Gilbert, Nicholas

Buonomo, Sara

Lazarova, Elena

2024-09-13T10:32:55Z

2024-09-13T10:32:55Z

2024-09-13

Large-scale chromatin organisation during interphase and mitosis has been extensively studied; however, the mitotic exit remains poorly understood. This thesis aims to elucidate the involvement of the essential protein Scaffold Attachment Factor A (SAF-A; also called HNRNPU) in chromatin organisation during the mitotic exit and its impact on cellular phenotypes, particularly in the context of HNRNPU-related neurodevelopmental disorder. SAF-A regulates interphase chromatin structures in a transcription-dependent manner. The protein can decompact gene-active regions by interacting with chromatin-associated RNAs and binding ATP to form protein oligomers. SAF-A's eviction from mitotic chromosomes suggests it might have a precise recruitment to gene-active regions for establishing and maintaining the RNA mesh after mitosis. Additionally, perturbations in SAF-A function may result in faulty chromatin architecture at gene-active regions, contributing to cellular phenotypes observed in patients with HNRNPU-related neurodevelopmental disorder. To investigate whether transcriptional events during telophase or in early G1 recruit SAF-A, I used a live cell imaging system and co-localisation analysis. Imaging cells going through mitosis showed that SAF-A is recruited to chromatin during cytokinesis and inhibiting transcription at that time delayed its recruitment to early G1. This allowed the examination of SAF-A's association with RNAs during mitosis and mitotic exit using synchronized cells. In comparison with monomeric SAF-A, more oligomeric SAF-A was associated with new and old RNA during mitosis and early G1, showing that the protein regulates large-scale chromatin organisation as soon as it is recruited during mitosis. Next, I investigated the interaction between SAF-A and RNA and whether SAF-A depletion affects the RNA mesh in interphase. Depleting SAF-A with RNAi treatment resulted in a reduced RNA mesh density and connectivity. Finally, the effect of SAF-A mutations, from patients with the HNRNPU-related neurodevelopmental disorder, on chromatin organisation was explored. Human cell lines expressing mutated SAF-A proteins were generated, and their impact on chromatin decompaction was quantified using FISH with each mutant having a different phenotype. This thesis sheds light on the behavior of SAF-A at the mitotic exit and provides insights into its role in large-scale chromatin organisation. Furthermore, it investigates the cellular phenotypes associated with SAF-A patient mutations, contributing to our understanding of the underlying mechanisms and potential therapeutic targets for HNRNPU-related neurodevelopmental disorder.

https://hdl.handle.net/1842/42154

http://dx.doi.org/10.7488/era/4875

en

The University of Edinburgh

Marenda, Mattia, Elena Lazarova, and Nick Gilbert. 2022. ‘The Role of SAF-A/hnRNP U in Regulating Chromatin Structure’. Current Opinion in Genetics & Development 72 (February):38–44. https://doi.org/10.1016/j.gde.2021.10.008

Marenda, Mattia, Elena Lazarova, Sebastian van de Linde, Nick Gilbert, and Davide Michieletto. 2021. ‘Parameter-Free Molecular Super-Structures Quantification in Single-Molecule Localization Microscopy’. Journal of Cell Biology 220 (5): e202010003. https://doi.org/10.1083/jcb.202010003

SAF-A

cytokinesis

SAF-A mutation

brain development

mitotic exit

Scaffold Attachment Factor A

faulty chromatin architecture

chromatin decompaction

Understanding SAF-A's role in chromatin organisation throughout the cell cycle and in a rare neurodevelopmental disorder

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy