Identification of dihydromyricetin as a natural DNA methylation inhibitor with rejuvenating activity in human skin.

Changes in DNA methylation patterning have been reported to be a key hallmark of aged human skin. The altered DNA methylation patterns are correlated with deregulated gene expression and impaired tissue functionality, leading to the well-known skin aging phenotype. Searching for small molecules, which correct the aged methylation pattern therefore represents a novel and attractive strategy for the identification of anti-aging compounds. DNMT1 maintains epigenetic information by copying methylation patterns from the parental (methylated) strand to the newly synthesized strand after DNA replication. We hypothesized that a modest inhibition of this process promotes the restoration of the ground-state epigenetic pattern, thereby inducing rejuvenating effects. In this study, we screened a library of 1800 natural substances and 640 FDA-approved drugs and identified the well-known antioxidant and anti-inflammatory molecule dihydromyricetin (DHM) as an inhibitor of the DNA methyltransferase DNMT1. DHM is the active ingredient of several plants with medicinal use and showed robust inhibition of DNMT1 in biochemical assays. We also analyzed the effect of DHM in cultivated keratinocytes by array-based methylation profiling and observed a moderate, but significant global hypomethylation effect upon treatment. To further characterize DHM-induced methylation changes, we used published DNA methylation clocks and newly established age predictors to demonstrate that the DHM-induced methylation change is associated with a reduction in the biological age of the cells. Further studies also revealed re-activation of age-dependently hypermethylated and silenced genes in vivo and a reduction in age-dependent epidermal thinning in a 3-dimensional skin model. Our findings thus establish DHM as an epigenetic inhibitor with rejuvenating effects for aged human skin.

Development of an epigenetic clock to predict visual age progression of human skin.

Aging is a complex process characterized by the gradual decline of physiological functions, leading to increased vulnerability to age-related diseases and reduced quality of life. Alterations in DNA methylation (DNAm) patterns have emerged as a fundamental characteristic of aged human skin, closely linked to the development of the well-known skin aging phenotype. These changes have been correlated with dysregulated gene expression and impaired tissue functionality. In particular, the skin, with its visible manifestations of aging, provides a unique model to study the aging process. Despite the importance of epigenetic age clocks in estimating biological age based on the correlation between methylation patterns and chronological age, a second-generation epigenetic age clock, which correlates DNAm patterns with a particular phenotype, specifically tailored to skin tissue is still lacking. In light of this gap, we aimed to develop a novel second-generation epigenetic age clock explicitly designed for skin tissue to facilitate a deeper understanding of the factors contributing to individual variations in age progression. To achieve this, we used methylation patterns from more than 370 female volunteers and developed the first skin-specific second-generation epigenetic age clock that accurately predicts the skin aging phenotype represented by wrinkle grade, visual facial age, and visual age progression, respectively. We then validated the performance of our clocks on independent datasets and demonstrated their broad applicability. In addition, we integrated gene expression and methylation data from independent studies to identify potential pathways contributing to skin age progression. Our results demonstrate that our epigenetic age clock, VisAgeX, specifically predicting visual age progression, not only captures known biological pathways associated with skin aging, but also adds novel pathways associated with skin aging.

Macrophages Are Polarized toward an Inflammatory Phenotype by their Aged Microenvironment in the Human Skin.

Aging of the skin is accompanied by cellular as well as tissue environmental changes, ultimately reducing the ability of the tissue to regenerate and adequately respond to external stressors. Macrophages are important gatekeepers of tissue homeostasis, and it has been reported that their number and phenotype change during aging in a site-specific manner. How aging affects human skin macrophages and what implications this has for the aging process in the tissue are still not fully understood. Using single-cell RNA-sequencing analysis, we show that there is at least a 50% increase of macrophages in human aged skin, which appear to have developed from monocytes and exhibit more proinflammatory M1-like characteristics. In contrast, the cell-intrinsic ability of aged monocytes to differentiate into M1 macrophages was reduced. Using coculture experiments with aged dermal fibroblasts, we show that it is the aged microenvironment that drives a more proinflammatory phenotype of macrophages in the skin. This proinflammatory M1-like phenotype in turn negatively influenced the expression of extracellular matrix proteins by fibroblasts, emphasizing the impact of the aged macrophages on the skin phenotype.

Modeling transcriptomic age using knowledge-primed artificial neural networks.

The development of 'age clocks', machine learning models predicting age from biological data, has been a major milestone in the search for reliable markers of biological age and has since become an invaluable tool in aging research. However, beyond their unquestionable utility, current clocks offer little insight into the molecular biological processes driving aging, and their inner workings often remain non-transparent. Here we propose a new type of age clock, one that couples predictivity with interpretability of the underlying biology, achieved through the incorporation of prior knowledge into the model design. The clock, an artificial neural network constructed according to well-described biological pathways, allows the prediction of age from gene expression data of skin tissue with high accuracy, while at the same time capturing and revealing aging states of the pathways driving the prediction. The model recapitulates known associations of aging gene knockdowns in simulation experiments and demonstrates its utility in deciphering the main pathways by which accelerated aging conditions such as Hutchinson-Gilford progeria syndrome, as well as pro-longevity interventions like caloric restriction, exert their effects.

Single-Cell RNA Profiling of Human Skin Reveals Age-Related Loss of Dermal Sheath Cells and Their Contribution to a Juvenile Phenotype.

The dermal sheath (DS) is a population of mesenchyme-derived skin cells with emerging importance for skin homeostasis. The DS includes hair follicle dermal stem cells, which exhibit self-renewal and serve as bipotent progenitors of dermal papilla (DP) cells and DS cells. Upon aging, stem cells exhibit deficiencies in self-renewal and their number is reduced. While the DS of mice has been examined in considerable detail, our knowledge of the human DS, the pathways contributing to its self-renewal and differentiation capacity and potential paracrine effects important for tissue regeneration and aging is very limited. Using single-cell RNA sequencing of human skin biopsies from donors of different ages we have now analyzed the transcriptome of 72,048 cells, including 50,149 fibroblasts. Our results show that DS cells that exhibit stem cell characteristics were lost upon aging. We further show that HES1, COL11A1, MYL4 and CTNNB1 regulate DS stem cell characteristics. Finally, the DS secreted protein Activin A showed paracrine effects on keratinocytes and dermal fibroblasts, promoting proliferation, epidermal thickness and pro-collagen production. Our work provides a detailed description of human DS identity on the single-cell level, its loss upon aging, its stem cell characteristics and its contribution to a juvenile skin phenotype.

Concomitant DNA methylation and transcriptome signatures define epidermal responses to acute solar UV radiation.

The simultaneous analysis of different regulatory levels of biological phenomena by means of multi-omics data integration has proven an invaluable tool in modern precision medicine, yet many processes ultimately paving the way towards disease manifestation remain elusive and have not been studied in this regard. Here we investigated the early molecular events following repetitive UV irradiation of in vivo healthy human skin in depth on transcriptomic and epigenetic level. Our results provide first hints towards an immediate acquisition of epigenetic memories related to aging and cancer and demonstrate significantly correlated epigenetic and transcriptomic responses to irradiation stress. The data allowed the precise prediction of inter-individual UV sensitivity, and molecular subtyping on the integrated post-irradiation multi-omics data established the existence of three latent molecular phototypes. Importantly, further analysis suggested a form of melanin-independent DNA damage protection in subjects with higher innate UV resilience. This work establishes a high-resolution molecular landscape of the acute epidermal UV response and demonstrates the potential of integrative analyses to untangle complex and heterogeneous biological responses.

Multi-omics network analysis reveals distinct stages in the human aging progression in epidermal tissue.

In recent years, reports of non-linear regulations in age- and longevity-associated biological processes have been accumulating. Inspired by methodological advances in precision medicine involving the integrative analysis of multi-omics data, we sought to investigate the potential of multi-omics integration to identify distinct stages in the aging progression from ex vivo human skin tissue. For this we generated transcriptome and methylome profiling data from suction blister lesions of female subjects between 21 and 76 years, which were integrated using a network fusion approach. Unsupervised cluster analysis on the combined network identified four distinct subgroupings exhibiting a significant age-association. As indicated by DNAm age analysis and Hallmark of Aging enrichment signals, the stages captured the biological aging state more clearly than a mere grouping by chronological age and could further be recovered in a longitudinal validation cohort with high stability. Characterization of the biological processes driving the phases using machine learning enabled a data-driven reconstruction of the order of Hallmark of Aging manifestation. Finally, we investigated non-linearities in the mid-life aging progression captured by the aging phases and identified a far-reaching non-linear increase in transcriptional noise in the pathway landscape in the transition from mid- to late-life.

The methylome of the marbled crayfish links gene body methylation to stable expression of poorly accessible genes.

BACKGROUND: The parthenogenetic marbled crayfish (Procambarus virginalis) is a novel species that has rapidly invaded and colonized various different habitats. Adaptation to different environments appears to be independent of the selection of genetic variants, but epigenetic programming of the marbled crayfish genome remains to be understood. RESULTS: Here, we provide a comprehensive analysis of DNA methylation in marbled crayfish. Whole-genome bisulfite sequencing of multiple replicates and different tissues revealed a methylation pattern that is characterized by gene body methylation of housekeeping genes. Interestingly, this pattern was largely tissue invariant, suggesting a function that is unrelated to cell fate specification. Indeed, integrative analysis of DNA methylation, chromatin accessibility and mRNA expression patterns revealed that gene body methylation correlated with limited chromatin accessibility and stable gene expression, while low-methylated genes often resided in chromatin with higher accessibility and showed increased expression variation. Interestingly, marbled crayfish also showed reduced gene body methylation and higher gene expression variability when compared with their noninvasive mother species, Procambarus fallax. CONCLUSIONS: Our results provide novel insights into invertebrate gene body methylation and its potential role in adaptive gene regulation.

Clonal genome evolution and rapid invasive spread of the marbled crayfish.

The marbled crayfish Procambarus virginalis is a unique freshwater crayfish characterized by very recent speciation and parthenogenetic reproduction. Marbled crayfish also represent an emerging invasive species and have formed wild populations in diverse freshwater habitats. However, our understanding of marbled crayfish biology, evolution and invasive spread has been hampered by the lack of freshwater crayfish genome sequences. We have now established a de novo draft assembly of the marbled crayfish genome. We determined the genome size at approximately 3.5 gigabase pairs and identified >21,000 genes. Further analysis confirmed the close relationship to the genome of the slough crayfish, Procambarus fallax, and also established a triploid AA'B genotype with a high level of heterozygosity. Systematic fieldwork and genotyping demonstrated the rapid expansion of marbled crayfish on Madagascar and established the marbled crayfish as a potent invader of freshwater ecosystems. Furthermore, comparative whole-genome sequencing demonstrated the clonality of the population and their genetic identity with the oldest known stock from the German aquarium trade. Our study closes an important gap in the phylogenetic analysis of animal genomes and uncovers the unique evolutionary history of an emerging invasive species.

Comprehensive DNA methylation analysis of the Aedes aegypti genome.

Aedes aegypti mosquitoes are important vectors of viral diseases. Mosquito host factors play key roles in virus control and it has been suggested that dengue virus replication is regulated by Dnmt2-mediated DNA methylation. However, recent studies have shown that Dnmt2 is a tRNA methyltransferase and that Dnmt2-dependent methylomes lack defined DNA methylation patterns, thus necessitating a systematic re-evaluation of the mosquito genome methylation status. We have now searched the Ae. aegypti genome for candidate DNA modification enzymes. This failed to reveal any known (cytosine-5) DNA methyltransferases, but identified homologues for the Dnmt2 tRNA methyltransferase, the Mettl4 (adenine-6) DNA methyltransferase, and the Tet DNA demethylase. All genes were expressed at variable levels throughout mosquito development. Mass spectrometry demonstrated that DNA methylation levels were several orders of magnitude below the levels that are usually detected in organisms with DNA methylation-dependent epigenetic regulation. Furthermore, whole-genome bisulfite sequencing failed to reveal any evidence of defined DNA methylation patterns. These results suggest that the Ae. aegypti genome is unmethylated. Interestingly, additional RNA bisulfite sequencing provided first evidence for Dnmt2-mediated tRNA methylation in mosquitoes. These findings have important implications for understanding the mechanism of Dnmt2-dependent virus regulation.

De Novo assembly and annotation of the freshwater crayfish Astacus astacus transcriptome.

We generated RNA-seq data to assemble the transcriptome of the noble crayfish (Astacus astacus) from four combined tissues (abdominal muscle, hepatopancreas, ovaries, green glands). A total of 194 million read pairs with a length of 100 bp were generated. The transcriptome was assembled de novo using Trinity software, producing 158,649 non-redundant transcripts. Lowly expressed transcripts were filtered out leaving 45,415 transcripts of which 14,559 were found to contain open reading frames with predicted gene function. The Transrate software revealed that 91% of the total reads were realigned to the assembly. Furthermore, BUSCO analysis indicated that our assembly is 64% complete. A total of 13,770 transcripts were assigned at least one GO term. This first de novo transcriptome assembly is an important foundation for future genomic research on the noble crayfish and adds to the general knowledge and further characterization of transcriptomes of non-model organisms.

The marbled crayfish as a paradigm for saltational speciation by autopolyploidy and parthenogenesis in animals.

The parthenogenetic all-female marbled crayfish is a novel research model and potent invader of freshwater ecosystems. It is a triploid descendant of the sexually reproducing slough crayfish, Procambarus fallax, but its taxonomic status has remained unsettled. By cross-breeding experiments and parentage analysis we show here that marbled crayfish and P. fallax are reproductively separated. Both crayfish copulate readily, suggesting that the reproductive barrier is set at the cytogenetic rather than the behavioural level. Analysis of complete mitochondrial genomes of marbled crayfish from laboratory lineages and wild populations demonstrates genetic identity and indicates a single origin. Flow cytometric comparison of DNA contents of haemocytes and analysis of nuclear microsatellite loci confirm triploidy and suggest autopolyploidisation as its cause. Global DNA methylation is significantly reduced in marbled crayfish implying the involvement of molecular epigenetic mechanisms in its origination. Morphologically, both crayfish are very similar but growth and fecundity are considerably larger in marbled crayfish, making it a different animal with superior fitness. These data and the high probability of a divergent future evolution of the marbled crayfish and P. fallax clusters suggest that marbled crayfish should be considered as an independent asexual species. Our findings also establish the P. fallax-marbled crayfish pair as a novel paradigm for rare chromosomal speciation by autopolyploidy and parthenogenesis in animals and for saltational evolution in general.

Characterization of genome methylation patterns in the desert locust Schistocerca gregaria.

DNA methylation is a widely conserved epigenetic modification. The analysis of genome-scale DNA methylation patterns in various organisms suggests that major features of animal methylomes are widely conserved. However, based on the variation of DNA methyltransferase genes in invertebrates, it has also been proposed that DNA methylation could provide a molecular mechanism for ecological adaptation. We have now analyzed the methylome of the desert locust, Schistocerca gregaria, which represents an organism with a high degree of phenotypic plasticity. Using genome-scale bisulfite sequencing, we show here that the S. gregaria methylome is characterized by CpG- and exon-specific methylation and thus shares two major features with other animal methylomes. In contrast to other invertebrates, however, overall methylation levels were substantially higher and a significant fraction of transposons was methylated. Additionally, genic sequences were densely methylated in a pronounced bimodal pattern, suggesting a role for DNA methylation in the regulation of locust gene expression. Our results thus uncover a unique pattern of genome methylation in locusts and provide an important foundation for investigating the role of DNA methylation in locust phase polyphenism.

The honey bee epigenomes: differential methylation of brain DNA in queens and workers.

In honey bees (Apis mellifera) the behaviorally and reproductively distinct queen and worker female castes derive from the same genome as a result of differential intake of royal jelly and are implemented in concert with DNA methylation. To determine if these very different diet-controlled phenotypes correlate with unique brain methylomes, we conducted a study to determine the methyl cytosine (mC) distribution in the brains of queens and workers at single-base-pair resolution using shotgun bisulfite sequencing technology. The whole-genome sequencing was validated by deep 454 sequencing of selected amplicons representing eight methylated genes. We found that nearly all mCs are located in CpG dinucleotides in the exons of 5,854 genes showing greater sequence conservation than non-methylated genes. Over 550 genes show significant methylation differences between queens and workers, revealing the intricate dynamics of methylation patterns. The distinctiveness of the differentially methylated genes is underscored by their intermediate CpG densities relative to drastically CpG-depleted methylated genes and to CpG-richer non-methylated genes. We find a strong correlation between methylation patterns and splicing sites including those that have the potential to generate alternative exons. We validate our genome-wide analyses by a detailed examination of two transcript variants encoded by one of the differentially methylated genes. The link between methylation and splicing is further supported by the differential methylation of genes belonging to the histone gene family. We propose that modulation of alternative splicing is one mechanism by which DNA methylation could be linked to gene regulation in the honey bee. Our study describes a level of molecular diversity previously unknown in honey bees that might be important for generating phenotypic flexibility not only during development but also in the adult post-mitotic brain.