Regulation of HOXA5 protein stability in neuronal cells

Details

Supervisors

Gofflot, Françoise ; Dumont, Patrick R.

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en chimie et bioindustries, à finalité spécialisée

Abstract

The tight spatiotemporal regulation of transcription factor (TFs) is crucial for proper neurodevelopment. While the spatial expression patterns of gene coding for developmental TFs are well characterised, the regulation of their activity over time - particularly via the protein stability - remains an overlooked feature. Yet, dynamic changes in TF stability may regulate their functional roles and/or active time window, thus emerging as a key mechanism in developmental timing. This master’s thesis aims at tackling this feature by investigating the regulation of the HOXA5 TF protein stability during neuronal maturation, given its known involvement in neurodevelopment and association with synaptogenesis at postnatal stages. To address this, we sought to identify HOXA5 interactors potentially involved in its stability regulation at both immature and mature neuronal stages, using the TurboID-based proximity labelling approach. The implementation of this technique, using an \textit{in vitro} neuronal differentiation model, was the primary objective of this work. The protocol was optimised at both stages, including transfection, doxycycline/biotin treatment, and streptavidin-based purification. An initial mass spectrometry (MS) analysis revealed a 10-fold increase in identified proteins in mature neurons transfected with TurboID, likely due to its whole-cell distribution and the increased volume and proteomic diversity of mature cells. Indeed subcellular localisation analysis reveals that TurboID is both nuclear and cytoplasmic, whereas TurboID-HOXA5 is strictly nuclear. To reduce background and match this localisation, a TurboID-NLS control was generated and included in a second MS analysis. This control showed improved nuclear confinement and yielded protein counts comparable to TurboID-HOXA5, supporting its use as a better background reference. Gene Ontology analyses confirmed the predominantly neuronal identity of mature populations. However, low protein input limited streptavidin saturation, likely contributing to contaminant copurification. Pooling multiple culture wells and biological triplicates are suggested to improve protein yield and data robustness in future experiments. From these optimisation data, several candidate interactors were identified in immature neurons: RBBP6 and RANBP2. Notably, IPO7 - a previously validated HOXA5 interactor involved in nuclear import - was also recovered. By optimising the proximity-labelling protocol, this work lays the foundation for robust MS analysis and, more broadly, for probing how HOXA5 interactors modulate its stability and how this regulation shapes neurodevelopmental processes.