bHLH proteins involved in Drosophila neurogenesis are mutually regulated at the level of stability

Abstract

Proneural bHLH activators are expressed in all neuroectodermal regions prefiguring events of central and peripheral neurogenesis. Drosophila Sc is a prototypical proneural activator that heterodimerizes with the E-protein Daughterless (Da) and is antagonized by, among others, the E(spl) repressors. We determined parameters that regulate Sc stability in Drosophila S2 cells. We found that Sc is a very labile phosphoprotein and its turnover takes place via at least three proteasome-dependent mechanisms. (i) When Sc is in excess of Da, its degradation is promoted via its transactivation domain (TAD). (ii) In a DNA-bound Da/Sc heterodimer, Sc degradation is promoted via an SPTSS phosphorylation motif and the AD1 TAD of Da; Da is spared in the process. (iii) When E(spl)m7 is expressed, it complexes with Sc or Da/Sc and promotes their degradation in a manner that requires the corepressor Groucho and the Sc SPTSS motif. Da/Sc reciprocally promotes E(spl)m7 degradation. Since E(spl)m7 is a direct target of Notch, the mutual destabilization of Sc and E(spl) may contribute in part to the highly conserved anti-neural activity of Notch. Sc variants lacking the SPTSS motif are dramatically stabilized and are hyperactive in transgenic flies. Our results propose a novel mechanism of regulation of neurogenesis, involving the stability of key players in the process.

Subcellular localisation modulates ubiquitylation and degradation of Ascl1

“…1C ), and then declined rapidly in differentiation medium to the point where it was undetectable by d13 (9 days in differentiation media). This is consistent with previous studies suggesting that Ascl1 protein levels are tightly regulated in co-ordination with neurogenesis 11 , 19 . As bHLH transcription factors are known to be regulated at the level of protein half-life, we next examined whether Ascl1 protein stability differs as NSCs progress through the differentiation programme, determining protein abundance after the addition of cycloheximide to block ongoing protein synthesis.…”

Regulation of the Drosophila ID protein Extra macrochaetae by proneural dimerization partners

“…Similar studies showed that Da itself was a stable protein ( Figure 3C,F ) although Da stability might be somewhat shortened by costransfection with Emc ( Figure 3D,F ). The half-life of Da alone was estimated at > 300 min, but that of Da co-transfected with Emc at 139 min ( Kiparaki et al, 2015 )( Figure 3D,F ). It would be interesting to investigate whether this difference affects Da levels in vivo when emc is mutated.…”

All in the family: proneural bHLH genes and neuronal diversity

“…Some distinctions between proneural bHLH genes must be due to their interactions with other regulatory factors, which explains, for example, how genes such as ato and Atoh1 can be responsible for different neuronal cell types in distinct tissues (Kiefer et al, 2005;Huang et al, 2014). There is also increasing evidence that, in addition to transcriptional regulation, the regulation of bHLH protein stability is important (Qu et al, 2013;Kiparaki et al, 2015;Weinberger et al, 2017;Li and Baker, 2018). Also, genes under constraint by multiple mechanisms may behave less effectively in gene replacement experiments.…”

“…Blocking autoregulation in this way is an effective means of permanently extinguishing gene expression and might be exploited by Notch signaling in many situations (Baker, 2004). In addition, there is increasing evidence that Notch signaling affects proneural protein stability, which would also negatively impact autoregulation (Kiparaki et al, 2015;Weinberger et al, 2017).…”