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The ubiquitin proteasome system (UPS) is known to be responsible for the rapid turnover of many transcription factors, where half-life is held to be critical for regulation of transcriptional activity. However, the stability of key transcriptional regulators of development is often very poorly characterised. Neurogenin 3 (Ngn3) is a basic helix-loop-helix transcription factor that plays a central role in specification and differentiation of endocrine cells of the pancreas and gut, as well as spermatogonia and regions of the brain. Here we demonstrate that Ngn3 protein stability is regulated by the ubiquitin proteasome system and that Ngn3 can be ubiquitylated on lysines, the N-terminus and, highly unusually, on non-canonical residues including cysteines and serines/threonines. Rapid turnover of Ngn3 is regulated both by binding to its heterodimeric partner E protein and by the presence of cdk inhibitors. We show that protein half-life does appear to regulate the activity of Ngn3 in vivo, but, unlike the related transcription factor c-myc, ubiquitylation on canonical sites is not a requirement for transcriptional activity of Ngn3. Hence, we characterise an important new level of Ngn3 post-translational control, which may regulate its transcriptional activity.
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23259061
???displayArticle.pmcLink???PMC3522888 ???displayArticle.link???Biol Open ???displayArticle.grants???[+]
Fig. 1. Ngn3 is degraded by ubiquitin-mediated proteolysis in interphase and mitosis.Degradation assays were performed in interphase (A) and mitotic (B) Xenopus egg extract using 35S-IVT Ngn3 in either the presence or absence of 100 µM Mg132 proteasome inhibitor. Samples were run on SDS-PAGE gels, analysed by autoradiography (A,B) and quantified by phosphorimaging (C). Degradation half-lives were calculated using first-order rate kinetics (D). In C, the solid line shows degradation in interphase extracts, and the dotted line shows degradation in mitotic extracts.
Fig. 2. Ubiquitylation of Ngn3 and mutants on canonical and non-canonical sites.Ubiquitylation assays were performed in interphase Xenopus egg extract using 35S-labeled IVT Ngn3 and mutants as labelled, in the presence of His-tagged ubiquitin (B,C). IVT protein levels were normalised, as demonstrated by the levels of starting material shown in A. Proteins were eluted from NTA-agarose beads using either non-reducing conditions (pH 6.8) (B) or highly reducing conditions (pH 10, 10% (v/v) β-mercaptoethanol) (C).
Fig. 3. Identification of Ngn3 ubiquitylation sites involved in degradation.Degradation assays were performed in interphase (A,B,C) and mitotic (D,E,F) Xenopus egg extract using 35S-IVT Ngn3 and mutants (including N-terminal acetylation (Ac), lysine to arginine (KO), and cysteine to alanines (CO) mutations). Samples were run on SDS-PAGE gels, analysed by autoradiography (A,D) and quantified by phosphorimaging (B,E). Degradation half-lives were calculated using first-order rate kinetics (C,F).
Fig. 4. Forms of Ngn3 lacking lysines are stabilised by Mg132.Degradation assays at a fixed point of 90 minutes were performed in interphase (A) and mitotic (B) Xenopus egg extract using 35S-IVT Ngn3 and mutants thereof, as labelled, in either the presence or absence of 100 µM Mg132 proteasome inhibitor. Samples were run on SDS-PAGE gels, analysed by autoradiography (A,B).
Fig. 5. Ngn3 is stabilised by E12 and Xic1 in interphase Xenopus egg extract.Degradation assays were performed in the presence of either (A) IVT E12 (at a 1:1 volume ratio to Ngn3) or IVT mix alone, or (B) 15 µM Xic1-MBP or 15 µM MBP as a control. Samples were run on SDS-PAGE gels, analysed by autoradiography (A,B) and quantified by phosphorimaging. Degradation half-lives were calculated using first-order rate kinetics (C).
Fig. 6. AcNgn3KO binds DNA less tightly than Ngn3.Ngn3, AcNgn3KO, Ngn2 and AcNgn2KO binding to E box-containing probe, with or without added E protein as indicated, were assayed by EMSA either in buffer alone (A) or in the presence of interphase egg extract (B), triangle representing two different amounts of each IVT (see Materials and Methods).
Fig. 7. Ngn3 and AcNgn3KO show similar activity in vivo.1 cell of a 2 cell-stage Xenopus embryo was injected with mRNA encoding GFP (as a control, A), Ngn3 (B,C), AcNgn3KO (D,E), Ngn2 (F) or AcNgn2KO (G) in the amounts as indicated, along with βgal mRNA as a lineage tracer. Expression of neural β tubulin at stage 15 was assayed by in situ hybridisation. A semi-quantitative scoring method for neural β tubulin expression was devised (H), where 0  =  no ectopic neurogenesis, 1  =  low levels of ectopic neurogenesis, 2  =  moderate ectopic neurogenesis and 3  =  extensive ectopic neurogenesis (number of embryos scored per condition: between 49 and 69).
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