Abstract
The proneural transcription factor Ascl1 is a master regulator of neurogenesis, coordinating proliferation and differentiation in the central nervous system. While its expression is well characterised, post-translational regulation is much less well understood. Here we demonstrate that a population of chromatin-bound Ascl1 can be found associated with short chains of ubiquitin while cytoplasmic Ascl1 harbours much longer ubiquitin chains. Only cytoplasmic ubiquitylation targets Ascl1 for destruction, which occurs by conjugation of ubiquitin to lysines in the basic helix-loop-helix domain of Ascl1 and requires the E3 ligase Huwe1. In contrast, chromatin-bound Ascl1 associated with short ubiquitin-chains, which can occur on lysines within the N-terminal region or the bHLH domain and is not mediated by Huwe1, is not targeted for ubiquitin-mediated destruction. We therefore offer further insights into post-translational regulation of Ascl1, highlighting complex regulation of ubiquitylation and degradation in the cytoplasm and on chromatin.
Highlights
During neurogenesis, the fate determination of neural stem cells (NSCs) is coordinated by the activity of proneural basic Helix-Loop-Helix transcription factors and their downstream target genes, acting in a forward genetic cascade during development[1,2]
Protocol established by Spiliotopoulos et al.[18], incorporating slow reduction of FGF with increasing BDNF concentration that promotes enhanced cell viability and a high conversion rate of NS cells to neurons (Fig. 1A). qPCR analysis confirmed that pro-proliferative genes decreased once NSCs were transferred into the differentiation medium (Supplementary Fig. S1A) with a concomitant upregulation of Neurod[1] and Tubb[3], consistent with neuronal differentiation (Supplementary Fig. S1B)
As basic Helix-Loop-Helix (bHLH) transcription factors are known to be regulated at the level of protein half-life, we examined whether Ascl[1] protein stability differs as NSCs progress through the differentiation programme, determining protein abundance after the addition of cycloheximide to block ongoing protein synthesis
Summary
The fate determination of neural stem cells (NSCs) is coordinated by the activity of proneural basic Helix-Loop-Helix (bHLH) transcription factors and their downstream target genes, acting in a forward genetic cascade during development[1,2]. This hierarchical order of activity occurs in transcription factor reprogramming models that can directly convert somatic cells into neurons[3,4]. Recent work demonstrates that it plays an additional role in neural progenitor maintenance during embryonic development, and it controls cycling of adult neural stem cells between an active state and quiescence[11]. We investigate ubiquitin-mediated degradation of Ascl[1] during proliferation and differentiation of mammalian neural stem cells (NSCs) and in mouse P19 embryonal carcinoma cells, which represent a classic model system that can respond to proneural protein overexpression by undergoing differentiation[16,17]
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