Epigenetic aging of human blood cells is influenced by the age of the host body.

Allogenic hematopoietic stem cell transplantation is a therapeutic procedure performed over a wide range of donor and recipient age combinations, representing natural experiments of how the age of the recipient affects aging in transplanted donor cells in vivo. We measured DNA methylation and epigenetic aging in donors and recipients and found that biological epigenetic clocks are accelerated in cells transplanted into an older body and decelerated in a younger body. This is the first evidence that the age of the circulating environment influences human epigenetic aging in vivo.

Single-cell m6A mapping in vivo using picoMeRIP-seq.

Current N6-methyladenosine (m6A) mapping methods need large amounts of RNA or are limited to cultured cells. Through optimized sample recovery and signal-to-noise ratio, we developed picogram-scale m6A RNA immunoprecipitation and sequencing (picoMeRIP-seq) for studying m6A in vivo in single cells and scarce cell types using standard laboratory equipment. We benchmark m6A mapping on titrations of poly(A) RNA and embryonic stem cells and in single zebrafish zygotes, mouse oocytes and embryos.

The RNA m6A landscape of mouse oocytes and preimplantation embryos.

Despite the significance of N6-methyladenosine (m6A) in gene regulation, the requirement for large amounts of RNA has hindered m6A profiling in mammalian early embryos. Here we apply low-input methyl RNA immunoprecipitation and sequencing to map m6A in mouse oocytes and preimplantation embryos. We define the landscape of m6A during the maternal-to-zygotic transition, including stage-specifically expressed transcription factors essential for cell fate determination. Both the maternally inherited transcripts to be degraded post fertilization and the zygotically activated genes during zygotic genome activation are widely marked by m6A. In contrast to m6A-marked zygotic ally-activated genes, m6A-marked maternally inherited transcripts have a higher tendency to be targeted by microRNAs. Moreover, RNAs derived from retrotransposons, such as MTA that is maternally expressed and MERVL that is transcriptionally activated at the two-cell stage, are largely marked by m6A. Our results provide a foundation for future studies exploring the regulatory roles of m6A in mammalian early embryonic development.

Post-acute symptoms 3-15 months after COVID-19 among unvaccinated and vaccinated individuals with a breakthrough infection.

OBJECTIVE: We aimed to describe post-acute sequelae of SARS-CoV-2 infection (PASC) related symptoms 3-15 months after a positive test in SARS-CoV-2 unvaccinated and vaccinated participants with a breakthrough infection. METHODS: Participants of the Norwegian COVID-19 cohort, without a positive SARS-CoV-2 test, completed a questionnaire about PASC-related symptoms between November 2020 and January 2021. About a year later, a second questionnaire (which also included the Everyday Memory Questionnaire [EMQ]-13) was completed by the same participants, most still without a positive SARS-CoV-2 test, but also by unvaccinated and vaccinated participants with a positive test 3-15 months before the questionnaire. Laboratory-confirmed SARS-CoV-2 status (positive or negative swab test determined by reverse transcriptase quantitative polymerase chain reaction) at the time of completing the questionnaire was ascertained from the Mandatory Norwegian Surveillance System for Communicable Diseases. RESULTS: No differences were found in the self-reported PASC symptoms, dyspnea, fatigue, smell/taste changes, concentration problems, or the EMQ-13 score between unvaccinated and vaccinated participants 3-15 months after the positive test. Fewer memory problems were reported among vaccinated than unvaccinated participants. CONCLUSION: SARS-CoV-2 vaccines offer minor protection against PASC symptoms, although fewer memory problems were reported among the vaccinated than the unvaccinated participants.

Cri du chat syndrome patients have DNA methylation changes in genes linked to symptoms of the disease.

BACKGROUND: Cri du chat (also called 5p deletion, or monosomy 5p) syndrome is a genetic disease caused by deletions of various lengths in the short (p) arm of chromosome 5. Genetic analysis and phenotyping have been used to suggest dose-sensitive genes in this region that may cause symptoms when a gene copy is lost, but the heterogeneity of symptoms for patients with similar deletions complicates the picture. The epigenetics of the syndrome has only recently been looked at with DNA methylation measurements of blood from a single patient, suggesting epigenetic changes in these patients. Here, we conduct the deepest epigenetic analysis of the syndrome to date with DNA methylation analysis of eight Cri du chat patients with sibling- and age-matched controls. RESULTS: The genome-wide patterns of DNA methylation in the blood of Cri du chat patients reveal distinct changes compared to controls. In the p-arm of chromosome 5 where patients are hemizygous, we find stronger changes in methylation of CpG sites than what is seen in the rest of the genome, but this effect is less pronounced in gene regulatory sequences. Gene set enrichment analysis using patient DNA methylation changes in gene promoters revealed enrichment of genes controlling embryonic development and genes linked to symptoms which are among the most common symptoms of Cri du chat syndrome: developmental delay and microcephaly. Importantly, this relative enrichment is not driven by changes in the methylation of genes on chromosome 5. CpG sites linked to these symptoms where Cri du chat patients have strong DNA methylation changes are enriched for binding of the polycomb EZH2 complex, H3K27me3, and H3K4me2, indicating changes to bivalent promoters, known to be central to embryonic developmental processes. CONCLUSIONS: Finding DNA methylation changes in the blood of Cri du chat patients linked to the most common symptoms of the syndrome is suggestive of epigenetic changes early in embryonic development that may be contributing to the development of symptoms. However, with the present data we cannot conclude about the sequence of events between DNA methylation changes and other cellular functions-the observed differences could be directly driving epigenetic changes, a result of other epigenetic changes, or they could be a reflection of other gene regulatory changes such as changed gene expression levels. We do not know which gene(s) on the p-arm of chromosome 5 that causes epigenetic changes when hemizygous, but an important contribution from this work is making the pool of possible causative genes smaller.

Prevention of covid-19 and other acute respiratory infections with cod liver oil supplementation, a low dose vitamin D supplement: quadruple blinded, randomised placebo controlled trial.

OBJECTIVE: To determine if daily supplementation with cod liver oil, a low dose vitamin D supplement, in winter, prevents SARS-CoV-2 infection, serious covid-19, or other acute respiratory infections in adults in Norway. DESIGN: Quadruple blinded, randomised placebo controlled trial. SETTING: Norway, 10 November 2020 to 2 June 2021. PARTICIPANTS: 34 601 adults (aged 18-75 years), not taking daily vitamin D supplements. INTERVENTION: 5 mL/day of cod liver oil (10 µg of vitamin D, n=17 278) or placebo (n=17 323) for up to six months. MAIN OUTCOME MEASURES: Four co-primary endpoints were predefined: the first was a positive SARS-CoV-2 test result determined by reverse transcriptase-quantitative polymerase chain reaction and the second was serious covid-19, defined as self-reported dyspnoea, admission to hospital, or death. Other acute respiratory infections were indicated by the third and fourth co-primary endpoints: a negative SARS-CoV-2 test result and self-reported symptoms. Side effects related to the supplementation were self-reported. The fallback method was used to handle multiple comparisons. RESULTS: Supplementation with cod liver oil was not associated with a reduced risk of any of the co-primary endpoints. Participants took the supplement (cod liver oil or placebo) for a median of 164 days, and 227 (1.31%) participants in the cod liver oil group and 228 (1.32%) participants in the placebo group had a positive SARS-CoV-2 test result (relative risk 1.00, multiple comparison adjusted confidence interval 0.82 to 1.22). Serious covid-19 was identified in 121 (0.70%) participants in the cod liver oil group and in 101 (0.58%) participants in the placebo group (1.20, 0.87 to 1.65). 8546 (49.46%) and 8565 (49.44%) participants in the cod liver oil and placebo groups, respectively, had ≥1 negative SARS-CoV-2 test results (1.00, 0.97 to 1.04). 3964 (22.94%) and 3834 (22.13%) participants in the cod liver oil and placebo groups, respectively, reported ≥1 acute respiratory infections (1.04, 0.97 to 1.11). Only low grade side effects were reported in the cod liver oil and placebo groups. CONCLUSION: Supplementation with cod liver oil in the winter did not reduce the incidence of SARS-CoV-2 infection, serious covid-19, or other acute respiratory infections compared with placebo. TRIAL REGISTRATION: ClinicalTrials.gov NCT04609423.

Breakthrough infections with the omicron and delta variants of SARS-CoV-2 result in similar re-activation of vaccine-induced immunity.

Background: Results showing that sera from double vaccinated individuals have minimal neutralizing activity against Omicron have been interpreted as indicating the need for a third vaccine dose for protection. However, there is little information about early immune responses to Omicron infection in double vaccinated individuals. Methods: We measured inflammatory mediators, antibodies to the SARS-CoV-2 spike and nucleocapsid proteins, and spike peptide-induced release of interferon gamma in whole blood in 51 double-vaccinated individuals infected with Omicron, in 14 infected with Delta, and in 18 healthy controls. The median time points for the first and second samples were 7 and 14 days after symptom onset, respectively. Findings: Infection with Omicron or Delta led to a rapid and similar increase in antibodies to the receptor-binding domain (RBD) of Omicron protein and spike peptide-induced interferon gamma in whole blood. Both the Omicron- and the Delta-infected patients had a mild and transient increase in inflammatory parameters. Interpretation: The results suggest that two vaccine doses are sufficient to mount a rapid and potent immune response upon infection in healthy individuals of with the Omicron variant. Funding: The study was funded by the Oslo University Hospital, and by grants from The Coalition for Epidemic Preparedness Innovations, Research Council of Norway (no 312780, 324272), South-Eastern Norway Regional Health Authority (no 2019067, 2021071, 10357, 2021047, 33612, 2021087, 2017092), EU Horizon 2020 grant no 848099, a philantropic donation from Vivaldi Invest A/S, and The European Virus Archive Global.

EWAS of post-COVID-19 patients shows methylation differences in the immune-response associated gene, IFI44L, three months after COVID-19 infection.

Although substantial progress has been made in managing COVID-19, it is still difficult to predict a patient's prognosis. We explored the epigenetic signatures of COVID-19 in peripheral blood using data from an ongoing prospective observational study of COVID-19 called the Norwegian Corona Cohort Study. A series of EWASs were performed to compare the DNA methylation profiles between COVID-19 cases and controls three months post-infection. We also investigated differences associated with severity and long-COVID. Three CpGs-cg22399236, cg03607951, and cg09829636-were significantly hypomethylated (FDR < 0.05) in COVID-19 positive individuals. cg03607951 is located in IFI44L which is involved in innate response to viral infection and several systemic autoimmune diseases. cg09829636 is located in ANKRD9, a gene implicated in a wide variety of cellular processes, including the degradation of IMPDH2. The link between ANKRD9 and IMPDH2 is striking given that IMPDHs are considered therapeutic targets for COVID-19. Furthermore, gene ontology analyses revealed pathways involved in response to viruses. The lack of significant differences associated with severity and long-COVID may be real or reflect limitations in sample size. Our findings support the involvement of interferon responsive genes in the pathophysiology of COVID-19 and indicate a possible link to systemic autoimmune diseases.

ALKBH5 regulates somatic cell reprogramming in a phase-specific manner.

Establishment of the pluripotency regulatory network in somatic cells by introducing four transcription factors [octamer binding transcription factor 4 (OCT4; also known as POU5F1), sex determining region Y (SRY)-box 2 (SOX2), Kruppel-like factor 4 (KLF4) and cellular myelocytomatosis (c-MYC)] provides a promising tool for cell-based therapies in regenerative medicine. Nevertheless, the mechanisms at play when generating induced pluripotent stem cells from somatic cells are only partly understood. Here, we show that the RNA-specific N6-methyladenosine (m6A) demethylase ALKBH5 regulates somatic cell reprogramming in a stage-specific manner through its catalytic activity. Knockdown or knockout of Alkbh5 in the early reprogramming phase impairs reprogramming efficiency by reducing the proliferation rate through arresting the cells at G2/M phase and decreasing the upregulation of epithelial markers. On the other hand, ALKBH5 overexpression at the early reprogramming phase has no significant impact on reprogramming efficiency, whereas overexpression at the late phase enhances reprogramming by stabilizing Nanog transcripts, resulting in upregulated Nanog expression. Our study provides mechanistic insight into the crucial dynamic role of ALKBH5, mediated through its catalytic activity, in regulating somatic cell reprogramming at the post-transcriptional level. This article has an associated First Person interview with the first author of the paper.

The use of public transport and contraction of SARS-CoV-2 in a large prospective cohort in Norway.

BACKGROUND: For many people public transport is the only mode of travel, and it can be challenging to keep the necessary distances in such a restricted space. The exact role of public transportation and risk of SARS-CoV-2 transmission is not known. METHODS: Participants (n = 121,374) were untested adult Norwegian residents recruited through social media who in the spring of 2020 completed a baseline questionnaire on demographics and the use of public transport. Incident cases (n = 1069) had a positive SARS-CoV-2 polymerase chain reaction test registered at the Norwegian Messaging System for Infectious Diseases by January 27, 2021. We investigated the association between the use of public transport and SARS-CoV-2 using logistic regression. Odds ratios (ORs) with 95% confidence intervals (CIs) adjusted for age, calendar time, gender, municipality, smoking, income level, fitness and underlying medical conditions were estimated. Frequency of the use of public transport was reported for 2 week-periods. RESULTS: Before lockdown, those who tested positive on SARS-CoV-2 were more likely to have used public transport 1-3 times (OR = 1.28, CI 1.09-1.51), 4-10 times (OR = 1.49, CI 1.26-1.77) and ≥ 11 times (OR = 1.50, CI 1.27-1.78, p for trend < 0.0001) than those who had not tested positive. CONCLUSION: The use of public transport was positively associated with contracting SARS-CoV-2 both before and after lockdown.

Screening bioactive food compounds in honey bees suggests curcumin blocks alcohol-induced damage to longevity and DNA methylation.

Various bioactive food compounds may confer health and longevity benefits, possibly through altering or preserving the epigenome. While bioactive food compounds are widely being marketed for human consumption as 'improving health and longevity' by counteracting harmful effects of poor nutrition and lifestyle, claimed effects are often not adequately documented. Using the honey bee (Apis mellifera) as a model species, we here employed a multi-step screening approach to investigate seven compounds for effects on lifespan and DNA methylation using ELISA and whole genome bisulfite sequencing (WGBS). A positive longevity effect was detected for valproic acid, isovaleric acid, and cyanocobalamin. For curcumin, we found that lifespan shortening caused by ethanol intake, was restored when curcumin and ethanol were co-administered. Furthermore, we identified region specific DNA methylation changes as a result of ethanol intake. Ethanol specific changes in DNA methylation were fully or partially blocked in honey bees receiving ethanol and curcumin together. Ethanol-affected and curcumin-blocked differentially methylated regions covered genes involved in fertility, temperature regulation and tubulin transport. Our results demonstrate fundamental negative effects of low dose ethanol consumption on lifespan and associated DNA methylation changes and present a proof-of-principle on how longevity and DNA methylation changes can be negated by the bioactive food component curcumin. Our findings provide a fundament for further studies of curcumin in invertebrates.

Persisting symptoms three to eight months after non-hospitalized COVID-19, a prospective cohort study.

Long-COVID-19 is a proposed syndrome negatively affecting the health of COVID-19 patients. We present data on self-rated health three to eight months after laboratory confirmed COVID-19 disease compared to a control group of SARS-CoV-2 negative patients. We followed a cohort of 8786 non-hospitalized patients who were invited after SARS-CoV-2 testing between February 1 and April 15, 2020 (794 positive, 7229 negative). Participants answered online surveys at baseline and follow-up including questions on demographics, symptoms, risk factors for SARS-CoV-2, and self-rated health compared to one year ago. Determinants for a worsening of self-rated health as compared to one year ago among the SARS-CoV-2 positive group were analyzed using multivariate logistic regression and also compared to the population norm. The follow-up questionnaire was completed by 85% of the SARS-CoV-2 positive and 75% of the SARS-CoV-2 negative participants on average 132 days after the SARS-CoV-2 test. At follow-up, 36% of the SARS-CoV-2 positive participants rated their health "somewhat" or "much" worse than one year ago. In contrast, 18% of the SARS-CoV-2 negative participants reported a similar deterioration of health while the population norm is 12%. Sore throat and cough were more frequently reported by the control group at follow-up. Neither gender nor follow-up time was associated with the multivariate odds of worsening of self-reported health compared to one year ago. Age had an inverted-U formed association with a worsening of health while being fit and being a health professional were associated with lower multivariate odds. A significant proportion of non-hospitalized COVID-19 patients, regardless of age, have not returned to their usual health three to eight months after infection.

Intestinal-epithelial LSD1 controls goblet cell maturation and effector responses required for gut immunity to bacterial and helminth infection.

Infectious and inflammatory diseases in the intestine remain a serious threat for patients world-wide. Reprogramming of the intestinal epithelium towards a protective effector state is important to manage inflammation and immunity and can be therapeutically targeted. The role of epigenetic regulatory enzymes within these processes is not yet defined. Here, we use a mouse model that has an intestinal-epithelial specific deletion of the histone demethylase Lsd1 (cKO mice), which maintains the epithelium in a fixed reparative state. Challenge of cKO mice with bacteria-induced colitis or a helminth infection model both resulted in increased pathogenesis. Mechanistically, we discovered that LSD1 is important for goblet cell maturation and goblet-cell effector molecules such as RELMß. We propose that this may be in part mediated by directly controlling genes that facilitate cytoskeletal organization, which is important in goblet cell biology. This study therefore identifies intestinal-epithelial epigenetic regulation by LSD1 as a critical element in host protection from infection.

KDM4A regulates the maternal-to-zygotic transition by protecting broad H3K4me3 domains from H3K9me3 invasion in oocytes.

The importance of germline-inherited post-translational histone modifications on priming early mammalian development is just emerging1-4. Histone H3 lysine 9 (H3K9) trimethylation is associated with heterochromatin and gene repression during cell-fate change5, whereas histone H3 lysine 4 (H3K4) trimethylation marks active gene promoters6. Mature oocytes are transcriptionally quiescent and possess remarkably broad domains of H3K4me3 (bdH3K4me3)1,2. It is unknown which factors contribute to the maintenance of the bdH3K4me3 landscape. Lysine-specific demethylase 4A (KDM4A) demethylates H3K9me3 at promoters marked by H3K4me3 in actively transcribing somatic cells7. Here, we report that KDM4A-mediated H3K9me3 demethylation at bdH3K4me3 in oocytes is crucial for normal pre-implantation development and zygotic genome activation after fertilization. The loss of KDM4A in oocytes causes aberrant H3K9me3 spreading over bdH3K4me3, resulting in insufficient transcriptional activation of genes, endogenous retroviral elements and chimeric transcripts initiated from long terminal repeats during zygotic genome activation. The catalytic activity of KDM4A is essential for normal epigenetic reprogramming and pre-implantation development. Hence, KDM4A plays a crucial role in preserving the maternal epigenome integrity required for proper zygotic genome activation and transfer of developmental control to the embryo.

Going low to reach high: Small-scale ChIP-seq maps new terrain.

Chromatin immunoprecipitation (ChIP) enables mapping of specific histone modifications or chromatin-associated factors in the genome and represents a powerful tool in the study of chromatin and genome regulation. Importantly, recent technological advances that couple ChIP with whole-genome high-throughput sequencing (ChIP-seq) now allow the mapping of chromatin factors throughout the genome. However, the requirement for large amounts of ChIP-seq input material has long made it challenging to assess chromatin profiles of cell types only available in limited numbers. For many cell types, it is not feasible to reach high numbers when collecting them as homogeneous cell populations in vivo. Nonetheless, it is an advantage to work with pure cell populations to reach robust biological conclusions. Here, we review (a) how ChIP protocols have been scaled down for use with as little as a few hundred cells; (b) which considerations to be aware of when preparing small-scale ChIP-seq and analyzing data; and (c) the potential of small-scale ChIP-seq datasets for elucidating chromatin dynamics in various biological systems, including some examples such as oocyte maturation and preimplantation embryo development. This article is categorized under: Laboratory Methods and Technologies > Genetic/Genomic Methods Developmental Biology > Developmental Processes in Health and Disease Biological Mechanisms > Cell Fates.

Histone Methylations Define Neural Stem/Progenitor Cell Subtypes in the Mouse Subventricular Zone.

Neural stem/progenitor cells (NSPCs) persist in the mammalian brain throughout life and can be activated in response to the physiological and pathophysiological stimuli. Epigenetic reprogramming of NPSC represents a novel strategy for enhancing the intrinsic potential of the brain to regenerate after brain injury. Therefore, defining the epigenetic features of NSPCs is important for developing epigenetic therapies for targeted reprogramming of NSPCs to rescue neurologic function after injury. In this study, we aimed at defining different subtypes of NSPCs by individual histone methylations. We found the three histone marks, histone H3 lysine 4 trimethylation (H3K4me3), histone H3 lysine 27 trimethylation (H3K27me3), and histone H3 lysine 36 trimethylation (H3K36me3), to nicely and dynamically portray individual cell types during neurodevelopment. First, we found all three marks co-stained with NSPC marker SOX2 in mouse subventricular zone. Then, CD133, Id1, Mash1, and DCX immunostaining were used to define NSPC subtypes. Type E/B, B/C, and C/A cells showed high levels of H3K27me3, H3K36me3, and H3K4me3, respectively. Our results reveal defined histone methylations of NSPC subtypes supporting that epigenetic regulation is critical for neurogenesis and for maintaining NSPCs.

5-hydroxymethylcytosine Marks Mammalian Origins Acting as a Barrier to Replication.

In most mammalian cells, DNA replication occurs once, and only once between cell divisions. Replication initiation is a highly regulated process with redundant mechanisms that prevent errant initiation events. In lower eukaryotes, replication is initiated from a defined consensus sequence, whereas a consensus sequence delineating mammalian origin of replication has not been identified. Here we show that 5-hydroxymethylcytosine (5hmC) is present at mammalian replication origins. Our data support the hypothesis that 5hmC has a role in cell cycle regulation. We show that 5hmC level is inversely proportional to proliferation; indeed, 5hmC negatively influences cell division by increasing the time a cell resides in G1. Our data suggest that 5hmC recruits replication-licensing factors, then is removed prior to or during origin firing. Later we propose that TET2, the enzyme catalyzing 5mC to 5hmC conversion, acts as barrier to rereplication. In a broader context, our results significantly advance the understating of 5hmC involvement in cell proliferation and disease states.

Epigenetic age is a cell-intrinsic property in transplanted human hematopoietic cells.

The age of tissues and cells can be accurately estimated by DNA methylation analysis. The multitissue DNA methylation (DNAm) age predictor combines the DNAm levels of 353 CpG dinucleotides to arrive at an age estimate referred to as DNAm age. Recent studies based on short-term observations showed that the DNAm age of reconstituted blood following allogeneic hematopoietic stem cell transplantation (HSCT) reflects the age of the donor. However, it is not known whether the DNAm age of donor blood remains independent of the recipient's age over the long term. Importantly, long-term studies including child recipients have the potential to clearly reveal whether DNAm age is cell-intrinsic or whether it is modulated by extracellular cues in vivo. Here, we address this question by analyzing blood methylation data from HSCT donor and recipient pairs who greatly differed in chronological age (age differences between 1 and 49 years). We found that the DNAm age of the reconstituted blood was not influenced by the recipient's age, even 17 years after HSCT, in individuals without relapse of their hematologic disorder. However, the DNAm age of recipients with relapse of leukemia was unstable. These data are consistent with our previous findings concerning the abnormal DNAm age of cancer cells, and it can potentially be exploited to monitor the health of HSCT recipients. Our data demonstrate that transplanted human hematopoietic stem cells have an intrinsic DNAm age that is unaffected by the environment in a recipient of a different age.

Positioning Europe for the EPITRANSCRIPTOMICS challenge.

The genetic alphabet consists of the four letters: C, A, G, and T in DNA and C,A,G, and U in RNA. Triplets of these four letters jointly encode 20 different amino acids out of which proteins of all organisms are built. This system is universal and is found in all kingdoms of life. However, bases in DNA and RNA can be chemically modified. In DNA, around 10 different modifications are known, and those have been studied intensively over the past 20 years. Scientific studies on DNA modifications and proteins that recognize them gave rise to the large field of epigenetic and epigenomic research. The outcome of this intense research field is the discovery that development, ageing, and stem-cell dependent regeneration but also several diseases including cancer are largely controlled by the epigenetic state of cells. Consequently, this research has already led to the first FDA approved drugs that exploit the gained knowledge to combat disease. In recent years, the ~150 modifications found in RNA have come to the focus of intense research. Here we provide a perspective on necessary and expected developments in the fast expanding area of RNA modifications, termed epitranscriptomics.