Sci Adv︱ heavyweight!

Written by Xingsen Zhao, edited by Xingsen Wang, Sizhen Wang In mammals, DNA methylation is a key epigenetic modification in gene expression regulation, parental imprinting, X chromosome inactivation, embryonic development, neurogenesis, cell reprogramming, etc.
Play an important role in the process [1-5]
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DNA methyltransferase 1 (DNMT1 for short), also known as DNA methylation maintenance enzyme, is responsible for converting the hemimethylated DNA on the genome into fully methylated DNA during the DNA replication process [6]
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 HSAN1E disease (hereditary sensory and autonomic neuropathy type 1E with dementia and hearing loss) and ADCA-DN disease (autosomal dominant cerebellar ataxia, deafness, and narcolepsy) are two neurodegenerative diseases.
Induced by mutations, HSAN1E disease manifests as hereditary sensory autonomic neuropathy accompanied by dementia and hearing loss, and ADCA-DN disease manifests as cerebellar ataxia, deafness, and narcolepsy (narcolepsy)
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In patients with HSAN1E and ADCA-DN, the heterozygous mutation site of the DNMT1 gene they carry is located in the RFTS (the replication foci targeting sequence) domain, and the RFTS domain of DNMT1 participates in the DNMT1 pair of replication forks and centromere chromatin Targeting [7-10]
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Although some studies have found that RFTS domain mutations can affect the function of DNMT1 protein, so far, how these mutations affect DNMT1 in the body and thus lead to neurodegenerative diseases is still unclear
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 On September 1, 2021, Professor Weng Jiemin's team from East China Normal University and Professor Li Xuekun's team from Zhejiang University published a research paper entitled "Mutation-induced DNMT1 cleavage drives neurodegenerative disease" online on "Science Advances"
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The researchers constructed two knock-in mouse models Dnmt1-M1 and Dnmt1-M2 that mimic the two DNMT1 gene mutations Y495C and D490E-P491Y carried by HSAN1E patients, and found that these two mice exhibited neurodegeneration of learning and memory impairment.
The phenotype of sexual diseases and revealed the possible pathogenesis of neurodegenerative diseases caused by mutations in the RFTS domain of DNMT1 protein
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The research team first constructed two Dnmt1-M1 and Dnmt1-M2 mice carrying DNMT1 gene mutations related to HSAN1E disease.
The mice with these two homozygous mutations died at about 10.
5 days after the embryo, and the heterozygotes could survive and be fertile.
And there is no obvious abnormality in growth and appearance (Figure 1)
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The DNMT1 protein level of Dnmt1-M1 and Dnmt1-M2 heterozygous mice is about 50% lower than that of normal wild-type (WT) mice
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The research team detected the level of DNA methylation in WT and mutant mouse embryos and adult mouse tissues, and found that the DNA methylation level in heterozygous embryos and adult mouse tissues decreased by about 3%-4%, which is in line with the report.
The decreased levels of DNA methylation in HSAN1E patients are consistent (Figure 1)
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Figure 1.
Lethal embryos homozygous for the RFTS mutation of DNMT1
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(Picture quoted from: W.
Wang et al.
, Science advances, 2021) Figure 2.
The DNMT1 protein level and DNA methylation level of heterozygotes knocked in by two gene mutations are reduced
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(Picture quoted from: W.
Wang et al.
, Science advances, 2021) In order to explore the phenotype and pathogenesis of DNMT1 mutant mice in neurodegenerative diseases, the research team isolated and cultured adult WT and DNMT1-M2 mice Neural stem cells (aNSCs)
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In vitro experiments found that the proliferation ability of DNMT1-M2 neural stem cells decreased, the number of differentiated neurons decreased, and astrocytes increased
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In vivo BrdU labeling experiments also found that the proliferation ability of DNMT1-M2 neural stem cells decreased
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Behavioral experiments such as water maze and eight-arm maze found that DNMT1-M2 mice showed poor learning and memory abilities
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These experimental results indicate that the adult neurogenesis of DNMT-M2 mice is abnormal and cognitive function declines
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This is consistent with the cognitive impairment phenotype of HSAN1E patients (Figure 3)
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Figure 3.
Adult DNMT1-M2 mutant mice exhibit learning and memory impairment
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(Picture quoted from: W.
Wang et al.
, Science advances, 2021) In order to reveal the reason for the decline in DNMT1 protein levels in Dnmt1-M1 and Dnmt1-M2 mice, the research team prepared wild-type, heterozygous and homozygous Dnmt1-M1 mouse embryonic stem cells were analyzed and it was found that the protein level of DNMT1 decreased in embryonic stem cells with DNMT1 mutation
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The embryonic stem cells were treated with the proteasome inhibitor MG132 and the lysosomal inhibitor CQ, respectively, and it was found that neither of these two inhibitors could bring the DNMT1 protein level of Dnmt1-M1 homozygotes back to the same level as that of control mice (Figure 4)
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This shows that although the mutant DNMT1 protein is indeed unstable compared to WT, it is not degraded through the proteasome or lysosomal pathway
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Figure 4.
The stability of the mutant DNMT1 protein decreases in homozygous mutant embryonic stem cells
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(Picture quoted from: W.
Wang et al.
, Science advances, 2021) The research team used the N-terminal region-specific antibody of DNMT1 to perform immunoprecipitation experiments from Dnmt1+/+ and Dnmt1M1/M1 mutant mouse embryonic stem cells (mESc).
DNMT1 truncated protein with a molecular weight of about 75-80 KDa was detected in Dnmt1M1/M1 and Dnmt1+/M2 cell lysates, but not in WT cell lysates
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This explains the decrease in DNMT1 protein levels in mutant mouse embryos, mutant mESc, and heterozygous mice
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The research team also tested the localization of DNMT1 in WT and Dnmt1 mutant cells, and found that the DNMT1 protein in mutant mESc, MEF (mouse embryo fibroblast) and aNSCs did not have cytoplasmic aggregation, but there was obvious nucleolar mislocation ( Figure 5)
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Figure 5 The mutant DNMT1 protein has a special proteolytic method and nuclear localization errors
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(Picture quoted from: W.
Wang et al.
, Science advances, 2021) In order to further determine whether the nucleolar-located DNMT1 is a full-length DNMT1 mutant or a truncated DNMT1, the research team constructed a series of expressions of different lengths of DNMT1 N The end domain of the plasmid, and the location of the over-expressed DNMT1 protein truncated body and the full-length DNMT1 mutant in HeLa cells were detected
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The results showed that the DNMT1 truncated protein was able to partially localize to the nucleolus, but the full-length DNMT1 mutant did not detect mislocalization (Figure 6)
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This indicates that the nucleolar-localized DNMT1 protein observed in DNMT1 mutant cells is most likely a truncated DNMT1
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Studies on Dnmt1 mutant mice and mouse-derived cells have shown that the cleavage of the mutant DNMT1 protein by proteases is a potential mechanism leading to DNMT1 functional defects in HSAN1E neurodegenerative diseases
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In order to further verify in human-derived cells, the research team used ABE-MAX single-base editing technology to construct a DNMT1-M1 mutant cell line in glioma HS683 cells
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The results showed that the proliferation of the edited HS683 cells was blocked and showed DNMT1 protein nucleolar localization (Figure 6)
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However, no truncated protein was detected in the DNMT1-M1 point mutant cells constructed in HeLa cells, and the mutant DNMT1 protein was basically normal in terms of subcellular localization, protein stability and DNA methyltransferase activity (Figure 7)
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 These findings indicate that the degradation of DNMT1 caused by mutations in the RFTS domain, rather than the mutation itself, leads to the functional defects of DNMT1 mutants
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Based on the above two neurodegenerative disease patients, only missense mutations in the RFTS region of DNMT1 were found, but no patients with nonsense mutations in this region and other regions were found, so the author proposed that The cause of the above neurodegenerative diseases is that, on the one hand, the specific shearing of the mutant DNMT1 protein leads to defects in its DNA methylation activity, on the other hand, the abnormal nucleolus localization of the corresponding truncated body may interfere with the normal function of the cell nucleolus (Such as ribosomal RNA transcription), which may eventually lead to defects in the development and differentiation of some nerve cells and apoptosis, leading to related neurodegenerative diseases
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Figure 6 Construction of cell line with abnormal nuclear localization of DNMT1 truncated body and mutation of DNMT1-M1
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(Picture quoted from: W.
Wang et al.
, Science advances, 2021) Figure 7.
The characteristics of the mutant DNMT1 protein in subcellular localization, protein stability and DNA methyltransferase activity are not significantly different from those of the wild-type protein
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(Picture quoted from: W.
Wang et al.
, Science advances, 2021) Conclusion and discussion of the article, inspiration and outlook The main findings of this research can be summarized as follows: 1.
The death of DNMT1 homozygous mutation is caused by the DNA methylation defect caused by the significant decrease of DNMT1 protein content
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2.
DNMT1 heterozygous mutant mice have neurogenesis disorders and learning and memory dysfunction
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3.
DNMT1 mutant protein has a special way of proteolysis
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4.
The truncated DNMT1 but not the mutant has abnormal nuclear localization, and may be a key factor leading to HSAN1E disease
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In summary, the nature of the DNMT1 mutant found in HSAN1E patients is not much different from that of wild-type DNMT1.
The mutant is cleaved by a special protease, which leads to instability, decreased protein levels and DNA methylation levels.
Misplaced truncations affect gene transcription and cell function, leading to disease
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Original link: https:// Professor Weng Jiemin from East China Normal University and Professor Li Xuekun from Zhejiang University are the co-corresponding authors of the paper, Dr.
Wencai Wang from East China Normal University and PhD from Zhejiang University Graduate student Zhao Xingsen is the co-first author of the paper
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This work was funded by the National Natural Science Foundation of China
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