1 Introduction
Ten-eleven translocation (TET) enzymes are important epigenetic factors that modulate gene transcription by hydroxylating DNA 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) for demethylation [
1–
3]. Although TET1, TET2, and TET3 share similar enzymatic actions, they differ in spatiotemporal expression patterns and nonredundant roles in gene expression, development, and diseases [
4–
6]. For example, TET1 and TET2 are highly expressed in embryonic stem cells (ESCs) [
4,
5], and TET3 is increasingly expressed during mouse development [
6]. Among the three TET members, TET2 is involved in the pathological and physiological processes of the central nervous system (CNS), such as neurogenesis [
7], depression [
8], and Alzheimer’s disease [
9]. The TET2-FOXO3A axis participates in the regulation of neurogenic gene expression in neural stem cells (NSC) during adult neurogenesis [
7], and TET2 knockdown affects neuronal survival [
10]. These findings imply the critical role of TET2 in the CNS.
Although many studies have mainly focused on the function of TET2 and its 5hmC catalytic activity in DNA [
1], issues about the regulation of TET2 itself still need to be addressed [
11]. TET2 is regulated by specific factors at the transcriptional, translational, and post-translational levels in different processes. For instance, approximately 30 miRNA molecules, including miR-7, miR-125b, miR-29b/c, miR-26, miR-101, miR142, and Let-7, repress TET2 expression, and regulate malignant hematopoiesis [
12]. TET2 is positively regulated by OCT4 upon the differentiation of mouse ESCs [
4]. High glucose levels impede AMPK-mediated phosphorylation TET2 at serine 99, destabilizing TET2 and dysregulating the tumor suppressive function of TET2 [
13]. IDAX (the inhibitor of disheveled and axin), which has been implicated in malignant renal cell carcinoma and colonic villous adenoma, downregulates TET2 protein through caspase activation [
14]. HIV-1 including viral protein R enhances viral replication by degrading TET2 and sustaining IL-6 expression through polyubiquitylation [
15]. Calpain 1, a member of calcium-dependent protease family, mediates TET2 degradation and affects global 5hmC level and the expression of certain lineage-specific genes in mESCs [
11]. However, the regulation of TET2 in neural differentiation is unclear.
Most members of the glucose-induced degradation-deficient (GID) complex have potential interactions with TET2 according to the mass spectrometry results. The GID ubiquitin ligase was initially found to regulate the polyubiquitin degradation of the key enzyme fructose-1,6-bisphosphatase from gluconeogenesis to glycolysis in yeast [
16] and is essential for maintaining normal cell proliferation [
17]. The major subunits of the human GID complex through comparative proteomics include RANBP9 (GID1), RMND5A (GID2), C17ORF29 (GID4), ARMC8 (GID5), WDR26 (GID7), TWA1 (GID8), MAEA (GID9), MKLN1, and RANBP10. Patients with
Wdr26 mutation show signs of mental retardation, speech retardation, and epilepsy [
18]. It is a plasma-derived candidate biomarker for patients with depression [
19]. Autism spectrum disorder is associated with the methylation level of
Armc8 [
20]. GID4 can identify the substrate and activate the ubquitin ligase [
21], and ARMC8 is necessary for GID4 binding [
17]. Hence, it is of great interest to interrogate the possible function of GID complex to TET2 in the CNS.
Here, we demonstrated that the protein level of TET2 decreased in contrast to mRNA levels during the differentiation of NSC. The GID complex interacted with TET2 and regulated the stability of TET2 protein during the differentiation of NSC, subsequently influencing the level of DNA 5hmC, regulating the differentiation direction of NSC, and participating in the fate determination of NSC.
2 Materials and methods
2.1 Reagents, antibodies, cell lines, and oligos
Lists of the reagents, antibodies, cell lines, and oligos used in this study are provided in the Supplemental Material.
2.2 Animals
All animal procedure protocols were approved by the ethical committee of Soochow University. C57BL/6 mice (25.0–30.0 g, 8–12 weeks old) were purchased from Shanghai Research Center for Model Organisms. Tet2flox/flox line was obtained from the Jackson Laboratory (stock number 017573, B6; 129S4-Tet2tm1.1laai/J). These floxed mutant mice possess loxp sites flanking the exon 3 of the Tet2 gene. Nestin-Cre mice were obtained from the Jackson Laboratory (stock number 003771, B6. Cg-Tg (Nes-Cre) 1 Kln/J). The Tet2 conditional knockout (cKO) mice were generated by crossing loxp-flanked Tet2 mice with Nestin-Cre mice. Wdr26+/− (C57BL/6) mice (stock number: KOAIP211129DY1) were generated by the CRISPR/Cas9 technology (Cyagen Biosciences Inc. Suzhou, China). Briefly, Cas9 mRNA and sgRNAs targeting the exons 2 and 3 of the mouse Wdr26 gene were generated by in vitro transcription and then microinjected into the fertilized eggs from C57BL/6 mice for knockout mouse production. The founder mice were confirmed by genotyping and DNA sequencing analysis, and positive founders were bred to the next generation. The mice were housed in groups of one to five per cage and subjected to a standard 12 h light–12 h dark cycle in a room maintained at 24 ± 2 °C with free access to water and chow. The morning of vaginal plug detection was designated as embryonic day (E) 0.
2.3 Cell culture, transfection, and differentiation
Mouse neuro2a neuroblastoma (N2a) cells, mouse hippocampal neuronal (HT22) cells, and human embryonic kidney (HEK293t) cells were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The cells were cultured in a humidified incubator at 37 °C with 5% CO2. For transfection, HEK293t cells were transfected at 70% confluency by using polyethyleneimine (PEI), and N2a cells were transfected at 70% confluency by using a PL transfection reagent according to the instructions. For differentiation induction, the N2a cells were induced by adding 20 μmol/L retinoic acid in DMEM supplemented with 1% FBS for 12 h.
2.4 Isolation, culture, transfection, and induced differentiation of neural stem cells
NSC were isolated from the dentate gyrus (DG) region of E14.5 mice and were cultured in DMEM/F12 supplemented with 2% B27, 20 ng/mL basic fibroblast growth factor, and 20 ng/mL epidermal growth factor (EGF). The NSC from each fetal mouse were cultured separately, and their genotypes were identified by tails individually. The primary neurosphere was cultured 5 days before experiment, and then neurosphere-derived single cells were inoculated into untreated 12-well plates at a density of 1 × 106 cells/mL. For NSC transfection, the cells were transfected with a lipofectamine stem transfection reagent according to the manufacturer’s protocol. The plates were coated with 10 μg/mL polyornithine (PLO) and 5 μg/mL laminin for 1 day each before the induction of differentiation. The NSC were digested with accutase and inoculated into a coated plate at a density of 1 × 106 cells/mL. The growth medium containing 1 μmol/L forskolin and 1 μmol/L retinoic acid was changed after 24 h. NSC-induced differentiation was induced and stopped after 48 h.
2.5 Primary neuron and astrocyte isolation and culture
The plates were coated with 0.1 mg/mL poly-D-lysine at 37 °C overnight before neuron isolation. Brain tissues were isolated from day 16 embryos, and the meninges were removed under a microscope. The tissues were digested with 0.125% trypsin at 37 °C for 10 min, and the reaction was terminated with DMEM supplemented with 10% FBS. The suspension was filtered with a 40 μm filter and centrifuged at 1000 rpm for 5 min. The cell precipitate was resuspended in the neurobasal medium supplemented with 2% B27 and 0.5 mmol/L L-glutamine. Neurons were cultured for 7 days, and half of the medium was replaced with fresh medium every 3.5 days.
For astrocyte isolation, the brains of the 2-day-old mice were removed, and the meninges were removed under a microscope. The tissue was trypsinized with 0.25% trypase at 37 °C and dissociated, and then the cells were plated in a DMEM containing 10% FBS. After 24 h, the culture medium was changed to a fresh DMEM containing 10% FBS. The culture medium was changed every 3 days. The cells were subcultured and purified on the 5th day at 85% confluency. After 10 days of cell growth, the cells were inoculated into the well plate for the follow-up experiment.
2.6 Immunoprecipitation (IP), protein extraction, and Western blot
For endogenous immunoprecipitation (IP), N2a cells at 70% confluency in three 90 mm dishes were lysed in 1 mL RIPA buffer (50 mmol/L Tris, pH 8.0; 0.1% SDS; 150 mmol/L NaCl; 1 mmol/L EDTA, pH 8.0; 1 mmol/L EGTA, pH 8.0; 0.5% deoxycholate, and 1% TritonX-100) containing 100× cocktail and 200× DTT at 4 °C under rotary agitation for 2 h. Approximately 5% input was obtained through 30 min of centrifugation at 13 200 rpm at 4 °C. Total protein was divided into two groups, and each group was not less than 2 mg. The protein supernatant was added after agarose beads were activated and were pre-cleared by rotating on the rotator for 2 h at 4 °C. The supernatant was moved to a new tube, and the same amounts of TET2 and IgG antibodies were added to the two tubes, which were then rotated at 4 °C overnight. The supernatant was transferred to the agarose beads and rotated at 1000 rpm at 4 °C for 15 h. The supernatant was removed after 3 min of centrifugation at 1000 rpm and 4 °C. A 2× loading buffer (50 μL) was added to the precipitate, and the samples were boiled at 95 °C for 10 min.
For exogenous IP, HEK293t cells were transiently transfected with target plasmids by using PEI. Three 90 mm dishes of HEK293t cells were used as one group. HA-WDR26 + VECTOR, HA-WDR26 + FLAG-TET2 (HA-ARMC8 + VECTOR, HA-ARMC8 + FLAG-TET2) were transfected for 48 h separately. Cell pellets were lysed with a lysis buffer (150 mmol/L NaCl; 25 mmol/L Tris-HCl, pH7.4; 10% glycerol; 0.5% TritonX-100; 2 mmol/L MgCl2) containing 100× cocktail and 200× DTT at 4 °C for 3 h. After 30 min of centrifugation at 4 °C and 13 200 rpm, 60 μL of supernatant was collected as input. The rest was placed in FLAG beads and rotated at 4 °C for 11 h. The supernatant was removed by a magnetic shelf, and the beads were cleaned twice with lysis buffer. Loading buffer (2×, 50 μL) was added to the precipitate, and the samples were boiled at 95 °C for 10 min.
For total protein extraction, cell pellets were lysed using RIPA buffer containing 50 mmol/L Tris (pH 8.0), 0.1% SDS, 150 mmol/L NaCl, 1 mmol/L EDTA (pH 8.0), 1 mmol/L EGTA (pH 8.0), 0.5% deoxycholate, and 1% TritonX-100 on ice for 30 min. Then, 15 min of centrifugation was performed at 4 °C and 12 000 rpm. The supernatant was collected and boiled for 10 min at 95 °C after 1/4 volume of 5× loading buffer was added. Approximately 30 μg of the protein aliquot of each sample was separated using standard SDS-PAGE and transferred to a PVDF membrane. After the transfer, the PVDF membrane was blocked with 10% nonfat milk for 1 h at RT and incubated overnight at 4 °C with a target primary antibody. Immunoreactive bands were detected using an ECL chemiluminescence reagent after incubation with a secondary antibody for 1 h. The details of the antibody and reagents used in this experiment are listed in the Supplemental Material.
2.7 RNA isolation and quantitative RT-PCR
Total RNA was extracted using RNAiso plus and was reverse transcribed using a reverse transcription kit according to the manufacturer’s protocol. Quantitative real-time PCR was performed using 2× SYBR Green PCR Master Mix with a 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). GAPDH was used as an endogenous control for real-time PCR amplification. For data analysis, fold change was calculated using the ΔΔCt method according to the threshold cycle value obtained from RT-PCR. The sequences of the primers used in this study are listed in the Supplemental Material.
2.8 Genomic DNA isolation and dot blot
Cell pellets were digested in 600 μL of DNA lysis buffer (100 mmol/L Tris-HCl, pH 8.5; 5 mmol/L EDTA; 0.2% SDS; 200 mmol/L NaCl) and mixed with 20 mg/mL proteinase K, and incubated at 55 °C overnight. An equal volume of phenol:chloroform:isoamyl alcohol solution (25:24:1, saturated with 10 mmol/L Tris, pH 8.0, and 1 mmol/L EDTA) was added on the second day. The mixture was vortexed for 1 min and separated through centrifugation for 10 min at 12 000 rpm at 4 °C. The aqueous layer solution was transferred to a new tube and precipitated with 600 μL of isopropanol. DNA pellets were obtained through 15 min of centrifugation at 12 000 rpm and 4 °C and washed with 70% ethanol twice. The DNA was air dried and eluted with nuclease-free water.
For dot blot, a 20 μL system containing 2000 ng of DNA, 10 μL of 2 M NaOH, and double-distilled water was prepared and left to stand at 4 °C for 20 min. 2 µL of each sample was spotted onto a nitrocellulose (NC) membrane and let the membrane dry at room temperature (RT). DNA was fixed to the membrane by incubating at 80 °C for 30 min. Then the NC membrane was blocked with 10% nonfat milk for 1 h at RT and incubated with 5hmC or 5mC antibody overnight at 4 °C. Immunoreactive signal was detected by the ECL chemiluminescence reagent after incubating with the secondary antibody for 1 h. And the dot signal was quantified by ImageJ software.
2.9 Immunofluorescence
The samples were fixed overnight at 4 °C in 4% PFA in PBS for 30 min, treated with 0.3% Triton X-100 in PBS (PBST) for 10 min and then blocked with 5% BSA in 0.3% PBST for 1 h at RT. Subsequently, the samples were incubated with sample-specific primary antibodies overnight at 4 °C, washed three times with PBS, and incubated with florescent-labeled secondary antibody containing DAPI for 1 h at RT. The samples were imaged with a fluorescence microscope (Axio Scope A1, Zeiss) after patching on the microslide.
2.10 Construction of N2a knockout cell lines by CRISPR Cas9
The sgRNA was designed using Benchling. The oligos were then annealed according to a standard protocol and ligated into the vector lentiCRISPR v2 (Addgene, #52961) and confirmed by sequencing. The plasmids were transfected into the N2a cell line at 60% confluency by using a PL transfection reagent for 36 h. Puromycin was added to the culture for the selection of transfected cells for 48 h after transfection. The surviving cells were subjected to picking single clones by limiting dilution. The sequences of the oligos used in this study are provided in the Supplemental Material.
2.11 Statistical analysis
All statistical analyses were performed using Prism 7.0 (GraphPad Software) and expressed as mean ± SEM. N numbers were reported in figure legends. The differences with different treatments were determined by t-test or one-way ANOVA followed by Tukey’s post hoc test. P < 0.05 was considered statistically significant.
3 Results
3.1 Proteasome pathway regulates the stability of TET2 in NSC
To investigate the function of
Tet2 in the progress of neural differentiation, we found that the expression levels of
Tet2 and DNA base 5hmC significantly decreased in the hippocampi of the
Tet2 conditional knockout (cKO) mice compared with the control mice, whereas levels of
Tet1 and
Tet3 did not change (
P < 0.01, 0.001, Fig. S1A–S1D). Immunofluorescence analysis for neural markers showed decreased neurogenesis and the enhanced proliferative capacity of NSC in the DG region of the adult
Tet2 cKO mice and increased NSC self-renewal capacity in the neurospheres isolated from the
Tet2 cKO mice at E14.5 (
P < 0.05, 0.01; Fig.1). We further extracted NSC from wild-type (WT) mice and induced the differentiation of NSC. After NSC differentiation, the mRNA level of
Tet2 was upregulated (
P < 0.05, Fig.2), whereas the protein level was downregulated (
P < 0.01, Fig.2 and 2C). To explore the reason for this inconsistency, we investigated the downregulation of TET2 after NSC differentiation. Given that the main pathways affecting the stability of TET2 include the caspase 3-dependent proteolytic cleavage pathway [
14], calpeptin-mediated degradation [
11], and proteasome-mediated ubiquitin degradation [
22], we transfected HEK293t cells with FLAG-TET2 plasmids and then treated them with proteasome inhibitor (MG132), calcium protease inhibitor (calpeptin), and apoptosis inhibitor (Z-VAD-FMK) successively to detect their effects on the expression of FLAG-TET2. The results showed that treatment with MG132 significantly increased the expression of FLAG-TET2 (
P < 0.05, Fig.2 and 2E). Then, we treated the nerve cells, N2a cells and HT22 neuronal cells with the inhibitors. Similarly, MG132 significantly inhibited the degradation of endogenous TET2 in the N2a and HT22 cells (
P < 0.05, 0.01, Fig.2–2I). These results indicated that proteasome-mediated degradation is the main pathway regulating the stability of TET2 in NSC.
3.2 TET2 interacts with the subunits of the GID complex
Previous study showed many TET2-interacting proteins in the mouse ESCs [
23]. We further analyzed and identified that the proteins interacting with TET2 were proteins involved in RNA splicing, translation initiation pathway, and protein degradation (Fig.3 and 3B). To identify the potential E3 ligase that may be involved in TET2 degradation, the proteins with potential interaction with TET2 were screened by cluster analysis. The members of the GID complex, including WDR26, ARMC8, MKLN1, RANBP10, RANBP9, and MAEA, were found to potentially interact with TET2 (Fig.3 and 3D). Among the subunits of the GID complex, WDR26 is the central subunit of mammalian GID complex and acts as a scaffold protein for substrate recruitment in the E3 ubiquitin ligase complex [
24]. ARMC8 is responsible for recruiting GID4 [
25], an activator of E3 ligase activity, to enable efficient E2 enzyme binding and catalytic core orientation toward the substrate.
To verify the interaction between the GID subunits and TET2, we first cotransfected FLAG-TET2 plasmids with HA-WDR26/HA-ARMC8 (two core subunits of the GID complex) separately in HEK293t cells and confirmed the interaction between WDR26/ARMC8 and TET2 (Fig.3, 3F, S2A and S2B) by co-immunoprecipitation. To gain further insight into the association between TET2 and GID complex, we induced the immunoprecipitation of soluble N2a cell extracts with an anti-TET2 antibody and then performed Western blot analysis of the immunoprecipitated proteins with anti-WDR26 or anti-ARMC8 antibody and revealed the presence of WDR26 and ARMC8 in the anti-TET2 immunoprecipitates (Fig.3). These results showed a potential role of the GID complex with WDR26 and ARMC8 as key subunits in the proteasome system that may be involved in the regulation of protein stability of TET2.
3.3 The protein levels of GID complex subunits rise reciprocally with TET2 level upon NSC differentiation
To explore the role of the GID complex in the CNS, we first extracted NSC, neurons from E14.5-day-old mice, and astrocytes from 2-day-old mice (Fig.4–4C). The expression of TET family members and GID complex members in the three kinds of neuronal cells were detected. RT-PCR results showed that the members of the TET family and GID complex showed similar expression patterns in different cells, that is, all of them were highly expressed in the neurons (Fig.4). After NSC differentiation, the mRNA level of
Tet2 was upregulated (
P < 0.05, Fig.5), protein level was downregulated (
P < 0.01, Fig.5 and 5C). By contrast, the mRNA and protein levels of the GID complex members were all upregulated (
P < 0.05, 0.01, Fig.5–5E). Consistently, after the differentiation of N2a cell, the mRNA level of
Tet2 was upregulated (
P < 0.05, Fig.5) and the protein level was downregulated (
P < 0.05, Fig.5 and 5H). However, the mRNA and protein levels of GID complex core subunits were upregulated (
P < 0.05, 0.01, Fig.5–5J). The expression levels of ARMC8 and WDR26 increased, whereas the expression level of TET2 decreased during N2a cell differentiation (Fig.5). The differentiation potential of the NSC decreased dramatically 2 weeks after the birth of the mice [
26]. Accordingly, we extracted the hippocampal tissues of 1-, 7-, 14-, 21-, and 30-day-old littermates and observed changes in TET2, WDR26, and ARMC8 levels during brain development. The expression levels of WDR26 and ARMC8 increased with age, but the expression level of TET2 decreased gradually with age from the second week (Fig.5).
3.4 TET2 protein levels are directly regulated by the GID complex
To directly investigate the relationship between the GID complex and TET2 during neuronal differentiation and the role of the GID complex in the regulation of the stability of TET2, we generated Armc8 KO, Wdr26 KO, and Tet2 KO N2a cell lines by using a CRISPR/Cas9 system. Western blots showed that TET2 level significantly increased in the Armc8 KO and Wdr26 KO cells (Fig.6 and 6B), whereas the deletion of Tet2 did not affect the expression levels of ARMC8 and WDR26 in the N2a cells (Fig.6). We further transfected ARMC8 or WDR26 overexpression plasmids to complement the deficiency in the Armc8 KO or Wdr26 KO cell lines. ARMC8 and WDR26 overexpression significantly decreased the level of TET2 (P < 0.05, 0.01, Fig.6–6G) but not the level of TET1 or TET3 (Fig. S2C and S2D). These results implied that TET2 is a novel downstream target of the GID E3 ligase complex during neuronal differentiation.
As GID complex is an enzyme with ubiquitin modification function, we subsequently explored the effect of the GID complex on TET2 ubiquitination. We examined the effect of the overexpression of the two core subunits of the GID complex on the ubiquitination of TET2 in N2a cells after the overexpression of HA-WDR26 or FLAG-ARMC8 plasmids. The results showed that ubiquitinated TET2 significantly increased after the overexpression of HA-WDR26 or FLAG-ARMC8 plasmid in the N2a cells (Fig.6 and 6K). To further verify the effect of the GID complex on the protein stability of TET2, we treated the Armc8 or Wdr26 KO N2a cells with cycloheximide (CHX), which can block protein synthesis, for 0, 1, 2, 4 and 8 h, respectively. The results showed that the half-life period of TET2 protein was about 2 h under normal conditions, and the degradation of TET2 protein decreased significantly after Wdr26 or Armc8 KO (Fig.6, 6J, 6L and 6M). Taken together, the TET2 protein was more stable in the Wdr26 or Armc8 KO N2a cells.
In addition,
Wdr26 haploinsufficiency causes the Skraban-Deardorff syndrome characterized by developmental delay or intellectual disability, characteristic facial features, hypotonia, epilepsy, and infant feeding difficulties [
18]. The mechanism by which
Wdr26 haploinsufficiency leads to these symptoms has yet to be elucidated. To determine whether TET2 stability is specifically regulated by the GID complex under physiologic conditions, we generated the heterozygous
Wdr26 knockout (
Wdr26+/−) mice (Fig. S1I and S1J). The Western blot results showed an increased expression of TET2 and no change in TET1 or TET3 expression in the prefrontal cortex of
Wdr26+/− mice (
P < 0.05, Fig.7–7D).
3.5 Armc8 or Wdr26 KO affected the process of neuronal differentiation
To elucidate the function and biological significance of the GID complex in regulating the protein stability of TET2. We first examined the changes in 5hmC and 5mC after Wdr26 or Armc8 was knocked out in the N2a cells. Dot blot showed that the level of 5hmC in Wdr26 or Armc8 KO N2a cells significantly decreased, whereas the level of 5mC had no significant effect (P < 0.05, Fig.8–8F). Given the effect of TET2 on neuronal differentiation, we induced the differentiation of Wdr26 or Armc8 knockout N2a cells and examined the effect of the GID complex key subunit deficiency on neuronal differentiation. The results showed that Armc8 or Wdr26 KO significantly increased the differentiation ratio of the N2a cells (Fig.9). The Wdr26 KO N2a cells had a higher proportion of differentiation and longer synapses (P < 0.0001, 0.01, Fig.9 and 9C). We knocked down Wdr26 or Armc8 in NSC derived from WT or Tet2 cKO mice (Fig.9 and 9F) and induced differentiation for 48 h. RT-PCR results showed that the reduction of Armc8 and Wdr26 increased the expression level of TUJ1 after WT NSC differentiation but not NSC from the Tet2 cKO mice (Fig.9 and 9G). In conclusion, these results suggested that the GID complex can regulate the level of 5hmC and further influence the differentiation of nerve cells by regulating the level of TET2.
4 Discussion
Here, the critical finding presented is the discovery of the GID complex, a new regulatory factor that regulates the stability of TET2. The GID complex involves neuronal differentiation homeostasis by regulating the protein level of TET2. As the focus of neurological disorders, stem cells need to undergo a rigorous and dynamic gene expression regulation for the determination of the maintenance and development of different organs [
27,
28]. Similarly, in the nervous system, NSC must carefully balance self-renewal and differentiation to maintain brain homeostasis and ensure the proper number of differentiated cell types needed to keep brain balance [
29]. NSC undergo symmetric division to ensure self-renewal and asymmetric division to pre-differentiate [
30]. The dys-differentiation or excessive self-renewal of NSC can lead to microcephaly [
31] and brain tumor [
32]. The premature differentiation of neurons may result in the insufficient pool reserve of neural progenitor cells and unbalanced distribution of other types of nerve cells, impede nerve cell migration, and impair the projection of brain regions [
29]. The transformation of NSC into other types of nerve cells is determined by many stem cell regulatory factors, including SOX2, NOTCH1, CYCLIN-B1, CYCLIN-D1, and MYT1 [
27]. Many genes, such as
Foxg1 [
33],
Mcph1 [
34], and
Nox [
35], affect brain development abnormalities or neuropsychiatric disorders caused by neural differentiation. Thus, many molecular mechanisms that influence neuronal differentiation need to be explored.
As an important member of epigenetic regulatory enzymes, the TET family promotes DNA demethylation by catalyzing DNA 5mC to 5hmC, 5-formylcytosine, and 5-carboxyl cytosine [
1]. All TET family members participate in neural differentiation in nonoverlapping roles.
Tet3 KO ESCs can be induced to differentiate into neural progenitors, they undergo rapid apoptosis and show greatly compromised terminal differentiation [
6]. Tet1 deficiency diminishes the adult SGZ neural progenitor pool but has no significant effect on the generation of neurons, astrocytes, and oligodendrocytes [
36]. Our results showed TET2 inhibits the differentiation of NSC into neurons. Understanding how different TET enzymes act on 5mC of specific-target genes for the cell specificity and functional specificity of different TET enzymes is necessary. This function may be mediated by recruiting additional chaperone proteins or upstream molecular regulation. In the exploration for the role of TET2 in neural differentiation, we first detected the expression of TET2 before and after neuronal differentiation. However, the function of one gene often depends on the actual protein output. After differentiation, the expression of
Tet2 mRNA was upregulated, whereas the level of TET2 protein was downregulated. For this normal physiologic process of neuronal differentiation, we will explore in greater depth the significance of decreased levels of TET2 in neuronal differentiation.
Current studies on the protein regulation of TET2 focus on the apoptosis, calpeptin, and ubiquitin degradation pathway. We found that proteasome has the most significant effect on the level of TET2 in nerve cells. By performing a co-immunoprecipitation/MS assay, we found that the GID complex physically interacts with and involves in the regulation of the stability of TET2. The GID complex regulates the cell cycle but has not been studied comprehensively in the CNS. Interestingly, GID subunits are involved in the development of many diseases related to neural differentiation, for example, the Skraban-Deardorff syndrome, a disease related to variations in
Wdr26 gene and characterized by a combination of symptoms: intellectual disability, seizures, abnormal gait, and distinctive facial features [
18]. Our results showed an increased expression of TET2 but no change in TET1 or TET3 in the
Wdr26 heterozygous recombinant mice. Whether this disease is related to TET2-induced changes in global 5hmC level in Skraban-Deardorff syndrome will be further investigated as many clinical features of this disease overlap with “transcriptomopathies” [
37]. Moreover,
Xenopus NSC
Armc8 knockout promotes neuronal differentiation [
38].
Mkln1 mutations may increase the risk of bipolar disorder [
39]. Given these close relationships, we investigated the effect of the GID complex on the expression of TET2 protein in neurons. In fact, the GID complex is strongly associated with many other diseases, such as cancer, which cause over 60% of tumor cases to show alterations in CTLH gene expression [
40]. These mechanisms are unclear. Therefore, further investigation is needed to examine whether the GID complex-TET2 axis plays a similar regulatory role in other cell types. In addition, we plan to investigate the regulatory role of the GID complex on TET2 in neuronal differentiation into diseases related to neuronal differentiation. We hope to find conditions that would use GID-TET2 as the potential target and provide insights into treatment strategies for these patients.
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