The cohesin-complex mediates sister chromatid cohesion from S-phase until mitosis and is involved in the formation of higher-order chromatin structure. To fulfill these vital functions, cohesin is loaded and positioned in the genome by mechanisms that are only poorly understood. In vitro, loading of cohesin on DNA only requires ATP and a loading-complex formed by Scc2-Scc4, while loading in vivo on chromatin is regulated by additional factors. For example, in Xenopus laevis oocytes, cohesin loading strictly depends on pre-replication complexes (pre-RCs), which are formed in telophase/G1. Mechanistic studies are required to understand how cohesin-loading occurs at the molecular level. I will first determine the mechanism by which Scc2-Scc4 loads cohesin on DNA. Using single-molecule FRET and optical tweezers, I will monitor the effect of Scc2-Scc4 on conformational changes of cohesin as it is loaded on a DNA template. After characterizing this minimal loading reaction, I will reconstitute cohesin-loading during telophase/G1 using a purified system. With these experiments I will address why and how loading of cohesin is regulated by the formation of pre-RCs.

Plasma cells (PCs) are antibody-producing cells that develop from activated B cells in an immune response. Antibodies produced by these cells are essential for the clearance of pathogens and long-term protection against recurrent infections. PCs can also be pathogenic in autoimmune disease, where self-recognising antibodies are produced, and cancer, in the form of multiple myeloma. An understanding of the molecular pathways that control PC function is therefore necessary for understanding human immunity and these pathologies. Interferon Regulatory Factor 4 (IRF4, gene name Irf4) is a transcription factor whose deletion in mouse models results in the loss of all PCs. Because of this, the genes that are regulated by IRF4, and hence which cellular pathways are required for PC survival, are unknown. Here, I propose to use a revolutionary approach to determine the target genes of IRF4. I will use targeted protein degradation to deplete IRF4 in mouse PCs, and then determine the immediate changes in transcription following IRF4 loss using a state-of-the-art RNA sequencing technique, known as SLAM-seq. The advantage of this approach is that I can analyze changes in transcription before the onset of the survival defect. I will then study the functional roles of the identified IRF4 target genes in PCs. This is a multidisciplinary project that combines molecular biology approaches with cell biology and immune physiology. In addition, this proposal allows for transfer of knowledge from myself, an expert in the regulation of cell survival, and the host institution, which will train me in molecular biology techniques and expertise in PC biology. IRF4 has been implicated in both autoimmune disease and multiple myeloma, and so this proposal addresses a basic research question that has translatable outcomes. Hence, this proposal is in line with the H2020 objective to increase the transfer of knowledge into tangible products, and contributes to the European knowledge-based economy.

The intestinal tract is the main site of nutrient absorption and faces constant exposure to complex environmental stimuli. Intestinal barrier integrity and functionality are safeguarded by the intestinal immune system, which must balance its pro- and anti-inflammatory activities to provide protection from pathogenic microorganisms while preventing unwanted reactivity to self-antigens, commensal microbes, and dietary components. The intestinal immune environment can be viewed as a multicellular network in which the identity and function of individual cells is intrinsically programmed by transcription factors (TFs) that regulate their gene expression. These cells in turn can respond to signals in their environment and impact the behavior of their neighbors, which over time drives changes in tissue physiology. Conventional genetic knockout approaches offer insufficient temporal resolution to dissect these regulatory layers. In this proposal, we will employ chemical genetic protein degradation using the auxin-inducible degron 2 (AID2) system to “deconstruct” the intestinal immune regulatory network. These studies will focus on the role of Foxp3+ regulatory T (Treg) cells, whose continuous immunosuppressive activity is required to prevent the onset of intestinal inflammation. In Aim 1, we will use newly generated mouse models to probe the direct gene regulatory functions of key TFs that define intestinal Treg cell subsets. In Aim 2, we will fluorescently label intestinal Treg cells and rapidly degrade the TFs that confer their suppressive activity to study their functionality in situ. Finally, in Aim 3, we will use a reversible mosaic protein degradation strategy to study how the signals that precede and promote intestinal Treg cell differentiation shape the developing intestinal immune system. Together, these studies will provide fundamentally new insights into the regulatory network underlying intestinal immune tolerance.

The DNA contained in each of our cells has a size of about 2 m and is fitted into the nucleus which is about six orders of magnitude smaller. It is unclear, how this extraordinary compaction is achieved and how the cell can still carry out highly regulated processes like gene expression, DNA replication, and DNA repair in such a dense environment. Cohesin is a protein that has been shown to play an important part in DNA compaction, especially in sister-chromatid cohesion. Recently, it has been observed that cohesin extrudes loops of DNA to achieve compaction, but how exactly it carries out its function is unknown. Fluorescence spectroscopy is a powerful tool to investigate conformational dynamics of biomolecules. MINFLUX is a recently developed method which localizes single molecules with a precision of a few nanometers. Here, I propose a new method based on MINFLUX which will allow to track fluorescent labels on large bio-molecular complexes with nanometer spatial and millisecond time resolution. The method will be used to study conformational dynamics of cohesin in vitro and investigate the mechanism of loop extrusion.

Most cancer patients are resistant to immunotherapies, especially patients with liver and bone metastases. This suggests that during metastasis, cells acquire additional immune evasion mechanisms that dictate susceptibility to immunotherapies. Current immunotherapies initiate T cell-mediated killing of cancer cells. However, combining them with agents that target other immune cells in the body, such as natural killer (NK) cells, may be more effective. Whether immune evasion mechanisms of metastatic cells differ between organs and whether these mechanisms can be targeted to improve immunotherapeutic strategies remains unknown. The first aim of this proposal is to leverage a molecular barcoding technique named CaTCH (CRISPRa tracing of clones in heterogenous cell populations) to search for new mechanisms of NK cell immune evasion. CaTCH enables tracing metastatic cancer cells in vivo and isolating clones in a barcode-specific manner for subsequent characterization and functional experiments. We engineered a human non-small-cell lung cancer model with a complex CaTCH barcoding library. Subsequently, we injected the cells into mice harboring NK cells and mice having no NK cells in an experimental metastasis assay. We identified several barcoded clones enriched in specific organs under immune pressure. I aim to investigate this dataset to find novel mechanisms of NK cell immune evasion in the metastatic environment. In the second aim, I will combine molecular barcoding with replicative history tracing of cells to identify cancer clones that evade immune-mediated dormancy in the metastatic niche. I will compare the transcriptional and molecular profiles of dividing cells high in abundance to dividing cells low in abundance to find novel mediators of immune-mediated dormancy. The successful implementation of these aims will uncover new immune evasion strategies of metastatic cells that will pave the way for novel immunotherapeutic strategies for cancer patients.