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Background There is a growing interest in generating precise predictions of survival to improve the assessment of health and life-improving interventions. We aimed to (a) test if observable characteristics may provide a survival prediction independent of chronological age; (b) identify the most relevant predictors of survival; and (c) build a metric of multidimensional age. Methods Data from 3 095 individuals aged ≥60 from the Swedish National Study on Aging and Care in Kungsholmen. Eighty-three variables covering 5 domains (diseases, risk factors, sociodemographics, functional status, and blood tests) were tested in penalized Cox regressions to predict 18-year mortality. Results The best prediction of mortality at different follow-ups (area under the receiver operating characteristic curves [AUROCs] 0.878–0.909) was obtained when 15 variables from all 5 domains were tested simultaneously in a penalized Cox regression. Significant prediction improvements were observed when chronological age was included as a covariate for 15- but not for 5- and 10-year survival. When comparing individual domains, we find that a combination of functional characteristics (ie, gait speed, cognition) gave the most accurate prediction, with estimates similar to chronological age for 5- (AUROC 0.836) and 10-year (AUROC 0.830) survival. Finally, we built a multidimensional measure of age by regressing the predicted mortality risk on chronological age, which displayed a stronger correlation with time to death (R = −0.760) than chronological age (R = −0.660) and predicted mortality better than widely used geriatric indices. Conclusions Combining easily accessible characteristics can help in building highly accurate survival models and multidimensional age metrics with potentially broad geriatric and biomedical applications.

SummaryWith the recent rapid advancements in sequencing technology, the analysis of omics data has become increasingly important for biomedical research. However, many laboratories lack the time or expertise to thoroughly analyze such data. Among omics assays, ATAC-Seq (Assay for transposase-accessiblechromatin usingsequencing) and mRNA-Seq (messengerRNA sequencing) are easy and cost-effective methods that are commonly used to define chromatin accessibility and gene expression levels, respectively. Here we introduce Cactus (Chromatinaccessibility and transcriptomicsunifyingsoftware), a pipeline that streamlines the individual or integrated analysis of ATAC-Seq and mRNA-Seq data. The pipeline is written in Nextflow for efficient scaling, caching, and parallelization, and all tools are packaged in Singularity/Docker containers or conda/Mamba virtual environments for simple installation and high reproducibility. Cactus conducts preprocessing on raw sequencing reads, followed by differential analysis between conditions. Results are split into Differential Analysis Subsets (DASs) based on significance threshold, direction of change, annotated genomic regions, and experiment type. Then, Cactus computes enrichments of entries from internal (i.e., DASs) and external (i.e., ontologies, pathways, DNA binding motifs, ChIP-Seq (Chromatin immunoprecipitationsequencing) binding sites, and chromatin states) databases in DASs and presents results in barplots and customizable heatmaps. The visualization of the results is simplified by the generation of merged PDFs, merged tables, and formatted Excel tables. In conclusion, Cactus can assist researchers in gaining comprehensive insights from chromatin accessibility and gene expression data in a quick, easy, and reproducible manner.Availability and implementationCactus can be installed via Nextflow pull jsalignon/cactus. Source code and documentation are available online (https://github.com/jsalignon/cactus).Contactjerome.salignon@ki.seorchristian.riedel@ki.se

SET-26, HCF-1, and HDA-1 are highly conserved chromatin factors with key roles in development and aging. Here we present mechanistic insights into how these factors regulate gene expression and modulate longevity inC. elegans. We show that SET-26 and HCF-1 cooperate to regulate a common set of genes, and both antagonize the histone deacetylase HDA-1 to limit longevity. We propose a model in which SET-26 recruits HCF-1 to chromatin in somatic cells, where they stabilize each other at the promoters of a subset of genes, particularly mitochondrial function genes, and regulate their expression. HDA-1 opposes SET-26 and HCF-1 on the regulation of a subset of their common target genes and in longevity. Our findings suggest that SET-26, HCF-1, and HDA-1 comprise a mechanism to fine-tune gene expression and longevity and likely have important implications for the mechanistic understanding of how these factors function in diverse organisms, particularly in aging biology.

Progeria syndromes are very rare, incurable premature aging conditions recapitulating most aging features. Here, we report the first whole genome, multiparametric CRISPR anti-aging screen, identifying 43 new genes that can reverse multiple aging phenotypes in progeria. The screen was implemented in fibroblasts from Néstor- Guillermo Progeria Syndrome (NGPS) patients, carrying a homozygous p.Ala12Thr mutation in barrier-to-autointegration factor (BAF A12T). The hits were enriched for genes involved in protein translation, protein and RNA transport and osteoclast formation. We further confirmed that BAF A12T drives increased protein translation and translational errors that could directly contribute to premature aging in patients. This work has highlighted the power of multiparametric whole genome synthetic rescue screens to identify new anti-aging genes and uncover novel biology behind progeria-associated cellular dysfunction.One-Sentence SummaryA whole genome multiparametric screen in progeria identifies new pathways that can reverse cellular aging phenotypes.

Aging is the single largest risk factor for most chronic diseases, and thus possesses large socioeconomic interest to continuously aging societies. Consequently, the field of aging research is expanding alongside a growing focus from the industry and investors in aging research. This year's 8th Annual Aging Research and Drug Discovery (ARDD) meeting was organized as a hybrid meeting from August 30th to September 3rd 2021 with more than 130 attendees participating on-site at the Ceremonial Hall at University of Copenhagen, Denmark, and 1800 engaging online. The conference comprised of presentations from 75 speakers focusing on new research in topics including mechanisms of aging and how these can be modulated as well as the use of AI and new standards of practices within aging research. This year, a longevity workshop was included to build stronger connections with the clinical community., Aging, 14 (2), ISSN:1945-4589

Chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) has become one of the most popular methods to study protein-DNA interactions and can be used, for instance, to identify the binding sites of transcription factors or to determine the distributions of histones with specific post-translational modifications throughout the genome. Although standard ChIP-seq protocols work well in most experimental systems, there are exceptions, and one of these is the popular model organism Caenorhabditis elegans. Even though this system is very amenable to genetic and cytological methods, biochemical approaches are challenging. This is due to both the animals' cuticle, which impairs lysis as well as penetration by cross-linkers, and the rather low protein and chromatin content per body weight. These issues have rendered standard ChIP-seq protocols inefficient in C. elegans and raised a need for their improvement. Here, we describe improved protocols, with the most important advances being the efficient breakage of the C. elegans cuticle by freeze-grinding and the use of a very sensitive sequencing library construction procedure, optimized for the relatively low DNA content per body weight of C. elegans. The protocols should therefore improve the reproducibility, sensitivity, and uniformity across tissues of ChIP-seq in this organism. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Growth and harvesting of synchronized Caenorhabditis elegans Basic Protocol 2: Chromatin immunoprecipitation (ChIP) Basic Protocol 3: Library construction for Illumina sequencing.

Objective The loss of forkhead box protein O1 (FoxO1) signaling in response to metabolic stress contributes to the etiology of type II diabetes, causing the dedifferentiation of pancreatic beta cells to a cell type reminiscent of endocrine progenitors. Lack of methods to easily model this process in vitro, however, have hindered progress into the identification of key downstream targets and potential inhibitors. We therefore aimed to establish such an in vitro cellular dedifferentiation model and apply it to identify novel agents involved in the maintenance of beta-cell identity. Methods The murine beta-cell line, Min6, was used for primary experiments and high-content screening. Screens encompassed a library of small-molecule drugs representing the chemical and target space of all FDA-approved small molecules with an automated immunofluorescence readout. Validation experiments were performed in a murine alpha-cell line as well as in primary murine and human diabetic islets. Developmental effects were studied in zebrafish and C. elegans models, while diabetic db/db mouse models were used to elucidate global glucose metabolism outcomes. Results We show that short-term pharmacological FoxO1 inhibition can model beta-cell dedifferentiation by downregulating beta-cell-specific transcription factors, resulting in the aberrant expression of progenitor genes and the alpha-cell marker glucagon. From a high-content screen, we identified loperamide as a small molecule that can prevent FoxO inhibitor-induced glucagon expression and further stimulate insulin protein processing and secretion by altering calcium levels, intracellular pH, and FoxO1 localization. Conclusions Our study provides novel models, molecular targets, and drug candidates for studying and preventing beta-cell dedifferentiation., Highlights • Pharmacological FoxO1 inhibition can model many aspects of beta cell dedifferentiation in vitro, including loss of beta cell identity and gain of progenitor and alpha cell markers. • Through a high content screen with this cellular dedifferentiation model, we identified the FDA-approved small molecule, loperamide, to counter FoxO inhibitor-mediated beta cell dedifferentiation. • Acute loperamide treatment increases FoxO1 nuclear translocation and insulin granule maturation through modulation of intracellular pH and free calcium levels. • Independently of pharmacological FoxO1 inhibition, loperamide treatment increases insulin secretion, protein and mRNA levels in human and murine diabetic pancreatic islets and strongly reduces the number of glucagon/insulin double positive cells in diabetic db/db mice.

Histones are the major proteinaceous component of chromatin in eukaryotic cells and an important part of the epigenome, affecting most DNA‐related events, including transcription, DNA replication, and chromosome segregation. The properties of histones are greatly influenced by their post‐translational modifications (PTMs), over 200 of which are known today. Given this large number, researchers need sophisticated methods to study histone PTMs comprehensively. In particular, mass spectrometry (MS)−based approaches have gained popularity, allowing for the quantification of dozens of histone PTMs at once. Using these approaches, even the study of co‐occurring PTMs and the discovery of novel PTMs become feasible. The success of MS‐based approaches relies substantially on obtaining pure and well‐preserved histones for analysis, which can be difficult depending on the source material. Caenorhabditis elegans has been a popular model organism to study the epigenome, but isolation of pure histones from these animals has been challenging. Here, we address this issue, presenting a method for efficient isolation of pure histone proteins from C. elegans at good yield. Further, we describe an MS pipeline optimized for accurate relative quantification of histone PTMs from C. elegans. We alkylate and tryptically digest the histones, analyze them by bottom‐up MS, and then evaluate the resulting data by a C. elegans−adapted version of the software EpiProfile 2.0. Finally, we show the utility of this pipeline by determining differences in histone PTMs between C. elegans strains that age at different rates and thereby achieve very different lifespans. © 2020 The Authors. Basic Protocol 1: Large‐scale growth and harvesting of synchronized C. elegans Basic Protocol 2: Nuclear preparation, histone extraction, and histone purification Basic Protocol 3: Bottom‐up mass spectrometry analysis of histone PTMs and histone variants

Aging is emerging as a druggable target with growing interest from academia, industry and investors. New technologies such as artificial intelligence and advanced screening techniques, as well as a strong influence from the industry sector may lead to novel discoveries to treat age-related diseases. The present review summarizes presentations from the 7th Annual Aging Research and Drug Discovery (ARDD) meeting, held online on the 1st to 4th of September 2020. The meeting covered topics related to new methodologies to study aging, knowledge about basic mechanisms of longevity, latest interventional strategies to target the aging process as well as discussions about the impact of aging research on society and economy. More than 2000 participants and 65 speakers joined the meeting and we already look forward to an even larger meeting next year. Please mark your calendars for the 8th ARDD meeting that is scheduled for the 31st of August to 3rd of September, 2021, at Columbia University, USA., Aging, 12 (24), ISSN:1945-4589

Aging is a complex phenomenon, where damage accumulation, increasing deregulation of biological pathways, and loss of cellular homeostasis lead to the decline of organismal functions over time. Interestingly, aging is not entirely a stochastic process and progressing at a constant rate, but it is subject to extensive regulation, in the hands of an elaborate and highly interconnected signaling network. This network can integrate a variety of aging-regulatory stimuli, i.e. fertility, nutrient availability, or diverse stresses, and relay them via signaling cascades into gene regulatory events - mostly of genes that confer stress resistance and thus help protect from damage accumulation and homeostasis loss. Transcription factors have long been perceived as the pivotal nodes in this network. Yet, it is well known that the epigenome substantially influences eukaryotic gene regulation, too. A growing body of work has recently underscored the importance of the epigenome also during aging, where it not only undergoes drastic age-dependent changes but also actively influences the aging process. In this review, we introduce the major signaling pathways that regulate age-related decline and discuss the synergy between transcriptional regulation and the epigenetic landscape.

SummaryAging is a major risk factor for human morbidity and mortality. Thus, the identification of compounds that defer aging, also known as ‘geroprotectors’, could greatly improve our health and promote a longer life. Here we screened for geroprotectors, employing the power of human transcriptomics to predict biological age. We used age-stratified human tissue transcriptomes to generate machine-learning-based classifiers capable of distinguishing transcriptomes from young versus old individuals. Then we applied these classifiers to transcriptomes induced by 1300 different compounds in human cell lines and ranked these compounds by their ability to induce a ‘youthful’ transcriptional state. Besides known geroprotectors, several new candidate compounds emerged from this ranking. Testing these in the model organismC. elegans, we identified two Hsp90 inhibitors, Monorden and Tanespimycin, which substantially extended the animals’ lifespan and improved their health. Hsp90 inhibition specifically induces the expression of heat shock proteins, known to improve protein homeostasis. Consistently, Monorden treatment improved the survival ofC. elegansunder proteotoxic stress, and its lifespan benefits were fully dependent on the master regulator of the cytosolic unfolded protein response, the transcription factor HSF-1. Taken together, we present an innovative transcriptomics-based screening approach to discover aging-preventive compounds and highlight Hsp90 inhibitors as powerful geroprotectors that could be of great value, to target the aging process in humans.

Aging strongly influences human morbidity and mortality. Thus, aging-preventive compounds could greatly improve our health and lifespan. Here we screened for such compounds, known as geroprotectors, employing the power of transcriptomics to predict biological age. Using age-stratified human tissue transcriptomes and machine learning, we generated age classifiers and applied these to transcriptomic changes induced by 1,309 different compounds in human cells, ranking these compounds by their ability to induce a "youthful" transcriptional state. Testing the top candidates in C. elegans, we identified two Hsp90 inhibitors, monorden and tanespimycin, which extended the animals' lifespan and improved their health. Hsp90 inhibition induces expression of heat shock proteins known to improve protein homeostasis. Consistently, monorden treatment improved the survival of C. elegans under proteotoxic stress, and its benefits depended on the cytosolic unfolded protein response-inducing transcription factor HSF-1. Taken together, our method represents an innovative geroprotector screening approach and was able to identify a class that acts by improving protein homeostasis. N.S. was supported by funding from the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership, the Centro de Excelencia Severo Ochoa, the CERCA Programme/Generalitat de Catalunya, and an award from the Glenn Foundation for Medical Research. E.A.A.N. was supported by the European Research Council (ERC) and the alumni chapter of Gooische Groningers facilitated by Ubbo Emmius Fonds. C.G.R. was supported by the Swedish Research Council (VR) grant 2015-03740, the COST grant BM1408 (GENiE), and an ICMC project grant

The ability to perceive and respond to harmful conditions is crucial for the survival of any organism. The transcription factor DAF-16/FOXO is central to these responses, relaying distress signals into the expression of stress resistance and longevity promoting genes. However, its sufficiency in fulfilling this complex task has remained unclear. Using C. elegans, we show that DAF-16 does not function alone but as part of a transcriptional regulatory module, together with the transcription factor HLH-30/TFEB. Under harmful conditions, both transcription factors translocate into the nucleus, where they often form a complex, co-occupy target promoters, and co-regulate many target genes. Interestingly though, their synergy is stimulus-dependent: They rely on each other, functioning in the same pathway, to promote longevity or resistance to oxidative stress, but they elicit heat stress responses independently, and they even oppose each other during dauer formation. We propose that this module of DAF-16 and HLH-30 acts by combinatorial gene regulation to relay distress signals into the expression of specific target gene sets, ensuring optimal survival under each given threat., The transcription factor DAF-16/FOXO is a downstream effector of insulin/insulin-like growth factor signaling and plays an important role in stress resistance and longevity. Here, the authors show that DAF-16/FOXO can form a complex with HLH-30/TFEB to synergistically regulate transcription of target genes in response to certain stress stimuli.

The DNA contains all the genetic information necessary to create a living organism. However, in eukaryotic cells large parts of the DNA are tightly packaged into chromatin structures inaccessible to transcription factors and other DNA-interacting proteins. This packaging, and its controlled opening to expose otherwise occluded DNA sequences, provides an important mean to regulate access to this genetic information. ATP-dependent chromatin remodelers have evolved to carry out this function. They bind to the smallest DNA packaging units, the nucleosomes, and utilize the energy derived from the hydrolysis of ATP to reposition them, eject them, or influence their assembly and composition. The action of chromatin remodeling enzymes can thus either increase or decrease the accessibility of the DNA to factors that regulate nuclear functions, thereby exerting control over subsequent events, in particular transcription, DNA replication, or DNA repair. This chapter reviews the four main families of ATP-dependent chromatin remodelers and their role in the biological processes of development, cancer, and aging.

Organisms are constantly challenged by stresses and privations and require adaptive responses for their survival. The forkhead box O (FOXO) transcription factor DAF-16 (hereafter referred to as DAF-16/FOXO) is a central nexus in these responses, but despite its importance little is known about how it regulates its target genes. Proteomic identification of DAF-16/FOXO-binding partners in Caenorhabditis elegans and their subsequent functional evaluation by RNA interference revealed several candidate DAF-16/FOXO cofactors, most notably the chromatin remodeller SWI/SNF. DAF-16/FOXO and SWI/SNF form a complex and globally co-localize at DAF-16/FOXO target promoters. We show that specifically for gene activation, DAF-16/FOXO depends on SWI/SNF, facilitating SWI/SNF recruitment to target promoters, to activate transcription by presumed remodelling of local chromatin. For the animal, this translates into an essential role for SWI/SNF in DAF-16/FOXO-mediated processes, in particular dauer formation, stress resistance and the promotion of longevity. Thus, we give insight into the mechanisms of DAF-16/FOXO-mediated transcriptional regulation and establish a critical link between ATP-dependent chromatin remodelling and lifespan regulation.

Summary Background In nematodes, plants, and fungi, RNAi is remarkably potent and persistent due to the amplification of initial silencing signals by RNA-dependent RNA polymerases (RdRPs). In Caenorhabditis elegans ( C. elegans ), the interaction between the RNA-induced silencing complex (RISC) loaded with primary small interfering RNAs (siRNAs) and the target messenger RNA (mRNA) leads to the recruitment of RdRPs and synthesis of secondary siRNAs using the target mRNA as the template. The mechanism and genetic requirements for secondary siRNA accumulation are not well understood. Results From a forward genetic screen for C. elegans genes required for RNAi, we identified rde-10 , and through proteomic analysis of RDE-10-interacting proteins, we identified a protein complex containing the new RNAi factor RDE-11, the known RNAi factors RSD-2 and ERGO-1, and other candidate RNAi factors. The RNAi defective genes rde-10 and rde-11 encode a novel protein and a RING-type zinc finger domain protein, respectively. Mutations in rde-10 and rde-11 genes cause dosage-sensitive RNAi deficiencies: these mutants are resistant to low dosage but sensitive to high dosage of double-stranded RNAs. We assessed the roles of rde-10 , rde-11 , and other dosage-sensitive RNAi-defective genes rsd-2 , rsd-6 , and haf-6 in both exogenous and endogenous small RNA pathways using high-throughput sequencing and qRT-PCR. These genes are required for the accumulation of secondary siRNAs in both exogenous and endogenous RNAi pathways. Conclusions The RDE-10/RDE-11 complex is essential for the amplification of RNAi in C. elegans by promoting secondary siRNA accumulation.

Hundreds of microRNAs (miRNAs) have been discovered in metazoans and plants, and understanding of their biogenesis has advanced dramatically; however, relatively little is known about the cofactors necessary for miRNA regulation of target gene expression. In Caenorhabditis elegans, the conserved miRNA let-7 and its paralogs, including mir-84, control the timing of stage-specific developmental events. To identify factors required for the activity of mir-84 and possibly other miRNAs, we screened for mutations that suppress the developmental defects caused by overexpression of mir-84. Mutations in the somi-1 gene prevent these defects without affecting the expression level of mir-84. Loss of somi-1 also causes phenotypes similar to deletion of mir-84, showing that somi-1 is necessary for the normal function of this miRNA. somi-1 encodes a zinc finger protein that localizes to nuclear foci and binds the promoters of let-60/RAS, lin-14, and lin-28, genes that may be targeted by mir-84 and similar miRNAs. Genetic evidence shows that somi-1 inhibits lin-14 and induction of the vulval precursors by the let-60/RAS pathway. Proteomic and genetic screens identified conserved chromatin-remodeling and homeodomain transcription factor complexes that work with somi-1 to regulate differentiation. Our results suggest that somi-1 coordinates a nuclear response that complements the activity of mir-84.

Segregation of chromosomes during meiosis depends on separase cleavage of Rec8, the meiosis-specific alpha-kleisin subunit of cohesin. We mapped Rec8 phosphorylation sites by mass spectrometry and show that, in fission yeast, Rec8 phosphorylation is required for proper chromosome disjunction during meiosis. We further show that the fission yeast casein kinase 1 (CK1) delta/epsilon isoforms Hhp1 and Hhp2 are required for full levels of Rec8 phosphorylation and for efficient removal of Rec8 at the onset of anaphase I. Our data are consistent with the model that Hhp1/Hhp2-dependent phosphorylation of Rec8 is required for separase-mediated cleavage of Rec8 during meiosis I.

Unlike the soma, which ages during the lifespan of multicellular organisms, the germ line traces an essentially immortal lineage. Genomic instability in somatic cells increases with age, and this decline in somatic maintenance might be regulated to facilitate resource reallocation towards reproduction at the expense of cellular senescence. Here we show that Caenorhabditis elegans mutants with increased longevity exhibit a soma-to-germline transformation of gene expression programs normally limited to the germ line. Decreased insulin-like signalling causes the somatic misexpression of the germline-limited pie-1 and pgl family of genes in intestinal and ectodermal tissues. The forkhead boxO1A (FOXO) transcription factor DAF-16, the major transcriptional effector of insulin-like signalling, regulates pie-1 expression by directly binding to the pie-1 promoter. The somatic tissues of insulin-like mutants are more germline-like and protected from genotoxic stress. Gene inactivation of components of the cytosolic chaperonin complex that induce increased longevity also causes somatic misexpression of PGL-1. These results indicate that the acquisition of germline characteristics by the somatic cells of C. elegans mutants with increased longevity contributes to their increased health and survival.

Tandem affinity purification (TAP) is a generic two-step affinity purification protocol for isolation of TAP-tagged proteins together with associated proteins. We used bacterial artificial chromosome to heterologously express TAP-tagged murine Sgo1 protein in human HeLa cells. This allowed us to test the functionality of the Sgo1-TAP protein by RNA interference-mediated depletion of the endogenous human Sgo1. Here, we present an optimized protocol for purification of TAP-tagged Sgo1 protein as well as KIAA1387 from HeLa cells with detailed instructions. The purification protocol can be completed in 1 day and it should be applicable to other proteins.

During sporulation and meiosis of budding yeast a developmental program determines the formation of the new plasma membranes of the spores. This process of prospore membrane (PSM) formation leads to the formation of meiotic daughter cells, the spores, within the lumen of the mother cell. It is initiated at the spindle pole bodies during meiosis II. Spore formation, but not meiotic cell cycle progression, requires the function of phospholipase D (PLD/Spo14). Here we show that PLD/Spo14 forms a complex with Sma1, a meiotically expressed protein essential for spore formation. Detailed analysis revealed that both proteins are required for early steps of prospore membrane assembly but with distinct defects in the respective mutants. In the Deltaspo14 mutant the initiation of PSM formation is blocked and aggregated vesicles of homogenous size are detected at the spindle pole bodies. In contrast, initiation of PSM formation does occur in the Deltasma1 mutant, but the enlargement of the membrane is impaired. During PSM growth both Spo14 and Sma1 localize to the membrane, and localization of Spo14 is independent of Sma1. Biochemical analysis revealed that Sma1 is not necessary for PLD activity per se and that PLD present in a complex with Sma1 is highly active. Together, our results suggest that yeast PLD is involved in two distinct but essential steps during the regulated vesicle fusion necessary for the assembly of the membranous encapsulations of the spores.

Chromosome segregation during mitosis and meiosis depends on the linkage of sister DNA molecules after replication. These links, known as sister-chromatid cohesion, are provided by a multi-subunit complex called cohesin. Recent papers suggest that chromatin-remodeling complexes also have a role in the generation of sister-chromatid cohesion. It remains unclear whether they do so by facilitating the recruitment of cohesin to specific chromosomal sequences or by modifying an event at replication forks giving rise to cohesion between sister DNAs.

One of the major determinants of aging in organisms ranging from worms to man are FOXO family transcription factors, which are downstream effectors of Insulin/IGF-1 signaling (IIS). The molecular mechanisms that actively promote DAF16/FOXO stability and function are unknown. Here we identify the deubiquitylating enzyme MATH-33 as an essential DAF-16 regulator in IIS, which stabilizes active DAF-16 protein levels and, as a consequence, influences DAF-16 functions, such as metabolism, stress response and longevity in C. elegans. MATH-33 associates with DAF-16 in cellulo and in vitro. MATH-33 functions as a deubiquitylase by actively removing ubiquitin moieties from DAF-16, thus counteracting the action of the RLE-1 E3-ubiquitin ligase. Our findings support a model in which MATH-33 promotes DAF-16 stability in response to decreased IIS by directly modulating its ubiquitylation state, suggesting that regulated oscillations in the stability of DAF-16 protein play an integral role in controlling processes such as metabolism and longevity.

Segregation of homologous maternal and paternal centromeres to opposite poles during meiosis I depends on post-replicative crossing over between homologous non-sister chromatids, which creates chiasmata and therefore bivalent chromosomes. Destruction of sister chromatid cohesion along chromosome arms due to proteolytic cleavage of cohesin's Rec8 subunit by separase resolves chiasmata and thereby triggers the first meiotic division. This produces univalent chromosomes, the chromatids of which are held together by centromeric cohesin that has been protected from separase by shugoshin (Sgo1/MEI-S332) proteins. Here we show in both fission and budding yeast that Sgo1 recruits to centromeres a specific form of protein phosphatase 2A (PP2A). Its inactivation causes loss of centromeric cohesin at anaphase I and random segregation of sister centromeres at the second meiotic division. Artificial recruitment of PP2A to chromosome arms prevents Rec8 phosphorylation and hinders resolution of chiasmata. Our data are consistent with the notion that efficient cleavage of Rec8 requires phosphorylation of cohesin and that this is blocked by PP2A at meiosis I centromeres.

The conserved DAF-16/FOXO transcription factors and SIR-2.1/SIRT1 deacetylases are critical for diverse biological processes, particularly longevity and stress response; and complex regulation of DAF-16/FOXO by SIR-2.1/SIRT1 is central to appropriate biological outcomes. Caenorhabditis elegans Host Cell Factor 1 (HCF-1) is a longevity determinant previously shown to act as a co-repressor of DAF-16. We report here that HCF-1 represents an integral player in the regulatory loop linking SIR-2.1/SIRT1 and DAF-16/FOXO in both worms and mammals. Genetic analyses showed that hcf-1 acts downstream of sir-2.1 to influence lifespan and oxidative stress response in C. elegans. Gene expression profiling revealed a striking 80% overlap between the DAF-16 target genes responsive to hcf-1 mutation and sir-2.1 overexpression. Subsequent GO-term analyses of HCF-1 and SIR-2.1-coregulated DAF-16 targets suggested that HCF-1 and SIR-2.1 together regulate specific aspects of DAF-16-mediated transcription particularly important for aging and stress responses. Analogous to its role in regulating DAF-16/SIR-2.1 target genes in C. elegans, the mammalian HCF-1 also repressed the expression of several FOXO/SIRT1 target genes. Protein–protein association studies demonstrated that SIR-2.1/SIRT1 and HCF-1 form protein complexes in worms and mammalian cells, highlighting the conservation of their regulatory relationship. Our findings uncover a conserved interaction between the key longevity determinants SIR-2.1/SIRT1 and HCF-1, and they provide new insights into the complex regulation of FOXO proteins., Author Summary The nematode C. elegans has been instrumental in identifying and characterizing genetic components that influence aging. Studies in worms have been successfully extended to complex mammalian organisms allowing for the identification of genetic factors that impact longevity in mammals. DAF-16/FOXO transcription factors are among the best characterized longevity factors, and their increased activity leads to a longer lifespan and improved stress resistance in many organisms. Elucidating how the activities of DAF-16/FOXO are regulated will provide new insights into the basic biology of aging and will aid future therapeutic developments aiming to improve healthy aging and alleviate age-related diseases in humans. We utilized both C. elegans and mammalian cell culture systems to dissect the functional and molecular interactions between two important DAF-16 regulators, HCF-1 and SIR-2.1/SIRT1. We demonstrated that HCF-1 and SIR-2.1/SIRT1 physically associate and antagonize each other to properly regulate DAF-16/FOXO-mediated expression of genes important for longevity and stress response. We further showed that the functional relationships among these three proteins are conserved in mammals. Our work implicates HCF-1 as an important player in the regulation of FOXO by SIRT1, and thereby a potential longevity determinant in humans, and prompts further characterization of HCF-1's functions in aging and age-related pathologies.

Summary Background Accurate chromosome segregation depends on the establishment of correct—amphitelic—kinetochore orientation. Merotelic kinetochore orientation is an error that occurs when a single kinetochore attaches to microtubules emanating from opposite spindle poles, a condition that hinders segregation of the kinetochore to a spindle pole in anaphase. To avoid chromosome missegregation resulting from merotelic kinetochore orientation, cells have developed mechanisms to prevent or correct merotelic attachment. A protein called Pcs1 has been implicated in preventing merotelic attachment in mitosis and meiosis II in the fission yeast S. pombe . Results We report that Pcs1 forms a complex with a protein called Mde4. Both Pcs1 and Mde4 localize to the central core of centromeres. Deletion of mde4 + , like that of pcs1 + , causes the appearance of lagging chromosomes during the anaphases of mitotic and meiosis II cells. We provide evidence that the kinetochores of lagging chromosomes in both pcs1 and mde4 mutant cells are merotelically attached. In addition, we find that lagging chromosomes in cells with defective centromeric heterochromatin also display features consistent with merotelic attachment. Conclusions We suggest that the Pcs1/Mde4 complex is the fission yeast counterpart of the budding yeast monopolin subcomplex Csm1/Lrs4, which promotes the segregation of sister kinetochores to the same pole during meiosis I. We propose that the Pcs1/Mde4 complex acts in the central kinetochore domain to clamp microtubule binding sites together, the centromeric heterochromatin coating the flanking domains provides rigidity, and both systems contribute to the prevention of merotelic attachment.

GFP has established itself as a highly useful tool throughout many areas of modern biology. Recently, the novel fluorescent protein drFP583, also termed DsRed or RFP, was cloned from a coral of the Discosoma genus. The protein is only weakly homologous to GFP and has a red emission spectrum, which makes drFP583 an attractive candidate for in vivo double labeling together with GFP variants. However, wildtype drFP583 has several drawbacks, including inefficient folding of the protein, extremely slow maturation of the chromophore, and tetramerization even in dilute solutions. Here we report on important improvements to this reporter that lead to higher levels of fluorescent drFP583 species in the cell. We further characterized our best mutant for applications in yeast and mammalian cell biology.