Nuclear Architecture, Biomolecular Condensates & Gene Regulation

Nuclear architecture is far from being random or static, genome organisation is dynamically regulated and highly structured, exerting profound effects on gene expression. The eukaryotic nucleus is partitioned into distinct functional compartments that segregate transcriptionally active and silent chromatin. Within these specialised nuclear domains, critical nuclear processes, including transcription, RNA processing, DNA replication, and DNA recombination, are spatially compartmentalised and coordinated. Notably, while intra- and interchromosomal interactions can boost transcription of specific genes via enhancers and super-enhancers, a higher-order spatial integration of transcription and splicing has been shown to enable efficient RNA processing and therefore selectively modulate gene expression (e.g. highly transcribed chromosomic regions clustering around nuclear speckles).

Notably, the study of biomolecular condensates and other membraneless organelles has shifted the paradigm of cellular organisation. Indeed, non-membrane bound compartments are widespread cellular structures that concentrate biomolecules without an enclosing lipid bilayer. Many of these compartments represent biomolecular condensates, which are dense, non-stoichiometric assemblies of macromolecules classically associated with liquid–liquid phase separation (LLPS). LLPS is a thermodynamic process whereby uniformly distributed biomolecules undergo “demixing” into a concentrated phase and a surrounding dilute phase. Biomolecular condensate assembly can also occur through physical transitions beyond classical LLPS. Within the nucleus, condensation gives rise to functional nuclear bodies, including the nucleolus, nuclear speckles, and transcription factories, driven by multivalent interactions between intrinsically disordered regions (IDRs), low-complexity domains, structured domains, and/or nucleic acids. Highly sensitive to environmental conditions, molecular crowding, and post-translational modifications, these condensates offer a rapid, reversible mechanism to tune nuclear biochemistry. By either concentrating proteins up to 100-fold to accelerate transcriptional and metabolic processes or sequestering factors to enforce gene silencing, biomolecular condensation represents an evolutionarily conserved strategy of spatial regulation across the tree of life- from ancient unicellular lineages to complex eukaryotes – and yet, their role in parasitic biology remains understudied (Aye, Cayla & Faria, 2026).

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Kinetoplastid Parasites: A Window into Novel Biology

Kinetoplastids, including Trypanosoma brucei and Leishmania mexicana exhibit atypical gene expression when compared to model eukaryotes.  Protein-coding genes are arranged in long polycistronic transcription units that are constitutively transcribed by RNA Polymerase II (Pol II) from defined transcription start site regions. Because Pol II transcribes these multigene units constitutively coupled with trans-splicing, individual gene expression is regulated mostly through mRNA processing, stability, decay, and translational control. Therefore, Kinetoplastids are likely to be exceptionally dependent on spatial compartmentalisation to coordinate post-transcriptional gene regulation. Notably, by operating outside the paradigms of standard opisthokont model systems, their distinct biology offers a framework to uncover core mechanisms of cellular architecture and test the functional boundaries of condensate-mediated control. 

Indeed, T. brucei for instance has developed specialised, non-membrane-bound assemblies to enable developmental transitions and navigate complex host-parasite interactions, offering a great model system for the study of membraneless organelles. A paramount example is the Expression Site Body (ESB). T. brucei survives extreme environmental shifts between its tsetse fly vector and mammalian host. In mammals, this extracellular parasite relies on antigenic variation to evade the host immune response by periodically switching its Variant Surface Glycoprotein (VSG) coat from a repertoire of over 2,500 genes. Crucially, surface antigen expression is restricted to a single sub-telomeric VSG Expression Site (VSG-ES) at a time - a phenomenon known as monogenic expression. While most of the parasite's genome undergoes constitutive Pol II transcription, Pol I-driven VSG expression is under exquisite transcriptional control. The ESB, a sub-nuclear membraneless compartment, serves as a specialised ‘transcription factory’, selectively harbouring the single active VSG-ES, which co-transcribes vital Expression Site-Associated Genes (ESAGs), while strictly excluding all inactive loci. Whilst its assembly and remodelling could be explained through a phase separation model, that has not been demonstrated. 

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Our Research

Our lab investigates how higher-order chromatin features and nuclear bodies shape gene expression in T. brucei and L. mexicana. We focus primarily on highly expressed virulence genes that mediate host-parasite interactions across both mammalian hosts and insect vectors. The divergent biology of kinetoplastids provides a rare opportunity to uncover universal principles of eukaryotic cellular organisation, post-translational modifications such as phosphorylation and SUMOylation, and the functional limits of condensate-driven regulation. Ultimately, our work sheds light on fundamental mechanisms of nuclear architecture while identifying parasite-specific vulnerabilities.

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The expression-site body – a fine example of extreme biology

Although the Expression Site Body (ESB) was described over 25 years ago, its precise composition and the mechanisms governing its assembly, maintenance, and single-allele choice remained elusive. Leveraging recent technological advances, we have made significant contributions in defining the molecular machinery of the ESB and the 3D nuclear context in which it operates. 

We identified VEX2, a large RNA:DNA helicase required for ESB integrity, and possibly assembly. Acting as an exclusion factor recruited in a transcription-dependent manner, VEX2 is essential for repression of VSG-ESs rather than activation (Faria et al, 2019). Our working model is that VEX2 enforces monogenic expression by compartmentalising transcriptional machinery to a single allele and restricting access to others. Further, VEX-mediated interchromosomal tethering of the active-VSG locus to the SL-array sustains monogenic high-level expression. The ESB sits in close spatial proximity to one of the two distinct Spliced Leader Array Body (SLAB) (Faria et al, 2021). This spatial clustering couples high-rate transcription with concentrated trans-splicing machinery, representing an extreme evolutionary adaptation of a fundamental eukaryotic principle.

Note that helicases are key regulators of phase transitions, controlling RNA flux between biomolecular condensates and the surrounding nuclear environment. Additionally, our single-cell transcriptomic analyses of VEX2-depleted cells have further begun to reveal the genomic determinants governing VSG activation hierarchies (Faria et al, 2023). Recently, we identified ESBX, a factor that integrates both activation and repression of VSG-ESs (Berazategui et al, 2026). Our hypothesis is that ESBX has a structural role at the ESB, enabling the coordinated function of positive and negative regulators, including VEX2. We have also discovered that the ESB integrates dedicated RNA processing machinery, comprising ESB2, ESB3, and ESAP1, to downregulate co-transcribed ESAGs (Lansink et al, 2026). By tethering specialised RNA decay machinery to the ESB, mediated by ESB2, an endonuclease, bloodstream T. brucei can immediately "filter" nascent transcripts from polycistronic units, enabling high-precision regulation of ESAG versus VSG abundance. These findings demonstrate that RNA decay can be coupled with transcription directly within the nucleus; indeed, specialised nuclear mRNA decay might be an underappreciated layer of gene regulation, further reshaping our view of how these parasites manage constitutive transcription.

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Our current interests

While the ESB serves as our primary paradigm for extreme spatial regulation, our research extends beyond this single domain. We are actively investigating other nuclear bodies and chromatin structures to understand how they globally shape nuclear organisation and gene expression, an emphasis we are expanding into the regulation of virulence genes in Leishmania.

Key ongoing questions in the lab include:

• What genomic features dictate initial antigen choice and maintain active-allele compartmentalisation?

• How do known ESB factors dynamically interact to mediate its assembly, disassembly, maintenance, and remodelling during the cell cycle and transcriptional or recombination-based switching?

• What are the molecular and biophysical mechanisms underpinning nuclear body assembly in kinetoplastid parasites? 

• How do conserved and species-specific nuclear bodies coordinate transcriptional and post-transcriptional gene regulation across different lifecycle stages and kinetoplastid species?

Resolving these fundamental questions will allow us to decipher the mechanisms of antigenic variation and host-parasite interactions. Ultimately, our findings will illuminate broad strategies of pathogen immune evasion and survival while revealing universal principles that govern eukaryotic gene regulation and nuclear organisation.

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The techniques…

Genetic Engineering

• CRISPR/Cas9

• RNAi & protein-degrons

• DiCre recombinase-based systems

Imaging

• Super-resolution fluorescence microscopy: 3D-SIM, UExM, PALM/STORM

• FRAP

• Cryo-EM

Proteomics

• AP-MS and XL-MS

• TurboID & APEX2 PL-MS

• Phosphoproteomics & DiGly Proteomics

Genomics

• WGS (ONT, PacBio, Illumina)

• RNA-Seq & single-cell RNA-Seq

• Hi-C / Micro-C

• ChIP-Seq, DRIP-Seq, and CLIP-Seq