Intragenic sequences in the trophectoderm harbour the greatest proportion of methylation errors in day 17 bovine conceptuses generated using assisted reproductive technologies.

BACKGROUND: Assisted reproductive technologies (ART) are widely used to treat fertility issues in humans and for the production of embryos in mammalian livestock. The use of these techniques, however, is not without consequence as they are often associated with inauspicious pre- and postnatal outcomes including premature birth, intrauterine growth restriction and increased incidence of epigenetic disorders in human and large offspring syndrome in cattle. Here, global DNA methylation profiles in the trophectoderm and embryonic discs of in vitro produced (IVP), superovulation-derived (SOV) and unstimulated, synchronised control day 17 bovine conceptuses (herein referred to as AI) were interrogated using the EmbryoGENE DNA Methylation Array (EDMA). Pyrosequencing was used to validate four loci identified as differentially methylated on the array and to assess the differentially methylated regions (DMRs) of six imprinted genes in these conceptuses. The impact of embryo-production induced DNA methylation aberrations was determined using Ingenuity Pathway Analysis, shedding light on the potential functional consequences of these differences. RESULTS: Of the total number of differentially methylated loci identified (3140) 77.3 and 22.7% were attributable to SOV and IVP, respectively. Differential methylation was most prominent at intragenic sequences within the trophectoderm of IVP and SOV-derived conceptuses, almost a third (30.8%) of the differentially methylated loci mapped to intragenic regions. Very few differentially methylated loci were detected in embryonic discs (ED); 0.16 and 4.9% of the differentially methylated loci were located in the ED of SOV-derived and IVP conceptuses, respectively. The overall effects of SOV and IVP on the direction of methylation changes were associated with increased methylation; 70.6% of the differentially methylated loci in SOV-derived conceptuses and 57.9% of the loci in IVP-derived conceptuses were more methylated compared to AI-conceptuses. Ontology analysis of probes associated with intragenic sequences suggests enrichment for terms associated with cancer, cell morphology and growth. CONCLUSION: By examining (1) the effects of superovulation and (2) the effects of an in vitro system (oocyte maturation, fertilisation and embryo culture) we have identified that the assisted reproduction process of superovulation alone has the largest impact on the DNA methylome of subsequent embryos.

Ontogeny, conservation and functional significance of maternally inherited DNA methylation at two classes of non-imprinted genes.

A functional role for DNA methylation has been well-established at imprinted loci, which inherit methylation uniparentally, most commonly from the mother via the oocyte. Many CpG islands not associated with imprinting also inherit methylation from the oocyte, although the functional significance of this, and the common features of the genes affected, are unclear. We identify two major subclasses of genes associated with these gametic differentially methylated regions (gDMRs), namely those important for brain and for testis function. The gDMRs at these genes retain the methylation acquired in the oocyte through preimplantation development, but become fully methylated postimplantation by de novo methylation of the paternal allele. Each gene class displays unique features, with the gDMR located at the promoter of the testis genes but intragenically for the brain genes. Significantly, demethylation using knockout, knockdown or pharmacological approaches in mouse stem cells and fibroblasts resulted in transcriptional derepression of the testis genes, indicating that they may be affected by environmental exposures, in either mother or offspring, that cause demethylation. Features of the brain gene group suggest that they might represent a pool from which many imprinted genes have evolved. The locations of the gDMRs, as well as methylation levels and repression effects, were also conserved in human cells.

5-Hydroxymethylation marks a class of neuronal gene regulated by intragenic methylcytosine levels.

We recently identified a class of neuronal gene inheriting high levels of intragenic methylation from the mother and maintaining this through later development. We show here that these genes are implicated in basic neuronal functions such as post-synaptic signalling, rather than neuronal development and inherit high levels of 5mC, but not 5hmC, from the mother. 5mC is distributed across the gene body and appears to facilitate transcription, as transcription is reduced in DNA methyltransferase I (Dnmt1) knockout embryonic stem cells as well as in fibroblasts treated with a methyltransferase inhibitor. However in adult brain, transcription is more closely associated with a gain in 5hmC, which occurs without a measurable loss of 5mC. These findings add to growing evidence that there may be a role for 5mC in promoting transcription as well as its classical role in gene silencing.

Genomic, Proteomic, and Phenotypic Biomarkers of COVID-19 Severity: Protocol for a Retrospective Observational Study.

BACKGROUND: Health organizations and countries around the world have found it difficult to control the spread of COVID-19. To minimize the future impact on the UK National Health Service and improve patient care, there is a pressing need to identify individuals who are at a higher risk of being hospitalized because of severe COVID-19. Early targeted work was successful in identifying angiotensin-converting enzyme-2 receptors and type II transmembrane serine protease dependency as drivers of severe infection. Although a targeted approach highlights key pathways, a multiomics approach will provide a clearer and more comprehensive picture of severe COVID-19 etiology and progression. OBJECTIVE: The COVID-19 Response Study aims to carry out an integrated multiomics analysis to identify biomarkers in blood and saliva that could contribute to host susceptibility to SARS-CoV-2 and the development of severe COVID-19. METHODS: The COVID-19 Response Study aims to recruit 1000 people who recovered from SARS-CoV-2 infection in both community and hospital settings on the island of Ireland. This protocol describes the retrospective observational study component carried out in Northern Ireland (NI; Cohort A); the Republic of Ireland cohort will be described separately. For all NI participants (n=519), SARS-CoV-2 infection has been confirmed by reverse transcription-quantitative polymerase chain reaction. A prospective Cohort B of 40 patients is also being followed up at 1, 3, 6, and 12 months postinfection to assess longitudinal symptom frequency and immune response. Data will be sourced from whole blood, saliva samples, and clinical data from the electronic care records, the general health questionnaire, and a 12-item general health questionnaire mental health survey. Saliva and blood samples were processed to extract DNA and RNA before whole-genome sequencing, RNA sequencing, DNA methylation analysis, microbiome analysis, 16S ribosomal RNA gene sequencing, and proteomic analysis were performed on the plasma. Multiomics data will be combined with clinical data to produce sensitive and specific prognostic models for severity risk. RESULTS: An initial demographic and clinical profile of the NI Cohort A has been completed. A total of 249 hospitalized patients and 270 nonhospitalized patients were recruited, of whom 184 (64.3%) were female, and the mean age was 45.4 (SD 13) years. High levels of comorbidity were evident in the hospitalized cohort, with cardiovascular disease and metabolic and respiratory disorders being the most significant (P<.001), grouped according to the International Classification of Diseases 10 codes. CONCLUSIONS: This study will provide a comprehensive opportunity to study the mechanisms of COVID-19 severity in recontactable participants. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/50733.

Folic acid intervention during pregnancy alters DNA methylation, affecting neural target genes through two distinct mechanisms.

BACKGROUND: We previously showed that continued folic acid (FA) supplementation beyond the first trimester of pregnancy appears to have beneficial effects on neurocognitive performance in children followed for up to 11 years, but the biological mechanism for this effect has remained unclear. Using samples from our randomized controlled trial of folic acid supplementation in second and third trimester (FASSTT), where significant improvements in cognitive and psychosocial performance were demonstrated in children from mothers supplemented in pregnancy with 400 µg/day FA compared with placebo, we examined methylation patterns from cord blood (CB) using the EPIC array which covers approximately 850,000 cytosine-guanine (CG) sites across the genome. Genes showing significant differences were verified using pyrosequencing and mechanistic approaches used in vitro to determine effects on transcription. RESULTS: FA supplementation resulted in significant differences in methylation, particularly at brain-related genes. Further analysis showed these genes split into two groups. In one group, which included the CES1 gene, methylation changes at the promoters were important for regulating transcription. We also identified a second group which had a characteristic bimodal profile, with low promoter and high gene body (GB) methylation. In the latter, loss of methylation in the GB is linked to decreases in transcription: this group included the PRKAR1B/HEATR2 genes and the dopamine receptor regulator PDE4C. Overall, methylation in CB also showed good correlation with methylation profiles seen in a published data set of late gestation foetal brain samples. CONCLUSION: We show here clear alterations in DNA methylation at specific classes of neurodevelopmental genes in the same cohort of children, born to FA-supplemented mothers, who previously showed improved cognitive and psychosocial performance. Our results show measurable differences at neural genes which are important for transcriptional regulation and add to the supporting evidence for continued FA supplementation throughout later gestation. This trial was registered on 15 May 2013 at www.isrctn.com as ISRCTN19917787.

The metabolic-epigenetic nexus in type 2 diabetes mellitus.

The prevalence of type 2 diabetes mellitus (T2DM) continues to rise globally. Yet the aetiology and pathophysiology of this noncommunicable, polygenic disease, is poorly understood. Lifestyle factors, such as poor dietary intake, lack of exercise, and abnormal glycaemia, are purported to play a role in disease onset and progression, and these environmental factors may disrupt specific epigenetic mechanisms, leading to a reprogramming of gene transcription. The hyperglycaemic cell per se, alters epigenetics through chemical modifications to DNA and histones via metabolic intermediates such as succinate, α-ketoglutarate and O-GlcNAc. To illustrate, α-ketoglutarate is considered a salient co-factor in the activation of the ten-eleven translocation (TET) dioxygenases, which drives DNA demethylation. On the contrary, succinate and other mitochondrial tricarboxylic acid cycle intermediates, inhibit TET activity predisposing to a state of hypermethylation. Hyperglycaemia depletes intracellular ascorbic acid, and damages DNA by enhancing the production of reactive oxygen species (ROS); this compromised cell milieu exacerbates the oxidation of 5-methylcytosine alongside a destabilisation of TET. These metabolic connections may regulate DNA methylation, affecting gene transcription and pancreatic islet ß-cell function in T2DM. This complex interrelationship between metabolism and epigenetic alterations may provide a conceptual foundation for understanding how pathologic stimuli modify and control the intricacies of T2DM. As such, this narrative review will comprehensively evaluate and detail the interplay between metabolism and epigenetic modifications in T2DM.

CandiMeth: Powerful yet simple visualization and quantification of DNA methylation at candidate genes.

BACKGROUND: DNA methylation microarrays are widely used in clinical epigenetics and are often processed using R packages such as ChAMP or RnBeads by trained bioinformaticians. However, looking at specific genes requires bespoke coding for which wet-lab biologists or clinicians are not trained. This leads to high demands on bioinformaticians, who may lack insight into the specific biological problem. To bridge this gap, we developed a tool for mapping and quantification of methylation differences at candidate genomic features of interest, without using coding. FINDINGS: We generated the workflow "CandiMeth" (Candidate Methylation) in the web-based environment Galaxy. CandiMeth takes as input any table listing differences in methylation generated by either ChAMP or RnBeads and maps these to the human genome. A simple interface then allows the user to query the data using lists of gene names. CandiMeth generates (i) tracks in the popular UCSC Genome Browser with an intuitive visual indicator of where differences in methylation occur between samples or groups of samples and (ii) tables containing quantitative data on the candidate regions, allowing interpretation of significance. In addition to genes and promoters, CandiMeth can analyse methylation differences at long and short interspersed nuclear elements. Cross-comparison to other open-resource genomic data at UCSC facilitates interpretation of the biological significance of the data and the design of wet-lab assays to further explore methylation changes and their consequences for the candidate genes. CONCLUSIONS: CandiMeth (RRID:SCR_017974; Biotools: CandiMeth) allows rapid, quantitative analysis of methylation at user-specified features without the need for coding and is freely available at https://github.com/sjthursby/CandiMeth.

Maternal folate nutrition and offspring health: evidence and current controversies.

Periconceptional folic acid (FA) is known to have a protective effect in the prevention of neural tube defects (NTD), leading to global recommendations for FA supplementation before and in early pregnancy. Maternal folate throughout pregnancy may have other roles in offspring health, including neurodevelopment and cognitive performance in childhood. Folate is essential for C1 metabolism, a network of pathways involved in several biological processes including nucleotide synthesis, DNA repair and methylation reactions. The evidence reviewed here shows a conclusive role for offspring health of maternal folate nutrition in early pregnancy and probable benefits in later pregnancy. Folate-mediated epigenetic changes in genes related to brain development and function offer a plausible biological basis to link maternal folate with effects in offspring brain, albeit this research is in its infancy. Mandatory FA fortification of food has proven to be highly effective in decreasing NTD cases in populations where it has been implemented, but this policy is controversial owing to concerns related to potential adverse effects of over-exposure to FA. In the absence of population-wide fortification, and given the generally poor compliance with current FA recommendations, optimising folate status of mothers in very early pregnancy for protection against NTD remains challenging. Thus, current policy in the UK, Ireland and elsewhere in Europe for the prevention of NTD (based on periconceptional FA supplementation only), has proven to be largely ineffective. This review addresses the evidence and the controversies that surround this area, as well as identifying the challenges in translating policy into practice.

A randomized controlled trial of folic acid intervention in pregnancy highlights a putative methylation-regulated control element at ZFP57.

BACKGROUND: Maternal blood folate concentrations during pregnancy have been previously linked with DNA methylation patterns, but this has been done predominantly through observational studies. We showed recently in an epigenetic analysis of the first randomized controlled trial (RCT) of folic acid supplementation specifically in the second and third trimesters (the EpiFASSTT trial) that methylation at some imprinted genes was altered in cord blood samples in response to treatment. Here, we report on epigenome-wide screening using the Illumina EPIC array (~ 850,000 sites) in these same samples (n = 86). RESULTS: The top-ranked differentially methylated promoter region (DMR) showed a gain in methylation with folic acid (FA) and was located upstream of the imprint regulator ZFP57. Differences in methylation in cord blood between placebo and folic acid treatment groups at this DMR were verified using pyrosequencing. The DMR also gains methylation in maternal blood in response to FA supplementation. We also found evidence of differential methylation at this region in an independent RCT cohort, the AFAST trial. By altering methylation at this region in two model systems in vitro, we further demonstrated that it was associated with ZFP57 transcription levels. CONCLUSIONS: These results strengthen the link between folic acid supplementation during later pregnancy and epigenetic changes and identify a novel mechanism for regulation of ZFP57. This trial was registered 15 May 2013 at www.isrctn.com as ISRCTN19917787.

Gene-specific DNA methylation in newborns in response to folic acid supplementation during the second and third trimesters of pregnancy: epigenetic analysis from a randomized controlled trial.

Background: Emerging evidence suggests that maternal folate status can impact cognitive development in childhood. Folate-dependent DNA methylation may provide a biological mechanism to link folate status during pregnancy with cognition in the offspring. Objective: The objective was to investigate the effect of continued folic acid (FA) supplementation beyond the first trimester of pregnancy on DNA methylation in cord blood of epigenetically controlled genes related to brain development and function. Design: Using available cord blood samples (n = 86) from the Folic Acid Supplementation in the Second and Third Trimesters (FASSTT) trial in pregnancy, we applied pyrosequencing techniques to analyze cord blood DNA at 9 candidate loci known to be regulated by methylation, including some previously implicated in observational studies: the widely dispersed retrotransposon long interspersed nuclear element-1 (LINE-1) and 8 single-copy loci (RBM46, PEG3, IGF2, GRB10, BDNF, GRIN3B, OPCML, and APC2). Results: The newborns of mothers who received ongoing FA (400 µg/d) through the second and third trimesters, compared with placebo, had significantly lower overall DNA methylation levels at LINE-1 (56.3% ± 1.7% compared with 57.2% ± 2.1%; P = 0.024), IFG2 (48.9% ± 4.4% compared with 51.2% ± 5.1%; P = 0.021), and BDNF (2.7% ± 0.7% compared with 3.1% ± 0.8%; P = 0.003). The effect of FA treatment on DNA methylation was significant only in female offspring for IGF2 (P = 0.028) and only in males for BDNF (P = 0.012). For GRB10 and GRIN3B, we detected no effect on overall methylation; however, individual cytosine-phosphate-guanine sites showed significant DNA methylation changes in response to FA. Conclusions: Continued supplementation with FA through trimesters 2 and 3 of pregnancy results in significant changes in DNA methylation in cord blood of genes related to brain development. The findings offer a potential biological mechanism linking maternal folate status with neurodevelopment of the offspring, but this requires further investigation using a genome-wide approach. This trial was registered at www.isrctn.com as ISRCTN19917787.

Depletion of DNMT1 in differentiated human cells highlights key classes of sensitive genes and an interplay with polycomb repression.

BACKGROUND: DNA methylation plays a vital role in the cell, but loss-of-function mutations of the maintenance methyltransferase DNMT1 in normal human cells are lethal, precluding target identification, and existing hypomorphic lines are tumour cells. We generated instead a hypomorphic series in normal hTERT-immortalised fibroblasts using stably integrated short hairpin RNA. RESULTS: Approximately two-thirds of sites showed demethylation as expected, with one-third showing hypermethylation, and targets were shared between the three independently derived lines. Enrichment analysis indicated significant losses at promoters and gene bodies with four gene classes most affected: (1) protocadherins, which are key to neural cell identity; (2) genes involved in fat homoeostasis/body mass determination; (3) olfactory receptors and (4) cancer/testis antigen (CTA) genes. Overall effects on transcription were relatively small in these fibroblasts, but CTA genes showed robust derepression. Comparison with siRNA-treated cells indicated that shRNA lines show substantial remethylation over time. Regions showing persistent hypomethylation in the shRNA lines were associated with polycomb repression and were derepressed on addition of an EZH2 inhibitor. Persistent hypermethylation in shRNA lines was, in contrast, associated with poised promoters. CONCLUSIONS: We have assessed for the first time the effects of chronic depletion of DNMT1 in an untransformed, differentiated human cell type. Our results suggest polycomb marking blocks remethylation and indicate the sensitivity of key neural, adipose and cancer-associated genes to loss of maintenance methylation activity.

Widespread recovery of methylation at gametic imprints in hypomethylated mouse stem cells following rescue with DNMT3A2.

BACKGROUND: Imprinted loci are paradigms of epigenetic regulation and are associated with a number of genetic disorders in human. A key characteristic of imprints is the presence of a gametic differentially methylated region (gDMR). Previous studies have indicated that DNA methylation lost from gDMRs could not be restored by DNMT1, or the de novo enzymes DNMT3A or 3B in stem cells, indicating that imprinted regions must instead undergo passage through the germline for reprogramming. However, previous studies were non-quantitative, were unclear on the requirement for DNMT3A/B and showed some inconsistencies. In addition, new putative gDMR has recently been described, along with an improved delineation of the existing gDMR locations. We therefore aimed to re-examine the dependence of methylation at gDMRs on the activities of the methyltransferases in mouse embryonic stem cells (ESCs). RESULTS: We examined the most complete current set of imprinted gDMRs that could be assessed using quantitative pyrosequencing assays in two types of ESCs: those lacking DNMT1 (1KO) and cells lacking a combination of DNMT3A and DNMT3B (3abKO). We further verified results using clonal analysis and combined bisulfite and restriction analysis. Our results showed that loss of methylation was approximately equivalent in both cell types. 1KO cells rescued with a cDNA-expressing DNMT1 could not restore methylation at the imprinted gDMRs, confirming some previous observations. However, nearly all gDMRs were remethylated in 3abKO cells rescued with a DNMT3A2 expression construct (3abKO + 3a2). Transcriptional activity at the H19/Igf2 locus also tracked with the methylation pattern, confirming functional reprogramming in the latter. CONCLUSIONS: These results suggested (1) a vital role for DNMT3A/B in methylation maintenance at imprints, (2) that loss of DNMT1 and DNMT3A/B had equivalent effects, (3) that rescue with DNMT3A2 can restore imprints in these cells. This may provide a useful system in which to explore factors influencing imprint reprogramming.

The interplay between DNA methylation, folate and neurocognitive development.

DNA methylation provides an attractive possible means for propagating the effects of environmental inputs during fetal life and impacting subsequent adult mental health, which is leading to increasing collaboration between molecular biologists, nutritionists and psychiatrists. An area of interest is the potential role of folate, not just in neural tube closure in early pregnancy, but in later major neurodevelopmental events, with consequences for later sociocognitive maturation. Here, we set the scene for recent discoveries by reviewing the major events of neural development during fetal life, with an emphasis on tissues and structures where dynamic methylation changes are known to occur. Following this, we give an indication of some of the major classes of genes targeted by methylation and important for neurological and behavioral development. Finally, we highlight some cognitive disorders where methylation changes are implicated as playing an important role.