Loss of ZC4H2 and RNF220 Inhibits Neural Stem Cell Proliferation and Promotes Neuronal Differentiation

The ubiquitin E3 ligase RNF220 and its co-factor ZC4H2 are required for multiple neural developmental processes through different targets, including spinal cord patterning and the development of the cerebellum and the locus coeruleus. Here, we explored the effects of loss of ZC4H2 and RNF220 on the proliferation and differentiation of neural stem cells (NSCs) derived from mouse embryonic cortex. We showed that loss of either ZC4H2 or RNF220 inhibits the proliferation and promotes the differentiation abilities of NSCs in vitro. RNA-Seq profiling revealed 132 and 433 differentially expressed genes in the ZC4H2(-/-)and RNF220(-/-)NSCs, compared to wild type (WT) NSCs, respectively. Specifically, Cend1, a key regulator of cell cycle exit and differentiation of neuronal precursors, was found to be upregulated in both ZC4H2(-/-)and RNF220(-/-)NSCs at the mRNA and protein levels. The targets of Cend1, such as CyclinD1, Notch1 and Hes1, were downregulated both in ZC4H2(-/-)and RNF220(-/-)NSCs, whereas p53 and p21 were elevated. ZC4H2(-/-)and RNF220(-/-)NSCs showed G0/G1 phase arrest compared to WT NSCs in cell cycle analysis. These results suggested that ZC4H2 and RNF220 are likely involved in the regulation of neural stem cell proliferation and differentiation through Cend1.