESS DNA viruses are yet to be identified.

G-quadruplex structures and CpG methylation cause drop-out of the maternal allele in polymerase chain reaction amplification of the imprinted MEST gene promoter.

We observed apparent non-Mendelian behaviour of alleles when genotyping a region in a CpG island at the 5' end of the maternally imprinted human MEST isoform. This region contains three single nucleotide polymorphisms (SNPs) in total linkage disequilibrium, such that only two haplotypes occur in the human population. Only one haplotype was detectable in each subject, never both, despite the use of multiple primers and several genotyping methods. We observed that this region contains motifs capable of forming several G-quadruplex structures. Circular dichroism spectroscopy and native polyacrylamide gel electrophoresis confirmed that at least three G-quadruplexes form in vitro in the presence of potassium ions, and one of these structures has a Tm of greater than 99°C in polymerase chain reaction (PCR) buffer. We demonstrate that it is the methylated maternal allele that is always lost during PCR amplification, and that formation of G-quadruplexes and presence of methylated cytosines both contributed to this phenomenon. This observed parent-of-origin specific allelic drop-out has important implications for analysis of imprinted genes in research and diagnostic settings.