Regulation of Αlpha-Synuclein Gene (SNCA) by Epigenetic Modifier TET1 in Parkinson Disease

Abstract

Purpose: Deregulation of SNCA encoding α-synuclein (α-SYN) has been associated with both the familial and sporadic forms of Parkinson disease (PD). Epigenetic regulation plays a crucial role in PD. The intron1 of SNCA harbors a large unmethylated CpG island. Ten-eleven translocation methylcytosine dioxygenase 1 (TET1), a CpG island binding protein, can repress gene expression by occupying hypomethylated CpG-rich promoters, and therefore SNCA could be a target for TET1. We investigated whether TET1 binds to SNCA-intron1 and regulates gene expression.

Methods: 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.

Results: 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 SNCA, and a CXXC domain of TET1 is the primary mediator for this repressive action independent of its hydroxymethylation activity. TET1 levels in PD patients are significantly lower than that in the controls.

Conclusion: We identified that TET1 acts as a repressor for SNCA by binding the intron1 regions of the gene. As a high level of α-SYN is strongly implicated in the pathogenesis of PD, discovering a repressor for the gene encoding α-SYN is highly important for developing novel therapeutic strategies for the disease.

Keywords: Parkinson disease; Repressor; TET1; Αlpha-synuclein.

Fig. 1.

The human SNCA gene showed both H3K4me3 and H3K27me3. (A) SNCA gene’s histone architecture in the NIH Roadmap epigenomics project dataset (http://www.epigenomebrowser.org) in the adult human brain’s substantia nigra tissues. It exhibited the presence of both H3K4me3 and H3K27me3 at the promoter/intron1 region of the gene. In the red box, the promoter region is magnified to show both marks’ presence. (B) Chromatin immunoprecipitation (ChIP) analysis of H3K4me3 and H3K27me3 enriched regions of SNCA-intron1 from undifferentiated/differentiated ReNcell VM. (C) Hypomethylated SNCA-intron1 in the neuronal cells. The 23 CpG dinucleotide containing SNCA-intron1 region was polymerase chain reaction amplified and sequenced. The horizontal axis represents each CpG residue, marked from 1 to 23. The open circles denote unmethylated cytosines, and the closed ones represent methylated cytosines. The vertical axis demonstrates the number of clones analyzed, 10 for undifferentiated ReN cells and 6 for differentiated cells. Position number 19 was not evaluated as this position is polymorphic and the cell lines have G allele instead of the C allele at that position. (D) A diagrammatic representation of the upstream regulatory region of SNCA. It showed the sequence of intron1 between noncoding (exon1b) and the first coding exon (exon2). The highlighted region in red is CpGs. The primer pair used to amplify the region of SNCA-intron1 in ChIP is underlined.

Fig. 2.

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 substantia nigra (SN) tissues using the chromatin immunoprecipitation (ChIP) assay. (C) ChIP results from HEK293T cells overexpressing a Flag-tagged DNA binding TET1-CXXC domain. (D) Western blot results from ReNcell VM overexpressing the TET1-CXXC domain. (E) Reverse transcription- polymerase chain reaction (RT-PCR), RT-quantitative PCR (qPCR), and Western blot (WB) results in TET1 knockdown ReNcell VM with small interfering RNA (siTET1) and short hairpin RNA targeting TET1 (shTET1). The values are shown as mean± standard error of the mean. TET1, ten-eleven translocation methylcytosine dioxygenase 1. The significance level was measured at ^*P<0.05.

Fig. 3.

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.

Fig. 4.

TET1 express reduces in postmortem human substantia nigra samples. (A) Western blot band of TET1 expression between postmortem control (n=8) and Parkinson disease (PD) (n=17) samples. (B) The quantification of the optical density of the TET1 band. The values are shown as mean±standard error of the mean. TET1, ten-eleven translocation methylcytosine dioxygenase 1. The significance level was measured at ^*P<0.05.