Notch1 mutations drive clonal expansion in normal esophageal epithelium but impair tumor growth.

NOTCH1 mutant clones occupy the majority of normal human esophagus by middle age but are comparatively rare in esophageal cancers, suggesting NOTCH1 mutations drive clonal expansion but impede carcinogenesis. Here we test this hypothesis. Sequencing NOTCH1 mutant clones in aging human esophagus reveals frequent biallelic mutations that block NOTCH1 signaling. In mouse esophagus, heterozygous Notch1 mutation confers a competitive advantage over wild-type cells, an effect enhanced by loss of the second allele. Widespread Notch1 loss alters transcription but has minimal effects on the epithelial structure and cell dynamics. In a carcinogenesis model, Notch1 mutations were less prevalent in tumors than normal epithelium. Deletion of Notch1 reduced tumor growth, an effect recapitulated by anti-NOTCH1 antibody treatment. Notch1 null tumors showed reduced proliferation. We conclude that Notch1 mutations in normal epithelium are beneficial as wild-type Notch1 favors tumor expansion. NOTCH1 blockade may have therapeutic potential in preventing esophageal squamous cancer.

Pseudoalignment tools as an efficient alternative to detect repeated transposable elements in scRNAseq data.

MOTIVATION: Transposable elements (TE) have played a major role in configuring the structures of mammalian genomes through evolution. In normal conditions, the expression of these elements is repressed by different epigenetic regulation mechanisms such as DNA methylation, histone modification and regulation by small RNAs. TE re-activation is associated with stemness potential acquisition, regulation of innate immunity and disease, such as cancer. However, the vast majority of current knowledge in the field is based on bulk expression studies, and very little is known on cell-type- or state-specific expression of TE-derived transcripts. Therefore, cost-efficient single-cell-resolution TE expression analytical approaches are needed. RESULTS: We have implemented an analytical approach based on pseudoalignment to consensus sequences to incorporate TE expression information to scRNAseq data. AVAILABILITY AND IMPLEMENTATION: All the data and code implemented are available as Supplementary data and in: https://github.com/jmzvillarreal/kallisto_TE_scRNAseq. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Cysteine and iron accelerate the formation of ribose-5-phosphate, providing insights into the evolutionary origins of the metabolic network structure.

The structure of the metabolic network is highly conserved, but we know little about its evolutionary origins. Key for explaining the early evolution of metabolism is solving a chicken-egg dilemma, which describes that enzymes are made from the very same molecules they produce. The recent discovery of several nonenzymatic reaction sequences that topologically resemble central metabolism has provided experimental support for a "metabolism first" theory, in which at least part of the extant metabolic network emerged on the basis of nonenzymatic reactions. But how could evolution kick-start on the basis of a metal catalyzed reaction sequence, and how could the structure of nonenzymatic reaction sequences be imprinted on the metabolic network to remain conserved for billions of years? We performed an in vitro screening where we add the simplest components of metabolic enzymes, proteinogenic amino acids, to a nonenzymatic, iron-driven reaction network that resembles glycolysis and the pentose phosphate pathway (PPP). We observe that the presence of the amino acids enhanced several of the nonenzymatic reactions. Particular attention was triggered by a reaction that resembles a rate-limiting step in the oxidative PPP. A prebiotically available, proteinogenic amino acid cysteine accelerated the formation of RNA nucleoside precursor ribose-5-phosphate from 6-phosphogluconate. We report that iron and cysteine interact and have additive effects on the reaction rate so that ribose-5-phosphate forms at high specificity under mild, metabolism typical temperature and environmental conditions. We speculate that accelerating effects of amino acids on rate-limiting nonenzymatic reactions could have facilitated a stepwise enzymatization of nonenzymatic reaction sequences, imprinting their structure on the evolving metabolic network.

Spatial competition shapes the dynamic mutational landscape of normal esophageal epithelium.

During aging, progenitor cells acquire mutations, which may generate clones that colonize the surrounding tissue. By middle age, normal human tissues, including the esophageal epithelium (EE), become a patchwork of mutant clones. Despite their relevance for understanding aging and cancer, the processes that underpin mutational selection in normal tissues remain poorly understood. Here, we investigated this issue in the esophageal epithelium of mutagen-treated mice. Deep sequencing identified numerous mutant clones with multiple genes under positive selection, including Notch1, Notch2 and Trp53, which are also selected in human esophageal epithelium. Transgenic lineage tracing revealed strong clonal competition that evolved over time. Clone dynamics were consistent with a simple model in which the proliferative advantage conferred by positively selected mutations depends on the nature of the neighboring cells. When clones with similar competitive fitness collide, mutant cell fate reverts towards homeostasis, a constraint that explains how selection operates in normal-appearing epithelium.

A single-progenitor model as the unifying paradigm of epidermal and esophageal epithelial maintenance in mice.

In adult skin epidermis and the epithelium lining the esophagus cells are constantly shed from the tissue surface and replaced by cell division. Tracking genetically labelled cells in transgenic mice has given insight into cell behavior, but conflicting models appear consistent with the results. Here, we use an additional transgenic assay to follow cell division in mouse esophagus and the epidermis at multiple body sites. We find that proliferating cells divide at a similar rate, and place bounds on the distribution cell cycle times. By including these results in a common analytic approach, we show that data from eight lineage tracing experiments is consistent with tissue maintenance by a single population of proliferating cells. The outcome of a given cell division is unpredictable but, on average, the likelihood of producing proliferating and differentiating cells is equal, ensuring cellular homeostasis. These findings are key to understanding squamous epithelial homeostasis and carcinogenesis.

Outcompeting p53-Mutant Cells in the Normal Esophagus by Redox Manipulation.

As humans age, normal tissues, such as the esophageal epithelium, become a patchwork of mutant clones. Some mutations are under positive selection, conferring a competitive advantage over wild-type cells. We speculated that altering the selective pressure on mutant cell populations may cause them to expand or contract. We tested this hypothesis by examining the effect of oxidative stress from low-dose ionizing radiation (LDIR) on wild-type and p53 mutant cells in the transgenic mouse esophagus. We found that LDIR drives wild-type cells to stop proliferating and differentiate. p53 mutant cells are insensitive to LDIR and outcompete wild-type cells following exposure. Remarkably, combining antioxidant treatment and LDIR reverses this effect, promoting wild-type cell proliferation and p53 mutant differentiation, reducing the p53 mutant population. Thus, p53-mutant cells can be depleted from the normal esophagus by redox manipulation, showing that external interventions may be used to alter the mutational landscape of an aging tissue.

Epidermal Tissue Adapts to Restrain Progenitors Carrying Clonal p53 Mutations.

Aging human tissues, such as sun-exposed epidermis, accumulate a high burden of progenitor cells that carry oncogenic mutations. However, most progenitors carrying such mutations colonize and persist in normal tissue without forming tumors. Here, we investigated tissue-level constraints on clonal progenitor behavior by inducing a single-allele p53 mutation (Trp53R245W; p53∗/wt), prevalent in normal human epidermis and squamous cell carcinoma, in transgenic mouse epidermis. p53∗/wt progenitors initially outcompeted wild-type cells due to enhanced proliferation, but subsequently reverted toward normal dynamics and homeostasis. Physiological doses of UV light accelerated short-term expansion of p53∗/wt clones, but their frequency decreased with protracted irradiation, possibly due to displacement by UV-induced mutant clones with higher competitive fitness. These results suggest multiple mechanisms restrain the proliferation of p53∗/wt progenitors, thereby maintaining epidermal integrity.

Nonenzymatic gluconeogenesis-like formation of fructose 1,6-bisphosphate in ice.

The evolutionary origins of metabolism, in particular the emergence of the sugar phosphates that constitute glycolysis, the pentose phosphate pathway, and the RNA and DNA backbone, are largely unknown. In cells, a major source of glucose and the large sugar phosphates is gluconeogenesis. This ancient anabolic pathway (re-)builds carbon bonds as cleaved in glycolysis in an aldol condensation of the unstable catabolites glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, forming the much more stable fructose 1,6-bisphosphate. We here report the discovery of a nonenzymatic counterpart to this reaction. The in-ice nonenzymatic aldol addition leads to the continuous accumulation of fructose 1,6-bisphosphate in a permanently frozen solution as followed over months. Moreover, the in-ice reaction is accelerated by simple amino acids, in particular glycine and lysine. Revealing that gluconeogenesis may be of nonenzymatic origin, our results shed light on how glucose anabolism could have emerged in early life forms. Furthermore, the amino acid acceleration of a key cellular anabolic reaction may indicate a link between prebiotic chemistry and the nature of the first metabolic enzymes.

Permeability-driven selection in a semi-empirical protocell model: the roots of prebiotic systems evolution.

The origin-of-life problem has been traditionally conceived as the chemical challenge to find the type of molecule and free-solution reaction dynamics that could have started Darwinian evolution. Different autocatalytic and 'self-replicative' molecular species have been extensively investigated, together with plausible synthetic pathways that might have led, abiotically, to such a minimalist scenario. However, in addition to molecular kinetics or molecular evolutionary dynamics, other physical and chemical constraints (like compartmentalization, differential diffusion, selective transport, osmotic forces, energetic couplings) could have been crucial for the cohesion, functional integration, and intrinsic stability/robustness of intermediate systems between chemistry and biology. These less acknowledged mechanisms of interaction and molecular control might have made the initial pathways to prebiotic systems evolution more intricate, but were surely essential for sustaining far-from-equilibrium chemical dynamics, given their functional relevance in all modern cells. Here we explore a protocellular scenario in which some of those additional constraints/mechanisms are addressed, demonstrating their 'system-level' implications. In particular, an experimental study on the permeability of prebiotic vesicle membranes composed of binary lipid mixtures allows us to construct a semi-empirical model where protocells are able to reproduce and undergo an evolutionary process based on their coupling with an internal chemistry that supports lipid synthesis.

Identifying ultrasensitive HGF dose-response functions in a 3D mammalian system for synthetic morphogenesis.

Nonlinear responses to signals are widespread natural phenomena that affect various cellular processes. Nonlinearity can be a desirable characteristic for engineering living organisms because it can lead to more switch-like responses, similar to those underlying the wiring in electronics. Steeper functions are described as ultrasensitive, and can be applied in synthetic biology by using various techniques including receptor decoys, multiple co-operative binding sites, and sequential positive feedbacks. Here, we explore the inherent non-linearity of a biological signaling system to identify functions that can potentially be exploited using cell genome engineering. For this, we performed genome-wide transcription profiling to identify genes with ultrasensitive response functions to Hepatocyte Growth Factor (HGF). We identified 3,527 genes that react to increasing concentrations of HGF, in Madin-Darby canine kidney (MDCK) cells, grown as cysts in 3D collagen cell culture. By fitting a generic Hill function to the dose-responses of these genes we obtained a measure of the ultrasensitivity of HGF-responsive genes, identifying a subset with higher apparent Hill coefficients (e.g. MMP1, TIMP1, SNORD75, SNORD86 and ERRFI1). The regulatory regions of these genes are potential candidates for future engineering of synthetic mammalian gene circuits requiring nonlinear responses to HGF signalling.

The Impact of Non-Enzymatic Reactions and Enzyme Promiscuity on Cellular Metabolism during (Oxidative) Stress Conditions.

Cellular metabolism assembles in a structurally highly conserved, but functionally dynamic system, known as the metabolic network. This network involves highly active, enzyme-catalyzed metabolic pathways that provide the building blocks for cell growth. In parallel, however, chemical reactivity of metabolites and unspecific enzyme function give rise to a number of side products that are not part of canonical metabolic pathways. It is increasingly acknowledged that these molecules are important for the evolution of metabolism, affect metabolic efficiency, and that they play a potential role in human disease-age-related disorders and cancer in particular. In this review we discuss the impact of oxidative and other cellular stressors on the formation of metabolic side products, which originate as a consequence of: (i) chemical reactivity or modification of regular metabolites; (ii) through modifications in substrate specificity of damaged enzymes; and (iii) through altered metabolic flux that protects cells in stress conditions. In particular, oxidative and heat stress conditions are causative of metabolite and enzymatic damage and thus promote the non-canonical metabolic activity of the cells through an increased repertoire of side products. On the basis of selected examples, we discuss the consequences of non-canonical metabolic reactivity on evolution, function and repair of the metabolic network.

The widespread role of non-enzymatic reactions in cellular metabolism.

Enzymes shape cellular metabolism, are regulated, fast, and for most cases specific. Enzymes do not however prevent the parallel occurrence of non-enzymatic reactions. Non-enzymatic reactions were important for the evolution of metabolic pathways, but are retained as part of the modern metabolic network. They divide into unspecific chemical reactivity and specific reactions that occur either exclusively non-enzymatically as part of the metabolic network, or in parallel to existing enzyme functions. Non-enzymatic reactions resemble catalytic mechanisms as found in all major enzyme classes and occur spontaneously, small molecule (e.g. metal-) catalyzed or light-induced. The frequent occurrence of non-enzymatic reactions impacts on stability and metabolic network structure, and has thus to be considered in the context of metabolic disease, network modeling, biotechnology and drug design.

Viability conditions for a compartmentalized protometabolic system: a semi-empirical approach.

In this work we attempt to find out the extent to which realistic prebiotic compartments, such as fatty acid vesicles, would constrain the chemical network dynamics that could have sustained a minimal form of metabolism. We combine experimental and simulation results to establish the conditions under which a reaction network with a catalytically closed organization (more specifically, an (M,R-system) would overcome the potential problem of self-suffocation that arises from the limited accessibility of nutrients to its internal reaction domain. The relationship between the permeability of the membrane, the lifetime of the key catalysts and their efficiency (reaction rate enhancement) turns out to be critical. In particular, we show how permeability values constrain the characteristic time scale of the bounded protometabolic processes. From this concrete and illustrative example we finally extend the discussion to a wider evolutionary context.

Size matters: influence of stochasticity on the self-maintenance of a simple model of metabolic closure.

A fundamental landmark in the emergence and maintenance of the first proto-biological systems must have been the formation of a "closed" metabolic organization, and this paper describes a stochastic analysis of a simple model of a system that is closed to efficient causation. Although it shows an absorbing barrier corresponding to the trivial solution that implies collapse and extinction, for certain values of the kinetic parameters it can also show a "coexistence state" in which there are non-null populations of its intermediates, which corresponds approximately to a non-trivial deterministic stable steady state. Depending on the initial conditions, fluctuations can drive the system either to the self-maintaining regime or to extinction, with different probabilities. Different lines of equal probability have been obtained and compared with the deterministic results, and the average time for reaching these states (characteristic time) has been estimated. The system shows strong dependence on volume size, and there is a critical volume below which it collapses very rapidly. The characteristic time is also affected by the volume, with faster responses for lower system volumes. All these results are discussed in the context of the origin of living organization.

Stochastic simulations of mixed-lipid compartments: from self-assembling vesicles to self-producing protocells.

The computational platform ENVIRONMENT, developed to simulate stochastically reaction systems in varying compartmentalized conditions [Mavelli and Ruiz-Mirazo: Philos Trans R Soc Lond B Biol Sci 362:1789-1802, 2007; Physical Biology 7(3): 036002, 2010], is here applied to study the dynamic properties and stability of model protocells that start producing their own lipid molecules (e.g., phospholipids), which get inserted in previously self-assembled vesicles, made of precursor amphiphiles (e.g., fatty acids). Attention is mainly focused on the changes that this may provoke in the permeability of the compartment, as well as in its eventual osmotic robustness.

A simple self-maintaining metabolic system: robustness, autocatalysis, bistability.

A living organism must not only organize itself from within; it must also maintain its organization in the face of changes in its environment and degradation of its components. We show here that a simple (M,R)-system consisting of three interlocking catalytic cycles, with every catalyst produced by the system itself, can both establish a non-trivial steady state and maintain this despite continuous loss of the catalysts by irreversible degradation. As long as at least one catalyst is present at a sufficient concentration in the initial state, the others can be produced and maintained. The system shows bistability, because if the amount of catalyst in the initial state is insufficient to reach the non-trivial steady state the system collapses to a trivial steady state in which all fluxes are zero. It is also robust, because if one catalyst is catastrophically lost when the system is in steady state it can recreate the same state. There are three elementary flux modes, but none of them is an enzyme-maintaining mode, the entire network being necessary to maintain the two catalysts.

Allele-specific gene expression is widespread across the genome and biological processes.

Allelic specific gene expression (ASGE) appears to be an important factor in human phenotypic variability and as a consequence, for the development of complex traits and diseases. In order to study ASGE across the human genome, we have performed a study in which genotyping was coupled with an analysis of ASGE by screening 11,500 SNPs using the Mapping 10 K Array to identify differential allelic expression. We found that from the 5,133 SNPs that were suitable for analysis (heterozygous in our sample and expressed in peripheral blood mononuclear cells), 2,934 (57%) SNPs had differential allelic expression. Such SNPs were equally distributed along human chromosomes and biological processes. We validated the presence or absence of ASGE in 18 out 20 SNPs (90%) randomly selected by real time PCR in 48 human subjects. In addition, we observed that SNPs close to -but not included in- segmental duplications had increased levels of ASGE. Finally, we found that transcripts of unknown function or non-coding RNAs, also display ASGE: from a total of 2,308 intronic SNPs, 1510 (65%) SNPs underwent differential allelic expression. In summary, ASGE is a widespread mechanism in the human genome whose regulation seems to be far more complex than expected.