ERC FUNDED PROJECTS

A common strategy of bacterial pathogens is active or passive uptake into host cells. There, they can localize within a bacterial containing vacuole (BCV) or access the host cytoplasm through BCV rupture. Hence, intracellular pathogens are often classified as vacuole-bound or cytoplasmic. Recently, this definition has been challenged by the discovery that many vacuole-bound pathogens, including Mycobacterium tuberculosis and Salmonella enterica, access the host cytoplasm, and by the insight that cytoplasmic bacteria, like Shigella flexneri or Listeria monocytogenes, do not always escape the BCV. Despite this increasing complexity, a precise understanding lacks for why and how a pathogen “chooses” between a BCV or the cytoplasm and yet this is very important: because of differential pathogen sensing in membrane-bound and cytoplasmic compartments, intracellular localization leads to induction of different host responses. Therefore, a comprehensive understanding of the processes controlling BCV integrity is not only essential, but can provide new therapeutic targets. Our previous research has implemented innovative fluorescence microscopy to track the invasion steps of pathogenic bacteria. We have further integrated a large-volume, correlative, light/electron microscopy (CLEM) workflow via focused ion beam scanning electron microscopy. This uncovered the subversion of host Rab cascades by Shigella to rupture its BCV. Starting with the Shigella model of epithelial cell invasion, we will delineate the precise molecular mechanisms controlling BCV integrity in different host cell types. We will analyze (i) the scaffolds of host pathways for membrane remodeling, (ii) their subversion by various pathogens, and (iii) their differential regulation depending on pathophysiological conditions. Together, this will allow development of novel, rational antimicrobial strategies and will yield fundamental insight into understudied cell biological mechanisms of membrane trafficking.

2 000 000 €

Start date: 2017-01-01, End date: 2021-12-31

Epigenetic alterations can be detected in all cancers and in essentially every patient. Despite their prevalence, the concrete functional roles of these alterations are not well understood, for two reasons: First, cancer samples tend to carry many correlated epigenetic alterations, making it difficult to statistically distinguish relevant driver events from those that co-occur for other reasons. Second, we lack tools for targeted epigenome editing that could be used to validate biological function in perturbation and rescue experiments. The proposed project strives to overcome these limitations through experimental and bioinformatic methods development, with the ambition of making and breaking cancer cells in vitro by introducing defined sets of epigenetic alterations. We will focus on leukemia as our “model cancer” (given its low mutation rate, frequent defects in epigenetic regulators, and availability of excellent functional assays), but the concepts and methods are general. In Aim 1, we will generate epigenome profiles for a human knockout cell collection comprising 100 epigenetic regulators and use the data to functionally annotate thousands of epigenetic alterations observed in large cancer datasets. In Aim 2, we will develop an experimental toolbox for epigenome programming using epigenetic drugs, CRISPR-assisted recruitment of epigenetic modifiers for locus-specific editing, and cell-derived guide RNA libraries for epigenome copying. Finally, in Aim 3 we will explore epigenome programming (methods from Aim 2) of candidate driver events (predictions from Aim 1) with the ultimate goal of converting cancer cells into non-cancer cells and vice versa. In summary, this project will establish a broadly applicable methodology and toolbox for dissecting the functional roles of epigenetic alterations in cancer. Moreover, successful creation of a cancer that is driven purely by epigenetic alterations could challenge our understanding of cancer as a genetic disease.

1 281 205 €

Start date: 2016-12-01, End date: 2021-11-30

Oligodendrocytes (OL) are glial cells that mediate myelination of neurons, a process that is defective in multiple sclerosis (MS). Although OL precursor cells (OPCs) can initially promote remyelination in MS, this regenerative mechanism eventually fails in progressive MS. OPCs go through several epigenetic states that ultimately define their potential to differentiate and myelinate. OPCs in progressive MS stall in a distinct epigenetic state, incompatible with differentiation and remyelination. We hypothesize that these OPCs regress to an epigenetic state reminiscent of the state of embryonic OPCs, which remain undifferentiated. In this proposal, we aim to uncover the causes behind the remyelination failure upon disease progression in MS. We will determine the epigenetic/transcriptional states of OPCs during development and in MS, using single cell and bulk RNA sequencing and quantitative proteomics. We will further investigate how the interplay between transcription factors (TFs), chromatin modifiers (ChMs) and non-coding RNAs (ncRNAs) contributes to the transition between epigenetic states of OPCs. The results will allow the identification of ChMs and ncRNAs that can modulate these states and thereby control OPC differentiation and myelination. We will use this knowledge to investigate whether we can reverse the epigenetic state of OPCs in MS, in order to promote their differentiation and remyelination. The unique combination of leading-edge techniques such as SILAC coupled with immunoprecipitation and mass-spectrometry, single-cell RNA sequencing, ChIP-Sequencing, among others, will allow us to provide insights into novel epigenetic mechanisms that might be underlying the effects of environmental and lifestyle risk factors for MS. Moreover, this project has the potential to lead to the discovery of new targets for epigenetic-based therapies for MS, which could provide major opportunities for improved clinical outcome of MS patients in the near future.

1 895 155 €

Start date: 2016-09-01, End date: 2021-08-31

Ion channel genes have long been linked to Mendelian focal epilepsies, but my recent finding of frequent mutations in DEPDC5 opens completely new perspectives. DEPDC5 is an inhibitor of the mTORC1 (mammalian target of rapamycin) signaling pathway, the master regulator of cell proliferation and growth. Mutations of this gene are found in a wide spectrum of focal epilepsy syndromes, with or without cortical malformations. I propose to examine the links between DEPDC5 and the mTORC1 pathway in cortical development and the genesis of epileptic activity. My proposal work will combine high-throughput sequencing, in vivo proteomics, biochemistry, electrophysiology, and animal behavior testing (video-EEG). Functional analyses will be made on human postoperative tissue and neuronal cultures from human iPSC and specific rodent models. These approaches will enable me to (1) ask if and how the mTORC1 signaling pathway may contribute to epileptogenesis and seizures in patients with DEPDC5 mutations, (2) attempt to explain the diversity of phenotypes, in particular the presence of cortical lesion by searching for somatic brain mutations in the gene, (3) explore neurobiology pathways and partners of DEPDC5, and (4) identify novel actors for inherited focal epilepsies. Our results will help us understand the genesis of epileptic networks, and more generally how defects in mTORC1 signaling cascade cause neurologic conditions. We anticipate genetic studies on germline and somatic mutations will have a significant clinical impact for genetic counseling and improved prognosis. The molecules and pathways that will be studied in this proposal differ completely from ion channels and receptors that have been so far associated with focal epilepsies. Thus I hope to provide a new orientation for the field, to identify novel genetic mechanisms and to provide an unbiased route to new molecular therapeutic targets.

1 998 760 €

Start date: 2016-10-01, End date: 2021-09-30