Organisms need strategies to survive when conditions are hard. For mammals, winter is particularly difficult - they have to invest large amounts of energy into keeping warm, while food availability is low. For this reason, many mammals migrate or hibernate. However, what to do if you are too small to migrate long distance, burn your energy fast, and cannot hibernate? The common shrew is such a mammal and has evolved an astonishing strategy: each individual shrinks in winter by up to 20% and then regrows in the spring by about 13%. This size change, thought to allow shrews to survive on fewer resources because of the smaller size and linked lower energy requirements, include not just overall size, but specifically organs that do not usually change size in fully grown animals, such as the brain, heart and liver.
The process of neurological degeneration and regeneration is of great interest, since many central nervous system diseases (e.g., Alzheimer’s, multiple sclerosis) involve degeneration, but ongoing research for therapies to reverse this process has been of limited success. As one of only a few recorded examples of mammalian brain regeneration, understanding how the shrew regrows its brain can accelerate research that leads to future therapies.
To answer the question of how the shrew shrinks and then regrows its brain, we will establish this unusual species as a new model, by studying the biological, molecular, biochemical and genetic processes behind this reversible size change. Besides establishing a database of information that can be mined and researched in years to come to discover the pathways that generate this cycle in the shrew, we will test a metabolic model of neurological change by artificially blocking molecular access to fats. Thus, the cross-disciplinary study of this wintering adaptation may help us understand more about regeneration in mammals in general, and the brain in particular.

A plausible explanation for why mitochondria have their own DNA (mtDNA) is to ensure colocalization of redox-genes and redox-regulation to the same membrane-bound organelle. While this distributed control system seems logical, very little is known about how such autonomous regulation takes place. A variety of methods that are widely applicable to study genomes fail to work on mtDNA, and sequence-to-function mapping of the mitochondrial genome is missing. I plan to create a modified, hypermutating DNA polymerase gamma (polG), the designated mtDNA replicase. polG is encoded in the nuclear genome and can therefore be easily manipulated. Hypermutator polG will be integrated into the nuclear genome of S. cerevisiae, which will introduce mutations in mtDNA without altering the nuclear genome. I will then use selection assays followed by next generation sequencing to study the ability of mtDNA yeast variants to adapt to respiratory conditions. Such experiment will highlight regulatory hotspots in mtDNA that will be mechanistically evaluated using genome-wide methods like native elongating transcript sequencing (NET-seq) and mitochondrial ribosome profiling that are the ‘bread and butter’ of the Churchman lab. Clear view of the fitness landscape associated with mtDNA will inform research into many deep questions in mitochondrial biology, ranging from ‘how selection shapes mitochondrial genomes?’ to ‘why do certain mtDNA mutations cause cancer?’.

Trypanosoma brucei is the causative agent of sleeping sickness. In the mammalian host, parasites exist in two major niches: the blood, and the brain. Recently, Dr. Figueiredo’s lab found a previously undiscovered third reservoir of parasites in the adipose tissue which were transcriptionally distinct from blood stream forms, and the genes upregulated included various markers relevant to parasite metabolism. My proposed work in Dr. Figueiredo’s lab is to implement in vivo imaging methods to a) investigate the parasite’s mechanisms of homing and crossing of the vascular endothelium into the adipose tissue, and to carefully define the adipose tissue niche that acts as reservoir for the parasites; and b) to elucidate host vasculature, and parasite factors involved in allowing such unique phenomenon, including generating parasite mutants deficient in adenylate cyclises, flagellar proteins, and mechano-sensing proteins among others. Using in vivo techniques and biophysical and molecular methods, I aim to investigate the biological significance of parasite residence in the adipose tissue – a location which could be clinically relevant for interventions in human and veterinary medicine.

Harmful algal blooms (HABs) have been long studied in marine and fresh water environments, yet most research to date has focused at the bulk scale. Despite the well-documented toxic ramifications of HABs on vertebrates and mammals, including humans, we still lack a biophysical understanding of the mechanisms by which toxins disperse during a bloom event. Using a combination of experiments and modelling, in this project I will explore the physico-chemical interplay underlying the release and transport of toxins at the scale of the microorganism. Towards this I will develop micro- and millifluidic experiments to generate ‘bloom-in-lab’ and visualize the dispersion of targeted toxins under three ecologically relevant cues: fluid flow, temperature and salinity. Specifically, I will study red-tide forming Heterosigma akashiwo, a globally distributed marine raphidophyte, known to release a range of toxins, including reactive oxygen-nitrogen species (RONS) during bloom conditions. Original data from high-speed and time-lapse microscopy on fluorescently labelled ‘target’ molecules will put forward first experimental quantifications of toxin release and transport under different micro-environmental cues, thereby informing a new mathematical model that will capture, at microscales, dispersion and auto-feedback dynamics during bloom events. Analysing the coupling between microbial biophysics, transport phenomena and biochemistry, this project will bring about a fundamental, mechanistic understanding of toxin transmission, and help develop accurate and comprehensive algorithms for predicting HAB formation, especially during the rapidly warming climate we encounter today.

Human medicine is on the verge of a new era where we face the possibility to precisely rewrite the genome to prevent, ameliorate and cure a wide range of immune-hematological diseases. Hematopoietic stem and progenitor cells (HSPC) have long been a preferred source for ex-vivo gene therapy, as gene correction in multipotent progenitors ensures a life-long supply of corrected progeny and polyclonal reconstitution of the bone marrow. Recently, programmable nucleases brought the possibility of genome editing (GE) within the reach of gene therapy by allowing locus-specific gene correction without the risk of aberrant transgene expression. However functional studies that accurately assess possible acute and long-lasting consequences of GE procedures in HSPC are needed to harness GE therapeutic potential. Indeed, despite recent advances in the generation of corrected HSPC by GE, the efficiency of the targeting process and the ability of edited cells to stably reconstitute the hematopoietic system upon transplantation remain limited, pointing to a loss of repopulating capacity of HSPC upon targeting. The goal of this project is to investigate the role of DNA damage response (DDR) pathways in GE strategies and uncover novel molecular players that could be exploited to increase the yield of long-term engrafting gene-edited HSPC. Our findings could be easily applied to gene correction strategies for a variety of inherited pathologies affecting the human immune-hematopoietic system and provide a foundation for more efficient modalities of editing HSPC at genomic sites of interest for both basic and translational research.

Areas of visual cortex integrate signals representing sensory input, ongoing motor actions, and internal states. While neurons in primary visual cortex (V1) were classically interpreted as feature detectors that signal the presence of a specific visual stimulus, recent evidence suggest that V1 circuits participate in the specific, topographical integration of sensorimotor signals. How sensory and sensorimotor computations are implemented in the circuitry of V1 and higher visual areas, and how these computations depend on the behavioral context, is unknown. I will combine novel circuit dissection tools, wide-field-of-view holographic stimulation, and virtual reality environments to approach these questions. I will selectively activate V1 ensembles with distinct functional properties during sensorimotor behavior using holographic stimulation and monitor the effects on the surrounding populations in V1 and higher visual areas. Furthermore, I will analyze the effects of holographic stimulation as a function of different behavioral parameters and during optogenetically induced variations in internal state. Finally, I will explore methods to target the expression of genetically encoded tools to distinct V1 ensembles, each with specific function during sensorimotor behavior, which will allow me to study if different ensembles are selectively connected to circuits throughout the brain, as well as to determine their selective impact on brain-wide dynamics. This project will shed new light onto the function of V1 during sensorimotor behavior, and yield fundamental insights into the mechanisms by which cortex can adapt its processing machinery to varying behavioral demands.

Transposable elements (TEs) are important drivers of genome evolution with the potential to rewire transcriptional networks but require constant surveillance to minimise deleterious effects. Major players in the transcriptional regulation of TEs are KRAB domain-containing zinc finger proteins (KZFPs) that co-evolved in response to new TE invasions and constitute the largest family of transcription factors encoded by the human genome.
Recent views have shifted from a simple arms race between TEs and their host genomes towards a more complex domestication process. This can involve the rewiring of gene regulatory networks using TE-derived regulatory elements, co-option of TE-derived genes, but also the exaptation of KZFPs to acquire new regulatory functions after the interaction with their target TE has become obsolete.
Such newly adapted functions for KZFPs can be mediated by novel protein interactors but can also involve the binding to, and regulation of RNA molecules, as zinc finger domains are not limited to interactions with DNA. Indeed, many of the evolutionary conserved KZFPs show weaker interactions with the TE-silencing machinery and instead display unique protein interactomes that indicate an RNA-based functionality.
This proposal lays out the experimental strategy to systematically identify RNA-binding KZFPs in the human genome and profile their corresponding RNA interactomes to elucidate the underlying regulatory mechanism. Exaptation of KZFPs to function as RNA-binding proteins extends their role from transposon control to post-transcriptional regulation of protein-coding and non-coding RNA molecules, thus providing intriguing new layers of gene regulation.

Cell division is a complex but fundamental life process. Among its many purposes, it is needed for a fertilized egg to develop into a human being, for our wounds to heal, for infections to clear and for our bodies to sustain life. Whenever a cell divides, its genetic information is duplicated and packaged into chromosomes which are then separated and equally distributed between the new daughter cells. For the newly formed cells and ultimately the body to be healthy, distribution of the chromosomes should be highly accurate. Accurate chromosome separation during cell division is driven by dynamic cellular cables that are connected to special chromosomal regions known as centromeres. Indeed, defects in centromere formation or function compromise chromosome separation and lead to daughter cells containing too many or too few chromosomes, a hallmark of cancerous cells.
When eggs are prepared for fertilization, a specialized form of cell division called meiosis separates the chromosomes. The accuracy of chromosome separation during meiosis determines whether a fertilized egg can develop into a healthy human being. Surprisingly, meiosis in humans and other mammals is highly prone to errors and often leads to eggs that contain the wrong number of chromosomes. Fertilization of such chromosomally abnormal eggs frequently leads to human embryo deaths and conditions such as Down’s syndrome. Complications arising from erroneous chromosome separation in eggs become even more frequent as women get older. Research in the field of meiosis has only scratched the surface of why chromosome separation in eggs is highly error-prone. Furthermore, the reasons behind the deterioration in the quality of this process as women get older largely remain unknown.
In this research proposal, we will test the hypothesis that defects in centromere function that accompany ageing may contribute to poor quality of eggs in older women. To achieve this, we will combine our unique but synergistic expertise in advanced light microscopy, genome editing and electron microscopy. Knowledge gained from this study will advance our understanding of why eggs of older women are often chromosomally abnormal. In the long-term, this work can potentially be exploited for treatments of human infertility.

To study how nervous systems arise and function, scientists use animal models in which it is possible to integrate research on fundamental processes across different levels of organization from genes to behavior, and from the zygote to the adult. The medicinal leech (genus Hirudo) provides one useful model, because its nervous system is much simpler and easier to work with than vertebrate or mammalian nervous sytems, even though it functions in a similar manner--Hirudo has been used to study phenomena of general importance such as: how glial cells function; how neurotransmitter are released; how synapses form and regenerate; and how neural circuits function to control behavior. Another leech (genus Helobdella) is used to study development and how development changes during evolution, giving rise to kinds of animals over hundreds of millions of years. These two leech species exhibit marked similarities of course, but also some differences. Technical considerations (small embryos for Hirudo; small adults for Helobdella) have made it difficult to integrate these two models, e.g. by applying molecular approaches in Hirudo or to study the adult nervous system in Helobdella. The goals of our project are: 1) to enhance the power of the Hirudo model by introducing newly-developed molecular approaches; 2) to implement approaches in Helobdella that will enable us to unite molecular and cellular approaches to developmental and behavioral neurobiology; 3) to develop new optical techniques for stimulating and recording neuronal activity without exogenous dyes or genetic manipulations.
The intellectual significance of the proposed work is twofold. First, it will enhance our abilities to answer fundamental questions regarding how nervous systems function and development by introducing cutting edge technical approaches to the cellularly simple, physiologically accessible leech models. Of equal importance, it will provide a new evolutionary perspective into neurobiology, by allowing us to examine similarities and differences between leech and other models, including arthropods, nematodes, and vertebrates which have all been evolving separately for more than half a billion years.

When an animal navigates in an environment, neurons located in the hippocampus fire selectively when the animal is in a particular place. Together, these ‘place cells’ form a map of the environment and report the position of the animal on this map. To construct its spatial map, the hippocampus relies on self-motion information (e.g. distance travelled or direction of motion) but also on sensory cues (e.g. visual, auditory or olfactory cues). The hippocampus thus gives special weight to sensory information, which is processed by cortical areas. Sensory areas of the cortex were classically thought to provide the hippocampus with purely sensory information, independently of the position of the animal in the environment. But we recently discovered that the activity of neurons in the primary visual cortex is influenced by the spatial context during navigation, suggesting that navigation signals modulate sensory processing in cortical areas. It is yet unknown which navigation signal influences visual cortex and how this phenomenon generalizes to other sensory modalities and other behavioral contexts where hippocampus is involved. By recording neurons in hippocampus and visual or auditory cortices while mice perform a task in a virtual environment, I will investigate three questions: 1) Are visual responses modulated by the route that the animal takes or by its absolute position? 2) Is there a similar modulation by spatial context in other sensory areas and how signals from different senses are integrated together during navigation? 3) Are sensory responses modulated in non-spatial contexts where the hippocampus still builds a representation?

Proteins have evolved to adopt many structures and perform diverse functions by exploring a sequence space spanned by twenty canonical amino acids (AAs). Whilst ten of the AAs were ‘obvious’ choices, as they abounded in the prebiotic world, the other ten were far less accessible prebiotically, thus provoking the question: Why (and how) were these AAs included in the genetic code, and was their inclusion prerequisite for protein evolution to be as successful as it has been? These seeming Gedankenexperiments are directly testable using an interdisciplinary approach we have devised.
Specifically, we propose to synthesize random protein libraries built from reduced (evolutionarily early) and alternative AA alphabets to compare the structure/function-forming potential of the proteinogenic and non-canonical yet prebiotically abundant AAs. The team of Stephen Fried will customize both commercial and home-made cell-free protein translation systems to express protein libraries composed of alternative AA alphabets. The team of Klara Hlouchova will use biophysical approaches to explore the structure-forming potential of the purified libraries. The capacity of the libraries to evince prebiotically-relevant functions will be assessed by the group of Kosuke Fujishima through selections that can relate genotype to phenotype (e.g., mRNA-display). Large sequence space (>10^12) will be analyzed in each experiment and because the same template libraries will be used, the outcomes of both the structural and functional studies will be directly comparable. This would not be possible without coordinated collaboration among the three teams.
Each team member enters the project with a key set of skills and scientific expertise. Klara and her team have a strong background in protein biochemistry, bioinformatics and experience with expression of protein libraries. Kosuke is an astrobiologist with strong experience in RNA molecular biology and his team takes a synthetic biology approach to studying peptide-RNA interactions. Stephen has experience in biophysics and synthetic biology and his newly started lab performs research in protein folding and engineering. This project relies on synergy of the above mentioned disciplines connected by our mutual interest in the origins of life, making it possible to address a broad fundamental biological question in a systematic way.

Non-canonical initiation of protein translation plays a central role during cellular stress, apoptosis and cell survival. Death-associated protein 5 (DAP5) acts as the major scaffolding initiation factor to promote cap-independent translation of cellular mRNAs such as p53, Bcl2, Apaf1 and XIAP. Potentially, translation of such transcripts can be initiated via internal ribosome entry sites (IRESs), an alternative to canonical cap-dependent translation. The structural and molecular mechanism of how DAP5 regulates IRES-driven translation is not understood. To resolve the inherent molecular processes, I will investigate IRES recognition by DAP5 in target mRNAs through novel NMR techniques, biophysical tools and mass spectrometry. My research is aimed to solve the first three-dimensional structure at atomic resolution of a cellular IRES along with its functional characterization. Based on this, the interaction between this IRES and DAP5 that is relevant for cell fate decisions will be studied structurally and biophysically. I will analyze not only the role of the structured MIF4G domain of DAP5, but also the so far unknown function of its disordered regions in IRES recognition. In order to shed light on regulatory principles of DAP5, I further aim to investigate structural effects on mRNA binding upon DAP5 phosphorylation. In a proteome-wide examination, I will identify additional factors of IRES-mediated translation to define a potential core complex associated with different mRNAs. My project thus comprehensively aims to decipher the mRNA recognition mechanism of DAP5 that controls IRES-driven, cap-independent translation linked to cell fate decisions.

Adaptive resistance is an emergent phenomenon in melanoma cells that allows tumor cells to adapt and escape treatment through a change in signaling state. The complex interplay between transcriptional and post-transcriptional regulation that gives rise to adaptive resistance is in large parts poorly understood. To unravel the underlying molecular mechanisms, in silico approaches involving statistical or kinetic models have to complement experimental analysis. Statistical models are well-suited to identify unknown molecular mechanisms but cannot describe emergent phenomena. In contrast, kinetic models intrinsically describe emergent phenomena but are challenging to apply when the underlying molecular mechanisms are unknown.
In this project, I propose a novel, integrated, data-driven approach to unravel the molecular mechanisms that give rise to adaptive resistance in melanoma. The approach combines kinetic and statistical modeling to harness the benefits of both approaches. The statistical modeling will be used to derive biological hypothesis to construct and extend kinetic models in an unbiased, data-driven, automated fashion. This will render the construction of kinetic models less dependent on prior knowledge. The constructed kinetic model will be able to quantitatively describe emergence of adaptive resistance and provide insight into underlying molecular resistance mechanisms.

Plants are the primary producers in land ecosystems, a central position that give them a fundamental ecological importance. In the wild, plants interact with multiple organisms, including bacteria and insects, to form rich interconnected networks. In particular, plants are colonised by communities of microbes, including both beneficial and pathogenic bacteria. For these microorganisms, it is crucial to spread between plants, which they often do by recruiting herbivorous insects as vectors. Plant pathogens may even manipulate the behaviour of the insect that carries them. However, the underlying mechanisms and evolutionary dynamics of this important behavioural component of plant-insect-bacteria systems remain unknown.

My work will address how bacteria attract and alter the behaviour of their insect vectors by combining three fields of biology: plant physiology, insect behaviour and bacterial genomics. This project follows two independent questions: 1) how pathogenic bacteria attract insect vectors and 2) whether bacteria that have colonized a vector can manipulate vector behaviour. To address these questions, I will
use the Sacptomyza-Arabidopsis-Pseudomonas system as a model of bacteria-facilitated herbivory
and build high-throughput experimental assays to score the behaviour of the drosophilid Scaptomyza. This quantitative approach will allow me to compare the effect of dozens of bacterial stains on the behaviour of their vector and ultimately to find the bacterial genes required for vector recruitment and manipulation. This approach could to transform a largely descriptive area of plant-insect-bacterial interactions into a high-throughput and mechanistic field.

Humans have an intimate bond with their gut microbes. These minute organisms, six orders of magnitude smaller than us, populate our gastrointestinal tract to form one of the densest ecosystems on earth. The gut microbiota, often referred to as ‘the forgotten organ’, has been shown to play important roles in human health; however, we still understand little on the molecular interactions among microbes and between microbes and the host. During my HFSP-supported postdoctoral training,I studied the role of gut microbes in complementing our immune system. We found that each microbe plays a unique melody on the host’s immune system and together the wealth of immune-modulatory gut microbes creates an exquisite harmony that complements our immune system. I also introduced a novel fluorescent-labeling method to enable, for the first time, to visualize live gut microbes, at real-time, in their natural habitat. For my CDA, I propose to combine my systems biology background with my postdoctoral training in immunology and microbiology, to study how the microbiota actively and mechanistically network and interact with the mammalian host to maintain homeostasis and confer protection from disease. My postdoctoral studies identified redundant immunomodulatory capacities of gut microbes across distant phyla.To me, these findings emphasize the importance in gut microbiota diversity. I hypothesize that the gut microbiota diversity is vital and physiological relevant, and that this diversity represents an intricate, delicate, evolutionarily balanced host–microbiota relationship that occupies a central role in health and disease. During my CDA, if granted, I would like to test this hypothesis.

Mitosis is key to the cell cycle, as it guarantees the accurate segregation of replicated chromosomes to daughter cells. This process relies on the mitotic spindle, a microtubule-based, dynamic, and bipolar structure. Spindle morphology varies greatly among species and cells to optimise its function. Size and architecture, in particular, are both essential for accurate chromosome segregation, cell division and cytokinesis. However, despite decades of study and the investigation of hundreds of proteins involved in spindle assembly, it remains unclear how spindle microtubule subpopulations organise into complex assemblies. Especially, how correct spindle morphometrics, at both the size and architectural levels, are established is poorly understood. Using the egg extracts of two Xenopus species of different spindle sizes and architectures, X. laevis and X. tropicalis, we will reconstruct microtubule structures assembled independently from the two major organising sites that contribute to spindle assembly, the spindle poles and chromatin. By combining cutting-edge fluorescence microscopy and electron tomography analyses, we aim to reveal the dynamic and ultrastructural bases of spindle substructure size and architecture. Our goal is then to extract quantitative parameters and combine them into physically realistic simulations to decipher the biophysical basis of the different architectures and scaling properties, and ultimately their implication for the regulation of spindle morphology. Altogether, this study at the frontier between cell biology and biophysics will not only unravel key mechanisms of microtubule organisation but also fundamental principles of spindle assembly.

The aerial courtship “dance” of mosquitoes has fascinated entomologists for over 150 years. This dance involves highly controlled variations in the frequency and intensity of flight-tones (i.e. sounds generated by the flapping wings) with concurrent changes in flight speed and direction, and enables recognition of conspecifics, display of fitness and transmission of mating interest. However, despite over a century and a half of research, significant knowledge gaps continue to exist in our understanding of this behavior. To decipher this courtship dance, entomologists have to integrate acoustic, energetic and flight information for untethered, free-flying mosquitoes, but the tools that can provide these data have, so far, not been available. In the current project, the two investigators combine their respective expertise in computational biomechanics and acoustics, and behavioral entomology, to generate unprecedented data and insights into the biomechanics and physics of courtship-associated acoustic communication in mosquitoes. In particular, by combining computational modeling with biological assays, the team will generate six-dimensional soundscapes of free-flying mosquitoes engaged in courtship and determine how these soundscapes are actively modified during courtship. We will also estimate for the first time, the energetic costs of courtship and mate-chasing, and the potential constraints this places on courtship behavior. Finally, the team will characterize the degree to which, carefully tailored exogenous sounds can alter and even disrupt courtship. The success of this novel approach could be transformative for future research into comparative auditory mechanisms of communication across a wide range of flying insects. In addition, the insights gleaned here could form the scientific foundation for novel insecticidal/surveillance traps and also lead to environmentally friendly strategies for diminishing mating success in mosquito species that are vectors for malaria, Zika fever and other devastating mosquito-borne diseases.

Mosquitoes rely on their exquisitely tuned sense of smell to efficiently home in on humans to blood feed. This epidemiologically important behavior is intricately gated by the internal physiological state of the mosquito. For instance, upon starvation the African malaria mosquito Anopheles gambiae reflexively exhibits heightened attraction towards human scent. Such state-dependent shifts in sensory perception are often evoked by neuromodulation of olfactory circuitry mediating attraction to food. Here, I propose to apply genome engineering coupled with multiphoton imaging in the mosquito nervous system to identify key olfactory circuits and neuromodulatory networks that control An. gambiae attraction to humans. To achieve this goal, I will initially characterize how whole human scent and its constituent odorants are represented in the primary olfactory processing center of the An. gambiae brain, the antennal lobes. Subsequently, I will determine if this pattern of neural representation is altered during fed and fasted states, as well as stages of malaria parasite infection previously shown to influence olfactory behavior in this mosquito species. Finally, I will evaluate a potential role for neuropeptide signaling in altering synaptic physiology during state-dependent changes in mosquito food search behavior.

Life requires that the brain accurately measure both internal (body core) and external (environmental) temperature. Environmental temperature detection allows animals to find suitable thermal climes and to perceive and learn to avoid painful stimuli. The mechanisms of environmental temperature detection are increasingly well understood, however the mechanisms by which the brain measures internal body temperature remain poorly defined. The vagus nerve is the dominant sensory system that monitors the state of the viscera, and vagal afferents are critical for commanding unconscious processes essential to life, such as keeping heart rate constant and controlling food digestion. But how the vagus nerve contributes to thermoregulation remains a mystery, and almost nothing is known about the specific molecules, cells, and pathways by which these afferents can trigger physiological and behavioural responses to temperature. I propose to identify the molecular and cellular mechanisms underlying thermodetection by vagal afferents innervating the gastrointestinal tract (GI) including the oesophagus, stomach and intestine. I will determine the molecular identity of vagal afferent cell types that measure core body temperature, determine the neurochemical signals that these thermosensitive vagal neurons use to communicate with the brain and test the hypothesis that these vagal cell types are essential for regulating temperature-dependent autonomic and behavioural functions in awake, behaving animals.

The redundant and long-distance activity of regulatory sequences such as enhancers controls tissue-specific gene expression during development. Enhancers are brought in proximity with their target genes through the 3D folding of chromatin in domains of specific interaction. Several factors including CTCF have been shown to be important for this process, but the role of repetitive sequences remains unknown. Here, I propose to investigate the impact of transposable elements (TEs) on gene regulation and the 3D organization of the genome. Focusing on one type of evolutionary young retrotransposon in mice, the MusD elements, I will address this question through three lines of research. (1) a locus-specific approach focused on a model of TE-associated limb malformation to investigate the impact on 3D chromatin folding and gene expression; (2) a genome-wide approach to gain insight into the impact of active TEs on gene regulation and (3) a targeted approach to identify a TE trans-acting factor responsible for silencing such elements. This will be achieved by combining cutting-edge genomic technologies with mouse embryo analysis and the generation of mutants through CRISPR/Cas9. These investigations are expected to reveal specific mechanisms by which transposable elements are able to influence chromatin folding and the expression of developmental genes. My work will shed light on the role of repetitive sequences in shaping the 3D architecture of the mammalian genomes thereby bringing new mechanistic insight into basic mechanisms of gene regulation and retrotransposon biology.