<> "The repository administrator has not yet configured an RDF license."^^ . <> . . "The impact of gene regulatory networks and genome topology on gene expression"^^ . "Gene regulation is the result of the combined action of activators and repressors through a network of transcription factors (TFs) that bind to multiple enhancers, contained within topologically associating domains (TADs). Due to the high complexity of these systems, which contain many regulatory elements (including multiple enhancers, insulators, promoters) and highly interacting components in a multi-level organization, it is challenging to assess the molecular function of TFs, and how they interact with each other and with different regulatory elements, to give rise to cellular phenotypes. In my thesis, I employed perturbation approaches (in both cis and trans) to disentangle the contributions of individual TFs and regulatory elements to genome architecture and transcriptional output. \r\nIn my first project, I focused on the mesoderm gene regulatory network (GRN), i.e. the ensemble of TFs, enhancers and their connections responsible for the development of the D. melanogaster mesoderm. Mesodermal enhancers show extensive time- and tissue- specific co-binding of the TFs Twist (Twi), and of its direct targets Tinman (Tin) and Myocyte enhancer factor 2 (Mef2). By depleting each factor at the precise stages when they drive mesoderm specification and differentiation, and applying single-cell ATAC-seq, I assessed the effect of time-specific depletion on cell identity, and determined the enhancers and target genes most involved in the maintenance of the correct developmental trajectory. This revealed new relationships between different TFs that co-bind to mesodermal enhancers, in addition to an exciting discovery that one mutant leads to a conversion of cell fates across germ layers, from mesoderm to ectoderm/neuronal-like fate, which is extremely rare. \r\nIn my second project, I examined the elements contributing to the formation of TAD boundaries. In Drosophila, TAD boundaries have multiple insulator proteins binding in combination, and they generally coincide with transcriptional and epigenetic domains. Therefore, whether boundaries have intrinsic features driving their function, or if they result exclusively from the context, remained unclear. By inserting a selection of endogenous boundaries in ectopic locations inside other TADs, we showed that most boundaries had an effect, often orientation- or context- dependent, proving that boundaries have intrinsic features, but require contextual elements to function. Their orientation specificity implied that directional elements like promoters or motifs are required for boundary function, and that they need to be present on both sides, i.e. in both boundaries, to make a functioning pair. Since motifs of known insulators or promoters could not explain boundary pairing, I uncovered a set of conservation-based motif pairs that are associated with working boundary pairs with > 90% specificity and present at nearly 50% of endogenous genome-wide boundaries. This motif set includes known insulator motifs and motifs for TFs previously not associated with boundary function, in addition to new motifs with unknown binding proteins. The motifs in each pair have a preferred orientation, and many are convergent or divergent. This goes against the current dogma for how Drosophila TADs are formed, and suggests that the presence of oriented motifs for insulator proteins is a conserved feature to create functional boundary pairs from flies to humans.\r\nIn summary, using perturbation-based approaches, I could contribute to our functional understanding of two diverse, complex systems: I directly assessed the impact of single TFs to enhancer state, TF interactions, and their connection to cellular phenotype within the mesoderm GRN. We could also provide a new perspective on the logic governing what it takes to make a functioning TAD boundary pair in Drosophila, converging on the model of boundary formation through oriented motif pairs from mammals to Drosophila."^^ . "2025" . . . . . . . "Martina"^^ . "Varisco"^^ . "Martina Varisco"^^ . . . . . . "The impact of gene regulatory networks and genome topology on gene expression (PDF)"^^ . . . "The impact of gene regulatory networks and genome topology on gene expression (Other)"^^ . . . . . . "The impact of gene regulatory networks and genome topology on gene expression (Other)"^^ . . . . . . "The impact of gene regulatory networks and genome topology on gene expression (Other)"^^ . . . . . . "The impact of gene regulatory networks and genome topology on gene expression (Other)"^^ . . . . . . "The impact of gene regulatory networks and genome topology on gene expression (Other)"^^ . . . . . "HTML Summary of #35198 \n\nThe impact of gene regulatory networks and genome topology on gene expression\n\n" . "text/html" . . . "570 Biowissenschaften, Biologie"@de . "570 Life sciences"@en . .