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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.)

To implant in the uterus, mammalian embryos form blastocysts comprising trophectoderm (TE) surrounding an inner cell mass (ICM), confined to the polar region by the expanding blastocoel. The mode of implantation varies between species. Murine embryos maintain a single layered TE until they implant in the characteristic thick deciduum, whereas human blastocysts attach via polar TE directly to the uterine wall. Using immunofluorescence (IF) of rapidly isolated ICMs, blockade of RNA and protein synthesis in whole embryos, or 3D visualization of immunostained embryos, we provide evidence of multi-layering in human polar TE before implantation. This may be required for rapid uterine invasion to secure the developing human embryo and initiate formation of the placenta. Using sequential fluorescent labeling, we demonstrate that the majority of inner TE in human blastocysts arises from existing outer cells, with no evidence of conversion from the ICM in the context of the intact embryo., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). 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).)

Background: Single-cell chromatin accessibility assays, such as scATAC-seq, are increasingly employed in individual and joint multi-omic profiling of single cells. As the accumulation of scATAC-seq and multi-omics datasets continue, challenges in analyzing such sparse, noisy, and high-dimensional data become pressing. Specifically, one challenge relates to optimizing the processing of chromatin-level measurements and efficiently extracting information to discern cellular heterogeneity. This is of critical importance, since the identification of cell types is a fundamental step in current single-cell data analysis practices., Results: We benchmark 8 feature engineering pipelines derived from 5 recent methods to assess their ability to discover and discriminate cell types. By using 10 metrics calculated at the cell embedding, shared nearest neighbor graph, or partition levels, we evaluate the performance of each method at different data processing stages. This comprehensive approach allows us to thoroughly understand the strengths and weaknesses of each method and the influence of parameter selection., Conclusions: Our analysis provides guidelines for choosing analysis methods for different datasets. Overall, feature aggregation, SnapATAC, and SnapATAC2 outperform latent semantic indexing-based methods. For datasets with complex cell-type structures, SnapATAC and SnapATAC2 are preferred. With large datasets, SnapATAC2 and ArchR are most scalable., (© 2024. The Author(s).)

Aging is the major risk factor for most human diseases and represents a major socio-economical challenge for modern societies. Despite its importance, the process of aging remains poorly understood. Epigenetic dysregulation has been proposed as a key driver of the aging process. Modifications in transcriptional networks and chromatin structure might be central to age-related functional decline. A prevalent feature described during aging is the overall reduction in heterochromatin, specifically marked by the loss of repressive histone modification, Histone 3 lysine 9 trimethylation (H3K9me3). However, the role of H3K9me3 in aging, especially in mammals, remains unclear. Here we show using a novel mouse strain, (TKOc), carrying a triple knockout of three methyltransferases responsible for H3K9me3 deposition, that the inducible loss of H3K9me3 in adulthood results in premature aging. TKOc mice exhibit reduced lifespan, lower body weight, increased frailty index, multi-organ degeneration, transcriptional changes with significant upregulation of transposable elements, and accelerated epigenetic age. Our data strongly supports the concept that the loss of epigenetic information directly drives the aging process. These findings reveal the importance of epigenetic regulation in aging and suggest that interventions targeting epigenetic modifications could potentially slow down or reverse age-related decline. Understanding the molecular mechanisms underlying the process of aging will be crucial for developing novel therapeutic strategies that can delay the onset of age-associated diseases and preserve human health at old age specially in rapidly aging societies., Competing Interests: COMPETING INTERESTS: A.O. is founder and shareholder of EPITERNA. A.O. is co-founder of Longevity Consultancy Group. S.H. is a founder of the non-profit Epigenetic Clock Development Foundation which licenses several patents from his former employer UC Regents. These patents list S.H. as inventor. The rest of the authors declares no competing interests.

Macrophages play crucial roles in organ-specific functions and homeostasis. In the adrenal gland, macrophages closely associate with sinusoidal capillaries in the aldosterone-producing zona glomerulosa. We demonstrate that macrophages preserve capillary specialization and modulate aldosterone secretion. Using macrophage-specific deletion of VEGF-A, single-cell transcriptomics, and functional phenotyping, we found that the loss of VEGF-A depletes PLVAP + fenestrated endothelial cells in the zona glomerulosa, leading to increased basement membrane collagen IV deposition and subendothelial fibrosis. This results in increased aldosterone secretion, called "haptosecretagogue" signaling. Human aldosterone-producing adenomas also show capillary rarefaction and basement membrane thickening. Mice with myeloid cell-specific VEGF-A deletion exhibit elevated serum aldosterone, hypokalemia, and hypertension, mimicking primary aldosteronism. These findings underscore macrophage-to-endothelial cell signaling as essential for endothelial cell specialization, adrenal gland function, and blood pressure regulation, with broader implications for other endocrine organs., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The intersection of metabolic processes and epigenetic regulation during embryogenesis is crucial yet not fully understood. Through a candidate RNAi screen in Caenorhabditis elegans , we identified metabolic enzymes ALDO-2 and PDHB-1 as potential epigenetic regulators. Mild alteration of the chromatin remodeler LET-418 /Mi2 activity rescues embryonic lethality induced by suppressing aldo-2 or pdhb-1 , suggesting a critical role for glucose and pyruvate metabolism in chromatin remodeling during embryogenesis. Given the conservation of central metabolic pathways and chromatin modifiers across species, our findings lay the foundation for future mechanistic investigations into the interplay between epigenetics and metabolism during development and upon disease., Competing Interests: The authors declare that there are no conflicts of interest present., (Copyright: © 2024 by the authors.)

Objectives: We have previously shown that lactate is an essential metabolite for macrophage polarisation during ischemia-induced muscle regeneration. Recent in vitro work has implicated histone lactylation, a direct derivative of lactate, in macrophage polarisation. Here, we explore the in vivo relevance of histone lactylation for macrophage polarisation after muscle injury., Methods: To evaluate macrophage dynamics during muscle regeneration, we subjected mice to ischemia-induced muscle damage by ligating the femoral artery. Muscle samples were harvested at 1, 2, 4, and 7 days post injury (dpi). CD45 + CD11b + F4/80 + CD64 + macrophages were isolated and processed for RNA sequencing, Western Blotting, and CUT&Tag-sequencing to investigate gene expression, histone lactylation levels, and histone lactylation genomic localisation and enrichment, respectively., Results: We show that, over time, macrophages in the injured muscle undergo extensive gene expression changes, which are similar in nature and in timing to those seen after other types of muscle-injuries. We find that the macrophage histone lactylome is modified between 2 and 4 dpi, which is a crucial window for macrophage polarisation. Absolute histone lactylation levels increase, and, although subtly, the genomic enrichment of H3K18la changes. Overall, we find that histone lactylation is important at both promoter and enhancer elements. Lastly, H3K18la genomic profile changes from 2 to 4 dpi were predictive for gene expression changes later in time, rather than being a reflection of prior gene expression changes., Conclusions: Our results suggest that histone lactylation dynamics are functionally important for the function of macrophages during muscle regeneration., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

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).)

Embryonic diapause in mammals is a temporary developmental delay occurring at the blastocyst stage. In contrast to other diapausing species displaying a full arrest, the blastocyst of the European roe deer (Capreolus capreolus) proliferates continuously and displays considerable morphological changes in the inner cell mass. We hypothesised that developmental progression also continues during this period. Here we evaluate the mRNA abundance of developmental marker genes in embryos during diapause and elongation. Our results show that morphological rearrangements of the epiblast during diapause correlate with gene expression patterns and changes in cell polarity. Immunohistochemical staining further supports these findings. Primitive endoderm formation occurs during diapause in embryos composed of around 3,000 cells. Gastrulation coincides with elongation and thus takes place after embryo reactivation. The slow developmental progression makes the roe deer an interesting model for unravelling the link between proliferation and differentiation and requirements for embryo survival., (© 2024. The Author(s).)

In obesity, sustained adipose tissue (AT) inflammation constitutes a cellular memory that limits the effectiveness of weight loss interventions. Yet, the impact of fasting regimens on the regulation of AT immune infiltration is still elusive. Here we show that intermittent fasting (IF) exacerbates the lipid-associated macrophage (LAM) inflammatory phenotype of visceral AT in obese mice. Importantly, this increase in LAM abundance is strongly p53 dependent and partly mediated by p53-driven adipocyte apoptosis. Adipocyte-specific deletion of p53 prevents LAM accumulation during IF, increases the catabolic state of adipocytes, and enhances systemic metabolic flexibility and insulin sensitivity. Finally, in cohorts of obese/diabetic patients, we describe a p53 polymorphism that links to efficacy of a fasting-mimicking diet and that the expression of p53 and TREM2 in AT negatively correlates with maintaining weight loss after bariatric surgery. Overall, our results demonstrate that p53 signalling in adipocytes dictates LAM accumulation in AT under IF and modulates fasting effectiveness in mice and humans., (© 2024. The Author(s).)

Human pluripotent stem cells (hPSCs) are of fundamental relevance in regenerative medicine. Naïve hPSCs hold promise to overcome some of the limitations of conventional (primed) hPSCs, including recurrent epigenetic anomalies. Naïve-to-primed transition (capacitation) follows transcriptional dynamics of human embryonic epiblast and is necessary for somatic differentiation from naïve hPSCs. We found that capacitated hPSCs are transcriptionally closer to postimplantation epiblast than conventional hPSCs. This prompted us to comprehensively study epigenetic and related transcriptional changes during capacitation. Our results show that CpG islands, gene regulatory elements, and retrotransposons are hotspots of epigenetic dynamics during capacitation and indicate possible distinct roles of specific epigenetic modifications in gene expression control between naïve and primed hPSCs. Unexpectedly, PRC2 activity appeared to be dispensable for the capacitation. We find that capacitated hPSCs acquire an epigenetic state similar to conventional hPSCs. Significantly, however, the X chromosome erosion frequently observed in conventional female hPSCs is reversed by resetting and subsequent capacitation.

Defects in insulin processing and granule maturation are linked to pancreatic beta-cell failure during type 2 diabetes (T2D). Phosphatidylinositol transfer protein alpha (PITPNA) stimulates activity of phosphatidylinositol (PtdIns) 4-OH kinase to produce sufficient PtdIns-4-phosphate (PtdIns-4-P) in the trans-Golgi network to promote insulin granule maturation. PITPNA in beta-cells of T2D human subjects is markedly reduced suggesting its depletion accompanies beta-cell dysfunction. Conditional deletion of Pitpna in the beta-cells of Ins-Cre, Pitpna flox/flox mice leads to hyperglycemia resulting from decreasing glucose-stimulated insulin secretion (GSIS) and reducing pancreatic beta-cell mass. Furthermore, PITPNA silencing in human islets confirms its role in PtdIns-4-P synthesis and leads to impaired insulin granule maturation and docking, GSIS, and proinsulin processing with evidence of ER stress. Restoration of PITPNA in islets of T2D human subjects reverses these beta-cell defects and identify PITPNA as a critical target linked to beta-cell failure in T2D., (© 2023. The Author(s).)

Background: Histone lactylation has been recently described as a novel histone post-translational modification linking cellular metabolism to epigenetic regulation., Results: Given the expected relevance of this modification and current limited knowledge of its function, we generate genome-wide datasets of H3K18la distribution in various in vitro and in vivo samples, including mouse embryonic stem cells, macrophages, adipocytes, and mouse and human skeletal muscle. We compare them to profiles of well-established histone modifications and gene expression patterns. Supervised and unsupervised bioinformatics analysis shows that global H3K18la distribution resembles H3K27ac, although we also find notable differences. H3K18la marks active CpG island-containing promoters of highly expressed genes across most tissues assessed, including many housekeeping genes, and positively correlates with H3K27ac and H3K4me3 as well as with gene expression. In addition, H3K18la is enriched at active enhancers that lie in proximity to genes that are functionally important for the respective tissue., Conclusions: Overall, our data suggests that H3K18la is not only a marker for active promoters, but also a mark of tissue specific active enhancers., (© 2022. The Author(s).)

Blastocyst complementation denotes a technique that aims to generate organs, tissues, or cell types in animal chimeras via injection of pluripotent stem cells (PSCs) into genetically compromised blastocyst-stage embryos. Here, we report on successful complementation of the male germline in adult chimeras following injection of mouse or rat PSCs into mouse blastocysts carrying a mutation in Tsc22d3, an essential gene for spermatozoa production. Injection of mouse PSCs into Tsc22d3-Knockout (KO) blastocysts gave rise to intraspecies chimeras exclusively embodying PSC-derived functional spermatozoa. In addition, injection of rat embryonic stem cells (rESCs) into Tsc22d3-KO embryos produced interspecies mouse-rat chimeras solely harboring rat spermatids and spermatozoa capable of fertilizing oocytes. Furthermore, using single-cell RNA sequencing, we deconstructed rat spermatogenesis occurring in a mouse-rat chimera testis. Collectively, this study details a method for exclusive xenogeneic germ cell production in vivo, with implications that may extend to rat transgenesis, or endangered animal species conservation efforts., Competing Interests: Conflicts of interest The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Scientific evidence suggests that not only murine scent communication is regulated by major urinary proteins, but that their expression may also vary in response to metabolism via a yet unknown mechanism. Major urinary proteins are expressed mainly in the liver, showing a sexually dimorphic pattern with substantially higher expression in males. Here, we investigate the metabolic implications of a major urinary protein knockout in twelve-week-old male and female C57BL/6N mice during ad libitum feeding. Despite both sexes of major urinary protein knockout mice displayed numerically increased body weight and visceral adipose tissue proportions compared to sex-matched wildtype mice, the main genotype-specific metabolic differences were observed exclusively in males. Male major urinary protein knockout mice exhibited plasma and hepatic lipid accumulation accompanied by a hepatic transcriptome indicating an activation of lipogenesis. These findings match the higher major urinary protein expression in male compared to female wildtype mice, suggesting a more distinct reduction in energy requirements in male compared to female major urinary protein knockout mice. The observed sex-specific anabolic phenotype confirms a role of major urinary protein in metabolism and, since major urinary proteins are not expressed in humans, suggests the major urinary protein knockout mouse as a potential alternative model for translational metabolism research which needs to be further elucidated., (© 2022. The Author(s).)

Transient MyoD overexpression in concert with small molecule treatment reprograms mouse fibroblasts into induced myogenic progenitor cells (iMPCs). However, the molecular landscape and mechanisms orchestrating this cellular conversion remain unknown. Here, we undertook an integrative multiomics approach to delineate the process of iMPC reprogramming in comparison to myogenic transdifferentiation mediated solely by MyoD. Using transcriptomics, proteomics, and genome-wide chromatin accessibility assays, we unravel distinct molecular trajectories that govern the two processes. Notably, only iMPC reprogramming is characterized by gradual up-regulation of muscle stem cell markers, unique signaling pathways, and chromatin remodelers in conjunction with exclusive chromatin opening in core myogenic promoters. In addition, we determine that the Notch pathway is indispensable for iMPC formation and self-renewal and further use the Notch ligand Dll1 to homogeneously propagate iMPCs. Collectively, this study charts divergent molecular blueprints for myogenic transdifferentiation or reprogramming and underpins the heightened capacity of iMPCs for capturing myogenesis ex vivo.

The acute stress response mobilizes energy to meet situational demands and re-establish homeostasis. However, the underlying molecular cascades are unclear. Here, we use a brief swim exposure to trigger an acute stress response in mice, which transiently increases anxiety, without leading to lasting maladaptive changes. Using multiomic profiling, such as proteomics, phospho-proteomics, bulk mRNA-, single-nuclei mRNA-, small RNA-, and TRAP-sequencing, we characterize the acute stress-induced molecular events in the mouse hippocampus over time. Our results show the complexity and specificity of the response to acute stress, highlighting both the widespread changes in protein phosphorylation and gene transcription, and tightly regulated protein translation. The observed molecular events resolve efficiently within four hours after initiation of stress. We include an interactive app to explore the data, providing a molecular resource that can help us understand how acute stress impacts brain function in response to stress., (© 2022. 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.)

DNA methylation represents one of the best characterized epigenetic modifications. In particular, global demethylation is a common feature of epigenetic reprogramming to naïve pluripotency in human and mouse pluripotent stem cells. In parallel to the global changes, several locus-specific changes to the DNA methylation landscape occur and also loss of imprinting has been observed in naïve human pluripotent stem cells. The current gold standard to assess and quantitively map DNA methylation is bisulfite sequencing. Various protocols are available for genome-wide bisulfite sequencing and here I describe an optimized method based on Post Bisulfite Adapter Tagging (PBAT) for low amounts of DNA or cells, with as little as 50 cells as minimum requirement, and with the possibility to process a large number of samples in parallel. I outline the basic bioinformatic steps needed to process raw Illumina sequencing data and then describe the inital steps of the analysis of DNA methylation datasets, including an assessment of imprint control regions., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Hydroxycarbamide (HC, hydroxyurea) is a cytoreductive drug inducing cell cycle blockade. However, emerging evidence suggests that HC plays a role in the modulation of transcription through the activity of transcription factors and DNA methylation. Examining the global mechanism of action of HC in the context of myeloproliferative neoplasms (MPNs), for which HC is the first-line treatment, will provide a better understanding of its molecular effects. To explore the effects of HC genome-wide, transcriptomic analyses were performed on two clinically relevant cell types at different stages of differentiation treated with HC in a murine MPN model. This study was replicated in MPN patients by profiling genome-wide gene expression and DNA methylation using patient blood samples collected longitudinally, before and following HC exposure. The effects of HC on the transcriptome were not only associated with cell cycle interruption but also with hematopoietic functions. Moreover, a group of genes were restored to normal expression levels in murine hematopoietic stem cells (HSCs) following drug treatment, including the master regulator of hematopoiesis, RUNX1 In humans, HC significantly modifies DNA methylation levels in HSCs at several distal regulatory regions, which we show to be associated with SPI1 binding sites and at the SPI1 locus itself. We have identified novel targets of HC that include pivotal transcription factors involved in hematopoiesis, and for the first time we report abnormal methylation patterns in MPN patients at the master regulator gene SPI1 and its distal binding sites, which HC is able to restore to normal levels., (© 2021 Contreras Castillo et al.; Published by Cold Spring Harbor Laboratory Press.)

DNA methylation plays a critical role during development, particularly in repressing retrotransposons. The mammalian methylation landscape is dependent on the combined activities of the canonical maintenance enzyme Dnmt1 and the de novo Dnmts, 3a and 3b. Here, we demonstrate that Dnmt1 displays de novo methylation activity in vitro and in vivo with specific retrotransposon targeting. We used whole-genome bisulfite and long-read Nanopore sequencing in genetically engineered methylation-depleted mouse embryonic stem cells to provide an in-depth assessment and quantification of this activity. Utilizing additional knockout lines and molecular characterization, we show that the de novo methylation activity of Dnmt1 depends on Uhrf1, and its genomic recruitment overlaps with regions that enrich for Uhrf1, Trim28 and H3K9 trimethylation. Our data demonstrate that Dnmt1 can catalyze DNA methylation in both a de novo and maintenance context, especially at retrotransposons, where this mechanism may provide additional stability for long-term repression and epigenetic propagation throughout development., (© 2021. The Author(s).)

Aims: Fibroblast activation protein (FAP) is upregulated at sites of tissue remodelling including chronic arthritis, solid tumours, and fibrotic hearts. It has also been associated with human coronary atherosclerotic plaques. Yet, the causal role of FAP in atherosclerosis remains unknown. To investigate the cause-effect relationship of endogenous FAP in atherogenesis, we assessed the effects of constitutive Fap deletion on plaque formation in atherosclerosis-prone apolipoprotein E (Apoe) or low-density lipoprotein receptor (Ldlr) knockout mice., Methods and Results: Using en face analyses of thoraco-abdominal aortae and aortic sinus cross-sections, we demonstrate that Fap deficiency decreased plaque formation in two atherosclerotic mouse models (-46% in Apoe and -34% in Ldlr knockout mice). As a surrogate of plaque vulnerability fibrous cap thickness was used; it was increased in Fap-deficient mice, whereas Sirius red staining demonstrated that total collagen content remained unchanged. Using polarized light, atherosclerotic lesions from Fap-deficient mice displayed increased FAP targets in terms of enhanced collagen birefringence in plaques and increased pre-COL3A1 expression in aortic lysates. Analyses of the Stockholm Atherosclerosis Gene Expression data revealed that FAP expression was increased in human atherosclerotic compared to non-atherosclerotic arteries., Conclusions: Our data provide causal evidence that constitutive Fap deletion decreases progression of experimental atherosclerosis and increases features of plaque stability with decreased collagen breakdown. Thus, inhibition of FAP expression or activity may not only represent a promising therapeutic target in atherosclerosis but appears safe at the experimental level for FAP-targeted cancer therapies., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: journals.permissions@oup.com.)

Objective: Adipose tissue-derived stem cells (ASCs) might play an important role in adipose microenvironment remodelling during tissue expansion through their response to hypoxia. We examined the cytokine profiles of hypoxic visceral ASCs (hypox-visASCs) from subjects with different metabolic risk, the interactions between cytokines as well as the impact of TNFα-induced death in the behavior of surviving hypoxic subcutaneous ASCs (hypox-subASCs) both at bulk population and single-cell level., Materials/methods: Visceral adipose tissue was processed to isolate the ASCs from 33 subjects grouped into normal weight, obese with and without metabolic syndrome. Multiplex assay was used to simultaneously measure multiple inflammatory, anti-inflammatory and angiogenic cytokines in hypox-visASCs from these patients and to elucidate cytokine profiles of hypox-subASCs upon stimulation with IL1β or TNFα and after TNFα-induced death. qPCR and single-cell RNA-sequencing were also performed to elucidate transcriptional impact in surviving hypox-subASCs after TNFα-induced apoptosis., Results: Hypox-visASCs from subjects without metabolic syndrome showed greater secretion levels of inflammatory, anti-inflammatory and angiogenic cytokines compared with those from patients with metabolic syndrome. While IL-1β stimulation was sufficient to increase the secretion levels of these cytokines in hypox-subASCs, TNFα-induced apoptosis also increased their levels and impacted on the expression levels of extracellular matrix proteins, acetyl-CoA producing enzymes and redox-balance proteins in surviving hypox-subASCs. TNFα-induced apoptosis under different glucose concentrations caused selective impoverishment of cell clusters and differentially influenced gene expression profiles of surviving hypox-subASCs., Conclusions: Immunoregulatory and angiogenic functions of hypox-visASCs from patients with metabolic syndrome could be insufficient to promote healthy adipose tissue expansion. TNFα-induced apoptosis may impact on functionality of hypox-subASC populations, whose differential metabolic sensitivity to death could serve to manipulate individual populations selectively in order to elucidate their role in shaping adipose heterogeneity and treating metabolic disorders., Competing Interests: Declaration of competing interest No potential conflicts of interest relevant to this article were reported., (Copyright © 2020. Published by Elsevier Inc.)

Genomic imprinting is an epigenetic phenomenon leading to parental allele-specific expression. Dosage of imprinted genes is crucial for normal development and its dysregulation accounts for several human disorders. This unusual expression pattern is mostly dictated by differences in DNA methylation between parental alleles at specific regulatory elements known as imprinting control regions (ICRs). Although several approaches can be used for methylation inspection, we lack an easy and cost-effective method to simultaneously measure DNA methylation at multiple imprinted regions. Here, we present IMPLICON, a high-throughput method measuring DNA methylation levels at imprinted regions with base-pair resolution and over 1000-fold coverage. We adapted amplicon bisulfite-sequencing protocols to design IMPLICON for ICRs in adult tissues of inbred mice, validating it in hybrid mice from reciprocal crosses for which we could discriminate methylation profiles in the two parental alleles. Lastly, we developed a human version of IMPLICON and detected imprinting errors in embryonic and induced pluripotent stem cells. We also provide rules and guidelines to adapt this method for investigating the DNA methylation landscape of any set of genomic regions. In summary, IMPLICON is a rapid, cost-effective and scalable method, which could become the gold standard in both imprinting research and diagnostics., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Satellite cells (SCs) are required for muscle repair following injury and are involved in muscle remodeling upon muscular contractions. Exercise stimulates SC accumulation and myonuclear accretion. To what extent exercise training at different mechanical loads drive SC contribution to myonuclei however is unknown., Results: By performing SC fate tracing experiments, we show that 8 weeks of voluntary wheel running increased SC contribution to myofibers in mouse plantar flexor muscles in a load-dependent, but fiber type-independent manner. Increased SC fusion however was not exclusively linked to muscle hypertrophy as wheel running without external load substantially increased SC fusion in the absence of fiber hypertrophy. Due to nuclear propagation, nuclear fluorescent fate tracing mouse models were inadequate to quantify SC contribution to myonuclei. Ultimately, by performing fate tracing at the DNA level, we show that SC contribution mirrors myonuclear accretion during exercise., Conclusions: Collectively, mechanical load during exercise independently promotes SC contribution to existing myofibers. Also, due to propagation of nuclear fluorescent reporter proteins, our data warrant caution for the use of existing reporter mouse models for the quantitative evaluation of satellite cell contribution to myonuclei.

The occurrence of repetitive genomic changes that provide a selective growth advantage in pluripotent stem cells is of concern for their clinical application. However, the effect of different culture conditions on the underlying mutation rate is unknown. Here we show that the mutation rate in two human embryonic stem cell lines derived and banked for clinical application is low and not substantially affected by culture with Rho Kinase inhibitor, commonly used in their routine maintenance. However, the mutation rate is reduced by >50% in cells cultured under 5% oxygen, when we also found alterations in imprint methylation and reversible DNA hypomethylation. Mutations are evenly distributed across the chromosomes, except for a slight increase on the X-chromosome, and an elevation in intergenic regions suggesting that chromatin structure may affect mutation rate. Overall the results suggest that pluripotent stem cells are not subject to unusually high rates of genetic or epigenetic alterations.

In contrast to conventional human pluripotent stem cells (hPSCs) that are related to post-implantation embryo stages, naive hPSCs exhibit features of pre-implantation epiblast. Naive hPSCs are established by resetting conventional hPSCs, or are derived from dissociated embryo inner cell masses. Here we investigate conditions for transgene-free reprogramming of human somatic cells to naive pluripotency. We find that Wnt inhibition promotes RNA-mediated induction of naive pluripotency. We demonstrate application to independent human fibroblast cultures and endothelial progenitor cells. We show that induced naive hPSCs can be clonally expanded with a diploid karyotype and undergo somatic lineage differentiation following formative transition. Induced naive hPSC lines exhibit distinctive surface marker, transcriptome, and methylome properties of naive epiblast identity. This system for efficient, facile, and reliable induction of transgene-free naive hPSCs offers a robust platform, both for delineation of human reprogramming trajectories and for evaluating the attributes of isogenic naive versus conventional hPSCs., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The unabated rise in obesity prevalence during the last 40 years has spurred substantial interest in understanding the reasons for this epidemic. Studies in mice and humans have demonstrated that obesity is a highly heritable disease; however genetic variations within specific populations have so far not been able to explain this phenomenon to its full extent. Recent work has demonstrated that environmental cues can be sensed by an organism to elicit lasting changes, which in turn can affect systemic energy metabolism by different epigenetic mechanisms such as changes in small noncoding RNA expression, DNA methylation patterns, as well as histone modifications. These changes can directly modulate cellular function in response to environmental cues, however research during the last decade has demonstrated that some of these modifications might be transmitted to subsequent generations, thus modulating energy metabolism of the progeny in an inter- as well as transgenerational manner. In this context, adipose tissue has become a focus of research due to its plasticity, which allows the formation of energy storing (white) as well as energy wasting (brown/brite/beige) cells within the same depot. In this Review, the effects of environmental induced obesity with a particular focus on adipose tissue are discussed., Competing Interests: The authors declare no conflict of interest.

The power of mouse embryonic stem (ES) cells to colonise the developing embryo has revolutionised mammalian developmental genetics and stem cell research. This power is vulnerable, however, to the cell culture environment, deficiencies in which can lead to cellular heterogeneity, adaptive phenotypes, epigenetic aberrations and genetic abnormalities. Here, we provide detailed methodologies for derivation, propagation, genetic modification and primary differentiation of ES cells in 2i or 2i+LIF media without serum or undefined serum substitutes. Implemented diligently, these procedures minimise variability and deviation, thereby improving the efficiency, reproducibility and biological validity of ES cell experimentation., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

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.)

Current molecular biology laboratories rely heavily on the purification and manipulation of nucleic acids. Yet, commonly used centrifuge- and column-based protocols require specialised equipment, often use toxic reagents, and are not economically scalable or practical to use in a high-throughput manner. Although it has been known for some time that magnetic beads can provide an elegant answer to these issues, the development of open-source protocols based on beads has been limited. In this article, we provide step-by-step instructions for an easy synthesis of functionalised magnetic beads, and detailed protocols for their use in the high-throughput purification of plasmids, genomic DNA, RNA and total nucleic acid (TNA) from a range of bacterial, animal, plant, environmental and synthetic sources. We also provide a bead-based protocol for bisulfite conversion and size selection of DNA and RNA fragments. Comparison to other methods highlights the capability, versatility, and extreme cost-effectiveness of using magnetic beads. These open-source protocols and the associated webpage (https://bomb.bio) can serve as a platform for further protocol customisation and community engagement., Competing Interests: TH and DB are directors of a small agricultural and biotech consultancy (TOTOGEN Ltd), at which some BOMB protocols were developed and have been donated to this project. SRH is director of an agricultural engineering firm (CENENG Ltd).

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.

Erasure of DNA methylation and repressive chromatin marks in the mammalian germline leads to risk of transcriptional activation of transposable elements (TEs). Here, we used mouse embryonic stem cells (ESCs) to identify an endosiRNA-based mechanism involved in suppression of TE transcription. In ESCs with DNA demethylation induced by acute deletion of Dnmt1, we saw an increase in sense transcription at TEs, resulting in an abundance of sense/antisense transcripts leading to high levels of ARGONAUTE2 (AGO2)-bound small RNAs. Inhibition of Dicer or Ago2 expression revealed that small RNAs are involved in an immediate response to demethylation-induced transposon activation, while the deposition of repressive histone marks follows as a chronic response. In vivo, we also found TE-specific endosiRNAs present during primordial germ cell development. Our results suggest that antisense TE transcription is a "trap" that elicits an endosiRNA response to restrain acute transposon activity during epigenetic reprogramming in the mammalian germline., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Execution of pluripotency requires progression from the naïve status represented by mouse embryonic stem cells (ESCs) to a state capacitated for lineage specification. This transition is coordinated at multiple levels. Non-coding RNAs may contribute to this regulatory orchestra. We identified a rodent-specific long non-coding RNA (lncRNA) linc1281, hereafter Ephemeron ( Eprn ), that modulates the dynamics of exit from naïve pluripotency. Eprn deletion delays the extinction of ESC identity, an effect associated with perduring Nanog expression. In the absence of Eprn , Lin28a expression is reduced which results in persistence of let-7 microRNAs, and the up-regulation of de novo methyltransferases Dnmt3a/b is delayed. Dnmt3a/b deletion retards ES cell transition, correlating with delayed Nanog promoter methylation and phenocopying loss of Eprn or Lin28a . The connection from lncRNA to miRNA and DNA methylation facilitates the acute extinction of naïve pluripotency, a pre-requisite for rapid progression from preimplantation epiblast to gastrulation in rodents. Eprn illustrates how lncRNAs may introduce species-specific network modulations.

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.)

Epigenetic memory, in particular DNA methylation, is established during development in differentiating cells and must be erased to create naïve (induced) pluripotent stem cells. The ten-eleven translocation (TET) enzymes can catalyze the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidized derivatives, thereby actively removing this memory. Nevertheless, the mechanism by which the TET enzymes are regulated, and the extent to which they can be manipulated, are poorly understood. Here we report that retinoic acid (RA) or retinol (vitamin A) and ascorbate (vitamin C) act as modulators of TET levels and activity. RA or retinol enhances 5hmC production in naïve embryonic stem cells by activation of TET2 and TET3 transcription, whereas ascorbate potentiates TET activity and 5hmC production through enhanced Fe 2+ recycling, and not as a cofactor as reported previously. We find that both ascorbate and RA or retinol promote the derivation of induced pluripotent stem cells synergistically and enhance the erasure of epigenetic memory. This mechanistic insight has significance for the development of cell treatments for regenenerative medicine, and enhances our understanding of how intrinsic and extrinsic signals shape the epigenome., Competing Interests: W.R. and S.B. serve as consultants for Cambridge Epigenetix Ltd.

Primordial germ cell (PGC) development is characterized by global epigenetic remodeling, which resets genomic potential and establishes an epigenetic ground state. Here we recapitulate PGC specification in vitro from naive embryonic stem cells and characterize the early events of epigenetic reprogramming during the formation of the human and mouse germline. Following rapid de novo DNA methylation during priming to epiblast-like cells, methylation is globally erased in PGC-like cells. Repressive chromatin marks (H3K9me2/3) and transposable elements are enriched at demethylation-resistant regions, while active chromatin marks (H3K4me3 or H3K27ac) are more prominent at regions that demethylate faster. The dynamics of specification and epigenetic reprogramming show species-specific differences, in particular markedly slower reprogramming kinetics in the human germline. Differences in developmental kinetics may be explained by differential regulation of epigenetic modifiers. Our work establishes a robust and faithful experimental system of the early events of epigenetic reprogramming and regulation in the germline., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)