Deciphering the role of nuclear architecture in gene expression regulation in Leishmania

Nuclear architecture and 3D genome organisation can have profound effects on gene expression regulation. For instance, intra and interchromosomal interactions can amplify transcription of specific genes (e.g. enhancers and super-enhancers). Additionally, transcription and splicing can be integrated in the 3D nuclear space: proximity to nuclear bodies that compartmentalise RNA processing machinery has been shown to increase gene expression in mammals and some lower eukaryotes. Conversely, the nuclear lamina has been shown to play important roles in anchoring specific genomic regions and heterochromatin formation. In this project, we will introduce and explore the concept of post-transcriptional enhancers, a unique nuclear body dedicated to RNA processing and gene-specific regulators of interchromosomal interactions - a fascinating window into novel biology. Leishmania species cause a spectrum of human diseases in tropical and subtropical regions of the world. These parasitic protozoa organise their genes into long polycistronic clusters, lack conventional enhancer sequences and have a limited repertoire of transcription factors, consequently displaying negligible control over transcription initiation. Gene expression regulation is therefore mostly post-transcriptional through regulation of RNA processing and stability. However, how chromatin organisation and nuclear compartmentalisation can influence those processes has never been investigated in these organisms. Therefore, we will characterise: its higher-order chromatin organisational features and whether and how these features impact the expression of highly expressed virulence genes involved in host-Leishmania interactions, either adapted to the insect or mammalian host; its major nuclear compartments, with a focus on nuclear bodies and the nuclear lamina, and how these shape genome organisation and gene expression; protein candidates as molecular tethers that sustain gene-specific interchromosomal interactions, which facilitate post-transcriptional enhancement of gene expression. To this end, we will use a combination of cutting-edge high resolution chromosome conformational capture (Pore-C), protein and DNA imaging (live-imaging, UExM, 3D-SIM, single-molecule FISH), chromatin-immunoprecipitation (CUT&RUN) and proteomics (miniTurbo & TurboID-based proximity labelling combined with highly sensitive LC-MS/MS) and gene editing (CRISPR/Cas9; DiCre-based conditional systems) techniques. Notably, chromosome conformational capture data available in Trypanosoma brucei and Trypanosoma cruzi, related parasites and causative agents of sleeping sickness and Chagas’ disease, respectively, shows similarities but also striking differences in genome organisation, mostly linked with expression of virulence genes. Moreover, T. brucei has four types of nuclear bodies involved in RNA processing (nucleolus, SLAB, NUFIP and ‘Cajal-like’), which we have extensively characterised by microscopy and proteomics. Most components have orthologs in Leishmania, however, our preliminary microscopy analysis points to a distinct nuclear organisation in Leishmania, including a single extra-nucleolar hub for mRNA processing. Despite the evolutionary proximity, these parasites undergo distinct developmental transitions as an adaptation to the different insect vectors and mammalian cells/tissues they infect and possess distinct virulence factors. Our work will be coordinated with collaborators undertaking similar investigations in T. cruzi and will be further integrated with available data in T. brucei so that we can assess whether distinct chromatin organisational features, and nuclear compartments might have evolved to accommodate parasite-specific gene expression programmes. Overall, the limited transcriptional control in Leishmania parasites renders them an exceptional model system to study mechanisms underpinning post-transcriptional regulation of gene expression. Moreover, ~1 million people develop some form of leishmaniasis every year, in the absence of a human vaccine and with a desperate need for new pharmacological interventions. Understanding their unorthodox biology is therefore of the uttermost importance.