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The HECT-domain ubiquitin ligase Huwe1 controls neural differentiation and proliferation by destabilizing the N-Myc oncoprotein

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Abstract

Development of the nervous system requires that timely withdrawal from the cell cycle be coupled with initiation of differentiation. Ubiquitin-mediated degradation of the N-Myc oncoprotein in neural stem/progenitor cells is thought to trigger the arrest of proliferation and begin differentiation. Here we report that the HECT-domain ubiquitin ligase Huwe1 ubiquitinates the N-Myc oncoprotein through Lys 48-mediated linkages and targets it for destruction by the proteasome. This process is physiologically implemented by embryonic stem (ES) cells differentiating along the neuronal lineage and in the mouse brain during development. Genetic and RNA interference-mediated inactivation of the Huwe1 gene impedes N-Myc degradation, prevents exit from the cell cycle by opposing the expression of Cdk inhibitors and blocks differentiation through persistent inhibition of early and late markers of neuronal differentiation. Silencing of N-myc in cells lacking Huwe1 restores neural differentiation of ES cells and rescues cell-cycle exit and differentiation of the mouse cortex, demonstrating that Huwe1 restrains proliferation and enables neuronal differentiation by mediating the degradation of N-Myc. These findings indicate that Huwe1 links destruction of N-Myc to the quiescent state that complements differentiation in the neural tissue.

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Figure 1: Huwe1 binds N-Myc in vivo and controls N-Myc stability.
Figure 2: Huwe1 ubiquitinates and directs N-Myc degradation by the proteasome.
Figure 3: Genetic inactivation of Huwe1 impairs N-Myc degradation.
Figure 4: Blockade of neuronal differentiation by genetic inactivation of Huwe1 is rescued by silencing of N-myc.
Figure 5: Conditional knockout of Huwe1 impairs neuronal differentiation in ES cells.
Figure 6: The Huwe1–N-Myc pathway in the developing brain.
Figure 7: Silencing of Huwe1 blocks differentiation of neural progenitors in vivo.

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Change history

  • 21 April 2009

    In the version of this article initially published, the control samples in Figure 7e were incorrect, the K63R label was misspelled in Figure 2c and the primer sequences were missing from the supplementary information. These errors have been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank Nicholas Sherman for mass spectrometry analyses, Simon Wing for anti-Huwe1 (Lasu1) antibodies, Xiaodong Wang for the pFastBac-1–Mule1, Wei Gu for V5-Huwe1–N and V5–Huwe1–C expression vectors, Richard Baer for pCMV–HA–ubiquitin expression plasmids and George DeMartino for the purified 26S proteasome. We are indebted to Austin Smith for 46C ES cells and for advice on neural differentiation in adherent culture, and to Allan Bradley for AB2.2 ES cells. We thank Yong-Seok Ho for preparation of recombinant Huwe1 proteins and technical support. This work was supported by grants from the NIH to A.L., A.I. and M.P. and an Emerald Foundation grant to D.G. D.G. is supported by a fellowship from Provincia di Benevento, Italy. J.H. is funded by the Australian NHMRC (ID:310616) and work in the F.G. laboratory is funded in part by an institutional grant from the Medical Research Council (UK).

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Contributions

A.L. conducted the initial purification of the N-Myc complexes; A.L., X.Z. and R.J. performed most of the experiments; D.G. performed the experiments shown in Fig. 2f under the supervision of M.P.; X.Z. and J.I.-T.H. performed the ex vivo electroporation experiments under the supervision of F. G.; M.P. provided reagents and suggestions; A.L. and A.I. coordinated the study and wrote the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Antonio Iavarone or Anna Lasorella.

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Supplementary Figures S1, S2, S3, S4, S5, S6, S7, S8, S9 (PDF 20846 kb)

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Zhao, X., Heng, JT., Guardavaccaro, D. et al. The HECT-domain ubiquitin ligase Huwe1 controls neural differentiation and proliferation by destabilizing the N-Myc oncoprotein. Nat Cell Biol 10, 643–653 (2008). https://doi.org/10.1038/ncb1727

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