Developing tools for studying the regulation of Ascl1 transcriptional activity by multisite phosphorylation

• Autor(a) principal: Silva, José Miguel Vieira
• Data de Publicação: 2022
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/36350
• topic: Ascl1; Electrostatic interactions; Neuronal differentiation; Phosphorylation; Single Molecule Tracking (SMT); Transcription factor

Proneural transcription factors (TFs) of the bHLH family, such as Ascl1, are master regulators of vertebrate neurogenesis, being both necessary and sufficient to promote a full program of neuronal differentiation. Ascl1 expression is restricted to multipotent neural stem (NS) cells and intermediate, neuronal committed, progenitors along the anterior/posterior axis of the developing brain and spinal cord. Ascl1 acts by promoting sequentially the proliferation and differentiation of neural stem/progenitor cells through activation of distinct sets of target genes, directly regulating multiple stages of neurogenesis. According to the current view, low Ascl1 activity promotes (and is compatible) with cell proliferation, whereas increased activity results in neuronal differentiation and cell cycle exit. Multisite phosphorylation of Ascl1 at six serine/proline (SP) sites outside its DNA-binding domain has been proposed as a major mechanism for reducing the activity of Ascl1 in proliferating progenitors. Ascl1 activity was shown to depend on the total number of phospho-residues, regardless of their position, a mechanism that is described by a “rheostat-like model”. The number of negatively charged phospho-residues modulates a protein electrostatic potential, a major determinant of TF-chromatin interactions in different cellular contexts. Nevertheless, the mechanisms whereby multisite-phosphorylation impacts how Ascl1 interacts with mitotic chromatin during cell division, or how it navigates the interphase genome searching for its target genes, remain unexplored. The goal of this work was to develop a set of tools for studying a major question: how changes in the phosphorylation status impact the dynamics of Ascl1 interactions with the genome. Here, we describe the generation of a cellular model that allows tracking of a neurogenic division, by labelling young neurons that express the pan-neuronal marker Doublecortin (DCX) with a fluorescent reporter, to be used when comparing Ascl1-chromatin interactions in proliferating versus differentiating progenitors. Also, we present our efforts in using CRISPR/Cas9 to generate NS cell models expressing phospho-mutant derivatives of Ascl1. Preliminary results suggest increased nuclear concentration of an Ascl1 derivative with alanine substitutions in all six SP sites, in line with its previously characterized increased neurogenic activity. While several studies have looked at Ascl1 chromatin interactions, by live cell- imaging or chromatin immunoprecipitation (CHIP), no studies have so far looked at Ascl1 properties at a single molecule level. Here we combined Total Internal Reflection Fluorescence (TIRF) microscopy with Highly Inclined Laminated Optical sheet (HILO) illumination, to establish Single-Molecule Tracking as an experimental approach to survey Ascl1 dynamics in live cells. To this end, we used genome editing to generate NS cells expressing transcriptionally active Halo-tagged Ascl1. Using this emerging technique to study TF kinetic properties, we were able to preliminarily distinguish Ascl1 subpopulations according to different binding dynamics.