G-quadruplex (G4) structures are stable four-stranded nucleic acids assemblies that can form in guanine-rich DNA. Their recent visualization in mammalian cells has established G4 structure formation throughout the genome and fueled research into understanding the biological implications of these structures in cell regulation. G4 sequence motifs are abundant and conserved in our genome. G4 structures form transiently and regulate numerous cellular processes such as DNA replication, transcription and telomere maintenance. Importantly, these structures also come at a cost as they are able to induce genomic instability in certain cellular conditions, for example in cancer cells that suffer from replication stress. Currently, the mechanisms that form and resolve G4 structures are unknown. Several helicases can unwind these stable DNA structures in vitro but it is unclear whether and how these helicases function in vivo. Understanding the biochemical mechanisms that resolve G4 structures is crucial to further understand their function and how they induce DNA mutations in the cell. In this project, I propose to decipher the molecular mechanisms of G4 structures unwinding. The groundbreaking nature of this proposal is the use of a unique method to follow G-quadruplex unfolding in time under near-physiological conditions in vitro. This method, which was recently established in the host laboratory, uses DNA replication stalling and bypass at defined G4 structures as a direct readout for G4 stability and unwinding. This gives me the opportunity to address important aspects that have not been studied under physiological conditions before: Aim 1: To determine the stability and unwinding properties of distinct G4 conformations. Aim 2: To identify molecular mechanisms and novel proteins in G4 unwinding. The results of this project will give important new insights into G4 regulation, an unexplored but important biological process.

Early animal embryos undergo a profound reprogramming of cell behaviour at the blastula stage in a major developmental transition known as the mid-blastula transition (MBT). Earlier studies demonstrated that MBT is an essential transition for embryonic development across various species, involving simultaneous changes in key cellular activities: activation of zygotic genome transcription (ZGA), cell cycle elongation, loss of division synchrony, and onset of cell motility. In contrast, little is known for mammalian embryos, which are often viewed as not having an MBT because of their slow initial cell cycle and early ZGA. However, MBT also exhibits a switch in the cell growth mode from reductive cleavage divisions to proliferative growth, and our recent findings suggest that this switch occurs at the blastocyst stage in mouse embryos. This project aims to identify the mammalian MBT, define its fundamental features and investigate the mechanisms that control its timing. Employing light-sheet microscopy to image mouse embryos at high spatiotemporal resolution, automatic cell segmentation and single-cell tracking, I will quantitatively characterise the cell volume dynamics in early embryogenesis. This will allow me to identify the transition from cleavage to proliferative growth and use this time reference to characterise novel MBT features. Specifically, I will focus on 18 hours within the blastocysts stage to test the hypothesis that mammalian MBT entails changes in cell fate, metabolism, spindle assembly and/or cell cycle control. Through temporally controlled perturbations I will determine if there is a causal link between these changes and identify the upstream regulator, the trigger for the mammalian MBT, as well as the mechanism by which the MBT timing is controlled. The identification and characterisation of mammalian MBT may provide a new definition and mechanism regulating this key transition in animal development.

Approximately forty million people across the world are blind, a condition with serious consequences for a person’s autonomy. Restoration of visual function in blind individuals is an important scientific goal with large societal benefits. In a large fraction of blind patients, the connection between the eye and the brain has degenerated so that restoration of a rudimentary form of vision can only be achieved in pathways downstream from the retina, like the visual cortex. PROVISO tests the feasibility of a new approach for a cortical visual prosthesis by implanting flexible polymer electrodes into the visual cortex that can be stimulated electrically to create a rudimentary form of vision. Weak electrical currents applied to an electrode in the visual cortex induce an artificial percept of light, called “phosphene”. Multiple phosphenes can be used to build up a shape, just as the lights of a matrix board along the highway generate letters. A promising approach to restore vision involves inserting tiny electrodes into the visual cortex, close to the neurons, so that weak currents result in phosphene perception. There are two problems that need to be solved before the prosthesis can become a treatment for blind individuals. The first problem is that the electrodes available for chronic implantation are made of silicon or metal, which causes a tissue response (gliosis) causing the interface with the tissue to degrade after several months. The second problem is coverage of the visual field. In humans, much of primary visual cortex (V1) is not located on the surface of the brain, but inside a sulcus, making it difficult to access. PROVISO will develop methods to implant flexible electrodes, that cause little tissue damage and remain functional across longer time scales, into the brain. These electrodes will be implanted deep into several brain areas, thereby providing a good coverage of the visual field.

DNA damage contributes to the ageing of tissues and causes mutations that drive cancer. However, the molecular mechanisms underlying many mutational processes are not understood, and neither is how much they contribute to disease. With a powerful new technology, I propose to close these major gaps for common ?clock? mutations of unknown origin. The recent genome sequencing revolution has revealed unexpected diversity in mutational patterns. One pattern in particular is intriguing as it behaves as a molecular ?clock?: the number of mutations increases over time and correlates with age. Although the textbook view of mutagenesis is that cell division is required to convert DNA damage into point mutations, surprisingly, non-dividing cells like neurons also accumulate ?clock? mutations as a function of time, indicating that ?clock? mutations arise without genome replication. Understanding how cells mutate independently of proliferation?and thus challenging the current paradigm of mutagenesis?is a fundamental question that has been hindered because assays to measure mutation require cell division. Excitingly, I have overcome this major obstacle by establishing a powerful strategy to sensitively detect mutations in single cells, genome-wide. My group will combine this approach with targeted genetic manipulations in cells and mice to answer two central questions. (1) How do cells mutate independently of proliferation? (2) What drives these mutations in tissues? My novel insights into endogenous DNA damage from mouse genetics and genomics, combined with the innovative sequencing strategy that I have established, uniquely position me to answer these long-standing questions. Together, the work proposed here will reveal the molecular mechanism(s) underlying the most common mutational process in humans: ?clock? mutation. The methods, data, and insights from these groundbreaking studies will directly impact cancer research and uncover novel sources of DNA damage during aging