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Reducing body weight to improve metabolic health and related comorbidities is a primary goal in treating obesity 1,2 . However, maintaining weight loss is a considerable challenge, especially as the body seems to retain an obesogenic memory that defends against body weight changes 3,4 . Overcoming this barrier for long-term treatment success is difficult because the molecular mechanisms underpinning this phenomenon remain largely unknown. Here, by using single-nucleus RNA sequencing, we show that both human and mouse adipose tissues retain cellular transcriptional changes after appreciable weight loss. Furthermore, we find persistent obesity-induced alterations in the epigenome of mouse adipocytes that negatively affect their function and response to metabolic stimuli. Mice carrying this obesogenic memory show accelerated rebound weight gain, and the epigenetic memory can explain future transcriptional deregulation in adipocytes in response to further high-fat diet feeding. In summary, our findings indicate the existence of an obesogenic memory, largely on the basis of stable epigenetic changes, in mouse adipocytes and probably other cell types. These changes seem to prime cells for pathological responses in an obesogenic environment, contributing to the problematic 'yo-yo' effect often seen with dieting. Targeting these changes in the future could improve long-term weight management and health outcomes., Competing Interests: Competing interests: M.B. received honoraria as a consultant and speaker from Amgen, AstraZeneca, Bayer, Boehringer-Ingelhiem, Lilly, Novo Nordisk and Sanofi. M.R. received honoraria as a consultant and speaker from AstraZeneca, Boehringer-Ingelheim, Lilly, Novo Nordisk and Sanofi. The other authors declare no competing interests., (© 2024. The Author(s).)

Background: DNA methylation (DNAm) has unique properties which makes it a potential biomarker for lifestyle-related exposures. Epigenetic clocks, particularly DNAm-based biological age predictors [epigenetic age (EA)], represent an exciting new area of clinical research and deviations of EA from chronological age [epigenetic age acceleration (EAA)] have been linked to overall health, age-related diseases, and environmental exposures., Objectives: This observational study investigates the relationships between biological aging and various dietary factors within the LifeLines-DEEP Cohort. These factors include diet quality, processed food consumption, dietary glycemic load, and intake of vitamins involved in maintaining the epigenetic homeostasis (vitamins B-9, B-12, B-6, B-2, and C)., Methods: Dietary records collected using food-frequency questionnaires were used to estimate diet quality [LifeLines Diet Score (LLDS)], measure the intake of unprocessed/ultraprocessed food according to the NOVA food classification system, and the adequacy of the dietary intake of vitamins B-9, B-12, B-2, B-6, and C. EA using Horvath, Hannum, Levine, and Horvath2 epigenetic clock models and DNAm-predicted telomere length (DNAm-TL) were calculated from DNAm data in 760 subjects. Associations between dietary factors and EAA were tested, adjusting for sex, energy intake, and body composition., Results: LLDS was associated with EAA (EAA_Horvath: β: -0.148; P = 1 × 10 -4 ; EAA_Hannum: β: -0.148; P = 9 × 10 -5 ; EAA_Levine: β: -0.174; P = 1 × 10 -5 ; and EAA_Horvath2: β: -0.176; P = 4 × 10 -6 ) and DNAm-TL (β: 0.116; P = 0.003). Particularly, EAA was associated with dietary glycemic load (EAA_Horvath: β: 0.476; P = 9 × 10 -10 ; EAA_Hannum: β: 0.565; P = 1 × 10 -13 ; EAA_Levine: β: 0.469; P = 5 × 10 -9 ; EAA_Horvath2: β: 0.569; P = 1 × 10 -13 ; and DNAmTL adjusted for age: β: -0.340; P = 2 × 10 -5 ) and different measures of food processing (NOVA classes 1 and 4). Positive EAA was also associated with inadequate intake of vitamin B-12 (EAA_Horvath: β: -0.167; P = 0.002; EAA_Hannum: β: -0.144; P = 0.007; and EAA_Horvath2: β: -0.126; P = 0.019) and C (EAA_Hannum: β: -0.136; P = 0.010 and EAA_Horvath2: β: -0.151; P = 0.005)., Conclusions: Our findings corroborate the hypothesis that nutrition plays a pivotal role in influencing epigenetic homeostasis, especially DNAm, thereby contributing to individual health trajectories and the pace of aging., Competing Interests: Conflict of interest The authors report no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Ageing is the accumulation of changes and decline of function of organisms over time. The concept and biomarkers of biological age have been established, notably DNA methylation-based clocks. The emergence of single-cell DNA methylation profiling methods opens the possibility of studying the biological age of individual cells. Here, we generate a large single-cell DNA methylation and transcriptome dataset from mouse peripheral blood samples, spanning a broad range of ages. The number of genes expressed increases with age, but gene-specific changes are small. We next develop scEpiAge, a single-cell DNA methylation age predictor, which can accurately predict age in (very sparse) publicly available datasets, and also in single cells. DNA methylation age distribution is wider than technically expected, indicating epigenetic age heterogeneity and functional differences. Our work provides a foundation for single-cell and sparse data epigenetic age predictors, validates their functionality and highlights epigenetic heterogeneity during ageing., (© 2024. The Author(s).)

The establishment of aging clocks highlighted the strong link between changes in DNA methylation and aging. Yet, it is not known if other epigenetic features could be used to predict age accurately. Furthermore, previous studies have observed a lack of effect of age-related changes in DNA methylation on gene expression, putting the interpretability of DNA methylation-based aging clocks into question. In this study, we explore the use of chromatin accessibility to construct aging clocks. We collected blood from 159 human donors and generated chromatin accessibility, transcriptomic, and cell composition data. We investigated how chromatin accessibility changes during aging and constructed a novel aging clock with a median absolute error of 5.27 years. The changes in chromatin accessibility used by the clock were strongly related to transcriptomic alterations, aiding clock interpretation. We additionally show that our chromatin accessibility clock performs significantly better than a transcriptomic clock trained on matched samples. In conclusion, we demonstrate that the clock relies on cell-intrinsic chromatin accessibility alterations rather than changes in cell composition. Further, we present a new approach to construct epigenetic aging clocks based on chromatin accessibility, which bear a direct link to age-related transcriptional alterations, but which allow for more accurate age predictions than transcriptomic clocks., (© 2023. The Author(s).)

A precise understanding of DNA methylation dynamics is of great importance for a variety of biological processes including cellular reprogramming and differentiation. To date, complex integration of multiple and distinct genome-wide datasets is required to realize this task. We present GwEEP (genome-wide epigenetic efficiency profiling) a versatile approach to infer dynamic efficiencies of DNA modifying enzymes. GwEEP relies on genome-wide hairpin datasets, which are translated by a hidden Markov model into quantitative enzyme efficiencies with reported confidence around the estimates. GwEEP predicts de novo and maintenance methylation efficiencies of Dnmts and furthermore the hydroxylation efficiency of Tets. Its design also allows capturing further oxidation processes given available data. We show that GwEEP predicts accurately the epigenetic changes of ESCs following a Serum-to-2i shift and applied to Tet TKO cells confirms the hypothesized mutual interference between Dnmts and Tets., Competing Interests: W.R. is a consultant and shareholder of Cambridge Epigenetix. All other authors declare no competing interests., (© 2022 The Authors.)

Conventional human embryonic stem cells are considered to be primed pluripotent but can be induced to enter a naive state. However, the transcriptional features associated with naive and primed pluripotency are still not fully understood. Here we used single-cell RNA sequencing to characterize the differences between these conditions. We observed that both naive and primed populations were mostly homogeneous with no clear lineage-related structure and identified an intermediate subpopulation of naive cells with primed-like expression. We found that the naive-primed pluripotency axis is preserved across species, although the timing of the transition to a primed state is species specific. We also identified markers for distinguishing human naive and primed pluripotency as well as strong co-regulatory relationships between lineage markers and epigenetic regulators that were exclusive to naive cells. Our data provide valuable insights into the transcriptional landscape of human pluripotency at a cellular and genome-wide resolution., (Crown Copyright © 2019. Published by Elsevier Inc. All rights reserved.)

Recent research has focused on environmental effects that control tissue functionality and systemic metabolism. However, whether such stimuli affect human thermogenesis and body mass index (BMI) has not been explored. Here we show retrospectively that the presence of brown adipose tissue (BAT) and the season of conception are linked to BMI in humans. In mice, we demonstrate that cold exposure (CE) of males, but not females, before mating results in improved systemic metabolism and protection from diet-induced obesity of the male offspring. Integrated analyses of the DNA methylome and RNA sequencing of the sperm from male mice revealed several clusters of co-regulated differentially methylated regions (DMRs) and differentially expressed genes (DEGs), suggesting that the improved metabolic health of the offspring was due to enhanced BAT formation and increased neurogenesis. The conclusions are supported by cell-autonomous studies in the offspring that demonstrate an enhanced capacity to form mature active brown adipocytes, improved neuronal density and more norepinephrine release in BAT in response to cold stimulation. Taken together, our results indicate that in humans and in mice, seasonal or experimental CE induces an epigenetic programming of the sperm such that the offspring harbor hyperactive BAT and an improved adaptation to overnutrition and hypothermia.

Much attention has focussed on the conversion of human pluripotent stem cells (PSCs) to a more naïve developmental status. Here we provide a method for resetting via transient histone deacetylase inhibition. The protocol is effective across multiple PSC lines and can proceed without karyotype change. Reset cells can be expanded without feeders with a doubling time of around 24 h. WNT inhibition stabilises the resetting process. The transcriptome of reset cells diverges markedly from that of primed PSCs and shares features with human inner cell mass (ICM). Reset cells activate expression of primate-specific transposable elements. DNA methylation is globally reduced to a level equivalent to that in the ICM and is non-random, with gain of methylation at specific loci. Methylation imprints are mostly lost, however. Reset cells can be re-primed to undergo tri-lineage differentiation and germline specification. In female reset cells, appearance of biallelic X-linked gene transcription indicates reactivation of the silenced X chromosome. On reconversion to primed status, XIST -induced silencing restores monoallelic gene expression. The facile and robust conversion routine with accompanying data resources will enable widespread utilisation, interrogation, and refinement of candidate naïve cells., Competing Interests: Competing interestsG.G. and A.Sm. are inventors on a patent filing by the University of Cambridge relating to human naïve pluripotent stem cells. W.R. is a consultant to, and shareholder in, Cambridge Epigenetix., (© 2017. Published by The Company of Biologists Ltd.)

Background: DNA methylation changes at a discrete set of sites in the human genome are predictive of chronological and biological age. However, it is not known whether these changes are causative or a consequence of an underlying ageing process. It has also not been shown whether this epigenetic clock is unique to humans or conserved in the more experimentally tractable mouse., Results: We have generated a comprehensive set of genome-scale base-resolution methylation maps from multiple mouse tissues spanning a wide range of ages. Many CpG sites show significant tissue-independent correlations with age which allowed us to develop a multi-tissue predictor of age in the mouse. Our model, which estimates age based on DNA methylation at 329 unique CpG sites, has a median absolute error of 3.33 weeks and has similar properties to the recently described human epigenetic clock. Using publicly available datasets, we find that the mouse clock is accurate enough to measure effects on biological age, including in the context of interventions. While females and males show no significant differences in predicted DNA methylation age, ovariectomy results in significant age acceleration in females. Furthermore, we identify significant differences in age-acceleration dependent on the lipid content of the diet., Conclusions: Here we identify and characterise an epigenetic predictor of age in mice, the mouse epigenetic clock. This clock will be instrumental for understanding the biology of ageing and will allow modulation of its ticking rate and resetting the clock in vivo to study the impact on biological age.

The molecular pathways that regulate gain and loss of DNA methylation during mammalian development need to be tightly balanced to maintain a physiological equilibrium. Here we explore the relative contributions of the different pathways and enzymatic activities involved in methylation homeostasis in the context of genome-wide and locus-specific epigenetic reprogramming in mammals. An adaptable epigenetic machinery allows global epigenetic reprogramming to concur with local maintenance of critical epigenetic memory in the genome, and appears to regulate the tempo of global reprogramming in different cell lineages and species., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Global demethylation is part of a conserved program of epigenetic reprogramming to naive pluripotency. The transition from primed hypermethylated embryonic stem cells (ESCs) to naive hypomethylated ones (serum-to-2i) is a valuable model system for epigenetic reprogramming. We present a mathematical model, which accurately predicts global DNA demethylation kinetics. Experimentally, we show that the main drivers of global demethylation are neither active mechanisms (Aicda, Tdg, and Tet1-3) nor the reduction of de novo methylation. UHRF1 protein, the essential targeting factor for DNMT1, is reduced upon transition to 2i, and so is recruitment of the maintenance methylation machinery to replication foci. Concurrently, there is global loss of H3K9me2, which is needed for chromatin binding of UHRF1. These mechanisms synergistically enforce global DNA hypomethylation in a replication-coupled fashion. Our observations establish the molecular mechanism for global demethylation in naive ESCs, which has key parallels with those operating in primordial germ cells and early embryos., (Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.)