Subhrangshu Guhathakurta and Min Kyung Song contributed equally to this study as co-first authors.
Deregulation of
The dopaminergic neuronal cell line, ReNcell VM, was used. Reverse transcription-polymerase chain reaction (RT-PCR), real time-quantitative PCR, Western blot, dot-blot, and Chromatin immunoprecipitation were conducted. The substantia nigra tissues of postmortem PD samples were used to confirm the level of TET1 expression.
In the human dopaminergic cell line, ReNcell VM, overexpression of the DNA-binding domain of TET1 (TET1-CXXC) led to significant repression of α-SYN. On the contrary, knocking down of TET1 led to significantly higher expression of α-SYN. However, overexpression of the DNA-hydroxymethylating catalytic domain of TET1 failed to change the expression of α-SYN. Altogether, we showed that TET1 is a repressor for
We identified that TET1 acts as a repressor for
- Ten-eleven translocation methylcytosine dioxygenase 1 (TET1) acts as a repressor for α-synuclein gene (
- Postmortem human Parkinson disease samples express low levels of TET1 compared to the control samples.
Parkinson disease (PD) is a progressive neurological disorder characterized by the loss of dopaminergic neurons in the
The ten-eleven translocation (TET) protein family includes 3 members (TET1-3), and all 3 TET proteins can subsequently oxidize the methyl group of 5-methylcytosine (5mC) to yield 3 different forms of 5-hydroxymethylcytosine (5hmC), 5-formylcytosine, and 5-carboxylcytosine [
This research investigates whether TET1 binds to
We used postmortem brain samples from the SN region (control [n=8], PD [n=17]). All human postmortem brain samples were procured from NIH Neurobiobank and were used in accordance with the principles of the Declaration of Helsinki of the World Medical Association. All the samples were ethnicity, age, and sex-matched. The demographic data of all participating samples are presented in
ReNcell VM cell line derived from human fetal ventral mesencephalon was obtained from EMD Millipore (SCC008). The cells were cultured following the previously described [
HEK293T cells were cultured in DMEM/high glucose medium (Thermo Fisher Scientific, SH30243FS) supplemented with 10% fetal bovine serum (Atlanta Biological Pvt. Ltd., Kolkata, India; S10350H). All cells were maintained in a humidified atmosphere with 5% CO2 at 37°C.
The CXXC domain (amino acid 528-674) of TET1 was amplified from cDNA of undifferentiated ReNcell VM. The 441 bp amplicon containing EcoRI/XhoI restriction enzyme sites was cloned into pAAV-IRES-hrGFP vector harboring 3X flag tag (Clontech Laboratories Inc., Mountain View, CA, USA). To generate the ReNcell VM stably overexpressing the TET1-CXXC domain, the cDNA encoding TET1-CXXC-flag from pAAV-IRES-hrGFP vector was subcloned into EcoRI/ApaI sites of pLVX-DsRed-Monomer-N1 vector (Clontech Laboratories Inc.). The catalytic domain of TET1 (amino acids 1418-2136) and its inactive catalytic form cloned in pAAV-EF1a-HA-hTET1CD-WPRE-PolyA vector were a kind gift from Hongjun Song (Addgene plasmids # 39454 and 39455). According to the manufacturer’s instructions, ReNcell VM or HEK293T cells were transfected with plasmid DNA using X-fect transfection reagent (Clontech Laboratories Inc., PT5003-2).
ReNcell VM stably expressing the TET1-CXXC domain were generated by lentiviral transduction. The pLVX-TET1-CXXC-DsRed-Monomer-N1 vector was cotransfected with lentiviral packaging plasmids pLP1, pLP2, and pLP/VSVG (Life Technologies, K4944-00) at a 1:1:1:1 ratio using X-fect transfection reagent in HEK293T cells. 48 hours after transfection, the lentiviral particles containing the medium were collected and centrifuged briefly at 500 g for 10 minutes to remove the remaining cell debris. Lentiviral particles were treated to ReNcell VM grown on 24 well plates. Following 48-hour treatment with lentiviral particles, the cells were positively selected under antibiotic puromycin (2 mg/mL) (Acros Organics, Veneto, Italy; 227420100).
Knocking down of the human TET1 gene was achieved by either lentiviral particles containing short hairpin RNA (shRNA) constructs or by small interfering RNA (siRNA), as mentioned in the text. The shRNA plasmid cocktails were purchased from Open Biosystems (RHS4531; now acquired by Horizon Discovery, Cambridge, UK). The shRNAs were embedded in miR30a backbone in pGIPZ vector; the sequences are shown in
Chromatin immunoprecipitation (ChIP) was performed following protocol for the EZ ChIP kit (Millipore, 17-371) with the following modifications as described in Guhathakurta et al. [
Total RNA was extracted from the ReNcell VM using Trizol reagent (Invitrogen, Waltham, MA, USA). cDNA was synthesized as described in the manufacturer’s protocol (GenDEPOT, Katy, TX, USA; R5600-050). The sequences of specific primers are shown in
Proteins (15–75 μg/sample) were loaded on 5 or 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. The membranes were blocked by 5% nonfat milk in Tris-buffered saline containing 0.05 % Tween-20 (TBS-T) for 1 hour at room temperature (RT) and incubated with respective primary antibodies at 4°C overnight. And then, the membrane was incubated with respective secondary antibodies for 1 hour at RT. The protein bands were visualized by Enhanced Chemiluminescent detection Reagents (Thermo Fisher Scientific, 45-010-090). A list of antibodies used in western blot is presented in
DNA Dot-blot for detecting 5hmC was performed following the protocol outlined by Ito et al. [
To understand the methylation status of the
Data are presented as mean ±standard error of the mean. Statistical analyses were conducted using GraphPad Prism v.9.3.1 (GraphPad Software Inc., San Diego, CA, USA). An unpaired Student t-test for cell line data and a Mann-Whitney t-test for human subjects were performed to compare the mean between the 2 groups. The data for methylation was prepared using Quantification Tool for Methylation Analysis (QUMA) software (
We first investigated the epigenetic structure of
It has been reported that TET1 binds to the unmethylated CGI-containing monovalent promoters (marked by H3K4me3 only) or bivalent promoters (marked by both H3K4me3 and H3K27me3) [
Flag-tagged DNA binding TET1-CXXC domain was overexpressed in HEK293T cells to determine its binding to this region. We found that the TET1-CXXC domain significantly binds to the
We showed that TET1 catalyzes 5mC to 5hmC conversion using its C-terminal catalytic domain in
Next, we examined the levels of TET1 in postmortem human SN brain samples. Interestingly, we found that the TET1 levels of PD subjects were significantly less than the control subjects (Mann-Whitney t-test, P<0.05) (
This study showed a novel mechanism for regulating α-SYN expression by TET1 in neuronal cells and postmortem brain samples. Our study is the first to describe the importance of TET1 in α-SYN regulation in PD pathogenesis. Epigenetic regulation of
TET1 is known to catalyze DNA hydroxymethylation and has been shown to act as both activator and repressor for gene expression [
Interestingly, we found that the CXXC-DNA binding domain of TET1 binds to this region and is enough to repress the gene expression without contribution from the catalytic domain, which is responsible for DNA hydroxymethylation. This result can be explained by the fact that CGI of the regulatory region of
Although the molecular mechanism of how the TET1-CXXC domain alone can inhibit gene expression is unknown, the CXXC domain might recruit other corepressors and repress the transcription altogether. We have also seen that overall TET1 levels in PD patients are significantly lower than in the controls. Our group has recently shown that occupancy of H3K4me3, a transcription initiation/favoring histone posttranslational modification, at the SNCA-promoter/intron1 region is significantly higher in PD patients, in turn, may make it less favorable for TET1 binding in the PD patients selectively [
Deregulated expression and aggregation of α-SYN are a long-standing observation in PD. However, the mechanism remains elusive. This report provided molecular and epigenetic insight into this gene’s deregulation in disease conditions. Although TET1 is known for its hydroxymethylating activity, we observed that the DNA binding domain of TET1 could repress SNCA expression by physically binding the intron1 region of the gene. This study links to how the epigenetic niche of
Supplementary Tables 1-4 and Figures 1-2 can be found via
The demographic information of postmortem brain samples
Sequences of short hairpin RNAs
List of primary and secondary antibodies
Overview of polymerase chain reaction primer sequences
Standardization of optimum shearing condition for chromatin immunoprecipitation (ChIP). Shearing of crosslinked chromatin for the ChIP experiments were done using rat brain sample. The optimal condition was reached after shearing it for 20 seconds pulse for 5 times with 30 seconds interval between each one. The reverse crosslinked sheared DNA fragments were ranged between 700 bp to 200 bp (lane 2). DNA ladder (DNA ladder in lane 1).
Results of full-length TET1 transfection. (A) Representative images 48 hours after transfection using X-fect, Lipofectamine 2000, and Fugene HD. (B) Representative FACS images 48 hours after transfection using magnetofection and nucleofection. The size of full-length TET1 is 11,983 bp. For transfection using several methods/reagents, including X-fect, Lipofectamine 2000, Fugene HD, magnetofection, and nucleofection, the transfection efficiency was as low as 0.5%–1%. FACS, fluorescence-activated cell sorter; GFP, green fluorescent protein; TET1, ten-eleven translocation methylcytosine dioxygenase 1.
This work was supported by the National Institute of Health (grant number 5R21NS088923-02); and Michael J Fox Foundation (Target Advancement award 2015) awarded to YSK.
All human postmortem brain samples were procured from NIH Neurobiobank and were used in accordance with the principles of the Declaration of Helsinki of the World Medical Association.
No potential conflict of interest relevant to this article was reported.
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·Funding acquisition:
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·Writing - original draft:
·Writing - review & editing:
Authors gratefully acknowledge (a) Human Brain and Spinal Fluid Resource Centre, UCLA, under NIH Neurobio bank; (b) Brain Endowment Bank of University of Miami, Miller School of Medicine; (c) Parkinson’s UK Brain Bank, and (d) Harvard Brain Tissue Resource Centre for providing all the human postmortem brain samples.
The human
TET1 is a repressor for α-synuclein (α-SYN). (A) Schematic representation of TET1, consisting of 2 principal domains, N-terminal CXXC and C-terminal catalytic domain, respectively. The CXXC domain contains 8 alternative cysteine residues from 528–674 amino acids (highlighted in red). The catalytic domain includes the cysteine-rich and double-stranded β-helix domains (DSBH). (B) Representative image for binding of TET1 to SNCA-intron1 of 2 independent human postmortem
TET1-catalytic domain does not play any role in regulating α-synuclein (α-SYN) in neuronal cells. (A) Representative Western blot images for overexpression of both the TET1-catalytic domain (cat. domain) and its inactive form (inactive cat. domain) in ReNcell VM. (B) Dot blot analysis of 5-hydroxymethylcytosine in the transfected ReNcell VM with either TET1-catalytic domain or its inactive domain. TET1, ten-eleven translocation methylcytosine dioxygenase 1.
TET1 express reduces in postmortem human