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Here we describe the LifeTime Initiative, which aims to track, understand and target human cells during the onset and progression of complex diseases, and to analyse their response to therapy at single-cell resolution. This mission will be implemented through the development, integration and application of single-cell multi-omics and imaging, artificial intelligence and patient-derived experimental disease models during the progression from health to disease. The analysis of large molecular and clinical datasets will identify molecular mechanisms, create predictive computational models of disease progression, and reveal new drug targets and therapies. The timely detection and interception of disease embedded in an ethical and patient-centred vision will be achieved through interactions across academia, hospitals, patient associations, health data management systems and industry. The application of this strategy to key medical challenges in cancer, neurological and neuropsychiatric disorders, and infectious, chronic inflammatory and cardiovascular diseases at the single-cell level will usher in cell-based interceptive medicine in Europe over the next decade. The LifeTime initiative is an ambitious, multidisciplinary programme that aims to improve healthcare by tracking individual human cells during disease processes and responses to treatment in order to develop and implement cell-based interceptive medicine in Europe., Author(s): Nikolaus Rajewsky [sup.1] [sup.2] [sup.3] [sup.4] , Geneviève Almouzni [sup.5] , Stanislaw A. Gorski [sup.1] , Stein Aerts [sup.6] [sup.7] , Ido Amit [sup.8] , Michela G. Bertero [sup.9] [...]

Two-dimensional materials and their heterostructures constitute a promising platform to study correlated electronic states, as well as the many-body physics of excitons. Transport measurements on twisted graphene bilayers have revealed a plethora of intertwined electronic phases, including Mott insulators, strange metals and superconductors.sup.1-5. However, signatures of such strong electronic correlations in optical spectroscopy have hitherto remained unexplored. Here we present experiments showing how excitons that are dynamically screened by itinerant electrons to form exciton-polarons.sup.6,7 can be used as a spectroscopic tool to investigate interaction-induced incompressible states of electrons. We study a molybdenum diselenide/hexagonal boron nitride/molybdenum diselenide heterostructure that exhibits a long-period moiré superlattice, as evidenced by coherent hole-tunnelling-mediated avoided crossings of an intralayer exciton with three interlayer exciton resonances separated by about five millielectronvolts. For electron densities corresponding to half-filling of the lowest moiré subband, we observe strong layer pseudospin paramagnetism, demonstrated by an abrupt transfer of all the (roughly 1,500) electrons from one molybdenum diselenide layer to the other on application of a small perpendicular electric field. Remarkably, the electronic state at half-filling of each molybdenum diselenide layer is resilient towards charge redistribution by the applied electric field, demonstrating an incompressible Mott-like state of electrons. Our experiments demonstrate that optical spectroscopy provides a powerful tool for investigating strongly correlated electron physics in the bulk and paves the way for investigating Bose-Fermi mixtures of degenerate electrons and dipolar excitons. Optical spectroscopy is used to probe correlated electronic states in a moiré heterostructure, showing many-body effects such as strong layer paramagnetism and an incompressible Mott-like state of electrons., Author(s): Yuya Shimazaki [sup.1] , Ido Schwartz [sup.1] , Kenji Watanabe [sup.2] , Takashi Taniguchi [sup.2] , Martin Kroner [sup.1] , Ataç Imamoglu [sup.1] Author Affiliations: (1) Institute for Quantum [...]

Organisms determine the transcription rates of thousands of genes through a few modes of regulation that recur across the genome1. In bacteria, the relationship between the regulatory architecture of a gene and its expression is well understood for individual model gene circuits2,3. However, a broader perspective of these dynamics at the genome scale is lacking, in part because bacterial transcriptomics has hitherto captured only a static snapshot of expression averaged across millions of cells4. As a result, the full diversity of gene expression dynamics and their relation to regulatory architecture remains unknown. Here we present a novel genome-wide classification of regulatory modes based on the transcriptional response of each gene to its own replication, which we term the transcription–replication interaction profile (TRIP). Analysing single-bacterium RNA-sequencing data, we found that the response to the universal perturbation of chromosomal replication integrates biological regulatory factors with biophysical molecular events on the chromosome to reveal the local regulatory context of a gene. Whereas the TRIPs of many genes conform to a gene dosage-dependent pattern, others diverge in distinct ways, and this is shaped by factors such as intra-operon position and repression state. By revealing the underlying mechanistic drivers of gene expression heterogeneity, this work provides a quantitative, biophysical framework for modelling replication-dependent expression dynamics.Single-cell expression data from bacteria are used to classify gene regulatory architectures in relation to gene expression dynamics and the cell cycle, revealing distinct categories of gene regulatory mechanisms. [ABSTRACT FROM AUTHOR]

Diploidy is a fundamental genetic feature in mammals, in which haploid cells normally arise only as post-meiotic germ cells that serve to ensure a diploid genome upon fertilization. Gamete manipulation has yielded haploid embryonic stem (ES) cells from several mammalian species (1-6), but haploid human ES cells have yet to be reported. Here we generated and analysed a collection of human parthenogenetic ES cell lines originating from haploid oocytes, leading to the successful isolation and maintenance of human ES cell lines with a normal haploid karyotype. Haploid human ES cells exhibited typical pluripotent stem cell characteristics, such as self-renewal capacity and a pluripotency-specific molecular signature. Moreover, we demonstrated the utility of these cells as a platform for loss-of-function genetic screening. Although haploid human ES cells resembled their diploid counterparts, they also displayed distinct properties including differential regulation of X chromosome inactivation and of genes involved in oxidative phosphorylation, alongside reduction in absolute gene expression levels and cell size. Surprisingly, we found that a haploid human genome is compatible not only with the undifferentiated pluripotent state, but also with differentiated somatic fates representing all three embryonic germ layers both in vitro and in vivo, despite a persistent dosage imbalance between the autosomes and X chromosome. We expect that haploid human ES cells will provide novel means for studying human functional genomics and development., Haploid genetics is useful for delineating genome function. Although haploid genetics has been mostly restricted to unicellular organisms, recent reports of haploid ES cells have extended it into animal species, [...]

MYC (also known as c-MYC) overexpression or hyperactivation is one of the most common drivers of human cancer. Despite intensive study, the MYC oncogene remains recalcitrant to therapeutic inhibition. MYC is a transcription factor, and many of its protumorigenic functions have been attributed to its ability to regulate gene expression programs (1-3). Notably, oncogenic MYC activation has also been shown to increase total RNA and protein production in many tissue and disease contexts (4-7). While such increases in RNA and protein production may endow cancer cells with pro-tumour hallmarks, this increase in synthesis may also generate new or heightened burden on MYC-driven cancer cells to process these macromolecules properly (8). Here we discover that the spliceosome is a new target of oncogenic stress in MYC-driven cancers. We identify BUD31 as a MYC-synthetic lethal gene in human mammary epithelial cells, and demonstrate that BUD31 is a component of the core spliceosome required for its assembly and catalytic activity. Core spliceosomal factors (such as SF3B1 and U2AF1) associated with BUD31 are also required to tolerate oncogenic MYC. Notably, MYC hyperactivation induces an increase in total precursor messenger RNA synthesis, suggesting an increased burden on the core spliceosome to process pre-mRNA. In contrast to normal cells, partial inhibition of the spliceosome in MYC-hyperactivated cells leads to global intron retention, widespread defects in pre-mRNA maturation, and deregulation of many essential cell processes. Notably, genetic or pharmacological inhibition of the spliceosome in vivo impairs survival, tumorigenicity and metastatic proclivity of MYC-dependent breast cancers. Collectively, these data suggest that oncogenic MYC confers a collateral stress on splicing, and that components of the spliceosome may be therapeutic entry points for aggressive MYC-driven cancers., To discover genes and cellular processes required to tolerate oncogenic MYC expression, we previously performed a genome-wide MYC-synthetic lethal screen in human mammary epithelial cells (HMECs) engineered with an inducible [...]

The Dirac cone underlies many unique electronic properties of graphene (1) and topological insulators, and its band structure--two conical bands touching at a single point--has also been realized for photons in waveguide arrays (2), atoms in optical lattices (3), and through accidental degeneracy (4,5). Deformation of the Dirac cone often reveals intriguing properties; an example is the quantum Hall effect, where a constant magnetic field breaks the Dirac cone into isolated Landau levels. A seemingly unrelated phenomenon is the exceptional point (6, 7), also known as the parity-time symmetry breaking point (8-11), where two resonances coincide in both their positions and widths. Exceptional points lead to counter-intuitive phenomena such as loss-induced transparency (12), unidirectional transmission or reflection (11,13,14), and lasers with reversed pump dependence (15) or single-mode operation (16,17). Dirac cones and exceptional points are connected: it was theoretically suggested that certain non-Hermitian perturbations can deform a Dirac cone and spawn a ring of exceptional points (18-20). Here we experimentally demonstrate such an 'exceptional ring' in a photonic crystal slab. Angle-resolved reflection measurements of the photonic crystal slab reveal that the peaks of reflectivity follow the conical band structure of a Dirac cone resulting from accidental degeneracy, whereas the complex eigenvalues of the system are deformed into a two-dimensional flat band enclosed by an exceptional ring. This deformation arises from the dissimilar radiation rates of dipole and quadrupole resonances, which play a role analogous to the loss and gain in parity-time symmetric systems. Our results indicate that the radiation existing in any open system can fundamentally alter its physical properties in ways previously expected only in the presence of material loss and gain., Closed and lossless physical systems are described by Hermitian operators, which guarantee realness of the eigenvalues and a complete set of eigenfunctions that are orthogonal to each other. On the [...]

CRISPR-Cas (clustered, regularly interspaced short palindromic repeats coupled with CRISPR-associated proteins) is a bacterial immunity system that protects against invading phages or plasmids. In the process of CRISPR adaptation, short pieces of DNA ('spacers') are acquired from foreign elements and integrated into the CRISPR array. So far, it has remained a mystery how spacers are preferentially acquired from the foreign DNA while the self chromosome is avoided. Here we show that spacer acquisition is replication-dependent, and that DNA breaks formed at stalled replication forks promote spacer acquisition. Chromosomal hotspots of spacer acquisition were confined by Chi sites, which are sequence octamers highly enriched on the bacterial chromosome, suggesting that these sites limit spacer acquisition from self DNA. We further show that the avoidance of self is mediated by the RecBCD double-stranded DNA break repair complex. Our results suggest that, in Escherichia coli, acquisition of new spacers largely depends on RecBCD-mediated processing of double-stranded DNA breaks occurring primarily at replication forks, and that the preference for foreign DNA is achieved through the higher density of Chi sites on the self chromosome, in combination with the higher number of forks on the foreign DNA. This model explains the strong preference to acquire spacers both from high copy plasmids and from phages., CRISPR-Cas is an adaptive defence system in bacteria and archaea that provides acquired immunity against phages and plasmids (1-6). It comprises multiple cas genes, as well as an array of [...]

Pluripotent stem cells provide a powerful system to dissect the underlying molecular dynamics that regulate cell fate changes during mammalian development. Here we report the integrative analysis of genome-wide binding data for 38 transcription factors with extensive epigenome and transcriptional data across the differentiation of human embryonic stem cells to the three germ layers. We describe core regulatory dynamics and show the lineage-specific behaviour of selected factors. In addition to the orchestrated remodelling of the chromatin landscape, we find that the binding of several transcription factors is strongly associated with specific loss of DNA methylation in one germ layer, and in many cases a reciprocal gain in the other layers. Taken together, our work shows context-dependent rewiring of transcription factor binding, downstream signalling effectors, and the epigenome during human embryonic stem cell differentiation., Human embryonic stem (ES) cells hold great promise for tissue engineering and disease modelling; yet a key challenge to deriving mature, functional cell types is understanding the molecular mechanisms that [...]

Mouse embryonic stem (ES) cells are isolated from the inner cell mass of blastocysts, and can be preserved in vitro in a naive innercell-mass-like configuration by providing exogenous stimulation with leukaemia inhibitory factor (LIF) and small molecule inhibition of ERK1/ERK2 and GSKβ signalling (termed 2i/LIF conditions) (1,2). Hallmarks of naive pluripotency include driving Oct4 (also known as Pou5f1) transcription by its distal enhancer, retaining a pre-inactivation X chromosome state, and global reduction in DNA methylation and in H3K27me3 repressive chromatin mark deposition on developmental regulatory gene promoters (3). Upon withdrawal of 2i/LIF, naive mouse ES cells can drift towards a primed pluripotent state resembling that of the post-implantation epiblast (4). Although human ES cells share several molecular features with naive mouse ES cells (5), they also share a variety of epigenetic properties with primed murine epiblast stem cells (EpiSCs) (6,7). These include predominant use of the proximal enhancer element to maintain OCT4 expression, pronounced tendency for X chromosome inactivation in most female human ES cells, increase in DNA methylation and prominent deposition of H3K27me3 and bivalent domain acquisition on lineage regulatory genes (7). The feasibility of establishing human ground state naive pluripotency in vitro with equivalent molecular and functional features to those characterized in mouse ES cells remains to be defined (1). Here we establish defined conditions that facilitate the derivation of genetically unmodified human naive pluripotent stem cells from already established primed human ES cells, from somatic cells through induced pluripotent stem (iPS) cell reprogramming or directly from blastocysts. The novel naive pluripotent cells validated herein retain molecular characteristics and functional properties that are highly similar to mouse naive ES cells, and distinct from conventional primed human pluripotent cells. This includes competence in the generation of cross-species chimaeric mouse embryos that underwent organogenesis following microinjection of human naive iPS cells into mouse morulas. Collectively, our findings establish new avenues for regenerative medicine, patient-specific iPS cell disease modelling and the study of early human development invitro and invivo., Naive 2i/LIF conditions are not sufficient to elicit a self-renewal response in human ES cells/human iPS cells and result in their differentiation (7). Nevertheless, the proof of concept for the [...]

CLP1 was the first mammalian RNA kinase to be identified. However, determining its in vivo function has been elusive. Here we generated kinase-dead Clpl ([Clp1.sup.K/K]) mice that show a progressive loss of spinal motor neurons associated with axonal degeneration in the peripheral nerves and denervation of neuromuscular junctions, resulting in impaired motor function, muscle weakness, paralysis and fatal respiratory failure. Transgenic rescue experiments show that CLP1 functions in motor neurons. Mechanistically, loss of CLP1 activity results in accumulation of a novel set of small RNA fragments, derived from aberrant processing of tyrosine pre-transfer RNA. These tRNA fragments sensitize cells to oxidative-stress-induced p53 (also known as TRP53) activation and p53-dependent cell death. Genetic inactivation of p53 rescues [Clp1.sup.K/K] mice from the motor neuron loss, muscle denervation and respiratory failure. Our experiments uncover a mechanistic link between tRNA processing, formation of a new RNA species and progressive loss of lower motor neurons regulated by p53., RNA molecules undergo co- and post-transcriptional processing, leading to mature, functional RNAs. In mammals and archaea CLP1 proteins are kinases that phosphorylate the 5' hydroxyl ends of RNA (1-3). Human [...]

Induced pluripotent stem cells (iPSCs) can be derived from somatic cells by ectopic expression of different transcription factors, classically Oct4 (also known as Pou5f1), Sox2, Klf4 and Myc (abbreviated as [...]

Although thousands of large intergenic non-coding RNAs (lincRNAs) have been identified in mammals, few have been functionally characterized, leading to debate about their biological role. To address this, we performed loss-of- function studies on most lincRNAs expressed in mouse embryonic stem (ES) cells and characterized the effects on gene expression. Here we show that knockdown of lincRNAs has major consequences on gene expression patterns, comparable to knockdown of well-known ES cell regulators. Notably, lincRNAs primarily affect gene expression in trans. Knockdown of dozens of lincRNAs causes either exit from the pluripotent state or upregulation of lineage commitment programs. We integrate lincRNAs into the molecular circuitry of ES cells and show that lincRNA genes are regulated by key transcription factors and that lincRNA transcripts bind to multiple chromatin regulatory proteins to affect shared gene expression programs. Together, the results demonstrate that lincRNAs have key roles in the circuitry controlling ES cell state., The mammalian genome encodes many thousands of large noncoding transcripts1 including a class of ~3,500 lincRNAs identified using a chromatin signature of actively transcribed genes2-4. These lincRNA genes have been [...]

The mature gut renews continuously and rapidly throughout adult life, often in a damage-inflicting micro-environment. The major driving force for self-renewal of the intestinal epithelium is the Wnt-mediated signalling pathway, and Wnt signalling is frequently hyperactivated in colorectal cancer (1). Here we show that casein kinase Iα (CKIα), a component of the β-catenin-destruction complex (1) ,is a critical regulator of the Wnt signalling pathway. Inducing the ablation of Csnk1a1 (the gene encoding CKIα) in the gut triggers massive Wnt activation, surprisingly without causing tumorigenesis. CKIα-deficient epithelium shows many of the features of human colorectal tumours in addition to Wnt activation, in particular the induction of the DNA damage response and cellular senescence, both of which are thought to provide a barrier against malignant transformation (2). The epithelial DNA damage response in mice is accompanied by substantial activation of p53, suggesting that the p53 pathway may counteract the pro-tumorigenic effects of Wnt hyperactivation. Notably, the transition from benign adenomas to invasive colorectal cancer in humans is typically linked to p53 inactivation, underscoring the importance of p53 as a safeguard against malignant progression (3); however, the mechanism of p53-mediated tumour suppression is unknown. We show that the maintenance of intestinal homeostasis in CKIα-deficient gut requires p53-mediated growth control, because the combined ablation of Csnk1a1 and either p53or its target gene p21 (also known as Waf1, Cip1, Sdi1 and Cdkn1a) triggered high-grade dysplasia with extensive proliferation. Unexpectedly, these ablations also induced non-proliferating cells to invade the villous lamina propria rapidly, producing invasive carcinomas throughout the small bowel. Furthermore, in p53-deficient gut, loss of heterozygosity of the gene encoding CKIα caused a highly invasive carcinoma, indicating that CKIα functions as a tumour suppressor when p53 is inactivated. We identified a set of genes (the p53-suppressed invasiveness signature, PSIS) that is activated by the loss of both p53 and CKIα and which probably accounts for the brisk induction of invasiveness. PSIS transcription and tumour invasion were suppressed by p21, independently of cell cycle control. Restraining tissue invasion through suppressing PSIS expression is thus a novel tumour-suppressor function of wild-type p53., To investigate the physiological roles of CKIα, we generated mice in which Csnk1a1 was either deleted from the germline or loxP flanked (floxed) and therefore able to be conditionally deleted [...]

Sex determination is a fundamental biological process, yet its mechanisms are remarkably diverse (1,2). In vertebrates, sex can be determined by inherited genetic factors or by the temperature experienced during embryonic development (2,3). However, the evolutionary causes of this diversity remain unknown. Here we show that live-bearing lizards at different climatic extremes of the species' distribution differ in their sex-determining mechanisms, with temperature-dependent sex determination in lowlands and genotypic sex determination in highlands. A theoretical model parameterized with field data accurately predicts this divergence in sex-determining systems and the consequence thereof for variation in cohort sex ratios among years. Furthermore, we show that divergent natural selection on sex determination across altitudes is caused by climatic effects on lizard life history and variation in the magnitude of between-year temperature fluctuations. Our results establish an adaptive explanation for intra-specific divergence in sex-determining systems driven by phenotypic plasticity and ecological selection, thereby providing a unifying framework for integrating the developmental, ecological and evolutionary basis for variation in vertebrate sex determination., Vertebrates exhibit both genotypic (GSD) and temperature-dependent sex determination (TSD) (1,2). The latter is particularly common in reptiles and both systems can co-occur within taxonomic families (3). In addition, some [...]

There is growing recognition that mammalian cells produce many thousands of large intergenic transcripts (1-4). However, the functional significance of these transcripts has been particularly controversial. Although there are some well-characterized examples, most (.95%) show little evidence of evolutionary conservation and have been suggested to represent transcriptional noise (5,6). Here we report a new approach to identifying large non-coding RNAs using chromatin-state maps to discover discrete transcriptional units intervening known protein-coding loci. Our approach identified ~1,600 large multi-exonic RNAs across four mouse cell types. In sharp contrast to previous collections, these large intervening non-coding RNAs (lincRNAs) show strong purifying selection in their genomic loci, exonic sequences and promoter regions, with greater than 95% showing clear evolutionary conservation. We also developed a functional genomics approach that assigns putative functions to each lincRNA, demonstrating a diverse range of roles for lincRNAs in processes fromembryonic stem cell pluripotency to cell proliferation. We obtained independent functional validation for the predictions for over 100 lincRNAs, using cell-based assays. In particular, we demonstrate that specific lincRNAs are transcriptionally regulated by key transcription factors in these processes such as p53, NFκB, Sox2, Oct4 (also known as Pou5f1) and Nanog. Together, these results define a unique collection of functional lincRNAs that are highly conserved and implicated in diverse biological processes., There are at present only about a dozen well-characterized lincRNAs in mammals, with transcript sizes ranging from 2.3 to 17.2 kilobases (kb) (7,8). These lincRNAs have distinctive biological roles through [...]

Author(s): Carsten Bahr [1, 2, 3]; Lisa von Paleske [1, 2, 3]; Veli V. Uslu [4]; Silvia Remeseiro [4]; Naoya Takayama [5, 6]; Stanley W. Ng [7]; Alex Murison [5, [...]

The 'certainty effect' (1,2) is a notable violation of expected utility theory by decision makers (3-6). It shows that people's tendency to select the safer of two prospects increases when [...]

Author(s): Amir Giladi (corresponding author) [1]; Ido Amit (corresponding author) [1] For more than a century, scientists have tried to characterize the different functions of the 10 trillion to 50 [...]

Author(s): Yuji Shiba (corresponding author) [1, 2]; Toshihito Gomibuchi [3]; Tatsuichiro Seto [3]; Yuko Wada [3]; Hajime Ichimura [3]; Yuki Tanaka [3]; Tatsuki Ogasawara [3]; Kenji Okada [3]; Naoko Shiba [...]

Author(s): Ohad Gafni; Leehee Weinberger; Abed AlFatah Mansour; Yair S. Manor; Elad Chomsky; Dalit Ben-Yosef; Yael Kalma; Sergey Viukov; Itay Maza; Asaf Zviran; Yoach Rais; Zohar Shipony; Zohar Mukamel; Vladislav [...]

Author(s): Yoach Rais; Asaf Zviran; Shay Geula; Ohad Gafni; Elad Chomsky; Sergey Viukov; Abed AlFatah Mansour; Inbal Caspi; Vladislav Krupalnik; Mirie Zerbib; Itay Maza; Nofar Mor; Dror Baran; Leehee Weinberger; [...]