In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Calcium phosphate (CaP) particle deposits are found in several inflammatory diseases including atherosclerosis and osteoarthritis. CaP, and other forms of crystals and particles, can promote inflammasome formation in macrophages leading to caspase-1 activation and secretion of mature interleukin-1β (IL-1β). Given the close association of small CaP particles with vascular smooth muscle cells (VSMCs) in atherosclerotic fibrous caps, we aimed to determine if CaP particles affected pro-inflammatory signalling in human VSMCs.

The class I phosphoinositide 3-kinase (PI3K) isoforms play important roles in platelet priming, activation, and stable thrombus formation. Class I PI3Ks predominantly regulate cell function through their catalytic product, the signaling phospholipid phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3], which coordinates the localization and/or activity of a diverse range of binding proteins. Notably, the complete repertoire of these class I PI3K effectors in platelets remains unknown, limiting mechanistic understanding of class I PI3K-mediated control of platelet function. We measured robust agonist-driven PtdIns (3,4,5)P3 generation in human platelets by lipidomic mass spectrometry (MS), and then used affinity-capture coupled to high-resolution proteomic MS to identify the targets of PtdIns (3,4,5)P3 in these cells. We reveal for the first time a diverse platelet PtdIns(3,4,5)P3 interactome, including kinases, signaling adaptors, and regulators of small GTPases, many of which are previously uncharacterized in this cell type. Of these, we show dual adaptor for phosphotyrosine and 3-phosphoinositides (DAPP1) to be regulated by Src-family kinases and PI3K, while platelets from DAPP1-deficient mice display enhanced thrombus formation on collagen in vitro. This was associated with enhanced platelet α/δ granule secretion and αIIbβ3 integrin activation downstream of the collagen receptor glycoprotein VI. Thus, we present the first comprehensive analysis of the PtdIns(3,4,5)P3 signalosome of human platelets and identify DAPP1 as a novel negative regulator of platelet function. This work provides important new insights into how class I PI3Ks shape platelet function.

In cycling human endometrium, menstruation is followed by rapid estrogen-dependent growth. Upon ovulation, progesterone and rising cellular cAMP levels activate the transcription factor Forkhead box O1 (FOXO1) in endometrial stromal cells (EnSCs), leading to cell cycle exit and differentiation into decidual cells that control embryo implantation. Here we show that FOXO1 also causes acute senescence of a subpopulation of decidualizing EnSCs in an IL-8 dependent manner. Selective depletion or enrichment of this subpopulation revealed that decidual senescence drives the transient inflammatory response associated with endometrial receptivity. Further, senescent cells prevent differentiation of endometrial mesenchymal stem cells in decidualizing cultures. As the cycle progresses, IL-15 activated uterine natural killer (uNK) cells selectively target and clear senescent decidual cells through granule exocytosis. Our findings reveal that acute decidual senescence governs endometrial rejuvenation and remodeling at embryo implantation, and suggest a critical role for uNK cells in maintaining homeostasis in cycling endometrium.

Mammalian genomes are spatially organized into compartments, topologically associating domains (TADs), and loops to facilitate gene regulation and other chromosomal functions. How compartments, TADs, and loops are generated is unknown. It has been proposed that cohesin forms TADs and loops by extruding chromatin loops until it encounters CTCF, but direct evidence for this hypothesis is missing. Here, we show that cohesin suppresses compartments but is required for TADs and loops, that CTCF defines their boundaries, and that the cohesin unloading factor WAPL and its PDS5 binding partners control the length of loops. In the absence of WAPL and PDS5 proteins, cohesin forms extended loops, presumably by passing CTCF sites, accumulates in axial chromosomal positions (vermicelli), and condenses chromosomes. Unexpectedly, PDS5 proteins are also required for boundary function. These results show that cohesin has an essential genome-wide function in mediating long-range chromatin interactions and support the hypothesis that cohesin creates these by loop extrusion, until it is delayed by CTCF in a manner dependent on PDS5 proteins, or until it is released from DNA by WAPL.

The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body. The Human Cell Atlas Project is an international collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles (such as gene expression profiles) and to connect this information with classical cellular descriptions (such as location and morphology). An open comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas, including a commitment to open data, code, and community.

V(D)J recombination is essential for the generation of diverse antigen receptor (AgR) repertoires. In B cells, immunoglobulin kappa (Igκ) light chain recombination follows immunoglobulin heavy chain (Igh) recombination. We recently developed the DNA-based VDJ-seq assay for the unbiased quantitation of Igh VH and DH repertoires. Integration of VDJ-seq data with genome-wide datasets revealed that two chromatin states at the recombination signal sequence (RSS) of VH genes are highly predictive of recombination in mouse pro-B cells. It is unknown whether local chromatin states contribute to Vκ gene choice during Igκ recombination. Here we adapt VDJ-seq to profile the Igκ VκJκ repertoire and present a comprehensive readout in mouse pre-B cells, revealing highly variable Vκ gene usage. Integration with genome-wide datasets for histone modifications, DNase hypersensitivity, transcription factor binding and germline transcription identified PU.1 binding at the RSS, which was unimportant for Igh, as highly predictive of whether a Vκ gene will recombine or not, suggesting that it plays a binary, all-or-nothing role, priming genes for recombination. Thereafter, the frequency with which these genes recombine was shaped both by the presence and level of enrichment of several other chromatin features, including H3K4 methylation and IKAROS binding. Moreover, in contrast to the Igh locus, the chromatin landscape of the promoter, as well as of the RSS, contributes to Vκ gene recombination. Thus, multiple facets of local chromatin features explain much of the variation in Vκ gene usage. Together, these findings reveal shared and divergent roles for epigenetic features and transcription factors in AgR V(D)J recombination and provide avenues for further investigation of chromatin signatures that may underpin V(D)J-mediated chromosomal translocations.

Blood coagulation functions as part of the innate immune system by preventing bacterial invasion, and it is critical to stopping blood loss (hemostasis). Coagulation involves the external membrane surface of activated platelets and leukocytes. Using lipidomic, genetic, biochemical, and mathematical modeling approaches, we found that enzymatically oxidized phospholipids (eoxPLs) generated by the activity of leukocyte or platelet lipoxygenases (LOXs) were required for normal hemostasis and promoted coagulation factor activities in a Ca- and phosphatidylserine (PS)-dependent manner. In wild-type mice, hydroxyeicosatetraenoic acid-phospholipids (HETE-PLs) enhanced coagulation and restored normal hemostasis in clotting-deficient animals genetically lacking p12-LOX or 12/15-LOX activity. Murine platelets generated 22 eoxPL species, all of which were missing in the absence of p12-LOX. Humans with the thrombotic disorder antiphospholipid syndrome (APS) had statistically significantly increased HETE-PLs in platelets and leukocytes, as well as greater HETE-PL immunoreactivity, than healthy controls. HETE-PLs enhanced membrane binding of the serum protein β2GP1 (β2-glycoprotein 1), an event considered central to the autoimmune reactivity responsible for APS symptoms. Correlation network analysis of 47 platelet eoxPL species in platelets from APS and control subjects identified their enzymatic origin and revealed a complex network of regulation, with the abundance of 31 p12-LOX-derived eoxPL molecules substantially increased in APS. In summary, circulating blood cells generate networks of eoxPL molecules, including HETE-PLs, which change membrane properties to enhance blood coagulation and contribute to the excessive clotting and immunoreactivity of patients with APS.

Migration of activated regulatory T (Treg) cells to inflamed tissue is crucial for their immune-modulatory function. While metabolic reprogramming during Treg cell differentiation has been extensively studied, the bioenergetics of Treg cell trafficking remains undefined. We have investigated the metabolic demands of migrating Treg cells in vitro and in vivo. We show that glycolysis was instrumental for their migration and was initiated by pro-migratory stimuli via a PI3K-mTORC2-mediated pathway culminating in induction of the enzyme glucokinase (GCK). Subsequently, GCK promoted cytoskeletal rearrangements by associating with actin. Treg cells lacking this pathway were functionally suppressive but failed to migrate to skin allografts and inhibit rejection. Similarly, human carriers of a loss-of-function GCK regulatory protein gene-leading to increased GCK activity-had reduced numbers of circulating Treg cells. These cells displayed enhanced migratory activity but similar suppressive function, while conventional T cells were unaffected. Thus, GCK-dependent glycolysis regulates Treg cell migration.

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 CDKN1A 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(INK4a), p21(CIP1), and p15(INK4b), 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.

There is limited knowledge about the metabolic reprogramming induced by cancer therapies and how this contributes to therapeutic resistance. Here we show that although inhibition of PI3K-AKT-mTOR signaling markedly decreased glycolysis and restrained tumor growth, these signaling and metabolic restrictions triggered autophagy, which supplied the metabolites required for the maintenance of mitochondrial respiration and redox homeostasis. Specifically, we found that survival of cancer cells was critically dependent on phospholipase A2 (PLA2) to mobilize lysophospholipids and free fatty acids to sustain fatty acid oxidation and oxidative phosphorylation. Consistent with this, we observed significantly increased lipid droplets, with subsequent mobilization to mitochondria. These changes were abrogated in cells deficient for the essential autophagy gene Accordingly, inhibition of PLA2 significantly decreased lipid droplets, decreased oxidative phosphorylation, and increased apoptosis. Together, these results describe how treatment-induced autophagy provides nutrients for cancer cell survival and identifies novel cotreatment strategies to override this survival advantage.

Pancreatic cancer is a high mortality form of cancer, with a median survival only six months. There are multiple associated risk factors associated, most importantly type 2 diabetes, obesity, pancreatitis and smoking. The relative rarity of the disease, however, has made it difficult to dissect causative risk factors, especially with related risk factors. A major unanswered question with important therapeutic implications is the effect of immunological responses on pancreatic cancer formation, with data from other cancers suggesting the potential for local immunological responses to either increase cancer development or increase cancer elimination. Due to the rarity and late diagnosis of pancreatic cancer direct epidemiological evidence is lacking, thus necessitating a reliance on animal models. Here we investigated the relationship between pancreatic autoimmunity and cancer by backcrossing the well characterised Ela1-Tag transgenic model of pancreatic cancer onto the pancreatic autoimmune susceptible NOD mouse strain. Through longitudinal magnetic resonance imaging we found that the NOD genetic background delayed the onset of pancreatic tumours and substantially slowed the growth rate of tumours after development. These results suggest that elevated autoimmune surveillance of the pancreas limits tumour formation and growth, identifying pancreatic cancer as a promising target for immune checkpoint blockade therapies that unleash latent autoimmunity.

BioModels serves as a central repository of mathematical models representing biological processes. It offers a platform to make mathematical models easily shareable across the systems modelling community, thereby supporting model reuse. To facilitate hosting a broader range of model formats derived from diverse modelling approaches and tools, a new infrastructure for BioModels has been developed that is available at http://www.ebi.ac.uk/biomodels. This new system allows submitting and sharing of a wide range of models with improved support for formats other than SBML. It also offers a version-control backed environment in which authors and curators can work collaboratively to curate models. This article summarises the features available in the current system and discusses the potential benefit they offer to the users over the previous system. In summary, the new portal broadens the scope of models accepted in BioModels and supports collaborative model curation which is crucial for model reproducibility and sharing.

Erasure of DNA methylation and repressive chromatin marks in the mammalian germline leads to risk of transcriptional activation of transposable elements (TEs). Here, we used mouse embryonic stem cells (ESCs) to identify an endosiRNA-based mechanism involved in suppression of TE transcription. In ESCs with DNA demethylation induced by acute deletion of Dnmt1, we saw an increase in sense transcription at TEs, resulting in an abundance of sense/antisense transcripts leading to high levels of ARGONAUTE2 (AGO2)-bound small RNAs. Inhibition of Dicer or Ago2 expression revealed that small RNAs are involved in an immediate response to demethylation-induced transposon activation, while the deposition of repressive histone marks follows as a chronic response. In vivo, we also found TE-specific endosiRNAs present during primordial germ cell development. Our results suggest that antisense TE transcription is a "trap" that elicits an endosiRNA response to restrain acute transposon activity during epigenetic reprogramming in the mammalian germline.

We achieve automated and reliable annotation of lipid species and their molecular structures in high-throughput data from chromatography-coupled tandem mass spectrometry using decision rule sets embedded in Lipid Data Analyzer (LDA; http://genome.tugraz.at/lda2). Using various low- and high-resolution mass spectrometry instruments with several collision energies, we proved the method's platform independence. We propose that the software's reliability, flexibility, and ability to identify novel lipid molecular species may now render current state-of-the-art lipid libraries obsolete.

The PI3K signaling pathway regulates cell growth and movement and is heavily mutated in cancer. Class I PI3Ks synthesize the lipid messenger PI(3,4,5)P3. PI(3,4,5)P3 can be dephosphorylated by 3- or 5-phosphatases, the latter producing PI(3,4)P2. The PTEN tumor suppressor is thought to function primarily as a PI(3,4,5)P3 3-phosphatase, limiting activation of this pathway. Here we show that PTEN also functions as a PI(3,4)P2 3-phosphatase, both in vitro and in vivo. PTEN is a major PI(3,4)P2 phosphatase in Mcf10a cytosol, and loss of PTEN and INPP4B, a known PI(3,4)P2 4-phosphatase, leads to synergistic accumulation of PI(3,4)P2, which correlated with increased invadopodia in epidermal growth factor (EGF)-stimulated cells. PTEN deletion increased PI(3,4)P2 levels in a mouse model of prostate cancer, and it inversely correlated with PI(3,4)P2 levels across several EGF-stimulated prostate and breast cancer lines. These results point to a role for PI(3,4)P2 in the phenotype caused by loss-of-function mutations or deletions in PTEN.

In vitro histone modification (HM) assays are used to characterize the activity of chromatin-modifying enzymes. These assays provide information regarding the modification sites on histones, the product specificity, and the impact of other histone or nucleotide modifications on enzyme activity. In particular, histone methyltransferase (HMT) assays have been instrumental in elucidating the activity and site specificity of many plant HMT enzymes. In this chapter, we describe a general protocol that can be used to perform HMT assays using different chromatin substrates, detection methods, and enzymes directly purified from plant material or heterologous sources.

A major limitation of embryo epigenotyping by chromatin immunoprecipitation analysis is the reduced amount of sample available from an embryo biopsy. We developed an in vitro system to expand trophectoderm cells from an embryo biopsy to overcome this limitation. This work analyzes whether expanded trophectoderm (EX) is representative of the trophectoderm (TE) methylation or adaptation to culture has altered its epigenome. We took a small biopsy from the trophectoderm (30-40 cells) of in vitro produced bovine-hatched blastocysts and cultured it on fibronectin-treated plates until we obtained ∼4 × 104 cells. The rest of the embryo was allowed to recover its spherical shape and, subsequently, TE and inner cell mass were separated. We examined whether there were DNA methylation differences between TE and EX of three bovine embryos using whole-genome bisulfite sequencing. As a consequence of adaptation to culture, global methylation, including transposable elements, was higher in EX, with 5.3% of quantified regions showing significant methylation differences between TE and EX. Analysis of individual embryos indicated that TE methylation is more similar to its EX counterpart than to TE from other embryos. Interestingly, these similarly methylated regions are enriched in CpG islands, promoters and transcription units near genes involved in biological processes important for embryo development. Our results indicate that EX is representative of the embryo in terms of DNA methylation, thus providing an informative proxy for embryo epigenotyping.

DNA methylation is an important epigenetic modification in many species that is critical for development, and implicated in ageing and many complex diseases, such as cancer. Many cost-effective genome-wide analyses of DNA modifications rely on restriction enzymes capable of digesting genomic DNA at defined sequence motifs. There are hundreds of restriction enzyme families but few are used to date, because no tool is available for the systematic evaluation of restriction enzyme combinations that can enrich for certain sites of interest in a genome. Herein, we present customised Reduced Representation Bisulfite Sequencing (cuRRBS), a novel and easy-to-use computational method that solves this problem. By computing the optimal enzymatic digestions and size selection steps required, cuRRBS generalises the traditional MspI-based Reduced Representation Bisulfite Sequencing (RRBS) protocol to all restriction enzyme combinations. In addition, cuRRBS estimates the fold-reduction in sequencing costs and provides a robustness value for the personalised RRBS protocol, allowing users to tailor the protocol to their experimental needs. Moreover, we show in silico that cuRRBS-defined restriction enzymes consistently out-perform MspI digestion in many biological systems, considering both CpG and CHG contexts. Finally, we have validated the accuracy of cuRRBS predictions for single and double enzyme digestions using two independent experimental datasets.

Mouse embryonic stem cells derived from the epiblast contribute to the somatic lineages and the germline but are excluded from the extra-embryonic tissues that are derived from the trophectoderm and the primitive endoderm upon reintroduction to the blastocyst. Here we report that cultures of expanded potential stem cells can be established from individual eight-cell blastomeres, and by direct conversion of mouse embryonic stem cells and induced pluripotent stem cells. Remarkably, a single expanded potential stem cell can contribute both to the embryo proper and to the trophectoderm lineages in a chimaera assay. Bona fide trophoblast stem cell lines and extra-embryonic endoderm stem cells can be directly derived from expanded potential stem cells in vitro. Molecular analyses of the epigenome and single-cell transcriptome reveal enrichment for blastomere-specific signature and a dynamic DNA methylome in expanded potential stem cells. The generation of mouse expanded potential stem cells highlights the feasibility of establishing expanded potential stem cells for other mammalian species.