RFTS Domain Mutation-induced DNMT1 Cleavage Drives Neurodegenerative Disease

In addition to accurately passing the genetic material DNA to the daughter cells,cells also need to accurately inherit the DNA methylation profile when they proliferate,which is critical to cell fate specification and function.DNMT1 is the key enzyme for maintaining DNA methylation pattern in mammalian cells.In recent years,mutations in the RFTS(Replication Foci Targeting Sequence)domain of DNMT1 have been identified in patients with HSAN1E and ADCA-DN(two adult-onset neurodegenerative diseases).However,how these mutations affect DNMT1 function in vivo and consequently lead to diseases are poorly understood.Therefore,we constructed mice models carrying point mutations identified in HSAN1E and ADCADN patients and investigated the potential mechanisms leading to neurodegenerative diseases.By CRISPR-Cas9 based gene editing techniques,we obtained mice with Dnmtl Y500C(Dnmtl-M1)mutation and P496Y(Dnmt1-M2)mutation identified in human HSAN1E patients.In addition,we obtained mice with Dnmtl C585R(Dnmtl-M6)mutation observed in ADCA-DN patients.We found all three types of homozygous mutant mice die around E10.5 days,and homozygous mutant embryos were virtually deficient of DNMT1 protein.All three types of heterozygote mice are viable,fertile and grossly normal in growth and morphology.However,analysis of DNA methylation revealed a reduced level of DNA methylation in various tissues from heterozygote mice as compared to wild-type mice.Importantly,we show that the heterozygote mice exhibit neurodegenerative phenotypes,including impairment of learning and memory and defect in proliferation and differentiation of neural stem cells.Furthermore,the levels of DNMT1 proteins in various tissues from heterozygote mice are only about half of that of wild type.We prepared Dnmt1+/+,Dnmt1+/M1 and Dnmt1M1/M1 mouse embryonic stem cells from corresponding embryos.We found that the mutation has no effect on gene expression of Dnmtl but markedly reduces the protein stability of the mutant DNMT1.However,reduced stability for mutant DNMT1 is not due to increased degradation by either proteasome or autophagy.Instead,we provide evidence that the mutant proteins were specifically degraded by a specific proteinase cleavage.The specific cleavage of mutant DNMT1 not only leads to degradation of mutant DNMT1,but also results in a truncated DNMT1 N-terminal domain that mislocalizes to and forms aggregates in the nucleolus.To investigate if RFTS mutation-induced DNMT1 cleavage also occurs in human neuronal cells,we generated DNMT1 Y495C mutation in HS683(human optic glioma cells)by base-editing technology.We found this mutation resulted in substantial DNMT1 nucleolus mislocalization and impaired cell proliferation.Notably,we also constructed the same DNMT1 Y495C mutation in HeLa cell line.In contrast to results in human optic glioma cells,DNMT1 Y495C mutation had no effect on DNMT1 protein stability,cell proliferation and DNA methylation and did not lead to specific proteinase cleavage of mutant proteins.We therefore conclude that the RFTS mutation-induced degradation of DNMT1,but not mutation itself,is responsible for functional defect of mutant DNMT1 in HSAN1E and ADCA-DN patients.In summary,we have generated mouse models for investigating the molecular mechanisms for DNMT1 RFTS mutation-induced neurodegenerative diseases.We provided evidences that RFTS mutations most likely result in DNMT1 inactivation via inducing specific proteinase cleavage of mutant DNMT1 proteins,which in turn leads to defects in neurogenesis and differentiation and eventually neurodegenerative diseases.