Transcriptional Alterations in X-Linked Dystonia-Parkinsonism Caused by the SVA Retrotransposon

Abstract

X-linked dystonia-parkinsonism (XDP) is a severe neurodegenerative disorder that manifests as adult-onset dystonia combined with parkinsonism. A SINE-VNTR-Alu (SVA) retrotransposon inserted in an intron of the TAF1 gene reduces its expression and alters splicing in XDP patient-derived cells. As a consequence, increased levels of the TAF1 intron retention transcript TAF1-32i can be found in XDP cells as compared to healthy controls. Here, we investigate the sequence of the deep intronic region included in this transcript and show that it is also present in cells from healthy individuals, albeit in lower amounts than in XDP cells, and that it undergoes degradation by nonsense-mediated mRNA decay. Furthermore, we investigate epigenetic marks (e.g., DNA methylation and histone modifications) present in this intronic region and the spanning sequence. Finally, we show that the SVA evinces regulatory potential, as demonstrated by its ability to repress the TAF1 promoter in vitro. Our results enable a better understanding of the disease mechanisms underlying XDP and transcriptional alterations caused by SVA retrotransposons.

Keywords: SVA; XDP; epigenetics; retrotransposon; splicing; transcription.

Figure 1

TAF1-32i transcript in cells of healthy controls and patients with XDP. (a) qPCR results of fibroblasts (n = 6 controls; n = 8 patients) and induced pluripotent stem cells (iPSC; n = 6 controls; n = 7 patients; n = 2 XDPΔSVA cell lines), normalized to GAPDH levels. XDPΔSVA refers to the patient-derived cell lines where the SVA was excised by CRISPR/Cas9. t test was performed on dCt values. (b) qPCR results (n = 10 female controls; n = 15 male controls; n = 50 patients) on blood-derived cDNA samples, relative to GAPDH. p values result from pair-wise Wilcoxon rank-sum test. To overcome possible batch effects, two independent samples were measured repeatedly in each batch, and all other samples were corrected according to the mean changes in measurement for these two samples. (c) Sanger sequencing showing the sequence of the intronic region included in the transcript and the scheme explaining the genomic locations, with genomic coordinates in the hg19/GRCh37 assembly. Blue squares represent canonical TAF1 exons, the violet square represents the intronic region within the transcript (not drawn to scale). (d) qPCR results showing increased levels of the TAF1-32i transcript in control (n = 2) and patient-derived cells (n = 3) after cycloheximide (CHX) treatment, as compared to the non-treated (nt) cells. (e) t test on dCt values, comparing non-treated and CHX-treated samples. Ctrl, control.

Figure 2

H3K36me3 levels at the intronic region included in the transcript. (a) USCS browser screenshot, showing chromatin state segmentation and H3K36me3 histone mark in different cell lines. Note that the strongest signal is present in NT2-D1 cells. The region marked in violet is included in the transcript, and its genomic coordinates are shown above. (b) ChIP-qPCR results from control (n = 2) and XDP (n = 3) iPSC lines. Results are calculated relative to the corresponding input sample and shown as % input. (c) ChIP-seq results showing the TAF1 locus in control (blue) and XDP-derived (red) iPSCs. The region highlighted in blue depicts TAF1 intron 32; the narrower region marked as IR indicates the intronic region retained in the TAF1-32i transcript, and the orange line marks the position in which the SVA is inserted.

Figure 3

DNA methylation in intron 32 of TAF1, proximal to the SVA insertion. (a) Long-read nanopore sequencing on DNA from different tissues and cell lines from a healthy control and a patient with XDP (i.e., blood, iPSCs, cerebellum). Arrows indicate the two CpGs selected for analysis by pyrosequencing. (b) Pyrosequencing results showing methylation levels at the two selected CpGs: chrX:70,659,134 and chrX:70,659,225 (hg19), in blood (n = 6 controls, n = 6 patients), cerebellum (n = 3 controls; n = 2 patients), frontal cortex (n = 3 controls; n = 2 patients), and iPSCs (n = 3 controls; n = 4 patients; n = 2 XDPΔSVA cell lines). Unpaired t test was performed on blood samples after testing for normality with Kolmogorov–Smirnov test. n.s., not significant.

Figure 4

Regulatory potential of the SVA in vitro. (a) UCSC browser screenshot of the TAF1 promoter region, showing DNase sensitivity, histone modifications and chromatin state segmentation in various cell types. In light blue (the lower part of the figure) are shown promoter fragments that were investigated in luciferase reporter assays, along with their genomic coordinates (hg19). Note that the most active TAF1 promoter region (fragment ≈ 400 bp) overlaps with the DNase-sensitive region, characteristic of open chromatin. (b) Relative luciferase activity (Firefly counts/Renilla TK counts) of different TAF1 promoter regions define the most active region (Fragment ≈ 400 bp). (c) Relative luciferase activity of the XDP-specific SVA-inserted sense or antisense (INV-inverted), as compared to a size-matched control. Maximum activity (100%) was exerted by the vector containing only the most active promoter region (TAF1 pro 400 bp), and p values are calculated relative to this sample using Kruskal–Wallis and Dunn’s multiple comparison tests.