Multi-omic QTL mapping in early developmental tissues reveals phenotypic and temporal complexity of regulatory variants underlying GWAS loci.

Most GWAS loci are presumed to affect gene regulation, however, only ∼43% colocalize with expression quantitative trait loci (eQTLs). To address this colocalization gap, we identify eQTLs, chromatin accessibility QTLs (caQTLs), and histone acetylation QTLs (haQTLs) using molecular samples from three early developmental (EDev) tissues. Through colocalization, we annotate 586 GWAS loci for 17 traits by QTL complexity, QTL phenotype, and QTL temporal specificity. We show that GWAS loci are highly enriched for colocalization with complex QTL modules that affect multiple elements (genes and/or peaks). We also demonstrate that caQTLs and haQTLs capture regulatory variations not associated with eQTLs and explain ∼49% of the functionally annotated GWAS loci. Additionally, we show that EDev-unique QTLs are strongly depleted for colocalizing with GWAS loci. By conducting one of the largest multi-omic QTL studies to date, we demonstrate that many GWAS loci exhibit phenotypic complexity and therefore, are missed by traditional eQTL analyses.

Human ESC-derived vascular cells promote vascular regeneration in a HIF-1α dependent manner.

Hypoxia-inducible factor (HIF-1α), a core transcription factor responding to changes in cellular oxygen levels, is closely associated with a wide range of physiological and pathological conditions. However, its differential impacts on vascular cell types and molecular programs modulating human vascular homeostasis and regeneration remain largely elusive. Here, we applied CRISPR/Cas9-mediated gene editing of human embryonic stem cells and directed differentiation to generate HIF-1α-deficient human vascular cells including vascular endothelial cells, vascular smooth muscle cells, and mesenchymal stem cells (MSCs), as a platform for discovering cell type-specific hypoxia-induced response mechanisms. Through comparative molecular profiling across cell types under normoxic and hypoxic conditions, we provide insight into the indispensable role of HIF-1α in the promotion of ischemic vascular regeneration. We found human MSCs to be the vascular cell type most susceptible to HIF-1α deficiency, and that transcriptional inactivation of ANKZF1, an effector of HIF-1α, impaired pro-angiogenic processes. Altogether, our findings deepen the understanding of HIF-1α in human angiogenesis and support further explorations of novel therapeutic strategies of vascular regeneration against ischemic damage.

A single-nucleus transcriptomic atlas of primate liver aging uncovers the pro-senescence role of SREBP2 in hepatocytes.

Aging increases the risk of liver diseases and systemic susceptibility to aging-related diseases. However, cell type-specific changes and the underlying mechanism of liver aging in higher vertebrates remain incompletely characterized. Here, we constructed the first single-nucleus transcriptomic landscape of primate liver aging, in which we resolved cell type-specific gene expression fluctuation in hepatocytes across three liver zonations and detected aberrant cell-cell interactions between hepatocytes and niche cells. Upon in-depth dissection of this rich dataset, we identified impaired lipid metabolism and upregulation of chronic inflammation-related genes prominently associated with declined liver functions during aging. In particular, hyperactivated sterol regulatory element-binding protein (SREBP) signaling was a hallmark of the aged liver, and consequently, forced activation of SREBP2 in human primary hepatocytes recapitulated in vivo aging phenotypes, manifesting as impaired detoxification and accelerated cellular senescence. This study expands our knowledge of primate liver aging and informs the development of diagnostics and therapeutic interventions for liver aging and associated diseases.

SGF29 nuclear condensates reinforce cellular aging.

Phase separation, a biophysical segregation of subcellular milieus referred as condensates, is known to regulate transcription, but its impacts on physiological processes are less clear. Here, we demonstrate the formation of liquid-like nuclear condensates by SGF29, a component of the SAGA transcriptional coactivator complex, during cellular senescence in human mesenchymal progenitor cells (hMPCs) and fibroblasts. The Arg 207 within the intrinsically disordered region is identified as the key amino acid residue for SGF29 to form phase separation. Through epigenomic and transcriptomic analysis, our data indicated that both condensate formation and H3K4me3 binding of SGF29 are essential for establishing its precise chromatin location, recruiting transcriptional factors and co-activators to target specific genomic loci, and initiating the expression of genes associated with senescence, such as CDKN1A. The formation of SGF29 condensates alone, however, may not be sufficient to drive H3K4me3 binding or achieve transactivation functions. Our study establishes a link between phase separation and aging regulation, highlighting nuclear condensates as a functional unit that facilitate shaping transcriptional landscapes in aging.

SIRT2 counteracts primate cardiac aging via deacetylation of STAT3 that silences CDKN2B.

Aging is a major risk factor contributing to pathophysiological changes in the heart, yet its intrinsic mechanisms have been largely unexplored in primates. In this study, we investigated the hypertrophic and senescence phenotypes in the hearts of aged cynomolgus monkeys as well as the transcriptomic and proteomic landscapes of young and aged primate hearts. SIRT2 was identified as a key protein decreased in aged monkey hearts, and engineered SIRT2 deficiency in human pluripotent stem cell-derived cardiomyocytes recapitulated key senescence features of primate heart aging. Further investigations revealed that loss of SIRT2 in human cardiomyocytes led to the hyperacetylation of STAT3, which transcriptionally activated CDKN2B and, in turn, triggered cardiomyocyte degeneration. Intra-myocardial injection of lentiviruses expressing SIRT2 ameliorated age-related cardiac dysfunction in mice. Taken together, our study provides valuable resources for decoding primate cardiac aging and identifies the SIRT2-STAT3-CDKN2B regulatory axis as a potential therapeutic target against human cardiac aging and aging-related cardiovascular diseases.

Genome-wide CRISPR activation screening in senescent cells reveals SOX5 as a driver and therapeutic target of rejuvenation.

Our understanding of the molecular basis for cellular senescence remains incomplete, limiting the development of strategies to ameliorate age-related pathologies by preventing stem cell senescence. Here, we performed a genome-wide CRISPR activation (CRISPRa) screening using a human mesenchymal precursor cell (hMPC) model of the progeroid syndrome. We evaluated targets whose activation antagonizes cellular senescence, among which SOX5 outperformed as a top hit. Through decoding the epigenomic landscapes remodeled by overexpressing SOX5, we uncovered its role in resetting the transcription network for geroprotective genes, including HMGB2. Mechanistically, SOX5 binding elevated the enhancer activity of HMGB2 with increased levels of H3K27ac and H3K4me1, raising HMGB2 expression so as to promote rejuvenation. Furthermore, gene therapy with lentiviruses carrying SOX5 or HMGB2 rejuvenated cartilage and alleviated osteoarthritis in aged mice. Our study generated a comprehensive list of rejuvenators, pinpointing SOX5 as a potent driver for rejuvenation both in vitro and in vivo.

CHIT1-positive microglia drive motor neuron ageing in the primate spinal cord.

Ageing is a critical factor in spinal-cord-associated disorders1, yet the ageing-specific mechanisms underlying this relationship remain poorly understood. Here, to address this knowledge gap, we combined single-nucleus RNA-sequencing analysis with behavioural and neurophysiological analysis in non-human primates (NHPs). We identified motor neuron senescence and neuroinflammation with microglial hyperactivation as intertwined hallmarks of spinal cord ageing. As an underlying mechanism, we identified a neurotoxic microglial state demarcated by elevated expression of CHIT1 (a secreted mammalian chitinase) specific to the aged spinal cords in NHP and human biopsies. In the aged spinal cord, CHIT1-positive microglia preferentially localize around motor neurons, and they have the ability to trigger senescence, partly by activating SMAD signalling. We further validated the driving role of secreted CHIT1 on MN senescence using multimodal experiments both in vivo, using the NHP spinal cord as a model, and in vitro, using a sophisticated system modelling the human motor-neuron-microenvironment interplay. Moreover, we demonstrated that ascorbic acid, a geroprotective compound, counteracted the pro-senescent effect of CHIT1 and mitigated motor neuron senescence in aged monkeys. Our findings provide the single-cell resolution cellular and molecular landscape of the aged primate spinal cord and identify a new biomarker and intervention target for spinal cord degeneration.

Hepatocyte reprogramming in liver regeneration: Biological mechanisms and applications.

The liver is one of the few organs that retain the capability to regenerate in adult mammals. This regeneration process is mainly facilitated by the dynamic behavior of hepatocytes, which are the major functional constituents in the liver. In response to liver injury, hepatocytes undergo remarkable alterations, such as reprogramming, wherein they lose their original identity and acquire properties from other cells. This phenomenon of hepatocyte reprogramming, coupled with hepatocyte expansion, plays a central role in liver regeneration, and its underlying mechanisms are complex and multifaceted. Understanding the fate of reprogrammed hepatocytes and the mechanisms of their conversion has significant implications for the development of innovative therapeutics for liver diseases. Herein, we review the plasticity of hepatocytes in response to various forms of liver injury, with a focus on injury-induced hepatocyte reprogramming. We provide a comprehensive summary of current knowledge on the molecular and cellular mechanisms governing hepatocyte reprogramming, specifically in the context of liver regeneration, providing insight into potential applications of this process in the treatment of liver disorders, including chronic liver diseases and liver cancer.

MAVS Antagonizes Human Stem Cell Senescence as a Mitochondrial Stabilizer.

Mitochondrial dysfunction is a hallmark feature of cellular senescence and organ aging. Here, we asked whether the mitochondrial antiviral signaling protein (MAVS), which is essential for driving antiviral response, also regulates human stem cell senescence. To answer this question, we used CRISPR/Cas9-mediated gene editing and directed differentiation techniques to generate various MAVS-knockout human stem cell models. We found that human mesenchymal stem cells (hMSCs) were sensitive to MAVS deficiency, as manifested by accelerated senescence phenotypes. We uncovered that the role of MAVS in maintaining mitochondrial structural integrity and functional homeostasis depends on its interaction with the guanosine triphosphatase optic atrophy type 1 (OPA1). Depletion of MAVS or OPA1 led to the dysfunction of mitochondria and cellular senescence, whereas replenishment of MAVS or OPA1 in MAVS-knockout hMSCs alleviated mitochondrial defects and premature senescence phenotypes. Taken together, our data underscore an uncanonical role of MAVS in safeguarding mitochondrial homeostasis and antagonizing human stem cell senescence.

Single-cell profiling reveals a potent role of quercetin in promoting hair regeneration.

Hair loss affects millions of people at some time in their life, and safe and efficient treatments for hair loss are a significant unmet medical need. We report that topical delivery of quercetin (Que) stimulates resting hair follicles to grow with rapid follicular keratinocyte proliferation and replenishes perifollicular microvasculature in mice. We construct dynamic single-cell transcriptome landscape over the course of hair regrowth and find that Que treatment stimulates the differentiation trajectory in the hair follicles and induces an angiogenic signature in dermal endothelial cells by activating HIF-1α in endothelial cells. Skin administration of a HIF-1α agonist partially recapitulates the pro-angiogenesis and hair-growing effects of Que. Together, these findings provide a molecular understanding for the efficacy of Que in hair regrowth, which underscores the translational potential of targeting the hair follicle niche as a strategy for regenerative medicine, and suggest a route of pharmacological intervention that may promote hair regrowth.

SESN1 is a FOXO3 effector that counteracts human skeletal muscle ageing.

Sarcopenia, a skeletal muscle disorder in which loss of muscle mass and function progresses with age, is associated with increased overall frailty, risk of falling and mortality in the elders. Here, we reveal that SESN1 safeguards skeletal muscle from ageing downstream of the longevity gene FOXO3, which we recently reported is a geroprotector in primate skeletal muscle. Knockdown of SESN1 mimicked the human myotube ageing phenotypes observed in the FOXO3-deficient human myotubes, whereas genetic activation of SESN1 alleviated human myotube senescence. Of note, SESN1 was identified as a protective secretory factor against muscle atrophy. Administration of recombinant SESN1 protein attenuated senescence of human myotubes in vitro and facilitated muscle regeneration in vivo. Altogether, we unveil a key role of SESN1 downstream of FOXO3 in protecting skeletal muscle from ageing, providing diagnostic biomarkers and intervention strategies for counteracting skeletal muscle ageing and related diseases.

m6A epitranscriptomic regulation of tissue homeostasis during primate aging.

How N6-methyladenosine (m6A), the most abundant mRNA modification, contributes to primate tissue homeostasis and physiological aging remains elusive. Here, we characterize the m6A epitranscriptome across the liver, heart and skeletal muscle in young and old nonhuman primates. Our data reveal a positive correlation between m6A modifications and gene expression homeostasis across tissues as well as tissue-type-specific aging-associated m6A dynamics. Among these tissues, skeletal muscle is the most susceptible to m6A loss in aging and shows a reduction in the m6A methyltransferase METTL3. We further show that METTL3 deficiency in human pluripotent stem cell-derived myotubes leads to senescence and apoptosis, and identify NPNT as a key element downstream of METTL3 involved in myotube homeostasis, whose expression and m6A levels are both decreased in senescent myotubes. Our study provides a resource for elucidating m6A-mediated mechanisms of tissue aging and reveals a METTL3-m6A-NPNT axis counteracting aging-associated skeletal muscle degeneration.

Single-cell transcriptomic atlas of mouse cochlear aging.

Progressive functional deterioration in the cochlea is associated with age-related hearing loss (ARHL). However, the cellular and molecular basis underlying cochlear aging remains largely unknown. Here, we established a dynamic single-cell transcriptomic landscape of mouse cochlear aging, in which we characterized aging-associated transcriptomic changes in 27 different cochlear cell types across five different time points. Overall, our analysis pinpoints loss of proteostasis and elevated apoptosis as the hallmark features of cochlear aging, highlights unexpected age-related transcriptional fluctuations in intermediate cells localized in the stria vascularis (SV) and demonstrates that upregulation of endoplasmic reticulum (ER) chaperon protein HSP90AA1 mitigates ER stress-induced damages associated with aging. Our work suggests that targeting unfolded protein response pathways may help alleviate aging-related SV atrophy and hence delay the progression of ARHL.

Single-nucleus profiling unveils a geroprotective role of the FOXO3 in primate skeletal muscle aging.

Age-dependent loss of skeletal muscle mass and function is a feature of sarcopenia, and increases the risk of many aging-related metabolic diseases. Here, we report phenotypic and single-nucleus transcriptomic analyses of non-human primate skeletal muscle aging. A higher transcriptional fluctuation was observed in myonuclei relative to other interstitial cell types, indicating a higher susceptibility of skeletal muscle fiber to aging. We found a downregulation of FOXO3 in aged primate skeletal muscle, and identified FOXO3 as a hub transcription factor maintaining skeletal muscle homeostasis. Through the establishment of a complementary experimental pipeline based on a human pluripotent stem cell-derived myotube model, we revealed that silence of FOXO3 accelerates human myotube senescence, whereas genetic activation of endogenous FOXO3 alleviates human myotube aging. Altogether, based on a combination of monkey skeletal muscle and human myotube aging research models, we unraveled the pivotal role of the FOXO3 in safeguarding primate skeletal muscle from aging, providing a comprehensive resource for the development of clinical diagnosis and targeted therapeutic interventions against human skeletal muscle aging and the onset of sarcopenia along with aging-related disorders.

A single-nucleus transcriptomic atlas of primate testicular aging reveals exhaustion of the spermatogonial stem cell reservoir and loss of Sertoli cell homeostasis.

The testis is pivotal for male reproduction, and its progressive functional decline in aging is associated with infertility. However, the regulatory mechanism underlying primate testicular aging remains largely elusive. Here, we resolve the aging-related cellular and molecular alterations of primate testicular aging by establishing a single-nucleus transcriptomic atlas. Gene-expression patterns along the spermatogenesis trajectory revealed molecular programs associated with attrition of spermatogonial stem cell reservoir, disturbed meiosis and impaired spermiogenesis along the sequential continuum. Remarkably, Sertoli cell was identified as the cell type most susceptible to aging, given its deeply perturbed age-associated transcriptional profiles. Concomitantly, downregulation of the transcription factor Wilms' Tumor 1 (WT1), essential for Sertoli cell homeostasis, was associated with accelerated cellular senescence, disrupted tight junctions, and a compromised cell identity signature, which altogether may help create a hostile microenvironment for spermatogenesis. Collectively, our study depicts in-depth transcriptomic traits of non-human primate (NHP) testicular aging at single-cell resolution, providing potential diagnostic biomarkers and targets for therapeutic interventions against testicular aging and age-related male reproductive diseases.

Resurrection of endogenous retroviruses during aging reinforces senescence.

Whether and how certain transposable elements with viral origins, such as endogenous retroviruses (ERVs) dormant in our genomes, can become awakened and contribute to the aging process is largely unknown. In human senescent cells, we found that HERVK (HML-2), the most recently integrated human ERVs, are unlocked to transcribe viral genes and produce retrovirus-like particles (RVLPs). These HERVK RVLPs constitute a transmissible message to elicit senescence phenotypes in young cells, which can be blocked by neutralizing antibodies. The activation of ERVs was also observed in organs of aged primates and mice as well as in human tissues and serum from the elderly. Their repression alleviates cellular senescence and tissue degeneration and, to some extent, organismal aging. These findings indicate that the resurrection of ERVs is a hallmark and driving force of cellular senescence and tissue aging.

A complex epigenome-splicing crosstalk governs epithelial-to-mesenchymal transition in metastasis and brain development.

Epithelial-to-mesenchymal transition (EMT) renders epithelial cells migratory properties. While epigenetic and splicing changes have been implicated in EMT, the mechanisms governing their crosstalk remain poorly understood. Here we discovered that a C2H2 zinc finger protein, ZNF827, is strongly induced during various contexts of EMT, including in brain development and breast cancer metastasis, and is required for the molecular and phenotypic changes underlying EMT in these processes. Mechanistically, ZNF827 mediated these responses by orchestrating a large-scale remodelling of the splicing landscape by recruiting HDAC1 for epigenetic modulation of distinct genomic loci, thereby slowing RNA polymerase II progression and altering the splicing of genes encoding key EMT regulators in cis. Our findings reveal an unprecedented complexity of crosstalk between epigenetic landscape and splicing programme in governing EMT and identify ZNF827 as a master regulator coupling these processes during EMT in brain development and breast cancer metastasis.

Large-scale chromatin reorganization reactivates placenta-specific genes that drive cellular aging.

Nuclear deformation, a hallmark frequently observed in senescent cells, is presumed to be associated with the erosion of chromatin organization at the nuclear periphery. However, how such gradual changes in higher-order genome organization impinge on local epigenetic modifications to drive cellular mechanisms of aging has remained enigmatic. Here, through large-scale epigenomic analyses of isogenic young, senescent, and progeroid human mesenchymal progenitor cells (hMPCs), we delineate a hierarchy of integrated structural state changes that manifest as heterochromatin loss in repressive compartments, euchromatin weakening in active compartments, switching in interfacing topological compartments, and increasing epigenetic entropy. We found that the epigenetic de-repression unlocks the expression of pregnancy-specific beta-1 glycoprotein (PSG) genes that exacerbate hMPC aging and serve as potential aging biomarkers. Our analyses provide a rich resource for uncovering the principles of epigenomic landscape organization and its changes in cellular aging and for identifying aging drivers and intervention targets with a genome-topology-based mechanism.

Heterochronic parabiosis induces stem cell revitalization and systemic rejuvenation across aged tissues.

The young circulatory milieu capable of delaying aging in individual tissues is of interest as rejuvenation strategies, but how it achieves cellular- and systemic-level effects has remained unclear. Here, we constructed a single-cell transcriptomic atlas across aged tissues/organs and their rejuvenation in heterochronic parabiosis (HP), a classical model to study systemic aging. In general, HP rejuvenated adult stem cells and their niches across tissues. In particular, we identified hematopoietic stem and progenitor cells (HSPCs) as one of the most responsive cell types to young blood exposure, from which a continuum of cell state changes across the hematopoietic and immune system emanated, through the restoration of a youthful transcriptional regulatory program and cytokine-mediated cell-cell communications in HSPCs. Moreover, the reintroduction of the identified rejuvenating factors alleviated age-associated lymphopoiesis decline. Overall, we provide comprehensive frameworks to explore aging and rejuvenating trajectories at single-cell resolution and revealed cellular and molecular programs that instruct systemic revitalization by blood-borne factors.

BMAL1 moonlighting as a gatekeeper for LINE1 repression and cellular senescence in primates.

Aging in humans is intricately linked with alterations in circadian rhythms concomitant with physiological decline and stem cell exhaustion. However, whether the circadian machinery directly regulates stem cell aging, especially in primates, remains poorly understood. In this study, we found that deficiency of BMAL1, the only non-redundant circadian clock component, results in an accelerated aging phenotype in both human and cynomolgus monkey mesenchymal progenitor cells (MPCs). Unexpectedly, this phenotype was mainly attributed to a transcription-independent role of BMAL1 in stabilizing heterochromatin and thus preventing activation of the LINE1-cGAS-STING pathway. In senescent primate MPCs, we observed decreased capacity of BMAL1 to bind to LINE1 and synergistic activation of LINE1 expression. Likewise, in the skin and muscle tissues from the BMAL1-deficient cynomolgus monkey, we observed destabilized heterochromatin and aberrant LINE1 transcription. Altogether, these findings uncovered a noncanonical role of BMAL1 in stabilizing heterochromatin to inactivate LINE1 that drives aging in primate cells.