Ubiquitin ligase COP1 coordinates transcriptional programs that control cell type specification in the developing mouse brain Kim Newtona,1, Debra L. Duggera, Arundhati Sengupta-Ghoshb, Ronald E. Ferrandoc,2, Felix Chuc,3, Janet Taoc, Wendy Lamc,4, Susan Hallerc, Sara Chanc, Susan Sac, Debra Dunlapc, Jeffrey Eastham-Andersonc, Hai Nguc, Jeffrey Hungc, Dorothy M. Frenchc,2, Joshua D. Websterc, Brad Bolond, Jinfeng Liue,5, Rohit Rejae, Sarah Kummerfelde,6, Ying-Jiun Chenf, Zora Modrusanf, Joseph W. Lewcockb,7, and Vishva M. Dixita,1 aDepartment of Physiological Chemistry, Genentech, South San Francisco, CA 94080; bDepartment of Neuroscience, Genentech, South San Francisco, CA 94080; cDepartment of Pathology, Genentech, South San Francisco, CA 94080; dGEMpath, Longmont, CO 80504; eDepartment of Bioinformatics and Computational Biology, Genentech, South San Francisco, CA 94080; and fDepartment of Molecular Biology, Genentech, South San Francisco, CA 94080 Contributed by Vishva M. Dixit, September 18, 2018 (sent for review March 23, 2018; reviewed by Wenyi Wei and Yuan Zhu) The E3 ubiquitin ligase CRL4COP1/DET1 is active in the absence of central nervous system because increased COP1 copy number is ERK signaling, modifying the transcription factors ETV1, ETV4, linked to autism in humans (12). ETV5, and c-JUN with polyubiquitin that targets them for protea- Potential substrates of CRL4COP1/DET1 in the brain include the somal degradation. Here we show that this posttranslational reg- transcription factors c-JUN, ETV1, ETV4, and ETV5. Activator ulatory mechanism is active in neurons, with ETV5 and c-JUN protein-1 (AP-1) transcription factor c-JUN is broadly expressed accumulating within minutes of ERK activation. Mice with consti- in the developing brain, regulates neuronal apoptosis in response to phosphorylation by upstream kinases (13), and is a substrate tutive photomorphogenesis 1 (Cop1) deleted in neural stem cells COP1/DET1 FBW7 showed abnormally elevated expression of ETV1, ETV4, ETV5, and of both CRL4 and the ubiquitin ligase SCF . c-JUN in the developing brain and spinal cord. Expression of c-JUN Posttranslational regulation of c-JUN is critical to neurogenesis BIOLOGY target genes Vimentin and Gfap was increased, whereas ETV5 and because deletion of Fbw7 from neural stem cells elevates c-JUN c-JUN both contributed to an expanded number of cells expressing and reduces cell viability (14). Regulation of c-JUN abundance DEVELOPMENTAL genes associated with gliogenesis, including Olig1, Olig2, and by FBW7 in the granule cell layer also plays an important role in development of the cerebellum (15). Sox10. The mice had subtle morphological abnormalities in the cerebral cortex, hippocampus, and cerebellum by embryonic day 18 and died soon after birth. Elevated c-JUN, ETV5, and ETV1 con- Significance tributed to the perinatal lethality, as several Cop1-deficient mice also lacking c-Jun and Etv5, or lacking Etv5 and heterozygous for The ubiquitin ligase CRL4COP1/DET1 modifies specific transcrip- Etv1, were viable. tion factor substrates with polyubiquitin so that they are de- graded. However, the Ras–MEK–ERK signaling pathway can COP1/DET1 COP1 | ETV1 | ETV4 | ETV5 | c-JUN inactivate CRL4 and thereby promote the rapid accu- mulation of these transcription factors. Here we show that onstitutive photomorphogenesis 1 (COP1; also called RFWD2) constitutive photomorphogenesis 1 (COP1) has a critical role in Cis the evolutionarily conserved substrate adaptor of the mouse brain development because its deletion from neural cullin-RING ubiquitin ligase CRL4COP1/DET1 (1). COP1 binds to stem cells stabilizes the transcription factors c-JUN, ETV1, ETV4, De-etiolated 1 (DET1), which in turn binds to Damage-specific and ETV5, leading to perturbation of normal gene expression patterns; anatomic anomalies in cerebral cortex, hippocampus, DNA binding protein 1 (DDB1) that is in complex with scaffold and cerebellum; and perinatal lethality. protein CUL4A and Ring-box 1 (2–4). Substrates of mammalian COP1/DET1 CRL4 include the transcription factors c-JUN, ETV1, Author contributions: K.N., D.L.D., A.S.-G., D.M.F., J.D.W., Z.M., J.W.L., and V.M.D. de- ETV4, ETV5, ETS1, ETS2, and C/EBPα, the metabolic enzyme signed research; D.L.D., A.S.-G., R.E.F., F.C., J.T., W.L., S.H., S.C., S.S., D.D., and Y.-J.C. acetyl-CoA carboxylase, and CREB regulated transcription performed research; J.E.-A., H.N., J.H., D.M.F., J.D.W., B.B., J.L., R.R., and S.K. analyzed coactivator 2 (2, 3, 5–8). Ubiquitination of these substrates targets data; and K.N. wrote the paper. them for proteasomal degradation. Reviewers: W.W., Harvard University; and Y.Z., Children’s National Medical Center. Studies with COP1-deficient mice suggest that regulation of Conflict of interest statement: All authors were employees of Genentech. c-JUN, ETV1, and ETV4 abundance contributes to tumor sup- This open access article is distributed under Creative Commons Attribution-NonCommercial- pression by COP1 (3, 9). For example, COP1 deficiency in mouse NoDerivatives License 4.0 (CC BY-NC-ND). prostate epithelial cells results in elevated ETV1, ETV4, and c-JUN Data deposition: The data reported in this paper have been deposited in the Gene Ex- and early prostate intraepithelial neoplasia (3). COP1 also has an pression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession nos. important role in regulating ETV1, ETV4, and ETV5 levels in GSE111564 and GSE111704). 1 pancreatic β-cells so that insulin secretion is not perturbed (10). To whom correspondence may be addressed. Email: [email protected] or dixit@gene. COP1/DET1 com. CRL4 activity is tightly regulated, with phosphoryla- 2 tion of DET1 by ERK contributing to inactivation of the ligase. Present address: Abbvie Biotherapeutics, South San Francisco, CA 94080. 3 Consequently, cells exposed to growth factors exhibit rapid Present address: Atreca, Redwood City, CA 94063. accumulation of CRL4COP1/DET1 substrates (4). 4Present address: Department of Pathology, Sutter Health, Palo Alto, CA 94301. Although COP1 deficiency in many tissues is deleterious, in- 5Present address: Department of Bioinformatics, Gilead Sciences, Foster City, CA 94404. COP1/DET1 appropriate CRL4 activity can also promote disease. 6Present address: Kinghorn Centre for Clinical Genomics, Garvin Institute of Medical Re- For example, C/EBPα degradation orchestrated by COP1 in search, Darlinghurst, NSW 2010, Australia. combination with the pseudokinase TRIB1 (or TRIB2) is linked 7Present address: Denali Therapeutics, South San Francisco, CA 94080. to impaired myeloid differentiation and the development of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. acute myeloid leukemia (7, 11). Inappropriate CRL4COP1/DET1 1073/pnas.1805033115/-/DCSupplemental. activity may also have consequences for the development of the www.pnas.org/cgi/doi/10.1073/pnas.1805033115 PNAS Latest Articles | 1of6 Downloaded by guest on October 3, 2021 ETS family members ETV1, ETV4, and ETV5 have several N roles in the developing nervous system (16–20), but less is known A fh/fh Cop1 about their posttranslational regulation in the brain. In de- Cop1 3xf/3xf veloping mouse cortex, Etv1 mRNA marks a subpopulation of exon 1loxP +/+ fh/fh 3xf/3xf 3xf/3xf early Cajal–Retzius neurons that are specified by FGF8 signal- Cop1Cop1 Det1Det1 Armc8 Flag-HA 97 ing, whereas, at later stages, it serves as a marker of layer 64 Flag DDB1 COP1 DET1 five cerebrocortical neurons (21–23). Etv4 and Etv5 are also IP Flag 64 expressed early in cortical development, but they show a distinct 64 COP1 64 DET1 pattern of expression to Etv1 (23, 24). Expression of Etv5 in the 51 DET1 51 ETV5 Det13xf/3xf ventricular zone of the mouse cerebral cortex is induced by the 64 Frt exon 5 ETV5 64 COP1 MAPKs MEK1 (also called MAP2K1) and MEK2 (MAP2K2), 51 and this is proposed to confer an astrocytic fate on neural 3xFlag stem and progenitor cells (18). MEK1 and MEK2 are part of 97 DDB1 – – 64 the RAF MEK ERK kinase cascade that is engaged by RAS B -- ETV5 GTPases. Consistent with this pathway regulating the switch ERKi BDNF -+ from neurogenesis to gliogenesis, deletion of the RAS negative -- COP1 C ERKi regulator neurofibromatosis 1 (NF1) from neural stem cells PTX -+ ETV5 promotes ERK-dependent gliogenesis at the expense of neu- Relative COP1 rogenesis in the olfactory bulb during perinatal stages (19). ETV Etv5 mRNA 1 2.1 0.9 0.9 ETV5 transcription factors have also been implicated in glioma initi- c-JUN Relative ation by oncogenic RAS (25). Whether ERK-dependent post- Relative Etv5 mRNA 1 0.8 0.7 0.9 1 1.7 1.1 1.8 translational mechanisms, potentially involving CRL4COP1/DET1, c-Jun mRNA p-RSK c-JUN Relative regulate expression of ETV1, ETV4, and/or ETV5 protein in * c-Jun mRNA 1 0.8 1.1 1.1 the brain has not been examined to our knowledge. We in- p-ERK vestigated how COP1 impacts brain development by deleting GAPDH Cop1 in neural stem and progenitor cells with Nestin.cre ERK transgenic mice, or in cells of the neocortex and hippocampus + IRES cre/ -- with Emx1 knock-in mice. D COP1 COP1 24 -- Results PTX -- COP1 Mediates Posttranslational Regulation of c-JUN, ETV1, and ETV5 COP1 During Brain Development. To determine whether COP1 and DET1 expressed in the developing mouse brain interact with ETV5 COP1/DET1 Relative known CRL4 substrates, epitope-tagged versions of Etv5 mRNA 0.9 1.4 1 1.1 COP1 and DET1 were affinity-purified from embryonic day 18.5 (E18.5) knock-in mouse brains (Fig. 1A). ETV5 and DET1 -tubulin copurified with Flag-HA-COP1. These interactions were specific because ETV5 and COP1 also copurified with DET1-3xFlag, but Fig. 1. Posttranslational regulation of ETV5 and c-JUN in neurons. (A) not with an unrelated protein, ARMC8-3xFlag.