Age‐related DNA methylation on Y chromosome and their associations with total mortality among Chinese males

In view of the sex differences in aging‐related diseases, sex chromosomes may play a critical role during aging process. This study aimed to identify age‐related DNA methylation changes on Y chromosome (ChrY). A two‐stage study design was conducted in this study. The discovery stage contained 419 Chinese males, including 205 from the Wuhan‐Zhuhai cohort panel, 107 from the coke oven workers panel, and 107 from the Shiyan panel. The validation stage contained 587 Chinese males from the Dongfeng‐Tongji sub‐cohort. We used the Illumina HumanMethylation BeadChip to determine genome‐wide DNA methylation in peripheral blood of the study participants. The associations between age and methylation levels of ChrY CpGs were investigated by using linear regression models with adjustment for potential confounders. Further, associations of age‐related ChrY CpGs with all‐cause mortality were tested in the validation stage. We identified the significant associations of 41 ChrY CpGs with age at false discovery rate (FDR) <0.05 in the discovery stage, and 18 of them were validated in the validation stage (p < 0.05). Meta‐analysis of both stages confirmed the robust positive associations of 14 CpGs and negative associations of 4 CpGs with age (FDR<0.05). Among them, cg03441493 and cg17816615 were significantly associated with all‐cause mortality risk [HR(95% CI) = 1.37 (1.04, 1.79) and 0.70 (0.54, 0.93), respectively]. Our results highlighted the importance of ChrY CpGs on male aging.     

Age patterns of intra‐pair DNA methylation discordance in twins: Sex difference in epigenomic instability and implication on survival

Aging is a biological process linked to specific patterns and changes in the epigenome. We hypothesize that age‐related variation in the DNA methylome could reflect cumulative environmental modulation to the epigenome which could impact epigenomic instability and survival differentially by sex. To test the hypothesis, we performed sex‐stratified epigenome‐wide association studies on age‐related intra‐pair DNA methylation discordance in 492 twins aged 56–80 years. We identified 3084 CpGs showing increased methylation variability with age (FDR < 0.05, 7 CpGs with p < 1e‐07) in male twins but no significant site found in female twins. The results were replicated in an independent cohort of 292 twins aged 30–74 years with 37% of the discovery CpGs successfully replicated in male twins. Functional annotation showed that genes linked to the identified CpGs were significantly enriched in signaling pathways, neurological functions, extracellular matrix assembly, and cancer. We further explored the implication of discovery CpGs on individual survival in an old cohort of 224 twins (220 deceased). In total, 264 CpGs displayed significant association with risk of death in male twins. In female twins, 175 of the male discovery CpGs also showed non‐random correlation with mortality. Intra‐pair comparison showed that majority of the discovery CpGs have higher methylation in the longer‐lived twins suggesting that loss of DNA methylation during aging contributes to increased risk of death which is more pronounced in male twins. In conclusion, age‐related epigenomic instability in the DNA methylome is more evident in males than in females and could impact individual survival and contribute to sex difference in human lifespan.     

Whole‐body senescent cell clearance alleviates age‐related brain inflammation and cognitive impairment in mice

Cellular senescence is characterized by an irreversible cell cycle arrest and a pro‐inflammatory senescence‐associated secretory phenotype (SASP), which is a major contributor to aging and age‐related diseases. Clearance of senescent cells has been shown to improve brain function in mouse models of neurodegenerative diseases. However, it is still unknown whether senescent cell clearance alleviates cognitive dysfunction during the aging process. To investigate this, we first conducted single‐nuclei and single‐cell RNA‐seq in the hippocampus from young and aged mice. We observed an age‐dependent increase in p16^Ink4a senescent cells, which was more pronounced in microglia and oligodendrocyte progenitor cells and characterized by a SASP. We then aged INK‐ATTAC mice, in which p16^Ink4a‐positive senescent cells can be genetically eliminated upon treatment with the drug AP20187 and treated them either with AP20187 or with the senolytic cocktail Dasatinib and Quercetin. We observed that both strategies resulted in a decrease in p16^Ink4a exclusively in the microglial population, resulting in reduced microglial activation and reduced expression of SASP factors. Importantly, both approaches significantly improved cognitive function in aged mice. Our data provide proof‐of‐concept for senolytic interventions'' being a potential therapeutic avenue for alleviating age‐associated cognitive impairment.     

Anti‐hsa‐miR‐59 alleviates premature senescence associated with Hutchinson‐Gilford progeria syndrome in mice

Hutchinson‐Gilford progeria syndrome (HGPS) is a lethal premature aging disorder without an effective therapeutic regimen. Because of their targetability and influence on gene expression, microRNAs (miRNAs) are attractive therapeutic tools to treat diseases. Here we identified that hsa‐miR‐59 (miR‐59) was markedly upregulated in HGPS patient cells and in multiple tissues of an HGPS mouse model (Lmna ^G609G/G609G ), which disturbed the interaction between RNAPII and TFIIH, resulting in abnormal expression of cell cycle genes by targeting high‐mobility group A family HMGA1 and HMGA2. Functional inhibition of miR‐59 alleviated the cellular senescence phenotype of HGPS cells. Treatment with AAV9‐mediated anti‐miR‐59 reduced fibrosis in the quadriceps muscle, heart, and aorta, suppressed epidermal thinning and dermal fat loss, and yielded a 25.5% increase in longevity of Lmna ^G609G/G609G mice. These results identify a new strategy for the treatment of HGPS and provide insight into the etiology of HGPS disease.     

Genetic signature of human longevity in PKC and NF‐κB signaling

Gene variants associated with longevity are also associated with protection against cognitive decline, dementia and Alzheimer''s disease, suggesting that common physiologic pathways act at the interface of longevity and cognitive function. To test the hypothesis that variants in genes implicated in cognitive function may promote exceptional longevity, we performed a comprehensive 3‐stage study to identify functional longevity‐associated variants in ~700 candidate genes in up to 450 centenarians and 500 controls by target capture sequencing analysis. We found an enrichment of longevity‐associated genes in the nPKC and NF‐κB signaling pathways by gene‐based association analyses. Functional analysis of the top three gene variants (NFKBIA, CLU, PRKCH) suggests that non‐coding variants modulate the expression of cognate genes, thereby reducing signaling through the nPKC and NF‐κB. This matches genetic studies in multiple model organisms, suggesting that the evolutionary conservation of reduced PKC and NF‐κB signaling pathways in exceptional longevity may include humans.     

Genome‐wide selective detection of Mile red‐bone goat using next‐generation sequencing technology

The ecotype population of goats (Capra hircus) was created by long‐term artificial selection and natural adaptation. Mile red‐bone goat is an indigenous breed with visible red bones, and its special bone structure has received extensive attention. This study aimed to identify genetic variants and candidate genes associated with specific bone phenotypes using next‐generation sequencing technology (NGS). The results revealed that 31,828,206 single nucleotide polymorphisms (SNPs) were obtained from 72 goats (20 Mile red‐bone goats and 52 common goats) by NGS. A total of 100 candidate genes were identified on the basis top 1% window interaction from nucleotide diversity (π), π ratio (π _A/π _B), and pairwise fixation index (F _ST). Exactly 77 known signaling pathways were enriched. Specifically, three coding genes (NMNAT2, LOC102172983, and PNLIP) were annotated in the vitamin metabolism signaling pathways, and NCF2 was annotated to the osteoclast (OC) differentiation pathway. Furthermore, 5862 reliable copy number variations (CNVs) were obtained, and 14 and 24 genes were annotated with the top 1‰ CNV based on F _ST (>0.490) and V _ST (>0.527), respectively. Several pathways related to bone development and metabolism of exogenous substances in vivo, including calcium signaling pathway, OC differentiation, and glycerophospholipid metabolism, were annotated. Specifically, six genes from 19 candidate CNVs, which were obtained by interaction of the top 1‰ CNVs with F _ST and V _ST, were annotated to mucin‐type O‐glycan biosynthesis and metabolic pathways. Briefly, the results implied that pseudopurpurin and specific genetic variants work together to contribute to the red‐bone color and specific bone structure of Mile red‐bone goat. This study is helpful to understanding the genetic basis of the unique bone phenotype of Mile red‐bone goats.     

Population differences in Chinook salmon (Oncorhynchus tshawytscha) DNA methylation: Genetic drift and environmental factors

Local adaptation and phenotypic differences among populations have been reported in many species, though most studies focus on either neutral or adaptive genetic differentiation. With the discovery of DNA methylation, questions have arisen about its contribution to individual variation in and among natural populations. Previous studies have identified differences in methylation among populations of organisms, although most to date have been in plants and model animal species. Here we obtained eyed eggs from eight populations of Chinook salmon (Oncorhynchus tshawytscha) and assayed DNA methylation at 23 genes involved in development, immune function, stress response, and metabolism using a gene‐targeted PCR‐based assay for next‐generation sequencing. Evidence for population differences in methylation was found at eight out of 23 gene loci after controlling for developmental timing in each individual. However, we found no correlation between freshwater environmental parameters and methylation variation among populations at those eight genes. A weak correlation was identified between pairwise DNA methylation dissimilarity among populations and pairwise F _ST based on 15 microsatellite loci, indicating weak effects of genetic drift or geographic distance on methylation. The weak correlation was primarily driven by two genes, GTIIBS and Nkef. However, single‐gene Mantel tests comparing methylation and pairwise F _ST were not significant after Bonferroni correction. Thus, population differences in DNA methylation are more likely related to unmeasured oceanic environmental conditions, local adaptation, and/or genetic drift. DNA methylation is an additional mechanism that contributes to among population variation, with potential influences on organism phenotype, adaptive potential, and population resilience.     

The genotype‐phenotype landscape of an allosteric protein

Allostery is a fundamental biophysical mechanism that underlies cellular sensing, signaling, and metabolism. Yet a quantitative understanding of allosteric genotype‐phenotype relationships remains elusive. Here, we report the large‐scale measurement of the genotype‐phenotype landscape for an allosteric protein: the lac repressor from Escherichia coli, LacI. Using a method that combines long‐read and short‐read DNA sequencing, we quantitatively measure the dose‐response curves for nearly 10⁵ variants of the LacI genetic sensor. The resulting data provide a quantitative map of the effect of amino acid substitutions on LacI allostery and reveal systematic sequence‐structure‐function relationships. We find that in many cases, allosteric phenotypes can be quantitatively predicted with additive or neural‐network models, but unpredictable changes also occur. For example, we were surprised to discover a new band‐stop phenotype that challenges conventional models of allostery and that emerges from combinations of nearly silent amino acid substitutions.     

NAD+ supplementation prevents STING‐induced senescence in ataxia telangiectasia by improving mitophagy

Senescence phenotypes and mitochondrial dysfunction are implicated in aging and in premature aging diseases, including ataxia telangiectasia (A‐T). Loss of mitochondrial function can drive age‐related decline in the brain, but little is known about whether improving mitochondrial homeostasis alleviates senescence phenotypes. We demonstrate here that mitochondrial dysfunction and cellular senescence with a senescence‐associated secretory phenotype (SASP) occur in A‐T patient fibroblasts, and in ATM‐deficient cells and mice. Senescence is mediated by stimulator of interferon genes (STING) and involves ectopic cytoplasmic DNA. We further show that boosting intracellular NAD⁺ levels with nicotinamide riboside (NR) prevents senescence and SASP by promoting mitophagy in a PINK1‐dependent manner. NR treatment also prevents neurodegeneration, suppresses senescence and neuroinflammation, and improves motor function in Atm^−/− mice. Our findings suggest a central role for mitochondrial dysfunction‐induced senescence in A‐T pathogenesis, and that enhancing mitophagy as a potential therapeutic intervention.     

ZMAT3 hypomethylation contributes to early senescence of preadipocytes from healthy first‐degree relatives of type 2 diabetics

Senescence of adipose precursor cells (APC) impairs adipogenesis, contributes to the age‐related subcutaneous adipose tissue (SAT) dysfunction, and increases risk of type 2 diabetes (T2D). First‐degree relatives of T2D individuals (FDR) feature restricted adipogenesis, reflecting the detrimental effects of APC senescence earlier in life and rendering FDR more vulnerable to T2D. Epigenetics may contribute to these abnormalities but the underlying mechanisms remain unclear. In previous methylome comparison in APC from FDR and individuals with no diabetes familiarity (CTRL), ZMAT3 emerged as one of the top‐ranked senescence‐related genes featuring hypomethylation in FDR and associated with T2D risk. Here, we investigated whether and how DNA methylation changes at ZMAT3 promote early APC senescence. APC from FDR individuals revealed increases in multiple senescence markers compared to CTRL. Senescence in these cells was accompanied by ZMAT3 hypomethylation, which caused ZMAT3 upregulation. Demethylation at this gene in CTRL APC led to increased ZMAT3 expression and premature senescence, which were reverted by ZMAT3 siRNA. Furthermore, ZMAT3 overexpression in APC determined senescence and activation of the p53/p21 pathway, as observed in FDR APC. Adipogenesis was also inhibited in ZMAT3‐overexpressing APC. In FDR APC, rescue of ZMAT3 methylation through senolytic exposure simultaneously downregulated ZMAT3 expression and improved adipogenesis. Interestingly, in human SAT, aging and T2D were associated with significantly increased expression of both ZMAT3 and the P53 senescence marker. Thus, DNA hypomethylation causes ZMAT3 upregulation in FDR APC accompanied by acquisition of the senescence phenotype and impaired adipogenesis, which may contribute to FDR predisposition for T2D.     

C‐terminal deletion‐induced condensation sequesters AID from IgH targets in immunodeficiency

In activated B cells, activation‐induced cytidine deaminase (AID) generates programmed DNA lesions required for antibody class switch recombination (CSR), which may also threaten genome integrity. AID dynamically shuttles between cytoplasm and nucleus, and the majority stays in the cytoplasm due to active nuclear export mediated by its C‐terminal peptide. In immunodeficient‐patient cells expressing mutant AID lacking its C‐terminus, a catalytically active AID‐delC protein accumulates in the nucleus but nevertheless fails to support CSR. To resolve this apparent paradox, we dissected the function of AID‐delC proteins in the CSR process and found that they cannot efficiently target antibody genes. We demonstrate that AID‐delC proteins form condensates both in vivo and in vitro, dependent on its N‐terminus and on a surface arginine‐rich patch. Co‐expression of AID‐delC and wild‐type AID leads to an unbalanced nuclear AID‐delC/AID ratio, with AID‐delC proteins able to trap wild‐type AID in condensates, resulting in a dominant‐negative phenotype that could contribute to immunodeficiency. The co‐condensation model of mutant and wild‐type proteins could be an alternative explanation for the dominant‐negative effect in genetic disorders.     

Microglial MERTK eliminates phosphatidylserine‐displaying inhibitory post‐synapses

Glia contribute to synapse elimination through phagocytosis in the central nervous system. Despite the important roles of this process in development and neurological disorders, the identity and regulation of the "eat‐me" signal that initiates glia‐mediated phagocytosis of synapses has remained incompletely understood. Here, we generated conditional knockout mice with neuronal‐specific deletion of the flippase chaperone Cdc50a, to induce stable exposure of phosphatidylserine, a well‐known "eat‐me" signal for apoptotic cells, on the neuronal outer membrane. Surprisingly, acute Cdc50a deletion in mature neurons causes preferential phosphatidylserine exposure in neuronal somas and specific loss of inhibitory post‐synapses without effects on other synapses, resulting in abnormal excitability and seizures. Ablation of microglia or the deletion of microglial phagocytic receptor Mertk prevents the loss of inhibitory post‐synapses and the seizure phenotype, indicating that microglial phagocytosis is responsible for inhibitory post‐synapse elimination. Moreover, we found that phosphatidylserine is used for microglia‐mediated pruning of inhibitory post‐synapses in normal brains, suggesting that phosphatidylserine serves as a general "eat‐me" signal for inhibitory post‐synapse elimination.     

Integrative analysis of clinical and epigenetic biomarkers of mortality

DNA methylation (DNAm) has been reported to be associated with many diseases and with mortality. We hypothesized that the integration of DNAm with clinical risk factors would improve mortality prediction. We performed an epigenome‐wide association study of whole blood DNAm in relation to mortality in 15 cohorts (n = 15,013). During a mean follow‐up of 10 years, there were 4314 deaths from all causes including 1235 cardiovascular disease (CVD) deaths and 868 cancer deaths. Ancestry‐stratified meta‐analysis of all‐cause mortality identified 163 CpGs in European ancestry (EA) and 17 in African ancestry (AA) participants at p < 1 × 10⁻⁷, of which 41 (EA) and 16 (AA) were also associated with CVD death, and 15 (EA) and 9 (AA) with cancer death. We built DNAm‐based prediction models for all‐cause mortality that predicted mortality risk after adjusting for clinical risk factors. The mortality prediction model trained by integrating DNAm with clinical risk factors showed an improvement in prediction of cancer death with 5% increase in the C‐index in a replication cohort, compared with the model including clinical risk factors alone. Mendelian randomization identified 15 putatively causal CpGs in relation to longevity, CVD, or cancer risk. For example, cg06885782 (in KCNQ4) was positively associated with risk for prostate cancer (Beta = 1.2, P _MR = 4.1 × 10⁻⁴) and negatively associated with longevity (Beta = −1.9, P _MR = 0.02). Pathway analysis revealed that genes associated with mortality‐related CpGs are enriched for immune‐ and cancer‐related pathways. We identified replicable DNAm signatures of mortality and demonstrated the potential utility of CpGs as informative biomarkers for prediction of mortality risk.     

LncRNA SNHG1 promotes sepsis‐induced myocardial injury by inhibiting Bcl‐2 expression via DNMT1

Myocardial injury is a frequently occurring complication of sepsis. This study aims to investigate the molecular mechanism of long noncoding RNA (lncRNA) small nucleolar RNA host gene 1 (SNHG1)‐mediated DNA methyltransferase 1/B‐cell lymphoma‐2 (DNMT1/Bcl‐2) axis in sepsis‐induced myocardial injury. Mice and HL‐1 cells were treated with lipopolysaccharide (LPS) to establish animal and cellular models simulating sepsis and inflammation. LncRNA SNHG1 was screened out as a differentially expressed lncRNA in sepsis samples through microarray profiling, and the upregulated expression of lncRNA SNHG1 was confirmed in myocardial tissues of LPS‐induced septic mice and HL‐1 cells. Further experiments suggested that silencing of lncRNA SNHG1 reduced the inflammation and apoptotic rate of LPS‐induced HL‐1 cells. LncRNA SNHG1 inhibited Bcl‐2 expression by recruiting DNMT1 to Bcl‐2 promoter region to cause methylation. Inhibition of Bcl‐2 promoter methylation reduced the inflammation and apoptotic rate of LPS‐induced HL‐1 cells. In vivo experiments substantiated that lncRNA SNHG1 silencing alleviated sepsis‐induced myocardial injury in mice. Taken together, lncRNA SNHG1 promotes LPS‐induced myocardial injury in septic mice by downregulating Bcl‐2 through DNMT1‐mediated Bcl‐2 methylation.