Some epigenetic marks are heritable in plants, such as cytosine DNA methylation in the CG dinucleotide context. Because epigenetic marks evolve at rates that are much faster than DNA, they are a potential agent for rapid adaptation to a changing environment, provided they impact fitness. Recent advances in sequencing technology have generated valuable resources, including genomes and more recently methylomes. These data have furthered our understanding about the repressive role of DNA methylation on transposable elements. Surprisingly, however, the role of gene body methylation (gbM) remains enigmatic. gbM is characterized by DNA methylation in the CG context within the body of genes – i.e., between the transcription start site and the transcription termination site – and it is associated with constitutive genes that have moderate to high expression levels. There are two explanations for this phenomenon. One is that gbM is a neutral consequence of transcription without any functional significance. Alternatively, gbM may be functional, in which case it will be subjected to natural selection. This project will search for footprints of selection acting on gbM levels, utilizing ~1000 methylomes available for Arabidopsis thaliana. I will adapt classical population genetic approaches, such as the McDonald-Kreitman test and analyses of the site frequency spectrum, to the study of gbM. By doing so, I will determine whether gbM is under selection in plants and also extend analytical approaches to population methylomic data. If selection on gbM level is detected, the study of the affected genes and their expression patterns may offer clues about gbM function.

The human microbiota produces a wide range of diffusible and cell-associated molecules, many of which are crucial for immune and metabolic homeostasis. The human immune system contains dendritic cells (DC), which sense bacteria-derived molecules through innate immune receptors and initiate an immune reaction that can be immunogenic or tolerogenic in tone. Previous studies have examined the effect of just a single or a few bacterial molecule(s) on the immune system. However, it is not yet understood how the immune system integrates a complex combination of numerous and diverse microbiota-derived molecules and 'decides' what kind of an immune response to mount. In this research, I will systematically elucidate the combinatorial effects of multiple microbiota-derived factors on DCs and identify genes in DCs underlying the integration of multiple signals. In Aim1, a large library of molecules from the microbiota and microenvironment will be generated. The library includes novel unpublished microbiota-derived molecules, in addition to commercially available microbial molecules and microenvironmental factors (i.e., tissue-specific cytokines). In Aim2, DCs will be cultured with combinatorial sets of molecules, and their transcriptional profiles and cytokine expression will be determined by multiplexed RNA-seq analysis. In Aim3, sets of genes required for DC responses will be identified by genome-wide CRISPRi/a screening and comprehensively analyzed using computational techniques. In Aim4, the results of the in vitro analysis will be tested in vivo using mouse experiments in which germ-free mice are colonized by genetically modified bacteria expressing multiple molecules.

The goal of my proposal is to harness protein homeostasis (proteostasis) to restore adult neural stem cell (NSC) function. Ageing is associated with a striking decrease in adult stem cell function in all tissues. In the nervous system, the number of NSCs and their ability to exit quiescence and to produce new neurones decline dramatically during ageing, which may underlie age-dependent decline in cognitive function. Similarly, disruptions in the proteostasis machinery are associated with accelerated ageing and neurodegenerative diseases. Thus, understanding the molecular mechanisms NSCs use to maintain proteostasis, and how they change with age, may help uncover ways to preserve their regenerative potential during ageing. Recent evidence in the Brunet laboratory has revealed that quiescent NSCs (qNSC) accumulate more aggregates than their activated counterparts, with even higher aggregation seen in old qNSCs. In the proposed project, I will carry out a genome-wide genetic screen to identify proteins involved in the formation of aggregates in qNSCs during ageing and will perform cutting-edge mass spectrometry to identify the proteins present in these aggregates. I will then use this knowledge to determine the function of the aggregates in NSCs, with the goal of restoring old NSC function. Completion of these studies will provide unique mechanistic insights into the regulation of protein aggregation during ageing in regenerative cells and their differentiated progeny. This work will pave the way for building new methods for the ‘rejuvenation’ of old cells and the restoration of proteome solubility, crucial steps in countering age-related decline and pathologies.

This project takes a novel approach to study how host organisms combat against pathogens. In general, hosts use resistance to attack and eliminate pathogens, but an alternative method is to employ tolerance, which protects tissues from damage without affecting pathogen burden. The molecular mechanisms of resistance are well established, but the regulation of tolerance is poorly known. Mammals activate tolerance mechanisms when they confront energy-constraining periods, such as infection or starvation. They temporarily abandon the maintenance of body temperature and engage in torpor, which protects organs from tissue damage. Since torpor enables organs to sustain stress without tissue damage, understanding the protective mechanisms might lead to discovery of novel treatment strategies against infections, which might be applicable to other human conditions as well. In the proposed work, I aim to study 1) how animals enter torpor, 2) what are the metabolic signatures of torpid animals and 3) how torpor affects organismal fitness during infections. To accomplish these goals, I will use lipopolysaccharide (LPS) to induce torpor in mice. First, I aim to identify which tissue senses LPS to induce torpor using tissue-specific knock-out mice for TLR4, which is the receptor for LPS. Next, I will perform untargeted metabolomics and transcriptomics to identify metabolic regulators of torpor. Finally, I will ask if torpor affects tissue damage during E. coli infection by assessing immunopathology of the infected animals. The proposed work will expand our understanding of tolerance mechanisms and will, possibly, provide novel strategies to treat infections.

G-protein coupled receptors (GPCRs) regulate virtually all physiological processes and represent the most common drug target. Apart from the classical signaling, involving the activation of heterotrimeric G-protein, GPCRs also initiate parallel signaling events by interacting with ß-arrestins. ß-arrestins serve as key junctions that receive input from receptors and route the signal to various effectors within the cell. The discovery of signal transduction through ß-arrestins underscores the multifaceted functionality of GPCRs and opens new horizons for future studies. Mapping the extensive GPCR-ß-arrestin-mediated signaling networks offers a possibility to design drugs with greater specificity of action and fewer side effects. However, a detailed structural understanding of interactions between GPCRs and ß-arrestins is still lacking. In this project, I will elucidate the molecular mechanisms of ß-arrestin-1 recruitment by the clinically important ß2-adrenergic receptor (ß2AR). To achieve this goal, I will employ a combination of structural and biophysical methods, such as X-ray crystallography, cryo-electron microscopy and double electron-electron resonance. The project will be performed in the laboratory of Professor Lefkowitz (Duke University, Durham, North Carolina, USA), a leading expert in GPCR biology. I aim to obtain the conformational snapshots of ß2AR-ß-arrestin complex at high resolution, identify the binding interface of the receptor and ß-arrestin and elucidate the dynamics of the complex upon ligand binding. These results will provide a holistic understanding of ß-arrestin recruitment by ß2AR and facilitate the development of novel pathway-specific drugs.

Phenotypic plasticity describes how a single genotype can generate a range of alternative phenotypes. The concept is exemplified by complex trait divergence between monozygotic co-twin pairs. Recently the Pospisilik lab identified a genetic circuit that regulates phenotypic plasticity by buffering against the emergence of alternate epigenetic states. The original Trim28(+/D9) line used for that work represents the first in vivo model for exploring the impact of mammalian polyphenism on complex trait biology and disease. Intriguingly, both gain- and loss- of function mutations in Trim28 have previously been associated with cancer. Here, I propose focusing on Trim28(+/D9) haploinsufficiency to assess for the first time the impact of bi-stable, epigenetically determined phenotypic variation on cancer. I will monitor tumour latency, progression and spectrum using in vivo imaging and assess tumour heterogeneity down to the single-cell level. In order to gain mechanistic insight, I will use unbiased in vivo shRNA-mediated oncosuppressor screening followed by molecular dissection using human and mouse organotypic cultures in vitro. My overarching scientific goal is to understand the non-genetic roots of cancer. The unique tools, knowledge and environment of the Pospisilik lab are ideal for assessing how non-genetic variation regulates cancer susceptibility, triggering and progression. If successful, these data will not only drive me much closer to these goals, they will reveal a first gene-gene-environment framework for understanding cancerogenesis in the context of phenotypic variation and epigenetic bi-stability.

In recent years there is a growing realization that many species are at risk of extinction due to habitat loss, while the spread of invasive species increases. These changes could influence the stability and function of ecosystems. Our ability to predict how perturbations, such as species extinctions and invasions, affect stability would help protect biodiversity, for example by restoring "keystone species" to sustain ecosystem structure and stability. The aim of the proposed project is to quantify and predict the impact of removal and addition of species on communities. Using a well-defined set of microbial species, we will construct synthetic communities composed of subsets of this set. We will study the effect of removal/addition of species from/to each subset on the species’ abundances. Specifically, the extinction of species as a result of the removal of other species would be of great interest since it will allow the identification of keystone species. These measurements would form a large dataset of experimentally validated co-occurrence networks, and would enable us to identify properties that make communities more vulnerable to the addition or removal of particular species. Next, we will test whether species’ pairwise interactions can be used to predict the community structure. For this aim, we will develop an experimental framework to map the pairwise interactions between all species, their directions and strengths. Finally, we will develop a mathematical model to predict extinction and invasion effects on a given ecosystem.

Non-coding segments - introns are removed from precursor messenger RNAs (pre-mRNAs) through a process known as pre-mRNA splicing, which is catalyzed by two large ribonucleoprotein (RNP) complexes, the ‘major’ and the ‘minor’ spliceosome. While recent structural studies of the major spliceosome have tremendously advanced our understanding of the basic splicing mechanism, the structure and mechanism of the minor spliceosome remains largely elusive. The minor spliceosome differs in its composition and specificity and no comparable structural insights are available. Therefore I propose to use cryo-electron microscopy (cryo-EM) to study the assembly of the minor spliceosome on its specific pre-mRNA substrates. To achieve this, I will extract endogenous complexes directly from human cells. Affinity tags that I will introduce into the genome by CRISPR/Cas9 gene editing techniques will enable me to purify this large RNP machine from nuclear extracts. I will reconstitute the minor spliceosome on substrate RNAs and analyze them for their composition and homogeneity by mass spectrometry and negative stain EM. Ultimately, I want to obtain atomic models of those complexes by cryo-electron microscopy. My studies will close the gap in knowledge between both spliceosomes in terms of structural insights and thus i) reveal new aspects about the evolution of the splicing mechanism, since the minor spliceosome might constitute a more simple system that is closer related to lower eukaryotes, and ii) provide a molecular understanding on diseases related to malfunctions of the minor spliceosome as well as opportunities for site-specific targeting of the major spliceosome in cancer.

Embryonic development and organ morphogenesis entail both the correct differentiation of cells into given lineages and their proper positioning along the body axes. During gastrulation in the zebrafish embryo, specification of the germ layers is concurrent to internalization movements, which reposition the future mesendoderm precursors underneath the prospective ectoderm. Nodal/TGF-ß signalling is important for both processes, raising the possibility that cell differentiation and tissue shaping may be coupled. Yet the existence, nature and spatiotemporal scale of the mutual feedback between these processes is still poorly understood. To address this fundamental question, I will follow three lines of research: (1) characterize with high spatiotemporal resolution the dynamics of Nodal-mediated mesendoderm fate acquisition and internalization along the dorsal-ventral margin of the germ ring; (2) dissect the molecular mechanism by which Nodal-mediated cell fate specification is linked to mesendoderm internalization; and, finally, (3) explore whether cell internalization feeds back onto mesendoderm fate specification, via mechanotransduction pathways. This exploratory research project will provide a map of cell specification with unprecedented cellular and tissue resolution and establish a framework to understand how gastrulation movements are coordinated with ongoing fate acquisition and embryonic axes establishment. Since there are striking similarities between the molecular mechanisms underlying tumour growth and metastasis and those governing gastrulation, this work is likely to provide key insights to the understanding of both embryonic development and cancer progression.

It has become increasingly evident that the transcriptional activity of a gene is intrinsically linked to its positioning within the nucleus. A distinctive subnuclear structure is the nucleolus, which is mainly formed upon transcription of ribosomal RNA (rRNA) genes. Hundreds to thousands of rRNA genes are tandemly arrayed in megabase-size rDNA clusters. The model plant Arabidopsis thaliana carries two unlinked rDNA clusters, with natural variation in rDNA cluster-activity, meaning that some strains express only one or the other cluster, and some both. Because large genomic regions adjacent to the actively transcribed rDNA cluster(s) –thus associated with the nucleolus– are expressed at lower levels than the genome average, I expect large-scale variation in gene activity. My goal is to investigate how variation in rDNA cluster-usage and its consequent reshaping of 3D genome architecture impact not only the expression of adjacent protein-coding genes, but also how genome-wide gene regulatory networks (GRNs) can adapt to such large-scale differences in gene expression. I propose to take full advantage of the diversity of genetic resources available in A. thaliana, including experimental populations, a large catalog of genomes and transcriptomes, as well as its accessibility for genetic engineering to pursue the following aims: (1) investigate the impact of rDNA-cluster usage on GRNs in natural strains, (2) swap rDNA expression patterns using crosses, and assess effects on GRNs, and (3) induce targeted inversions and translocations using CRISPR-Cas9 technology to directly test the functional consequences of gene-positioning with respect to active rDNA clusters.

A primary feature of highly-social animals, like humans, is the tendency to help one another. These behaviors, where one animal helps another without immediate benefit to itself, are known as prosocial behaviors. While there has been considerable research into how the brain controls social behaviors driven by fear or aggression, there has been almost no investigation of brain mechanisms of prosocial behaviors– when one animal aids another. We will conduct the first mechanistic investigation of the microcircuits underlying prosocial behaviors. To this end, we will utilize the natural food-sharing behaviors of Egyptian fruit bats; where provider-bats feed scrounger-bats; and will conduct wireless single-unit electrophysiological recordings from multiple freely-behaving bats living and interacting together in a colony. We will investigate two brain areas: (i) Medial prefrontal cortex, specifically infralimbic cortex– a brain area linked in humans to prosocial behaviors– where we will investigate neural correlates of prosocial feeding events. (ii) Hippocampus– an area involved in social memory and strongly connected with the prefrontal cortex– where we will investigate how other animals are recognized, a key component of prosocial behaviors. We will further introduce ethological manipulations to the freely-behaving colony to determine how natural variations, such as changes in competition or resource-availability, impacts prosocial behaviors and their neural representations. Finally we will switch a provider bat to a scrounger, by adding weight to create temporary ‘handicap’– and investigate the changes in the underlying neural representations of both oneself and others.

Skeletal muscle has the capacity to regenerate after injury. This process involves an orchestra of cells and factors that sequentially intervene to repair muscle fibers. Muscle regeneration is particularly vital for muscle disorders and aged muscle in which myofibers are constantly degrading. In contrast to the classical approach in studying muscle regeneration, this project focuses on exploring the intrinsic capacity of damaged myofiber to self-repair. We observed that after local injury, nearby nuclei migrate to the damaged site where they become internalized, re-organize chromatin and begin rotating. This cell response coincides with repair of the injury and has never been previously described. Interestingly this process is impaired in aged myofibers that are incapable of repair post injury. We therefore propose to study the intrinsic capacity of myofibers to repair. We will first explore the underlying mechanisms driving nuclear migrations to the damaged site after local injury. We will then establish how chromatin is re-organized for myofiber self-repair. Finally, we will investigate the failing mechanisms in aged myofibers. The difference between intrinsic myofiber repair and regeneration most likely stems from the severity of the injury, with minor damage triggering the intrinsic repair program. The capacity of myofibers to self-repair may be the first line of defense to muscle injury. Exploring the mechanisms of this newly observed process could reveal novel therapeutic strategies to maintain myofiber integrity. This could delay the involvement of the multi-cellular regenerative process, usually overwhelmed or diminished in muscle disorders and old age.

What makes us human? Understanding the evolutionary emergence of human traits is important both for fundamental knowledge and our capacity to treat diverse diseases. In this project I aim to understand molecular mechanisms governing human speech development. FoxP2 is a highly conserved transcription factor, dubbed the ‘speech gene’ because families with mutations in this protein have speech deficits. Two amino acids variants in FoxP2 have been subject to strong selection in our lineage. Introducing them into mice increases vocalization. Yet these mutations do not localize to FoxP2’s DNA-binding domain. Rather, they bracket a long prion-like domain near its N-terminus. Preliminary results show that these variants enhance FoxP2’s capacity to form ‘infectious’ prion-like aggregates. Remarkably, FoxP2 aggregates arise naturally, and in a developmentally regulated manner in the neural crest. I aim to characterize the physiological function of FoxP2 prion-like aggregation during neural crest differentiation, identify the developmental consequences of the evolutionarily selected mutations in this protein, and ultimately identify molecular mechanism(s) by which FoxP2 regulates cellular differentiation. I will test the importance of FoxP2’s prion domain using a series of powerful genetic and cell biological approaches. Subsequently, I will investigate the regulation of prion formation during development. Finally, as a long-term goal I will test if aggregation alone, or templated propagation of the prion per se, is required for normal differentiation. If fulfilled, the aims of this proposal thus have the potential to transform our understanding of human development and evolution.

Dendritic cells, a type of white blood cell, are the sentinels of the immune system. Upon infection with a pathogenic microbe, such as a bacteria or fungus, dendritic cells can ingest the microbe by a process call phagocytosis. Dendritic cells then kill the ingested microbe by huge amounts of hydrogen peroxide, a disinfectant, and subsequently degrade the microbe by enzymes. This releases protein fragments from the microbes which can be presented to T cells, another type of white blood cell. T cells thereby learn to recognize the microbe and can mount a pathogen-specific immune response.
Dendritic cells need to be able to ingest many different types of microbes with a large variation in motilities, sizes and shapes, ranging from very small viruses to fungal hyphens. Although it is well-known that many types of pathogens use their shape and motility to try to avert immune responses, the underlying cell biological mechanisms are still largely unknown. The goal of this project is to address how microbe shape and motility affect their processing by dendritic cells for T cell activation.
We plan to chemically engineer bacteria-sized artificial particles with well-defined but highly irregular shapes, such as star-shaped particles. These particles will be equipped with chemical engines that are fuelled by hydrogen peroxide (produced by the dendritic cells) to generate motion. We expect that the shape and forces exerted by these particles will affect their transport and processing within the crowded environment of dendritic cells. The behaviour of these particles will be studied in well-defined dense suspensions that mimic the complex environment within dendritic cells. These biophysical experiments will be combined with experiments with dendritic cells isolated from the blood of healthy volunteers. This will allow a full quantitative understanding of how pathogen shape and motility affect their fate in dendritic cells.
This study will lead to a better understanding of how pathogens use their shape and motilities to avoid clearance by the immune system. Moreover, understanding how microbe shape and motilities affect immune responses will allow us to generate particles that elicit very fast and strong immune responses or, conversely, no (or very slow) immune responses, which will aid in the design of improved vaccines and carriers for drug delivery.

Animals exploit acoustic signals for a wide range of behaviors, from sound communication to predator localization, which require the discrimination and identification of different sound sources in the auditory scene. Echolocating bats, for example, rely not only on sound to navigate the environment, but also to communicate with conspecifics. Therefore, bats must segregate self-generated echoes from other acoustic signals in the environment, including those produced by conspecifics. Past research has yielded a wealth of data on the neural mechanisms of biological sonar in bats, but far less is known about how the bat auditory system differentiates between echolocation and social communication signals. My research aims to address this question by recording and analyzing midbrain inferior colliculus responses to sound stimuli that carry echo information from the environment or social information from conspecifics. Experiment 1 will compare neural activity in bats listening to playbacks of their own echolocation signals, those of conspecifics, and acoustic communication signals. Experiment 2 will investigate the dynamics of neural activity evoked by actively echolocating bats engaged in a target-tracking task. Experiment 3 will employ novel methods to characterize auditory activity evoked by sonar echoes and social communication signals in the free-flying bat as it navigates around objects in its physical environment. Comparative studies are key to identifying specializations and general principles of neural systems, and this project will advance a deeper understanding of auditory processing in animals that rely on sound to guide behavior.

It is estimated that about 40% of plant species can reproduce asexually, suggesting that this mode of reproduction is beneficial to allow plant survival under certain environmental conditions. Despite its prevalence, we know very little about how asexual reproduction contributes to the environmental stress response in plants. The central hypothesis of this proposal is that environmental stress induces epigenetic changes that can be stably propagated to the asexual progeny and potentially contribute to plant adaptation. To test this hypothesis, I will analyze the phenotypic, transcriptional and epigenetic changes in response to stress using Arabidopsis lyrata (A. lyrata), a plant species that asexually produces novel ramets from its root. In particular, I will investigate stress memory in novel ramets produced from parental plants that have been exposed to environmental stress (heat, salt, and cold stress). I will generate and analyze epigenetic profiles of ramets propagated over five generations under stress or non-stress conditions to identify stress-induced epimutations. I will furthermore investigate the signal transduction between maternal plants and ramets attached to each other through roots. Transcriptomic and phenotypic analyses will reveal how stress treatment of parental plants affects the attached ramets. Finally, I will test whether induced epimutations have adaptive value by investigating whether clones derived from stress-treated maternal plants are better adapted to stress. The proposed study will not only reveal a potential novel mechanism to adapt to environmental conditions, but also provide the basis for the generation of stress-adapted crops and trees.

Despite a century of study, little is known about the mechanisms underlying bacterial cell death and survival during starvation, even for the simple model organism E. coli. In this project, we will address long-standing questions on how starvation results in cell death, i.e. which molecular processes collapse irrevocably and why. Our preliminary data indicates that cell death of E. coli during starvation is due to turgor pressure induced lysis. I will approach this hypothesis with two objectives: 1. Understanding cell wall dynamics during starvation-induced lysis and 2. Elucidating the role of turgor pressure during lysis and its dependence on metabolic state. In objective 1, I will quantify the role of cell wall remodeling in starvation-induced lysis. I will capture rare lysis events using microscopy, genetically titrate key cell wall hydrolases and metabolic enzymes involved in cell wall turnover, as well as investigate the trade-off between optimal starvation survival and swift growth resumption. In objective 2, I will characterize the energy-dependence of osmoregulation (ion pumps and channels) and its effect on cell wall remodeling. Theory and experiments will be combined, eventually resulting in a mathematical model that will be capable of predicting starvation dynamics under diverse environmental and genetic perturbations. The project will also reveal new avenues to fight dormant and recurring infections, as it will identify the essential processes that enable long-term survival, which therefore constitute important drug targets.

The cerebellum is a particularly attractive model system for studying brain function since it has a relatively simple and well-defined circuit and its computations are directly related to behavior. Key functions of the cerebellum are the fine coordination of movements and motor learning. Influential early theoretical work predicted how input from mossy fibers is recoded in granule cells in the input layer of the cerebellar cortex to increase the computational performance and to achieve separation of similar patterns. However, due to the limited possibility to simultaneously record inputs and outputs of this granule cell layer the mechanisms employed are still not fully understood. Likewise, it is unknown how mossy fiber inputs or the transformations in the granule cell layer change during motor learning. Finally, whether expectation mismatches, important to the fine coordination of movements and motor learning, are encoded in granule cells is unclear. Therefore, simultaneous two-photon in vivo imaging of two different calcium indicators in 100-300 mossy fibers and granule cells in awake behaving mice will be employed. A water-droplet-reaching task will be used to assess pattern separation between similar movements, contributions from different precerebellar regions to information processing, and alterations in mossy fiber input and computations during learning. A lever-press task introducing force fields will be used to generate an expectation mismatch. The proposed study aims to significantly improve the understanding of computations, including learning-related adaptations, in the cerebellar cortex to shed light on the basic processing performed in neuronal circuits.

During embryonic development a series of gene expression programs are executed in a tightly controlled, quantitative manner. A multitude of developmental processes must progress in a highly coordinated fashion governed by complex gene-regulatory networks. To elucidate the regulatory principles that allow the coordinated execution of multiple developmental processes I propose to investigate the interplay between X-chromosome inactivation and cellular differentiation, which occur during early embryogenesis of mammals and can be recapitulated in female mouse embryonic stem cells in cell culture. X-inactivation is initiated by up-regulation of the long non-coding RNA Xist, which will mediate gene silencing of the entire chromosome in cis. Xist up-regulation is thought to be triggered by down-regulation of pluripotency factors that repress Xist in undifferentiated cells. More recently however I discovered that X-inactivation will also feed back into the differentiation process, suggesting a more complex interplay between these two events. I propose to combine several experimental and theoretical approaches to elucidate the structure and function of the underlying regulatory network. To this end, we will perform CRISPR-based screens to identify key regulators that mediate these interactions and perform quantitative perturbation experiments of the identified factors. We will use these data sets to discriminate between alternative topologies of the underlying regulatory network and to develop a quantitative predictive mathematical model. In this way I hope to gain a quantitative understanding of a complex regulatory network that governs early mammalian development.

Cancer cells undergo metabolic reprograming to sustain proliferation and to enable metastatic outgrowth, but this results in oxidative stress limiting cancer progression. Cancer cells are able to overcome their distorted redox balance, however, mechanisms providing redox tolerance during cancer progression are not well understood. Redox tolerance is largely provided by cysteine-derived glutathione, the major cellular antioxidant. Despite the clear biological relevance of cysteine metabolism, the role of de novo cysteine synthesis (transsulfuration) for cancer progression remains elusive. Our objective is to unravel the contribution of the transsulfuration pathway to redox control in cancer cells. We hypothesize that metastasizing cancer cells circumvent the need for cysteine import by activating the transsulfuration machinery to overcome oxidative stress-induced cell death. In this research program, we will explore whether induction of transsulfuration can sustain redox control in cancer cells under cysteine deprivation, identify factors that drive or limit utilization of the transsulfuration pathway, and define the role of the transsulfuration pathway and its regulating factors for cancer progression. To reach these aims, we will employ cell culture and in vivo models, state-of-the-art metabolomics, CRISPR/Cas9 screening, and test the validity of the findings in human cancer specimen. My approach will create a better understanding of transsulfuration in the context of cancer and elicit novel concepts of redox control mechanisms. As result, this research program will shed light on unprecedented tumour vulnerabilities that could be exploited as anti-cancer therapy.