Deregulation of α-synuclein in Parkinson's disease: Insight from epigenetic structure and transcriptional regulation of SNCA

Abstract

Understanding regulation of α-synuclein has long been a central focus for Parkinson's disease (PD) researchers. Accumulation of this protein in the Lewy body or neurites, mutations in the coding region of the gene and strong association of α-synuclein encoding gene multiplication (duplication/triplication) with familial form of PD have indicated the importance of this molecule in pathogenesis of the disease. Several years of research identified many potential faulty pathways associated with accumulation of α-synuclein inside dopaminergic neurons and its transmission to neighboring ones. Concurrently, an appreciable body of research is growing to understand the epigenetic and genetic deregulation of α-synuclein that might contribute to the disease pathology. Completion of the ENCODE (Encyclopedia of DNA Elements) project and recent advancement made in the epigenetic and trans factor mediated regulation of each gene, has tremendously accelerated the need to carefully understand the epigenetic structure of the gene (SNCA) encoding α-synuclein protein in order to decipher the regulation and contribution of α-synuclein to the pathogenesis of PD. We have also analyzed the detailed epigenetic structure of this gene with knowledge from ENCODE database, which may open new avenues in α-synuclein research. Interestingly, we have found that the gene contains several transcriptionally activate histone modifications and associated potential transcription factor binding sites in the non-coding areas that strongly suggest alternative regulatory pathways. Altogether this review will provide interesting insight of α-synuclein gene regulation from epigenetic, genetic and post-transcriptional perspectives and their potential implication in the PD pathogenesis.

Keywords: DNA methylation; Epigenetics; Gene regulation; Histone post translational modification; Parkinson's disease; α-synuclein.

Fig. 1. CpG distribution in the SNCA 5′ regulatory region

Human SNCA gene has 6 exons with two alternative non-coding exon 1s. The translation start codon is located in exon 2. The vertical bars represent each exon. The sequence from +121 to −2,648 with respect to ATG (+1) (marked in green), encompassing both non-coding exons, intron1 and exon 2, shows very high density of CG bases as highlighted in orange. The presence of the CpG island was predicted using the CpGPLOT program (http://www.ebi.ac.uk/Tools/seqstats/emboss_cpgplot/) with default parameter; the predicted island of 591 base pairs is highlighted in grey. The black dashed marked box represents the region in intron1 whose CpG methylation status has been analyzed by most of the studies (A). Comparative CpG density between human, mouse and rat α-SYN gene (−2,648 to +1) was done using the same program as mentioned above. Analysis revealed a significant difference in CpG base distribution between human and other species when the same region of the gene was tested. Only human showed the presence of a CpG island >200 bp, and the ratio between the observed CpG distribution over expected region ranged mostly in between 1.0 to 1.5. The ratio went down to 0.8 when the other two species were analyzed for CpG distribution for the same region (B). The cut-off for the ratio was set at 0.6.

Fig. 2. Differential epigenetic landscape of SNCA promoter in different cell lines

SNCA promoter and upstream region was analyzed for methylation status of its CpG rich area and occupancy of different transcription related histone marks. The entire analyses is based on the data from ENCODE using UCSC genome browser. ENCODE data showed the presence of a CpG island of around 861 bp with the starting base slightly upstream when compared to that of our analysis using CpGPLOT program as shown in Figure 1 (−2052 to −1191 bp in case of ENCODE analysis Vs −1777 to −1187 bp using CpGPLOT program; all positions are designated considering ATG as +1) (A). Twenty-two CpG sites distributed over the SNCA promoter/intron1 area were analyzed using 450K methylation bead array in the ENCODE database. The relative position of those 22 CpG sites within exon 1a to exon 2 is shown by downward vertical bars with round head. The methylation statuses of all 22 CpGs for 6 different cell lines are summarized in the figure: The blue ball represents unmethylated cytosines, pink represents methylated cytosines, and purple shows partially methylated cytosines. The ENCODE database showed that GM12878, H1ESC, HUVEC and K562 remains majorly unmethylated for respective CpGs, HeLa-S3 exhibits a heavily methylated pattern, and HepG2 cells demonstrates partial methylation across the 22 CpG sites. Different histone PTMs associated with transcriptional activity were also assessed from ENCODE database for similar areas of SNCA across different cell lines, as demonstrated by the matrix representation (B). H3K4me1/2/3, H3K9ac, H3K27ac represent active state of chromatin where as H3K27me3 and absence of any histone PTMs (designated as “None”) are associated with inactive state of chromatin. Each cell type shows possession of one or more different types of histone PTMs in similar or slightly different areas in the promoter, and their presence is designated by +. A549, human adenocarcinomic alveolar basal epithelial cell line; DND-41, human T cell leukemic cell line; GM12878, human lymphoblastoid cell line; H1ESC, human embryonic cell line; HMEC, human mammary epithelial cell line; HSMC, human skeletal muscle and myoblast; HSMMtube, skeletal muscle myotubes differentiated from HSMM cell line; HUVEC, human umbilical vein endothelial cells; HeLa-S3, clonal derivative from HeLa cells; HepG2, human liver hepatocellular cells; IMR90, human fetal lung cell line; K562, chronic myelogenous leukemia cell line; NH-A, normal human astrocyte cells; NHDF-AD, normal human dermal fibroblast cells; NHEK, normal human epidermal keratinocytes; NHLF, normal human fibroblast cells.

Figure 3. SNCA gene regulation depends on its non-coding region

SNCA gene is around 114 kb long which encompasses 6 exons and one very long intron4/5 of around 93 kb. A polymorphic repeat-based enhancer element is present at around 10 kb upstream of ATG known as NACP-Rep1. The intron 4/5 area harbors two distinct regions where transcriptionally active marks H3K27ac (red peaks) and H3K4me1 (green peaks) signals are present. Several transcription factors along with polymerase IIA (Pol IIA) bind to those areas cell type specifically, some of which are shown in the figure. The entire gene harbors thousands of SNPs; of which some significantly demonstrate a repeated association with PD are shown in the figure with their respective IDs and relative position. Two SNPs which were significantly associated with the disease (rs11931074, rs356219) are located around 5.7 and 7.6 kb downstream of the stop codon respectively. The 3′-UTR region of SNCA is approximately 2,529 nucleotides and harbors several motifs and microRNA (miR) binding sites. Features of the long 3′-UTR of SNCA and its regulatory elements are shown: regions in red depict alternative poly (A) signals; arrows labeled a through e are indicative of micro-RNA binding sites (miR-7, miR-34b, miR-34b, miR-153 and miR-34C respectively). Asterisks illustrate miR-7 and miR-153 binding sites that result in significant repression of SNCA. The double-sided arrow represents the LINE region of the SNCA-3′-UTR and dark-blue areas represent the conserved regions of 3′-UTR across the species. Locations of the two SNPs in the UTR that are implicated in PD are shown by the triangles labeled with their respective IDs. The 3′-UTR motifs and conserved sequences are depicted according to Sotiriou et al. (2009). The binding sites of all the microRNAs shown here are adopted from references as discussed in the text (Doxakis, 2010; Kim et al., 2013; Choi et al., 2014; Kabaria et al., 2015). The locations of the H3K27ac/H3K4me1 peaks are adopted from ENCODE. The peak heights, distribution and area under the peaks are not in exact scale.