Inactivation of DET1 causes neurological defects and lethality in mice and humans II

COP1 and DET1 are components of an E3 ubiquitin ligase that is conserved from plants to humans. Mammalian COP1 binds to DET1 and is a substrate adaptor for the CUL4A-DDB1-RBX1 RING E3 ligase. Its transcription factor substrates, including c-Jun, ETV4, and ETV5, are targeted for proteasomal degradation to effect rapid transcriptional changes in response to cues such as growth factor deprivation. Here we show that a homozygous DET1R26W mutation that is linked to lethal developmental abnormalities in humans disrupts DET1 binding to DDB1 and compromises E3 ligase function. Human-induced pluripotent stem cells bearing the homozygous DET1R26W mutation expressed ETV4 and ETV5 highly, had alterations in mitochondrial protein expression, and exhibited impaired neuronal differentiation. Mice lacking Det1 died during embryogenesis, while Det1 deletion just in neural stem cells elicited hydrocephalus, cerebellar dysplasia, and neonatal lethality. Our findings highlight an important role for DET1 in the neurological development of mice and humans. Overall design: WT human iPSCs and a DET1 p.R26W knock-in clone were cultured in mTeSR Plus media on Matrigel-coated dishes, with media refreshed every other day. At ~75% confluency, cells were rinsed with DPBS, treated with Accutase, neutralized with mTeSR Plus, filtered through 40 µm strainers, and centrifuged at 300g for 5 minutes. iPSCs were resuspended in induction media, counted, and seeded at 30,000 cells/cm² on iMatrix-511-coated dishes. Induction media was replaced daily from days 1 to 4. On day 5, neurons were detached, dissociated, strained, counted, and replated onto PDL- (50 µg/mL) and iMatrix-coated dishes at 20,000 cells/cm² in maturation media. Neurons were grown for two more days before collection on day 7. The experiment was performed in triplicate.