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Gladyshev, V. N.

Publications and source records attributed to Gladyshev, V. N..

At least 73 records · Page 4Linked to original sources

Ultra-cheap and scalable epigenetic age predictions with TIME-Seq

Epigenetic "clocks" based on DNA methylation (DNAme) have emerged as the most robust and widely employed aging biomarkers, but conventional methods for applying them are expensive and laborious. Here, we develop Tagmentation-based Indexing for Methylation Sequencing (TIME-Seq), a highly multiplexed and scalable method for low-cost epigenetic clocks. Using TIME-Seq, we applied multi-tissue and tissue-specific epigenetic clocks to over 1,600 mouse DNA samples. We also discovered a novel approach for age prediction from shallow sequencing (e.g., 10,000 reads) by adapting scAge for bulk measurements. In benchmarking experiments, TIME-Seq performed favorably against prevailing methods and could quantify the effects of interventions thought to accelerate, slow, and reverse aging in mice. Finally, we built and validated a highly accurate human blood clock from 1,056 demographically representative individuals. Our methods increase the scalability and reduce the cost of epigenetic age predictions by more than 100-fold, enabling accurate aging biomarkers to be applied in more large-scale animal and human studies.

genomics↗

Cost-effective epigenetic age profiling in shallow methylation sequencing data

There is a critical need for robust, high-throughput assays of biological aging trajectories. Among various approaches, epigenetic aging clocks emerged as reliable molecular trackers of the aging process. However, current methods for epigenetic age profiling are inherently costly and lack throughput. Here, we leverage the scAge framework for accurate prediction of biological age from very few bisulfite sequencing reads in bulk samples, thereby enabling drastic (100-1,000-fold) reduction in sequencing costs per sample. Our approach permits age assessment based on distinct assortments of CpG sites in different samples, without the need for targeted site enrichment or specialized reagents. We demonstrate the efficacy of this method to quantify the age of mouse blood samples across independent cohorts, identify the effect of calorie restriction as an attenuator of the aging process, and discern rejuvenation upon cellular reprogramming. We propose that this framework may be used for epigenetic age prediction in extremely high-throughput applications, enabling robust, large-scale and inexpensive interventions testing and age profiling.

genomics↗

Systematic identification of yeast mutants with increased rates of cell death reveals rapid stochastic necrosis associated with cell division

Cell death plays a major role in development, pathology and aging and can be triggered by various types of acute stimuli which arrest cell growth. However, little is known about chronic cell death in the context of continuing cell division. Here, we performed a genome-wide search for mutants with this type of death in dividing bakers yeast by assaying staining with phloxine B, which accumulates in dead cells. This screen yielded 83 essential and 43 non-essential gene mutants. Three contrasting types of spatial distribution of dead cells in colonies were observed, which corresponded to gene ontology enrichment for (i) DNA replication and repair, RNA processing, chromatin organization, and nuclear transport; (ii) mitosis and cytokinesis; and (iii) vesicular transport and glycosylation/cell wall homeostasis. To study dynamics of cell death in these mutants, we developed methods for analyzing the death of newborn cells (DON) and cell death in real time using microfluidics-based microscopy. These revealed rapid stochastic necrosis during bud generation or cytokinesis without prior division arrest. Increased death during division was associated with common sensitivity to plasma membrane and cell-wall perturbing agents, and could be mitigated by neutral pH stabilization of the medium. This suggests a common downstream type of cell death caused by a wide range of genetic perturbations.

cell biology↗

Rilmenidine mimics caloric restriction via the nischarin I1-imidazoline receptor to extend lifespan in C. elegans

Caloric restriction increases lifespan across species and has health benefits in humans. Because complying with a low-calorie diet is challenging, here we investigated pharmacological interventions mimicking the benefits of caloric restriction. Searching for compounds that elicit a similar gene expression signature to caloric restriction, we identified rilmenidine, an I1-imidazoline receptor agonist and prescription medication for the treatment of hypertension. We then show that treating C. elegans with rilmenidine at young and older ages increases lifespan. We also demonstrate that the stress-resilience, healthspan, and lifespan benefits upon rilmenidine treatment in worms are mediated by the I1-imidazoline receptor nish-1, implicating this receptor as a potential longevity target. Furthermore, we show that rilmenidine treatment increased ERK phosphorylation via NISH-1. Consistent with the shared caloric-restriction-mimicking gene signature, supplementing rilmenidine to caloric restricted C. elegans, genetic reduction of TORC1 function, or rapamycin treatment did not further increase lifespan. The rilmenidine-induced longevity required the transcription factors FOXO/DAF-16 and NRF1,2,3/SKN-1, both important for caloric restriction-mediated longevity. Furthermore, we find that autophagy, but not AMPK signaling, was needed for rilmenidine-induced longevity. Lastly, we find that treating mice with rilmenidine showed transcriptional changes in liver and kidney similar to caloric restriction. Overall, our findings reveal rilmenidine as a caloric restriction mimetic and as a novel geroprotective compound.

pharmacology and toxicology↗

Intergenerational hormesis is regulated by heritable 18S rRNA methylation

Heritable non-genetic information can regulate a variety of complex phenotypes. However, what specific non-genetic cues are transmitted from parents to their descendants are poorly understood. Here, we perform metabolic methyl-labelling experiments to track the heritable transmission of methylation from ancestors to their descendants in the nematode Caenorhabditis elegans. We find that methylation is transmitted to descendants in proteins, RNA, DNA and lipids. We further find that in response to parental starvation, fed naive progeny display reduced fertility, increased heat stress resistance, and extended longevity. This intergenerational hormesis is accompanied by a heritable increase in N6-dimethyl adenosine (m6,2A) on the 18S ribosomal RNA at adenosines 1735 and 1736. We identified the conserved DIMT-1 as the m6,2A methyltransferase in C. elegans and find that dimt-1 is required for the intergenerational hormesis phenotypes. This study provides the first labeling and tracking of heritable non-genetic material across generations and demonstrates the importance of rRNA methylation for regulating the heritable response to starvation.

genetics↗

A standard knockout procedure alters expression of adjacent loci at the translational level

The S. cerevisiae gene deletion collection is widely used for functional gene annotation and genetic interaction analyses. However, the standard G418-resistance cassette used to produce knockout mutants delivers strong regulatory elements into the target genetic loci. To date, its side effects on the expression of neighboring genes have never been systematically assessed. Here, using ribosome profiling data, RT-qPCR, and reporter expression, we investigated perturbations induced by the KanMX module. Our analysis revealed significant alterations in the transcription efficiency of neighboring genes and, more importantly, severe impairment of their mRNA translation, leading to changes in protein abundance. In the "head-to-head" orientation of the neighbor and the deleted gene, knockout often led to a shift of the transcription start site of the neighboring gene, introducing new uAUG codon(s) into the expanded 5 untranslated region (5 UTR). In the "tail-to-tail" arrangement, knockout led to activation of alternative polyadenylation signals in the neighboring gene, thus altering its 3 UTR. These events may explain the so-called neighboring gene effect (NGE), i.e. false genetic interactions of the deleted genes. We estimate that in as much as [~]1/5 of knockout strains the expression of neighboring genes may be substantially (>2-fold) deregulated at the level of translation.

molecular biology↗

Ectopic cervical thymi and no thymic involution until midlife in naked mole-rats

Immunosenescence is a hallmark of aging and manifests as increased susceptibility to infection, autoimmunity, and cancer in the elderly. One component of immunosenescence is thymic involution, age-associated shrinkage of the thymus, observed in all vertebrates studied to date. The naked mole-rat (Heterocephalus glaber) has become an attractive animal model in aging research due to its extreme longevity and resistance to disease. Here we show that naked mole rats display no thymic involution up to 11 years of age. Furthermore, we found large ectopic cervical thymi in addition to the canonical thoracic thymus, both being identical in their cell composition. The developmental landscape in naked mole-rat thymi revealed overt differences from the murine T cell compartment, most notably a decrease of CD4+/CD8+ double-positive cells and lower abundance of cytotoxic effector T cells. Our observations suggest that naked mole rats display a delayed immunosenescence. Therapeutic interventions aimed at reversing thymic aging remain limited, underscoring the importance of understanding the cellular and molecular mechanisms behind a sustained immune function in the naked mole rat.

immunology↗

Naked mole-rat hematopoietic stem and progenitors are highly quiescent with an inherent myeloid bias

Naked mole-rats (NMRs) are the longest-lived rodents yet their stem cell characteristics remain enigmatic. Here we comprehensively mapped the NMR hematopoietic landscape and identified unique features likely contributing to longevity. Adult NMRs form red blood cells in spleen and marrow, which is a neotenic trait. A myeloid bias towards granulopoiesis in concert with decreased B-lymphopoiesis defines the marrow composition, resembling fetal leukopoiesis. Very similar to primates, the primitive stem cell compartment is marked by CD34 and THY1. Remarkably, stem and progenitor respiration rates are as low as in human cells, while NMR cells show a strong expression signature for fatty acid metabolism. The pool of quiescent stem cells is higher than in mice, and the cell cycle of hematopoietic cells is prolonged. Our work provides a platform to study immunology and stem cell biology in an animal model of exceptional longevity. TeaserJuvenile features of hematopoiesis shape the blood system of the longest-lived rodent.

cell biology↗

Redox regulation by TXNRD3 during epididymal maturation underlies capacitation-associated mitochondrial activation and sperm motility in mice

During epididymal transit, redox remodeling protects mammalian spermatozoa, preparing them for survival in the subsequent journey to fertilization. However, molecular mechanisms of redox regulation in sperm development and maturation remain largely elusive. In this study, we report that TXNRD3, a thioredoxin reductase family member particularly abundant in elongating spermatids at the site of mitochondrial sheath formation, regulates redox homeostasis to support male reproduction. Using Txnrd3-/- mice, our biochemical, ultrastructural, and live cell imaging analyses revealed impairments in sperm morphology and motility under conditions of TXNRD3 deficiency. We reveal that mitochondria develop more defined cristae during capacitation in wild type sperm. Absence of TXNRD3 alters redox status in both the head and tail during sperm maturation and capacitation, resulting in defective mitochondrial ultrastructure and activity under capacitating conditions. These findings provide insights into molecular mechanisms of redox homeostasis and bioenergetics during sperm maturation, capacitation, and fertilization.

physiology↗

Epigenetic predictors of maximum lifespan and other life history traits in mammals

Maximum lifespan of a species is the oldest that individuals can survive, reflecting the genetic limit of longevity in an ideal environment. Here we report methylation-based models that accurately predict maximum lifespan (r=0.89), gestational time (r=0.96), and age at sexual maturity (r=0.87), using cytosine methylation patterns collected from over 12,000 samples derived from 192 mammalian species. Our epigenetic maximum lifespan predictor corroborated the extended lifespan in growth hormone receptor knockout mice and rapamycin treated mice. Across dog breeds, epigenetic maximum lifespan correlates positively with breed lifespan but negatively with breed size. Lifespan-related cytosines are located in transcriptional regulatory regions, such as bivalent chromatin promoters and polycomb-repressed regions, which were hypomethylated in long-lived species. The epigenetic estimators of maximum lifespan and other life history traits will be useful for characterizing understudied species and for identifying interventions that extend lifespan.

evolutionary biology↗

DNA Methylation Networks Underlying Mammalian Traits

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.

evolutionary biology↗

Profiling epigenetic age in single cells

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.

genomics↗

Epigenetic clocks reveal a rejuvenation event during embryogenesis followed by aging

The notion that germline cells do not age goes back to the 19th century ideas of August Weismann. However, being in a metabolically active state, they accumulate damage and other age-related changes over time, i.e., they age. For new life to begin in the same young state, they must be rejuvenated in the offspring. Here, we developed a new 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 (the ground zero) marks the beginning of organismal aging.

developmental biology↗

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

While cancer is an age-related disease, many cancer studies utilize younger animal models. Here, we uncover how a cancer, B-cell lymphoma, develops as a consequence of a naturally aged system. We show that this malignancy is associated with increased cell size, splenomegaly, and a newly discovered age-associated clonal B-cell (ACBC) population. Driven by exogenous c-Myc activation, hypermethylated promoters and somatic mutations, ACBC cells clonally expand independent of germinal centers (IgM+) and show increased biological age and hypomethylation in partially methylated domains related to mitotic solo-CpGs. Epigenetic changes in transformed mouse B cells are enriched for changes observed in human B-cell lymphomas. Mechanistically, the data suggest that cancerous ACBC cells originate from age-associated B cells, in part involving CD22 protein signaling fostered by the aging microenvironment. Transplantation assays demonstrate that ACBC evolve to become self-sufficient and support malignancy when transferred into young recipients. Inhibition of mTOR or c-Myc in old mice attenuates premalignant changes in B cells during aging and emerges as a therapeutic strategy to delay the onset of age-related lymphoma. Together, we show how aging contributes to B-cell lymphoma through a previously unrecognized mechanism involving cell-intrinsic changes and the aged microenvironment, characterize a model that captures the origin and progression of spontaneous cancer during aging and identify candidate interventions against age-associated lymphoma.

cancer biology↗

Universal DNA methylation age across mammalian tissues

Aging is often perceived as a degenerative process resulting from random accrual of cellular damage over time. Despite 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, we hypothesized that one can build pan-mammalian clocks that measure age in all mammalian species. To address this, we generated data using 11,754 methylation arrays, each profiling up to 36 thousand cytosines in highly-conserved stretches of DNA, from 59 tissue-types derived from 185 mammalian species. From these methylation profiles, we constructed three age predictors, each with a single mathematical formula, termed universal pan-mammalian clocks that are accurate in estimating the age (r>0.96) of any mammalian tissue. Deviations between epigenetic age and chronological age relate to mortality risk in humans, mutations that affect the somatotropic axis in mice, and caloric restriction. We characterized specific cytosines, whose methylation levels change with age across most mammalian species. These cytosines are greatly enriched in polycomb repressive complex 2-binding sites, are located in regions that gradually lose chromatin accessibility with age and are proximal to genes that play a role in mammalian development, cancer, human obesity, and human longevity. Collectively, these results 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 compelling evidence. SUMMARYThis study identifies and characterizes evolutionarily conserved cytosines implicated in the aging process across mammals and establishes pan mammalian epigenetic clocks.

developmental biology↗

Evolution of Natural Lifespan Variation and Molecular Strategies of Extended Lifespan

To understand the genetic basis and selective forces acting on longevity, it is useful to examine lifespan variation among closely related species, or ecologically diverse isolates of the same species, within a controlled environment. In particular, this approach may lead to understanding mechanisms underlying natural variation in lifespan. Here, we analyzed 76 ecologically diverse wild yeast isolates and discovered a wide diversity of replicative lifespan. Phylogenetic analyses pointed to genes and environmental factors that strongly interact to modulate the observed aging patterns. We then identified genetic networks causally associated with natural variation in replicative lifespan across wild yeast isolates, as well as genes, metabolites and pathways, many of which have never been associated with yeast lifespan in laboratory settings. In addition, a combined analysis of lifespan-associated metabolic and transcriptomic changes revealed unique adaptations to interconnected amino acid biosynthesis, glutamate metabolism and mitochondrial function in long-lived strains. Overall, our multi-omic and lifespan analyses across diverse isolates of the same species shows how gene-environment interactions shape cellular processes involved in phenotypic variation such as lifespan.

genetics↗

The genome of North American beaver provides insights into the mechanisms of its longevity and cancer resistance

The North American beaver (Castor canadensis) is an exceptionally long-lived and cancer-resistant rodent species, and thus an excellent model organism for comparative genomic studies of longevity. Here, we utilize a significantly improved beaver genome assembly to assess evolutionary changes in gene coding sequences, copy number, and expression. We found that the beaver Aldh1a1, a stem cell marker gene encoding an enzyme required for detoxification of ethanol and aldehydes, is expanded (~10 copies vs. two in mouse and one in human). We also show that the beaver cells are more resistant to ethanol, and beaver liver extracts show higher ability to metabolize aldehydes than the mouse samples. Furthermore, Hpgd, a tumor suppressor gene, is uniquely duplicated in the beaver among rodents. Our evolutionary analysis identified beaver genes under positive selection which are associated with tumor suppression and longevity. Genes involved in lipid metabolism show positive selection signals, changes in copy number and altered gene expression in beavers. Several genes involved in DNA repair showed a higher expression in beavers which is consistent with the trend observed in other long-lived mammals. In summary, we identified several genes that likely contribute to beaver longevity and cancer resistance, including increased ability to detoxify aldehydes, enhanced tumor suppression and DNA repair, and altered lipid metabolism.

genomics↗

An NMR-based biosensor to measure stereo-specific methionine sulfoxide reductase (MSR) activities in vitro and in vivo.

Oxidation of protein methionines to methionine-sulfoxides (MetOx) is associated with several age-related diseases. In healthy cells, MetOx is reduced to methionine by two families of conserved methionine sulfoxide reductase enzymes, MSRA and MSRB that specifically target the S- or R-diastereoisomers of methionine-sulfoxides, respectively. To directly interrogate MSRA and MSRB functions in cellular settings, we developed an NMR-based biosensor that we call CarMetOx to simultaneously measure both enzyme activities in single reaction setups. We demonstrate the suitability of our strategy to delineate MSR functions in complex biological environments that range from native cell lysates to zebrafish embryos. Thereby, we establish differences in substrate specificities between prokaryotic and eukaryotic MSRs and introduce CarMetOx as a highly sensitive tool for studying therapeutic targets of oxidative stress-related human diseases and redox regulated signaling pathways. Our approach further extends high-resolution in-cell NMR measurements of exogenously delivered biomolecules to an entire multicellular organism.

biophysics↗