Expression of the transcription factors OCT4, SOX2, KLF4, and cMYC (OSKM) reprograms somatic cells into induced pluripotent stem cells (iPSCs). Reprogramming is a slow and inefficient process, ...suggesting the presence of safeguarding mechanisms that counteract cell fate conversion. One such mechanism is senescence. To identify modulators of reprogramming-induced senescence, we performed a genome-wide shRNA screen in primary human fibroblasts expressing OSKM. In the screen, we identified novel mediators of OSKM-induced senescence and validated previously implicated genes such as
We developed an innovative approach that integrates single-cell RNA sequencing (scRNA-seq) with the shRNA screen to investigate the mechanism of action of the identified candidates. Our data unveiled regulation of senescence as a novel way by which mechanistic target of rapamycin (mTOR) influences reprogramming. On one hand, mTOR inhibition blunts the induction of cyclin-dependent kinase (CDK) inhibitors (CDKIs), including p16
, p21
, and p15
, preventing OSKM-induced senescence. On the other hand, inhibition of mTOR blunts the senescence-associated secretory phenotype (SASP), which itself favors reprogramming. These contrasting actions contribute to explain the complex effect that mTOR has on reprogramming. Overall, our study highlights the advantage of combining functional screens with scRNA-seq to accelerate the discovery of pathways controlling complex phenotypes.
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.
To effectively test rejuvenation techniques on in vivo model organisms, we have developed two novel design strategies that use mean methylation over regions, rather than individual CpGs (an approach which we show is ineffective when applied to external test datasets). 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), increased robustness on low coverage data and negative age acceleration in calorie‐restricted mice.
DNA methylation rates have previously been found to broadly correlate with maximum lifespan in mammals, yet no precise relationship has been observed. We developed a statistically robust framework to ...compare methylation rates at conserved age-related sites across mammals. We found that methylation rates negatively scale with maximum lifespan in both blood and skin. The emergence of explicit scaling suggests that methylation rates are, or are linked to, an evolutionary constraint on maximum lifespan acting across diverse mammalian lineages.
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.
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•Tfap2b and its target genes specify McSCs with mixed pigment cell identities•Melanocyte regeneration from McSCs functionally depends on Tfap2b•tfap2b specifies ErbB-dependent McSCs at the stem cell niche•Fate mapping reveals Tfap2b-McSCs have multifate potential for adult pigment cells
Brombin et al. use scRNA sequencing to identify the transcriptome of McSCs in zebrafish and discover that tfap2b is a functional marker of this cell type. Fate mapping analyses reveal that these embryonically established stem cells have multifate potential in the adult pattern.
DNA modifications, especially methylation, are known to play a crucial part in many regulatory processes in the cell. Recently, 5-hydroxymethylcytosine (5hmC) was discovered, a DNA modification ...derived as an intermediate of 5-methylcytosine (5mC) oxidation. Efforts to gain insights into function of this DNA modification are underway and several methods were recently described to assess 5hmC levels using sequencing approaches. Here we integrate adaptation based multiplexing and high-efficiency library prep into the oxidative Bisulfite Sequencing (oxBS-seq) workflow reducing the starting amount and cost per sample to identify 5hmC levels genome-wide.
Cellular senescence is a cell fate triggered in response to stress and is characterized by stable cell-cycle arrest and a hypersecretory state. It has diverse biological roles, ranging from tissue ...repair to chronic disease. The development of new tools to study senescence in vivo has paved the way for uncovering its physiological and pathological roles and testing senescent cells as a therapeutic target. However, the lack of specific and broadly applicable markers makes it difficult to identify and characterize senescent cells in tissues and living organisms. To address this, we provide practical guidelines called “minimum information for cellular senescence experimentation in vivo” (MICSE). It presents an overview of senescence markers in rodent tissues, transgenic models, non-mammalian systems, human tissues, and tumors and their use in the identification and specification of senescent cells. These guidelines provide a uniform, state-of-the-art, and accessible toolset to improve our understanding of cellular senescence in vivo.
This primer introduces the “minimal information on cellular senescence experimentation in vivo” (MICSE) guidelines, which aim to address the challenges in identifying and characterizing senescent cells. It provides an overview of senescence markers in various systems and offers a practical toolset to enhance our understanding of cellular senescence in vivo.
Progressive fibrosis is a feature of aging and chronic tissue injury in multiple organs, including the kidney and heart. Glioma-associated oncogene 1 expressing (Gli1
) cells are a major source of ...activated fibroblasts in multiple organs, but the links between injury, inflammation, and Gli1
cell expansion and tissue fibrosis remain incompletely understood. We demonstrated that leukocyte-derived tumor necrosis factor (TNF) promoted Gli1
cell proliferation and cardiorenal fibrosis through induction and release of Indian Hedgehog (IHH) from renal epithelial cells. Using single-cell-resolution transcriptomic analysis, we identified an "inflammatory" proximal tubular epithelial (iPT) population contributing to TNF- and nuclear factor κB (NF-κB)-induced IHH production in vivo. TNF-induced Ubiquitin D (
) expression was observed in human proximal tubular cells in vitro and during murine and human renal disease and aging. Studies using pharmacological and conditional genetic ablation of TNF-induced IHH signaling revealed that IHH activated canonical Hedgehog signaling in Gli1
cells, which led to their activation, proliferation, and fibrosis within the injured and aging kidney and heart. These changes were inhibited in mice by
deletion in
-expressing cells or by pharmacological blockade of TNF, NF-κB, or Gli1 signaling. Increased amounts of circulating IHH were associated with loss of renal function and higher rates of cardiovascular disease in patients with chronic kidney disease. Thus, IHH connects leukocyte activation to Gli1
cell expansion and represents a potential target for therapies to inhibit inflammation-induced fibrosis.
Characterising associations between the methylome, proteome and phenome may provide insight into biological pathways governing brain health. Here, we report an integrated DNA methylation and ...phenotypic study of the circulating proteome in relation to brain health. Methylome-wide association studies of 4058 plasma proteins are performed (N = 774), identifying 2928 CpG-protein associations after adjustment for multiple testing. These are independent of known genetic protein quantitative trait loci (pQTLs) and common lifestyle effects. Phenome-wide association studies of each protein are then performed in relation to 15 neurological traits (N = 1,065), identifying 405 associations between the levels of 191 proteins and cognitive scores, brain imaging measures or APOE e4 status. We uncover 35 previously unreported DNA methylation signatures for 17 protein markers of brain health. The epigenetic and proteomic markers we identify are pertinent to understanding and stratifying brain health.
Clonal haemopoiesis of indeterminate potential (CHIP) in healthy individuals was initially observed through an increased skewing in X chromosome inactivation and occurs from age 60 onwards with up to ...20% of 90 year olds being CHIP carriers. More recently, several groups reported that CHIP is driven by somatic mutations (Genovese et al, 2014), with the most prevalent CHIP mutations in the DNMT3A,TET2 and JAK2 genes, previously described as drivers of myeloid malignancies. CHIP is associated with an increased risk for haematological cancers and age-related disease (Jaiswal et al, 2014). We recently reported evidence of accelerated epigenetic age in individuals with CHIP (Robertson et al, 2019).
In this study, using error-corrected sequencing and a new framework for extracting fitness effects from longitudinal data, we quantified the growth potential or fitness effect within each individual, allowing us to determine which specific variants are potentially the most pathogenic. We show that gene-specific fitness differences can outweigh inter-individual variation allowing the use of this quantitative biomarker as a proxy for disease progression. Further, we develop a novel approach, a non-neutral growth filter, that better segregates between naturally drifting populations of cells and fast growing clones. Our framework nuances the classification of variants with a low fitness to accurately detect potentially harmful variants on a patient-basis before they become dominant. Our study overcomes limitations in inter-individual variability and accuracy of clonal fitness prediction by using longitudinal time-course data sampled over up to 21 years in late age.
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