Epigenetic clocks based on DNA methylation (DNAm) accurately predict age, but their biological underpinnings remain unclear. One primary mechanism by which DNAm might influence gene regulation is by modulating transcription factor binding activity. This study investigates the regulatory potential of predictive CpGs in established epigenetic clocks. Our analysis reveals that generally most CpGs used by epigenetic clocks do not overlap known transcription factor binding sites (TFBS), indicating that clock accuracy is not primarily driven by changes in TF binding dynamics. However, analysis of CpGs within TFBSs identifies key transcription factors potentially involved in aging, including ZBED1, NFE2, and CEBPB, which are enriched for age-associated CpGs, while RELA, IKZF1, and STAT3 significantly protected against methylation changes. Leveraging TFBS-associated and age-correlated CpGs, combined with noise-stabilizing feature engineering steps, we developed an alternative TFMethyl Clock model that provides competitive predictions of chronological age. Age-predictive CpGs selected by our model enrich for target genes involved in interleukin-1β production and fatty-acid metabolism, while being enriched at TFBSs of NR2C2. Furthermore, approximately three-fourths of these target genes exhibit significant age-related changes, suggesting deeper insights into possible methylation-driven aging processes. Our findings demonstrate that incorporating regulatory information into epigenetic clocks may provide mechanistic insights into the aging process while improving the interpretability and predictive power.

DNA methylation is a critical epigenetic mark across numerous species, and identifying differentially methylated regions (DMRs) is essential for understanding genome regulation. Most existing DMR detection methods require predefined sample conditions, limiting the discovery of new epigenetic patterns, especially when group identities are unknown or uncertain, as is common in clinical settings. Additionally, only a very few approaches enable comparisons across multiple conditions. To address this significant gap, we present metilene3, a method for rapid, multi-condition DMR detection that operates in both supervised and unsupervised modes, using user-provided labels or autonomously clustering unlabeled samples. By segmenting the genome based on multiple pairwise methylation difference signals, metilene3 enables sample classification and DMR-anchored inference of epigenetic relationships. Using simulated and diverse human datasets, we show that metilene3 accurately detects DMRs, robustly clusters samples, and holds the potential to reveal new regulatory elements and sample stratifications. Specifically, in a pancreatic tissue dataset, metilene3 identifies DMRs enriched for key transcription factors involved in pancreatic cancer development, hinting towards an altered NFKB-NFAT regulatory program. Together, metilene3 provides a fast, interpretable framework for exploring heterogeneous methylomes and discovering epigenetic patterns across complex biological and clinical datasets.

The transcriptional interference model suggests that RNA polymerases elongating through overlapping transcription units mutually inhibit transcription and disrupt associated cis-regulatory elements. As a longstanding fundamental concept of gene regulation, the idea of reciprocal inhibition between sense and antisense transcription has been supported by a significant body of research. However, despite the model's biophysical plausibility and historical significance, evidence from large-scale transcriptome studies raises questions about its universal applicability. In particular, the new data indicate that a measurable influence of transcriptional interference is absent from the majority of loci with overlapping transcription. Here we highlight key aspects of overlapping transcription and propose potential solutions to this emerging puzzle. Gaining a better understanding of the molecular mechanisms that render loci sensitive or resistant to interference could lead to groundbreaking insights into the biology of gene regulation.

DNA methylation is a widely studied epigenetic mark and a powerful biomarker of cell type, age, environmental exposures, and disease. Whole-genome sequencing following selective conversion of unmethylated cytosines into thymines via bisulfite treatment or enzymatic methods remains the reference method for DNA methylation profiling genome-wide. While numerous software tools facilitate processing of DNA methylation sequencing reads, a comprehensive benchmarking study has been lacking. In this study, we systematically compared complete computational workflows for processing DNA methylation sequencing data using a dedicated benchmarking dataset generated with five whole-genome profiling protocols. As an evaluation reference, we employed accurate locus-specific measurements from our previous benchmark of targeted DNA methylation assays. Based on this experimental gold-standard assessment and multiple performance metrics, we identified workflows that consistently demonstrated superior performance and revealed major workflow development trends. To ensure the long-term utility of our benchmark, we implemented an interactive workflow execution and data presentation platform, adaptable to user-defined criteria and readily expandable to future software.

Aging-related cognitive decline is associated with changes across different tissues and the gut microbiome, including dysfunction of the gut-brain axis. However, only few studies have linked multi-organ alterations to cognitive decline during aging. Here we report a multi-omics analysis integrating metabolomics, transcriptomics, DNA methylation, and metagenomics data from hippocampus, liver, colon, and fecal samples of mice, correlated with cognitive performance in the Barnes Maze spatial learning task across different age groups. We identified 734 molecular features associated with cognitive rank within individual data layers, of which 227 features remain when integrating all data layers with each other. Among the single-layer predictors, several host and microbial features were highlighted, with host-associated markers being predominant. Host features associated with cognitive function mainly belong to innate and adaptive inflammatory activity (inflammaging) and developmental processes. Our findings suggest that cognitive decline in aging is tightly coupled to systemic, age-associated inflammation, potentially initiated by microbiome-driven gastrointestinal inflammatory activity, emphasizing a link between peripheral tissue alterations and brain function.

The olm (Proteus anguinus), with a predicted maximum lifespan of more than 100 years, is the longest-lived amphibian, which in addition possesses a range of unique adaptations to its dark, subterranean cave habitat. To assess the underlying molecular signatures, we present the first comprehensive transcriptome of the olm. Our study provides gene expression data across six organs and comparative genomics analyses, accessible via an interactive web server: http://comp-pheno.de/olm . The data uncover significant organ-specific gene expression, with the brain showing the highest number of organ-specific expressed genes. Our findings reveal significantly more genes under strong negative selection than positive selection, particularly in brain-specific expressed genes. Processes under positive selection in the olm resemble those in other long-lived species.

Embryonic macrophages emerge before the onset of definitive hematopoiesis, seed into discrete tissues and contribute to specialized resident macrophages throughout life. Presence of embryonic macrophages in the bone marrow and functional impact on hematopoietic stem cells (HSC) or the niche remains unclear. Here we show that bone marrow macrophages consist of two ontogenetically distinct cell populations from embryonic and adult origin. Newborn mice lacking embryonic macrophages have decreased HSC numbers in the bone marrow suggesting an important function for embryo-derived macrophages in orchestrating HSC trafficking around birth. The establishment of a normal cellular niche space in the bone marrow critically depends on embryonic macrophages that are important for the development of mesenchymal stromal cells, but not other non-hematopoietic niche cells, providing evidence for a specific role for embryo-derived macrophages in the establishment of the niche environment pivotal for the establishment of a normally sized HSC pool.

Convergent transcription, that is, the collision of sense and antisense transcription, is ubiquitous in mammalian genomes and believed to diminish RNA expression. Recently, antisense transcription downstream of promoters was found to be surprisingly prevalent. However, functional characteristics of affected promoters are poorly investigated. Here we show that convergent transcription marks an unexpected positively co-regulated promoter constellation. By assessing transcriptional dynamic systems, we identified co-regulated constituent promoters connected through a distinct chromatin structure. Within these cis-regulatory domains, transcription factors can regulate both constituting promoters by binding to only one of them. Convergent promoters comprise about a quarter of all active transcript start sites and initiate 5′-overlapping antisense RNAs—an RNA class believed previously to be rare. Visualization of nascent RNA molecules reveals convergent cotranscription at these loci. Together, our results demonstrate that co-regulated convergent promoters substantially expand the cis-regulatory repertoire, reveal limitations of the transcription interference model and call for adjusting the promoter concept. Genome-wide analysis and genetic manipulation at loci regulated by p53, E2F4 and RFX7 show that convergent promoters with similar epigenetic features can be co-regulated and simultaneously expressed in the same direction.

Methylation-based epigenetic clocks are among the most accurate tools for predicting chronological age. Although DNA methylation (DNAm) at genomic CpG sites is linked to various regulatory mechanisms, the biological interpretability of epigenetic clocks remains surprisingly limited. One primary mechanism by which DNAm is thought to influence gene regulation is by modulating transcription factor binding activity. In this study, we examine established epigenetic clocks to assess the regulatory potential of their predictive CpGs during the aging process. Our analysis reveals that generally most CpG sites used by epigenetic clocks do not overlap known transcription factor binding sites (TFBSs), indicating that changes in TFBS dynamics may not account for prediction accuracy of these models. On the other hand, by identifying age-associated CpGs that overlap TFBSs, we identified transcription factors that may be involved in the aging process. Specifically, the TFBSs of ZBED1, NFE2, CEBPB, FOXP1, EGR1, SP1, PAX5, and MAZ were particularly enriched for age-associated CpGs, while RBPJ, NFIC, RELA, IKZF1, STAT3, and USF2 were significantly protected against methylation changes. By focusing on TFBS-associated CpGs, combined with additional feature selection and engineering steps, we developed an alternative, TFMethyl Clock model, outperforming several existing approaches. Target genes of model-selected, age-predictive CpGs are enriched in the interleukin-1b production and long- chain fatty acid metabolism pathways. In contrast, these CpGs themselves are enriched mainly at binding sites of NR2C2 TF. Furthermore, approximately three-fourths of the target genes downstream of age- predictive CpGs exhibit significant age-related changes, suggesting that our approach captures deeper insights into possible methylation-driven biological aging processes. Our findings demonstrate that incorporating regulatory loci into the design of epigenetic predictors may provide mechanistic insights into the aging process while maintaining or even improving the predictive power. ### Competing Interest Statement The authors have declared no competing interest.

Pioneer transcription factors can bind to closed chromatin, initiating its opening and subsequent gene activation. However, the specific features that enable transcription factors to activate particular loci remain largely undefined. Here, we show that the transcription factor p53 unexpectedly initiates epigenetic remodeling at the majority of its binding sites and drives transcription at select loci. Our quantitative epigenetic data reveal that p53 establishes new enhancers, while quantitative transcription initiation analyses indicate that high local p53 abundance and sequence-specific binding are key features of sites where p53 successfully induces transcription. Surprisingly, we observed a spatial overlap between p53 binding sites and transcription initiation sites, suggesting a decoupling of these two events. Our results reveal that p53 activity unfolds across three distinct layers: histone modification, nucleosome eviction, and transcription initiation, with the latter driven by dynamic rather than static p53 DNA binding. These insights expand our understanding of the function of p53 by showing it not only actively initiates transcription but also broadly remodels chromatin. Overall, these findings offer a conceptual framework to explore how transcription factors regulate chromatin states and gene expression.

An important hallmark of aging is the loss of proteostasis, which can lead to the formation of protein aggregates and mitochondrial dysfunction in neurons. Although it is well known that protein synthesis is finely regulated in the brain, especially at synapses, where mRNAs are locally translated in an activity-dependent manner, little is known as to the changes in the synaptic proteome and transcriptome during aging. Therefore, this work aims to elucidate the relationship between the transcriptome and proteome at the soma and synaptic levels during aging. Proteomic and transcriptomic data analysis reveal that, in young animals, proteins and transcripts are correlated and synaptic regulation is driven by changes in the soma. During aging, there is a decoupling between transcripts and proteins and between somatic and synaptic compartments. Furthermore, the soma-synapse gradient of ribosomal genes changes upon aging, that is, ribosomal transcripts are less abundant and ribosomal proteins are more abundant in the synaptic compartment of old mice with respect to younglings. Additionally, transcriptomics data highlight a difference in the splicing of certain synaptic mRNA with aging. Taken together, our data provide a valuable resource for the study of the aging synapse.

The detection of circular RNA molecules (circRNAs) is typically based on short-read RNA sequencing data processed using computational tools. Numerous such tools have been developed, but a systematic comparison with orthogonal validation is missing. Here, we set up a circRNA detection tool benchmarking study, in which 16 tools detected more than 315,000 unique circRNAs in three deeply sequenced human cell types. Next, 1,516 predicted circRNAs were validated using three orthogonal methods. Generally, tool-specific precision is high and similar (median of 98.8%, 96.3% and 95.5% for qPCR, RNase R and amplicon sequencing, respectively) whereas the sensitivity and number of predicted circRNAs (ranging from 1,372 to 58,032) are the most significant differentiators. Of note, precision values are lower when evaluating low-abundance circRNAs. We also show that the tools can be used complementarily to increase detection sensitivity. Finally, we offer recommendations for future circRNA detection and validation. This study describes benchmarking and validation of computational tools for detecting circRNAs, finding most to be highly precise with variations in sensitivity and total detection. The study also finds over 315,000 putative human circRNAs.

The traditional view of aging as a gradual, progressive process is increasingly being challenged. A growing body of evidence suggests the existence of abrupt transitions in the aging process, marked by sudden molecular shifts. Interestingly, the data indicates that such transitions occur not only in late life but also throughout the entire lifespan. Further research on the nature of such events could enhance our understanding of aging and pave the way for novel therapeutic strategies, including personalized medicine. We propose that these abrupt molecular shifts could serve as biomarkers, dividing the lifespan into distinct stages and providing the foundation for a much-needed staging system for aging. Furthermore, we argue that the sudden changes may be the hallmarks of aging tipping points, that is, points in time where aging processes are quickly amplified after surpassing critical biological thresholds.

BACKGROUND: Chronic stress significantly contributes to mood and anxiety disorders. Previous data suggest a correlative connection between vitamin B12 supplementation, depression, and stress resilience. However, the underlying mechanisms are still poorly understood. METHODS: Using the chronic variable stress mouse model coupled with RNA sequencing, we identified vitamin B12- induced transcriptional changes related to stress resilience. Using viral-mediated gene transfer and in vivo epigenome editing, we revealed a functional pathway linking vitamin B12, DNA methylation (DNAme), and depression-like symptoms. RESULTS: We identified Ttr (transthyretin) as a key sex-specific target of vitamin B12 in chronic stress. Accordingly, TTR expression was increased postmortem in the prefrontal cortex of male but not female patients with depression. Virally altered Ttr in the prefrontal cortex functionally contributed to stress- and depression-related behaviors, changes in dendritic spine morphology, and gene expression. In stressed mice, vitamin B12 reduced DNAme in the Ttr promoter region. Importantly, using in vivo epigenome editing to alter DNAme in the brains of living mice for the first time, we established a direct causal link between DNAme and Ttr and stress-associated behaviors. CONCLUSIONS: Using state-of-the-art techniques, this study uncovered a mechanistic link between vitamin B12 supplementation, Ttr, and markers of chronic stress and depression, encouraging further studies into dietary interventions for mood disorders.

Burkitt lymphoma (BL) is an aggressive germinal center B-cell-derived malignancy. Historically, sporadic, endemic, and immunodeficiency-associated variants were distinguished, which differ in the frequency of Epstein-Barr virus (EBV) positivity. Aiming to identify subgroups based on DNA methylation patterns, we here profiled 96 BL cases, 17 BL cell lines, and six EBV-transformed lymphoblastoid cell lines using Illumina BeadChip arrays. DNA methylation analyses clustered the cases into four subgroups: two containing mostly EBV-positive cases (BL-mC1, BL-mC2) and two containing mostly EBV-negative cases (BL-mC3, BL-mC4). The subgroups BL-mC1/2, enriched for EBV-positive cases, showed increased DNA methylation, epigenetic age, and, in part, proliferation history compared to BL-mC3/4. CpGs hypermethylated in EBV-positive BLs were enriched for polycomb repressive complex 2 marks, while the CpGs hypomethylated in EBV-negative BLs were linked to, for example, B-cell receptor signaling. EBV-associated hypermethylation affected regulatory regions of genes frequently mutated in BL (e.g., CCND3, TP53) and impacted superenhancers. This finding suggests that hypermethylation may compensate for the lower mutational burden of pathogenic drivers in EBV-positive BLs. Though minor, significant differences were also observed between EBV-positive endemic and sporadic cases (e.g., at the SOX11 and RUNX1 loci). Our findings suggest that EBV status, rather than epidemiological variants, drives the DNA methylation-based subgrouping of BL.

The Greenland shark ( Somniosus microcephalus ) is the longest-lived vertebrate known, with an estimated lifespan of ∼ 400 years. Here, we present a chromosome-level assembly of the 6.45 Gb Greenland shark, rendering it one of the largest non-tetrapod genomes sequenced so far. Expansion of the genome is mostly accounted for by a substantial expansion of transposable elements. Using public shark genomes as a comparison, we found that genes specifically duplicated in the Greenland shark form a functionally connected network enriched for DNA repair function. Furthermore, we identified a unique insertion in the conserved C-terminal region of the key tumor suppressor p53. We also provide a public browser to explore its genome. ### Competing Interest Statement The authors have declared no competing interest.

Growing evidence suggests that somatic mutations may be a major cause of the aging process. However, it remains to be tested whether the predictions of the theory also apply to species with longer life spans than humans.Hydrais a genus of freshwater polyps with remarkable regeneration abilities and a potentially unlimited life span under laboratory conditions. By genome sequencing of single cells and whole animals, we found that the mutation rates inHydra’s stem cells are even slightly higher than in humans or mice. A potential explanation for this deviation from the prediction of the theory may lie in the adaptability offered by a higher mutation rate, as we were able to show that the genome of the widely studiedHydra magnipapillata strain 105 has undergone a process of strong positive selection since the strain's cultivation 50 years ago. This most likely represents a rapid adaptation to the drastically altered environmental conditions associated with the transition from the wild to laboratory conditions. Processes under positive selection in captive animals include pathways associated withHydra’s simple nervous system, its nucleic acid metabolic process, cell migration, and hydrolase activity.

The transcription factor regulatory factor X 7 (RFX7) has been identified as a tumor suppressor that is recurrently mutated in lymphoid cancers and appears to be dysregulated in many other cancers. RFX7 is activated by the well-known tumor suppressor p53 and regulates several other known tumor suppressor genes. However, what other factors regulate RFX7 and its target genes remains unclear. Here, reporter gene assays were used to identify that RFX7 regulates the tumor suppressor gene PDCD4 through direct interaction with its X-box promoter motif. We utilized mass spectrometry to identify factors that bind to DNA together with RFX7. In addition to RFX7, we also identified RFX5, RFXAP, RFXANK, and ANKRA2 that bind to the X-box motif in the PDCD4 promoter. We demonstrate that ANKRA2 is a bona fide direct p53 target gene. We used transcriptome analyses in two cell systems to identify genes regulated by ANKRA2, its sibling RFXANK, and RFX7. These results revealed that ANKRA2 functions as a critical cofactor of RFX7, whereas RFXANK regulates largely distinct gene sets.

Circadian clock disruption and lack of sleep impair health of cells and organs, but mechanisms remain elusive. Here, we used multi-omics, molecular and functional assays in three model systems ( C. elegans , human retinal cells and mouse brain) to identify the DREAM complex as a ubiquitous health repressor acting downstream of clock impairment. We show that clock dysfunction aberrantly activates DREAM leading to simultaneous de-regulation of multiple cellular processes. The affected activities including translation, stress responses and OXPHOS are basic and ubiquitous, explaining pan-cellular decline induced by impaired clock and sleep. Accordingly, inhibition of DREAM restores healthy homeostasis and alleviates de-regulation of adaptive mechanisms in clock-impaired nematodes and human cells. This restorative effect occurs amid persisting clock dysfunction, suggesting that DREAM ablation uniquely uncouples circadian clock impairments from their negative effects on health. Our study coins DREAM as a new therapeutic target for preserving organismal fitness under re-current circadian dysfunction. ### Competing Interest Statement ME and ISV are listed as inventors on a patent application, which is based on some of the described findings.

Chronic stress is a major risk factor for depression, a leading cause of disability and suicide. Because current antidepressants work slowly, have common side effects, and are only effective in a minority of patients, there is an unmet need to identify the underlying molecular mechanisms. Here, we identify the receptor for neuropeptides B and W, Npbwr1, as a key regulator of depressive-like symptoms. Npbwr1 is increased in the nucleus accumbens of chronically stressed mice and postmortem in patients diagnosed with depression. Using viral-mediated gene transfer, we demonstrate a causal link between Npbwr1, dendritic spine morphology, the biomarker Bdnf, and depressive-like behaviors. Importantly, microinjection of the synthetic antagonist of Npbwr1, CYM50769, rapidly ameliorates depressive-like behavioral symptoms and alters Bdnf levels. CYM50769 is selective, well tolerated, and shows effects up to 7 days after administration of a single dose. In summary, these findings advance our understanding of mood and chronic stress and warrant further investigation of CYM50769 as a potential fast-acting antidepressant.