2021 Articles

Epigenetic clocks reveal a rejuvenation event during embryogenesis followed by aging

Kerepesi C, Zhang B, Lee SL, Trapp A, Gladyshev VN

Science Advances

Abstract The notion that the germ line does not age goes back to the 19th-century ideas of August Weismann. However, being metabolically active, the germ line accumulates damage and other changes over time, i.e., it ages. For new life to begin in the same young state, the germ line must be rejuvenated in the offspring. Here, we developed a multi-tissue epigenetic clock and applied it, together with other aging clocks, to track changes in biological age during mouse and human prenatal development. This analysis revealed a significant decrease in biological age, i.e., rejuvenation, during early stages of embryogenesis, followed by an increase in later stages. We further found that pluripotent stem cells do not age even after extensive passaging and that the examined epigenetic age dynamics is conserved across species. Overall, this study uncovers a natural rejuvenation event during embryogenesis and suggests that the minimal biological age (ground zero) marks the beginning of organismal aging. More Information

Profiling epigenetic age in single cells

Trapp A, Kerepesi C, Gladyshev VN


Abstract DNA methylation of a defined set of CpG dinucleotides emerged as a critical and precise biomarker of the aging process. Multi-variate machine learning models, known as epigenetic clocks, can exploit quantitative changes in the methylome to predict the age of bulk tissue with remarkable accuracy. However, intrinsic sparsity and digitized methylation in individual cells have so far precluded the assessment of aging in single cell data. Here, we present scAge, a probabilistic approach to determine the epigenetic age of single cells, and validate our results in mice. scAge tissue-specific and multi-cell type single cell clocks correctly recapitulate chronological age of the original tissue, while uncovering the inherent heterogeneity that exists at the single-cell level. The data suggest that while tissues age in a coordinated fashion, some cells age more or less rapidly than others. We show that individual embryonic stem cells exhibit an age close to zero, that certain stem cells in a tissue show a reduced age compared to their chronological age, and that early embryogenesis is associated with the reduction of epigenetic age of individual cells, the latter supporting a natural rejuvenation event during gastrulation. scAge is both robust against the low coverage that is characteristic of single cell sequencing techniques and is flexible for studying any cell type and vertebrate organism of interest. This study demonstrates for the first time the potential for accurate epigenetic age profiling at single-cell resolution. More Information

The Ground Zero of Organismal Life and Aging

Gladyshev VN.

Trends in Molecular Medicine, 27(1), 11-19

Abstract Cells may naturally proceed or be forced to transition to a state with a radically lower biological age, that is, be rejuvenated. Examples are the conversion of somatic cells to induced pluripotent stem cells and rejuvenation of the germline with each generation. We posit that these processes converge to the same ‘ground zero’, the mid-embryonic state characterized by the lowest biological age where both organismal life and aging begin. It may also be related to the phylotypic state. The ground zero model clarifies the relationship between aging, development, rejuvenation, and de-differentiation, which are distinct throughout life. By extending the rejuvenation phase during early embryogenesis and editing the genome, it may be possible to achieve the biological age at the ground zero lower than that achieved naturally. More Information

James R. Mitchell (1971-2020)

Ristow M, Lee CH, De Bock K, Gladyshev VN, Hotamisligil GS, Manning BD.

Cell Metabolism, 33(3), 458-461

Abstract We are deeply saddened by the task of writing this memoriam for our beloved friend and colleague James (Jay) Mitchell, who passed away after a cycling accident on November 17 at the age of 49. Jay was absolutely unmatched in his creativity and ability to cross fields from molecular biology to physiology, from nutrition to genetics, and from epidemiology to surgery. He was among the first to crack the molecular code of the benefits of dietary restriction and defined unique pathways that opened many translational opportunities. He was a “science whisperer” in all stages of his career; this was his magical trademark. He was soft spoken but fiercely determined, incredibly resilient yet very kind and thoughtful, expressed strength with humility and content, and could relate to anyone in any walk of life and in any intellectual or artistic domain. He leaves behind his wife Elisabeth; his three sons, Lucas, Darius, and Ian; and an irreplaceable legacy as a husband, father, brother, son, friend, teacher, colleague, and most importantly, an invaluable human treasure. More Information

Naked mole rat TRF1 safeguards glycolytic capacity and telomere replication under low oxygen

Augereau A, Mariotti M, Pousse M, Filipponi D, Libert F, Beck B, Gorbunova V, Gilson E, Gladyshev VN.

Science Advances, 7(8), eabe0174

Abstract The naked mole rat (NMR), a long-lived and cancer-resistant rodent, is highly resistant to hypoxia. Here, using robust cellular models wherein the mouse telomeric protein TRF1 is substituted by NMR TRF1 or its mutant forms, we show that TRF1 supports maximal glycolytic capacity under low oxygen, shows increased nuclear localization and association with telomeres, and protects telomeres from replicative stress. We pinpoint this evolutionary gain of metabolic function to specific amino acid changes in the homodimerization domain of this protein. We further find that NMR TRF1 accelerates telomere shortening. These findings reveal an evolutionary strategy to adapt telomere biology for metabolic control under an extreme environment. More Information

Translation elongation rate varies among organs and decreases with age

Gerashchenko MV, Peterfi Z, Yim SH, Gladyshev VN.

Nucleic Acids Research, 49(2), e9

Abstract There has been a surge of interest towards targeting protein synthesis to treat diseases and extend lifespan. Despite the progress, few options are available to assess translation in live animals, as their complexity limits the repertoire of experimental tools to monitor and manipulate processes within organs and individual cells. It this study, we developed a labeling-free method for measuring organ- and cell-type-specific translation elongation rates in vivo. It is based on time-resolved delivery of translation initiation and elongation inhibitors in live animals followed by ribosome profiling. It also reports translation initiation sites in an organ-specific manner. Using this method, we found that the elongation rates differ more than 50% among mouse organs and determined them to be 6.8, 5.0 and 4.3 amino acids per second for liver, kidney, and skeletal muscle, respectively. We further found that the elongation rate is reduced by 20% between young adulthood and mid-life. Thus, translation, a major metabolic process in cells, is tightly regulated at the level of elongation of nascent polypeptide chains. More Information

Aging predisposes B cells to malignancy by activating c-Myc and perturbing the genome and epigenome

Shindyapina AV*, Castro JP*, Barbieri A, Strelkova OS, Paulo JA, Kerepesi C, Petrashen AP, Mariotti M, Meer M, Hu Y, Losyev G, Indzhykulian AA, Gygi SP, Sedivy JM, Manis JP, Gladyshev VN


Abstract Age is the single major risk factor for human cancer; however, naturally occurring cancers are rarely studied in aged animal models. Laboratory mouse strains spontaneously develop cancer with age and some predominantly die from B-cell lymphoma. Here, we uncover how B-cell lymphoma develops as a consequence of the aging immune system. We found that aged B cells undergo clonal expansions driven by genetic and epigenetic changes and established cell and spleen size as early markers of malignant transformation. High-throughput and omics assays of aged B cells and the use of mouse models revealed that c-Myc is a master regulator of B cell size and clonal expansion. A single-cell RNA-seq analysis suggested that clonal B cells originate from age-associated B cells, memory B cells that accumulate during aging. Further studies showed that c-Myc becomes activated in B cells in response to the aging microenvironment. Thus, c-Myc, aging environment, somatic mutations and the epigenome cooperate to give rise to clonal age-accelerated B cells, which we named Myc+ cells. We further show the relevance of this model to aged human B cells in blood and spleen. This study characterized a first mouse model that captures a natural transition of B cells to a prevalent type of cancer during aging. More Information

Assessing Ribosome Distribution Along Transcripts with Polarity Scores and Regression Slope Estimates

Vorontsov IE, Egorov AA, Anisimova AS, Eliseeva IA, Makeev VJ, Gladyshev VN, Dmitriev SE, Kulakovskiy IV.

Trends in Molecular Medicine, 2252, 269-294

Abstract During translation, the rate of ribosome movement along mRNA varies. This leads to a non-uniform ribosome distribution along the transcript, depending on local mRNA sequence, structure, tRNA availability, and translation factor abundance, as well as the relationship between the overall rates of initiation, elongation, and termination. Stress, antibiotics, and genetic perturbations affecting composition and properties of translation machinery can alter the ribosome positional distribution dramatically. Here, we offer a computational protocol for analyzing positional distribution profiles using ribosome profiling (Ribo-Seq) data. The protocol uses papolarity, a new Python toolkit for the analysis of transcript-level short read coverage profiles. For a single sample, for each transcript papolarity allows for computing the classic polarity metric which, in the case of Ribo-Seq, reflects ribosome positional preferences. For comparison versus a control sample, papolarity estimates an improved metric, the relative linear regression slope of coverage along transcript length. This involves de-noising by profile segmentation with a Poisson model and aggregation of Ribo-Seq coverage within segments, thus achieving reliable estimates of the regression slope. The papolarity software and the associated protocol can be conveniently used for Ribo-Seq data analysis in the command-line Linux environment. Papolarity package is available through Python pip package manager. The source code is available at https://github.com/autosome-ru/papolarity. More Information

Measuring Organ-Specific Translation Elongation Rate in Mice

Gerashchenko MV, Gladyshev VN.

Methods in Molecular Biology, 2252, 189-200

Abstract Modern methods of genome editing enable the rapid generation of mouse models to study the regulation of protein synthesis. At the same time, few options are available to study translation in rodents as the animal’s complexity severely limits the repertoire of experimental tools. Here we describe a method to monitor translation in mice and other small animals. The technique is based on a ribosome profiling and specifically tailored toward measuring translation elongation. However, it can be easily applied for short upstream reading frames discovery. The advantage of this method is the ability to study translation in fully developed animals without extracting and subculturing cells, therefore, maintaining unperturbed physiological conditions. More Information

Development of a novel fluorescent biosensor for dynamic monitoring of metabolic methionine redox status in cells and tissues

Choi DW, Roh YJ, Kim S, Lee HM, Kim M, Shin D, Park JH, Cho Y, Park HH, Ok YS, Kang D, Kim JH, Tarrago L, Danial NN, Gladyshev VN, Min PK, Lee BC.

Biosensors and Bioelectronics, 178, 113031

Abstract Aberrant production of reactive oxygen species (ROS) leads to tissue damage accumulation, which is associated with a myriad of human pathologies. Although several sensors have been developed for ROS quantification, their applications for ROS-related human physiologies and pathologies still remain problematic due to the unstable nature of ROS. Herein, we developed Trx1-cpYFP-fRMsr (TYfR), a genetically-encoded fluorescent biosensor with the remarkable specificity and sensitivity toward fMetRO (free Methionine-R-sulfoxide), allowing for dynamic quantification of physiological levels of fMetRO, a novel indicator of ROS and methionine redox status in vitro and in vivo. Moreover, using the sensor, we observed a significant fMetRO enrichment in serum from patients with acute coronary syndrome, one of the most severe cardiovascular diseases, which becomes more evident following percutaneous coronary intervention. Collectively, this study proposes that fMetRO is a novel biomarker of tissue damage accumulation in ROS-associated human pathologies, and that TYfR is a promising tool for quantifying fMetRO with potentials in versatile applications. More Information

Universal DNA methylation age across mammalian tissues

Mammalian Methylation Consortium.


Abstract Aging is often perceived as a degenerative process caused by random accrual of cellular damage over time. In spite of this, age can be accurately estimated by epigenetic clocks based on DNA methylation profiles from almost any tissue of the body. Since such pan-tissue epigenetic clocks have been successfully developed for several different species, it is difficult to ignore the likelihood that a defined and shared mechanism instead, underlies the aging process. To address this, we generated 10,000 methylation arrays, each profiling up to 37,000 cytosines in highly-conserved stretches of DNA, from over 59 tissue-types derived from 128 mammalian species. From these, we identified and characterized specific cytosines, whose methylation levels change with age across mammalian species. Genes associated with these cytosines are greatly enriched in mammalian developmental processes and implicated in age-associated diseases. From the methylation profiles of these age-related cytosines, we successfully constructed three highly accurate universal mammalian clocks for eutherians, and one universal clock for marsupials. The universal clocks for eutherians are similarly accurate for estimating ages (r>0.96) of any mammalian species and tissue with a single mathematical formula. Collectively, these new observations support the notion that aging is indeed evolutionarily conserved and coupled to developmental processes across all mammalian species – a notion that was long-debated without the benefit of this new and compelling evidence. More Information

DNA Methylation Networks Underlying Mammalian Traits

Haghani A, Lu AT, Li CZ, Robeck TR, Belov K, Breeze CE, Brooke RT, Clarke S, Faulkes CG, Fei Z, Ferguson SH, Finno CJ, Gladyshev VN, Gorbunova V, Goya RG, Hogan AN, Hogg CJ, Hore TA, Kiaris H, Kordowitzki P, Banks G, Koski WR, Mozhui K, Naderi A, Ostrander EA, Parsons KM, Plassais J, Robbins J, Sears KE, Seluanov A, Steinman KJ, Szladovits B, Thompson MJ, Villar D, Wang N, Wilkinson GS, Young BG, Zhang J, Zoller JA, Ernst J, Yang XW, Raj K, S. Horvath


Abstract Epigenetics has hitherto been studied and understood largely at the level of individual organisms. Here, we report a multi-faceted investigation of DNA methylation across 11,117 samples from 176 different species. We performed an unbiased clustering of individual cytosines into 55 modules and identified 31 modules related to primary traits including age, species lifespan, sex, adult species weight, tissue type and phylogenetic order. Analysis of the correlation between DNA methylation and species allowed us to construct phyloepigenetic trees for different tissues that parallel the phylogenetic tree. In addition, while some stable cytosines reflect phylogenetic signatures, others relate to age and lifespan, and in many cases responding to anti-aging interventions in mice such as caloric restriction and ablation of growth hormone receptors. Insights uncovered by this investigation have important implications for our understanding of the role of epigenetics in mammalian evolution, aging and lifespan. More Information