Title Promoter Affiliations Abstract "UNRAVELING THE ROLE OF CHROMATIN REGULATORS DURING EARLY HUMAN EMBRYONIC DEVELOPMENT" "Vincent Pasque" "Stem Cell and Developmental Biology" "Chromatin regulation is essential for lineage specification during embryonic development. However, despite advances in our understanding of chromatin processes in mammalian development, little is known about chromatin regulation in early human embryos. Here, I will use functional perturbations in human stem cell-based embryo models, and single-cell technologies, to understand the role of chromatin regulators in early human embryonic lineage specification. Human pluripotent stem cells have the remarkable ability to form 3D-embryo-like structures, called blastoids, which resemble early human embryos and provide an exciting new platform to study embryogenesis. To study chromatin regulation in blastoids, I will localise and compare candidate chromatin regulator proteins in human blastoids and embryos. Subsequently, I will deplete chromatin regulators during blastoid development, and quantify the effects on lineage specification using molecular analysis. I will use single-cell transcriptomics and epigenomics to analyse the effects of chromatin regulator depletion on the chromatin landscape and gene expression of blastoids. This approach will allow me to identify and validate cis-regulatory regions whose dysregulation may be linked to diseases. Unravelling the role of chromatin regulators in early human embryogenesis will revolutionise our understanding of human embryonic development and thus provide a foundation for future research and infertility treatments." "The regulatory effect of 3D chromatin organization on genes resistant to reprogramming" "Vincent Pasque" "Stem Cell and Developmental Biology" "To understand how cells in our body resist changes in gene expression would be instrumental for better understanding the causes of diseases and designing improved treatments. We have recently identified a possible link between resistance to transcriptional reprogramming and 3D chromatin organization. We hypothesize that genes with low resistance to transcriptional reprogramming occupy accessible 3D positions in the nucleus, while more resistant genes are embedded within more topologically restricted chromatin. However, further studies are needed to elucidate the role of 3D genome organization in the stability of gene silencing and resistance to transcriptional activation. We will use Hi-C, a method allowing the analysis of the spatial organization of chromatin, to define the relationship between the resistance to transcriptional reprogramming and genome organization. We will then study dynamic changes in chromatin topology during induced pluripotent stem cell reprogramming using single-cell Hi-C. This will reveal the relationship between the kinetics of chromatin domain formation and the timing of transcriptional activation. Furthermore, we will identify how modulating chromatin organization affects the resistance to transcriptional reprogramming using genome editing coupled with transcriptomic approaches. Altogether, we aim to better understand the effects of chromatin organization on gene expression, and ultimately gene regulation, cellular identity and diseases." "HIV nuclear import and chromatin reading as novel drug targets" "Zeger Debyser" "Molecular Virology and Gene Therapy" "The replication cycle of HIV is a complicated interplay between viral and cellular proteins. Until now  antiretroviral therapy mainly targets the viral proteins although the interaction between the viral and cellular proteins could be interesting new drug targets. This strategy is mainly of interest because cellular proteins are much less prone to mutations which gives the virus less opportunity to develop drug resistance.  My laboratory has a track record in research on cellular cofactors of the viral enzyme integrase.  In 2003 Cherepanov et al. discovered LEDGF/p75 as an interaction partner of integrase and in 2008 Christ et al. found transportin-SR2 in a yeast-two-hybrid screen as an interaction partner of  integrase. Transportin-SR2 plays an important role in the nuclear import of the virus and LEDGF/p75 tethers the viral DNA to actively transcribed chromatin where it can integrate. Both proteins form interesting new targets for the treatment of HIV. Next to its role in HIV replication, LEDGF/p75 plays a role in mixed lineage leukemia. This type of cancer is driven by translocations involving the MLL1-gene and mainly affects children. Targeting the chromatin reading function of LEDGF/p75 could therefore be an interesting drug target in the treatment of both HIV and mixed lineage leukemia.In the first chapter of this PhD thesis I focus on the TRN-IN interaction and try to gain more insight into their binding mode. Since nuclear import is generally considered as a bottleneck in the viral replication cycle this could be an interesting new target for antiretroviral therapy. Previous studies have already revealed that mainly the catalytic core domain (CCD) and the C-terminal domain (CTD) of integrase interact with transportin-SR2. The aim of this work was to gain more insight into this protein complex and to pinpoint the important regions in TRN-SR2 that are necessary for integrase binding. In collaboration with the bio-crystallography lab I revealed that one TRN-SR2 molecule can bind a CCD-CTD dimer. Next, I divided transportin-SR2 into small peptides, each corresponding to a single HEAT repeat and used AlphaScreen binding assays to determine if these peptides could still bind integrase. This study revealed that it is mainly the N-terminal region of TRN-SR2 that interacts with integrase, principally through HEAT repeats 4, 10 and 11. Based on these results in combination with small-angle X-ray scattering data for the complex of TRN-SR2 with a truncated integrase, I propose a model of the complex in which the nuclear import of the pre-integration complex can proceed in parallel with nuclear transport of its endogenous cargoes.In the second part of this thesis the aim was to identify novel antivirals that do not suffer from cross-resistance with existing drugs. This chapter describes the development and use of an AlphaScreen-based high-throughput screening cascade for inhibitors of the TRN-IN interaction. In total, 25608 small molecules were tested and after eliminating false positives and nonspecific protein-protein interaction inhibitors two active compound series were discovered. Although the inhibitory effect of these compounds in a multiple and single round antiviral activity assay was only moderate, they significantly reduced the nuclear import of fluorescently labelled HIV particles. This study shows that it is possible to use the AlphaScreen technology as a high throughput platform to screen for a novel class of inhibitors. These results again confirm the important role of the TRN-IN interaction in nuclear import. Our hit compounds represent the first small molecule inhibitors of this step in the viral replication cycle and hold promise for future drug development.The last part of this work focuses on the protein-chromatin interaction between LEDGF/p75 and the trimethylated lysine 36 on histone 3 (H3K36me3). The PWWP domain of LEDGF/p75 plays an important role in tethering the HIV pre-integration complex to the host chromatin. Also in the case of mixed lineage leukemia, LEDGF/p75 is responsible for targeting the leukemogenic MLL-fusion/MENIN complex to actively transcribed genes. Targeting the LEDGF/p75-H3K36me3 interaction could therefore have beneficial therapeutic effects in the treatment of both HIV and mixed lineage leukemia. This part of the thesis describes the development and use of a fragment-based drug discovery campaign. Molecular modelling was used to virtually screen over 4 million compounds for binding to the PWWP domain and the top 525 molecules were selected. An AlphaScreen-based screen was next used to pick initial hit compounds from this small compound library. All compounds were also tested in a nano-DSF set-up in which the intrinsic tryptophan fluorescence of the PWWP domain is monitored upon thermal denaturation in the presence and absence of compounds. Initial results indicate that some of our compounds can indeed inhibit the LEDGF/p75-H3K36me3 interaction, although the effect is only moderate. In parallel we have therefore worked on resolving the crystal structure of the PWWP domain. This structural information could guide further chemical optimization of our compounds.In brief, this work aims to discover and validate novel targets for drug discovery targeting HIV and/or mixed lineage leukemia. Protein-protein and protein-chromatin interactions can be interesting new targets since the risk for cross-resistance with already existing drugs is rather low. However, the main challenge in targeting these interactions is to develop selectivity against the specific interaction while keeping interference with other proteins to a minimum." "Deciphering the role of chromatin organisation in alternative isoform choice in the human brain." "Valeriya Malysheva" "VIB CMN - Computational Neurobiology" "Isoform expression is highly cell type-specific, drives cell fate trajectories, profoundly influences drug responses and its dysregulation is a cause of disease. Despite research traditionally focusing on alternative splicing, the use of alternative transcription start sites (TSSs) and termination sites (TTSs) accounts for the majority of transcriptional diversity in humans. Yet little is known about what governs alternative TSS and TTS isoform choice. Our preliminary data show chromatin organisation, via 3D isoform-enhancer interactions, likely plays a crucial role, but no in-depth research has been done. We hypothesise that chromatin organisation is a central coordinator of isoform choice. As the brain contains the widest isoform diversity it is ideal for addressing this question. I will use experimental methods including the chromatin organisation technology patented by Prof. Malysheva, which is the only method capable of detecting isoform-level 3D chromatin organisation interactions in rare cell populations at high resolution, as well as computational methods for integrative regulatory network inference to identify the drivers of isoform choice in the major cell types of the human frontal cortex. This project will reveal the ground rules of isoform choice, which, in the long run, will be a game-changer for understanding the role of isoforms in health and disease and shift the regulatory paradigm from gene-centric to isoform-centric." "Investigating the role of chromatin accessibility in the global transcription regulation in Leishmania quiescent cellusing multiomic single cell approaches" "Martha Stas, Malgorzata Domagalska" "Department of Biomedical Sciences, Management, Trypanosoma, Molecular Parasitology" "Quiescence is a physiological state allowing micro-organisms to survive many environmental insults and is characterized by a reversible cell division arrest and metabolic downregulation. It is essential for the perpetuation of pathogen populations, contributes to the success of parasitism and might explain several (sub-)clinical features like asymptomatic infections, relapses and treatment failure (without drug resistance). Quiescence was recently demonstrated in Leishmania, but is still understudied.One of the common features of quiescent cells is chromatin condensation, which is likely a mechanism to downregulate transcription, protect the genome, and keep the cell at minimal metabolic activity. It is currently unknown whether changes in chromatin conformation occur in Leishmania quiescent cells. In this project focusing on Leishmania donovani, we will investigate the chromatin accessibility during the entry, maintenance and exit of quiescence and correlate it with the observed transcriptional changes. We will consider two quiescence models based on starvation and exposure to drugs, respectively. These will be studied at single cell level by applying a cutting edge method combining an assay for transposase-accessible chromatin using sequencing (ATACseq) andRNAseq, allowing the simultaneous measurements in the same cells across thousands of cells." "Chromatin structure analysis of rare likely pathogenic inherited Copy Number Variants" "Catia Attanasio" "Laboratory of Gene Regulation and Disease, Department of Human Genetics" "Chromatin structure has been shown to play important roles in the orchestration of gene expression programs during development. Spatio-temporal specific cis-regulatory sequences often lie at a long distance from the gene(s) they regulate, requiring spatial chromatin folding to move them in close proximity of their target promoters and fine-tune the time, place and level of gene expression. Interestingly, several whole-genome chromatin interaction analyses have also recently revealed the existence of topologically associated domains (TADs) which seem to act as fundamental regulatory units of the genome by delimiting the scope of action of regulatory elements in the 3D nuclear space. In other words, TADs organize the genome into regulatory islands by defining regions that interact more frequently with themselves than with the rest of the genome. Few studies have reported that the alteration of TADs by structural genomic rearrangements could disrupt the normal activity of TADs and play significant roles in human diseases. Copy-Number Variants (CNVs) are DNA sequence deletions or insertions of ≥50bp which have been shown to play important roles in human biology. The overall contribution of CNVs to human phenotypic variability and diseases is, however, still largely unclear. Few studies have now reported that deletion or duplication of DNA segments could alter gene expression regulation by disrupting, duplicating or shifting TADs boundaries causing gene(s) misexpression and diseases. Here, we propose to investigate the contribution of rare likely pathogenic CNVs into human disorders by analyzing theirs effect on chromatin architecture, and by extension on gene regulatory programs, in patients’ and controls’ derived cells. This project is part of a larger research effort that will combine our analyses with deep phenotyping, whole genome sequencing and evolutionary analyses in selected families to assess the contribution of rare likely pathogenic CNVs to phenotypic variability of developmental disorders." "Targeting the chromatin for innovative cancer therapy." "Zeger Debyser" "Molecular Virology and Gene Therapy, Intellectual Property" "Changes in the chromatin environment (mutations, translocations, epigenetic changes) are initiating factors of uncontrolled cell growth/oncogenesis. Chroma Target wishes to develop new, more specific therapeutic strategies (with minor side effects) and lead compounds against a subset of cancers with poor prognosis from an improved understanding of the malignant chromatin environment. Comparing omica data will also enable personalized medicine through identification of specific biomarkers." "Targetting the chromatin for innovative cancer therapy" "Pieter Van Vlierberghe" "Department of Biomolecular Medicine, Department of Pediatrics and medical genetics" "Changes in chromatin environment (mutations, translocations, epigenetic changes) are initiating factors of uncontrolled cell growth / oncogenesis. Chroma Target wishes from an improved understanding of the malignant chromatin environment new, more specific therapeutic strategies (less side effects) and lead compounds against a subset of cancers with a poor prognosis (T-cell progenitor acute lymphoblastic leukemia (ETP-ALL), MLL fusion develop mediated leukemia, medulloblastoma). Comparing 'omics' data will also allow gepersonaliiseerde medicine through identification of specific biomarkers." "LEDGF/p75 chromatin tethering as a target to treat Jpo2 and MLL dependent malignancies" "Zeger Debyser" "Molecular Virology and Gene Therapy" "When all links between genes associated with disease development (diseaseome) and virushost interactions (infectome) are mapped, a network emerges revealing common genetic origin of many diseases and viral infections. In addition, interaction nodes or protein hubs appear with a central function in many disorders or infections. In the proposed project we study ‘Lens Epithelium Derived Growth Factor/p75’ (LEDGF/p75), originally discovered in our research group in 2003 as a cellular cofactor of the human immunodeficiency virus type 1 (HIV-1), the causative agent of the acquired immunodeficiency syndrome (AIDS). Subsequent research revealed that LEDGF/p75 has a similar role in MLL mediated acute leukemia. In both cases LEDGF/p75 tethers a protein(-DNA) complex to the chromatin. We could show that disruption of this chromosomal localization was sufficient to block HIV replication as well as to hold back the development of MLL mediated acute leukemia. These results feed our hypothesis that LEDGF/p75 is a chromatin tethering hub and a valid target for several diseases. LEDGF/p75 is also known to bind Jpo2, a protein brought in connection with several mental disorders and the development of medullobalstoma, the most common malignant brain tumor in children. In this project we will investigate the molecular mechanism of LEDGF/p75 tethering, explore the Jpo2-LEDGF/p75 interaction as a new target to treat Jpo2 mediated diseases and develop new drugs that can inhibit the MLLLEDGF/ p75 interaction." "Exploration of the therapeutic potential of protein phosphatases involved in chromatin signaling." "Mathieu Bollen" "Biochemistry, Molecular and Structural Biology, Laboratory of Biosignaling & Therapeutics" "Our research group has a long-standing expertise in protein phosphatase 1 (PP1) and chromatin signaling and we want to develop chromatin-associated PP1 as a successful therapeutic target. Specific research objectives of this project are: 1. To delineate the role of PP1 in the dynamics of histone phosphorylation. 2. To identify the protein that target PP1 to histones. 3. To explore how PP1 cross-talks with other histone-modifying enzymes. 4. To select PP1 holoenzymes with a high therapeutic potential. 5. To determine the 3D-structues of selected PP1 holoenzymes. 6. To screen for small-molecule effectors of selected PP1 holoenzymes."