PRC2-AgeIndex as a universal biomarker of aging and rejuvenation.

DNA methylation (DNAm) is one of the most reliable biomarkers of aging across mammalian tissues. While the age-dependent global loss of DNAm has been well characterized, DNAm gain is less characterized. Studies have demonstrated that CpGs which gain methylation with age are enriched in Polycomb Repressive Complex 2 (PRC2) targets. However, whole-genome examination of all PRC2 targets as well as determination of the pan-tissue or tissue-specific nature of these associations is lacking. Here, we show that low-methylated regions (LMRs) which are highly bound by PRC2 in embryonic stem cells (PRC2 LMRs) gain methylation with age in all examined somatic mitotic cells. We estimated that this epigenetic change represents around 90% of the age-dependent DNAm gain genome-wide. Therefore, we propose the "PRC2-AgeIndex," defined as the average DNAm in PRC2 LMRs, as a universal biomarker of cellular aging in somatic cells which can distinguish the effect of different anti-aging interventions.

Clearance of senescent macrophages ameliorates tumorigenesis in KRAS-driven lung cancer.

The accumulation of senescent cells in the tumor microenvironment can drive tumorigenesis in a paracrine manner through the senescence-associated secretory phenotype (SASP). Using a new p16-FDR mouse line, we show that macrophages and endothelial cells are the predominant senescent cell types in murine KRAS-driven lung tumors. Through single cell transcriptomics, we identify a population of tumor-associated macrophages that express a unique array of pro-tumorigenic SASP factors and surface proteins and are also present in normal aged lungs. Genetic or senolytic ablation of senescent cells, or macrophage depletion, result in a significant decrease in tumor burden and increased survival in KRAS-driven lung cancer models. Moreover, we reveal the presence of macrophages with senescent features in human lung pre-malignant lesions, but not in adenocarcinomas. Taken together, our results have uncovered the important role of senescent macrophages in the initiation and progression of lung cancer, highlighting potential therapeutic avenues and cancer preventative strategies.

Region-based epigenetic clock design improves RRBS-based age prediction.

Recent studies suggest that epigenetic rejuvenation can be achieved using drugs that mimic calorie restriction and techniques such as reprogramming-induced rejuvenation. To effectively test rejuvenation in vivo, mouse models are the safest alternative. However, we have found that the recent epigenetic clocks developed for mouse reduced-representation bisulphite sequencing (RRBS) data have significantly poor performance when applied to external datasets. We show that the sites captured and the coverage of key CpGs required for age prediction vary greatly between datasets, which likely contributes to the lack of transferability in RRBS clocks. To mitigate these coverage issues in RRBS-based age prediction, we present two novel design strategies that use average methylation over large regions rather than individual CpGs, whereby regions are defined by sliding windows (e.g. 5 kb), or density-based clustering of CpGs. We observe improved correlation and error in our regional blood clocks (RegBCs) compared to published individual-CpG-based techniques when applied to external datasets. The RegBCs are also more robust when applied to low coverage data and detect a negative age acceleration in mice undergoing calorie restriction. Our RegBCs offer a proof of principle that age prediction of RRBS datasets can be improved by accounting for multiple CpGs over a region, which negates the lack of read depth currently hindering individual-CpG-based approaches.

Tfap2b specifies an embryonic melanocyte stem cell that retains adult multifate potential.

Melanocytes, the pigment-producing cells, are replenished from multiple stem cell niches in adult tissue. Although pigmentation traits are known risk factors for melanoma, we know little about melanocyte stem cell (McSC) populations other than hair follicle McSCs and lack key lineage markers with which to identify McSCs and study their function. Here we find that Tfap2b and a select set of target genes specify an McSC population at the dorsal root ganglia in zebrafish. Functionally, Tfap2b is required for only a few late-stage embryonic melanocytes, and is essential for McSC-dependent melanocyte regeneration. Fate mapping data reveal that tfap2b+ McSCs have multifate potential, and are the cells of origin for large patches of adult melanocytes, two other pigment cell types (iridophores and xanthophores), and nerve-associated cells. Hence, Tfap2b confers McSC identity in early development, distinguishing McSCs from other neural crest and pigment cell lineages, and retains multifate potential in the adult zebrafish.

Cellular reprogramming and epigenetic rejuvenation.

Ageing is an inevitable condition that afflicts all humans. Recent achievements, such as the generation of induced pluripotent stem cells, have delivered preliminary evidence that slowing down and reversing the ageing process might be possible. However, these techniques usually involve complete dedifferentiation, i.e. somatic cell identity is lost as cells are converted to a pluripotent state. Separating the rejuvenative properties of reprogramming from dedifferentiation is a promising prospect, termed epigenetic rejuvenation. Reprogramming-induced rejuvenation strategies currently involve using Yamanaka factors (typically transiently expressed to prevent full dedifferentiation) and are promising candidates to safely reduce biological age. Here, we review the development and potential of reprogramming-induced rejuvenation as an anti-ageing strategy.

Epigenetic age prediction.

Advanced age is the main common risk factor for cancer, cardiovascular disease and neurodegeneration. Yet, more is known about the molecular basis of any of these groups of diseases than the changes that accompany ageing itself. Progress in molecular ageing research was slow because the tools predicting whether someone aged slowly or fast (biological age) were unreliable. To understand ageing as a risk factor for disease and to develop interventions, the molecular ageing field needed a quantitative measure; a clock for biological age. Over the past decade, a number of age predictors utilising DNA methylation have been developed, referred to as epigenetic clocks. While they appear to estimate biological age, it remains unclear whether the methylation changes used to train the clocks are a reflection of other underlying cellular or molecular processes, or whether methylation itself is involved in the ageing process. The precise aspects of ageing that the epigenetic clocks capture remain hidden and seem to vary between predictors. Nonetheless, the use of epigenetic clocks has opened the door towards studying biological ageing quantitatively, and new clocks and applications, such as forensics, appear frequently. In this review, we will discuss the range of epigenetic clocks available, their strengths and weaknesses, and their applicability to various scientific queries.

Kidney Single-Cell Atlas Reveals Myeloid Heterogeneity in Progression and Regression of Kidney Disease.

BACKGROUND: Little is known about the roles of myeloid cell subsets in kidney injury and in the limited ability of the organ to repair itself. Characterizing these cells based only on surface markers using flow cytometry might not provide a full phenotypic picture. Defining these cells at the single-cell, transcriptomic level could reveal myeloid heterogeneity in the progression and regression of kidney disease. METHODS: Integrated droplet- and plate-based single-cell RNA sequencing were used in the murine, reversible, unilateral ureteric obstruction model to dissect the transcriptomic landscape at the single-cell level during renal injury and the resolution of fibrosis. Paired blood exchange tracked the fate of monocytes recruited to the injured kidney. RESULTS: A single-cell atlas of the kidney generated using transcriptomics revealed marked changes in the proportion and gene expression of renal cell types during injury and repair. Conventional flow cytometry markers would not have identified the 12 myeloid cell subsets. Monocytes recruited to the kidney early after injury rapidly adopt a proinflammatory, profibrotic phenotype that expresses Arg1, before transitioning to become Ccr2+ macrophages that accumulate in late injury. Conversely, a novel Mmp12+ macrophage subset acts during repair. CONCLUSIONS: Complementary technologies identified novel myeloid subtypes, based on transcriptomics in single cells, that represent therapeutic targets to inhibit progression or promote regression of kidney disease.

Partial reprogramming induces a steady decline in epigenetic age before loss of somatic identity.

Induced pluripotent stem cells (IPSCs), with their unlimited regenerative capacity, carry the promise for tissue replacement to counter age-related decline. However, attempts to realize in vivo iPSC have invariably resulted in the formation of teratomas. Partial reprogramming in prematurely aged mice has shown promising results in alleviating age-related symptoms without teratoma formation. Does partial reprogramming lead to rejuvenation (i.e., "younger" cells), rather than dedifferentiation, which bears the risk of cancer? Here, we analyse the dynamics of cellular age during human iPSC reprogramming and find that partial reprogramming leads to a reduction in the epigenetic age of cells. We also find that the loss of somatic gene expression and epigenetic age follows different kinetics, suggesting that they can be uncoupled and there could be a safe window where rejuvenation can be achieved with a minimized risk of cancer.

Wilms Tumor 1b defines a wound-specific sheath cell subpopulation associated with notochord repair.

Regenerative therapy for degenerative spine disorders requires the identification of cells that can slow down and possibly reverse degenerative processes. Here, we identify an unanticipated wound-specific notochord sheath cell subpopulation that expresses Wilms Tumor (WT) 1b following injury in zebrafish. We show that localized damage leads to Wt1b expression in sheath cells, and that wt1b+cells migrate into the wound to form a stopper-like structure, likely to maintain structural integrity. Wt1b+sheath cells are distinct in expressing cartilage and vacuolar genes, and in repressing a Wt1b-p53 transcriptional programme. At the wound, wt1b+and entpd5+ cells constitute separate, tightly-associated subpopulations. Surprisingly, wt1b expression at the site of injury is maintained even into adult stages in developing vertebrae, which form in an untypical manner via a cartilage intermediate. Given that notochord cells are retained in adult intervertebral discs, the identification of novel subpopulations may have important implications for regenerative spine disorder treatments.

cGAS surveillance of micronuclei links genome instability to innate immunity.

DNA is strictly compartmentalized within the nucleus to prevent autoimmunity; despite this, cyclic GMP-AMP synthase (cGAS), a cytosolic sensor of double-stranded DNA, is activated in autoinflammatory disorders and by DNA damage. Precisely how cellular DNA gains access to the cytoplasm remains to be determined. Here, we report that cGAS localizes to micronuclei arising from genome instability in a mouse model of monogenic autoinflammation, after exogenous DNA damage and spontaneously in human cancer cells. Such micronuclei occur after mis-segregation of DNA during cell division and consist of chromatin surrounded by its own nuclear membrane. Breakdown of the micronuclear envelope, a process associated with chromothripsis, leads to rapid accumulation of cGAS, providing a mechanism by which self-DNA becomes exposed to the cytosol. cGAS is activated by chromatin, and consistent with a mitotic origin, micronuclei formation and the proinflammatory response following DNA damage are cell-cycle dependent. By combining live-cell laser microdissection with single cell transcriptomics, we establish that interferon-stimulated gene expression is induced in micronucleated cells. We therefore conclude that micronuclei represent an important source of immunostimulatory DNA. As micronuclei formed from lagging chromosomes also activate this pathway, recognition of micronuclei by cGAS may act as a cell-intrinsic immune surveillance mechanism that detects a range of neoplasia-inducing processes.

Luminogenic cytochrome P450 assays.

Luminogenic cytochrome P450 (CYP) assays couple CYP enzyme activity to firefly luciferase luminescence in a technology called P450-Glo(TM) (Promega). Luminogenic substrates are used in assays of human CYP1A1, -1A2, -1B1, -2C8, -2C9, -2C19, -2D6, -2J2, -3A4, -3A7, -4A11, -4F3B, -4F12 and -19. The assays detect dose-dependent CYP inhibition by test compounds against recombinant CYP enzymes or liver microsomes. Induction or inhibition of CYP activities in cultured hepatocytes is measured in a nonlytic approach that leaves cells intact for additional analysis. Luminogenic CYP assays offer advantages of speed and safety over HPLC and radiochemical-based methods. Compared with fluorogenic methods the approach offers advantages of improved sensitivity and decreased interference between optical properties of test compound and CYP substrate. These homogenous assays are sensitive and robust tools for high-throughput CYP screening in early drug discovery.