G-quadruplexes in an SVA retrotransposon cause aberrant TAF1 gene expression in X-linked dystonia parkinsonism

Abstract

G-quadruplexes (G4s) are non-canonical nucleic acid structures that form in guanine (G)-rich genomic regions. X-linked dystonia parkinsonism (XDP) is an inherited neurodegenerative disease in which a SINE-VNTR-Alu (SVA) retrotransposon, characterised by amplification of a G-rich repeat, is inserted into the coding sequence of TAF1, a key partner of RNA polymerase II. XDP SVA alters TAF1 expression, but the cause of this outcome in XDP remains unknown. To assess whether G4s form in XDP SVA and affect TAF1 expression, we first characterised bioinformatically predicted XDP SVA G4s in vitro. We next showed that highly stable G4s can form and stop polymerase amplification at the SVA region from patient-derived fibroblasts and neural progenitor cells. Using chromatin immunoprecipitazion (ChIP) with an anti-G4 antibody coupled to sequencing or quantitative PCR, we showed that XDP SVA G4s are folded even when embedded in a chromatin context in patient-derived cells. Using the G4 ligands BRACO-19 and quarfloxin and total RNA-sequencing analysis, we showed that stabilisation of the XDP SVA G4s reduces TAF1 transcripts downstream and around the SVA, and increases upstream transcripts, while destabilisation using the G4 unfolder PhpC increases TAF1 transcripts. Our data indicate that G4 formation in the XDP SVA is a major cause of aberrant TAF1 expression, opening the way for the development of strategies to unfold G4s and potentially target the disease.

Graphical Abstract

Figure 1.

XDP SVA G4 prediction and characterisation in vitro. (A) Schematic representation of the XDP SVA retrotransposon antisense insertion within the TAF1 gene. Each bar corresponds to a pG4 predicted by QGRS (43). Putative G4 sequences are clustered in the VNTR (green bars) and HEX (red bars) regions. (B) CD melting analysis of the Hex sequence. Increasing the temperature leads to loss of the G4 parallel topology, as indicated by the arrow. (C) DMS footprinting analysis of the folded (F) and unfolded (U) Hex sequence was performed to identify the putative Gs involved in G-tetrad formation. M stands for sequence marker for adenines and Gs obtained with the Maxam and Gilbert protocol. The black (unfolded) and red (folded) densitograms indicate the degree of protection of each G. (D) The Hex sequence according to the DMS footprinting output. Bases protected from DMS alkylation/cleavage are shown in red. The font size is proportional to the degree of protection. (E) CD analysis of the Hex G4, alone (red) or with G4 ligands, B19 (blue) and Q (green). (Left panel) CD spectra were recorded at 25°C: they represent a parallel G4 topology with a positive peak at 265 nm. Both compounds show an increase in molar ellipticity, indicating stabilisation of the G4 structure. (Right panel) Melting curves showing stabilisation of the G4 structure mediated by the two compounds. (F) Taq pol stop assay (left panel). The Hex sequence and negative control templates were treated in the absence or presence of G4-stabilising conditions (KCl and G4 ligand B19 or Q). Under the latter conditions, only the full-length product of the Hex template was reduced. At the same time, stop bands were more intense in the presence of B19 or Q, indicating the ability of the stabilized G4 structure to interfere with enzyme activity. Quantification of bands in the Taq pol stop assay (right panel). Full-length and stop bands were quantified from both no G4 and Hex templates in different conditions of KCl and G4 ligands. Error bars represent standard deviation of three independent experiments. Statistical analyses were performed using ANOVA test with multiple comparisons setting the condition without KCl as reference (ns = P ≥ 0.05, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P ≤ 0.0001).

Figure 2.

G4 stabilisation reduces polymerase activity at the VNTR and HEX domains of the XDP SVA. (A) Schematic illustration of the PCR stop assay. Under G4-inducing conditions, such as increasing KCl and G4 ligand concentrations, G4 folding of the G-rich sequences in the XDP SVA retrotransposon would block PCR amplification. The same conditions would not affect amplification of a non-G-rich template. (B) Representative SVA PCR stop assay agarose gel. Genomic DNA (gDNA) was extracted from XDP-affected, NMC and healthy wild-type (WT) cells, folded at different KCl concentrations, and used as PCR template. (C) Quantification of the gel bands shown in panel (B). Intensity of the bands from the PCR reactions was quantified with ImageJ and normalised on the sample without KCl. G4 stabilisation by increasing KCl concentration correlates with a decrease in PCR product in gDNA containing the SVA insertion, but not in controls. Error bars represent standard deviation (n = 2). (D) Representative agarose gel of the nested PCR stop assay for each SVA domain at increasing concentrations of KCl. Only the two putative domains where pG4s were predicted, i.e. VNTR and HEX, show less amplification product at 100 mM KCl. Amplification of the Alu and SINE domains is not reduced. (E) Quantification of the gel bands shown in panel (D). Only HEX and VNTR amplification is impaired at increasing KCl concentrations, confirming the presence of G4 structure within the two domains. Error bars represent standard deviation (n = 2).

Figure 3.

G4s form in the XDP SVA in XDP hFib and NPCs. (A) Plot profiles of G4 ChIP-seq from hFib. Representative G4-ChIP-seq plot profiles showing G4 enrichment in SVA retrotransposons compared with the input signal (left panel). G4-ChIP-seq plot profiles at gene TSS (right panel) corroborate literature data in other cell lines (50). Input profile is shown in black, fNMC in orange, fXDP in pink and fWT in grey. All three G4-ChIP profiles are enriched compared with the input. (B) G4-ChIP-seq coverage in the SVA F retrotransposon family. The bar plot represents the read coverage obtained in the input, fNMC, fXDP and fWT profiles. The increase in G4-ChIP-seq coverage confirms that SVA F retrotransposons, such as the XDP SVA, contain G4s that are folded in cells. (C) G4-ChIP-qPCR at the XDP SVA HEX region in hFib and NPCs. Results are reported as enrichment over the G4-negative region ESR1. The XDP SVA HEX region is shown in red and the G4-negative region TMCC1 in black. Only SVA carrier cells show enrichment of the HEX, which is higher compared with the negative ctrl TMCC1, confirming the presence of G4s in XDP cells within the XDP SVA HEX. Significance levels were calculated using ANOVA test (n = 3) considering ctrl hFib or NPCs as reference for HEX and TMCC1 genes (ns = P ≥ 0.05, * P < 0.05, ** P < 0.01).

Figure 4.

TAF1 transcript levels in XDP cells. (A) TAF1 transcript levels in NPCs untreated (NT) or treated with G4 ligands B19 and Q from RNA-seq analysis. Statistical analysis was performed with the non-parametric simple t-test (n = 3). Transcript levels of different exons of TAF1 upon treatment with G4 ligands (B19 5 μM or Q 3 μM) in NPCs (B) and hFib (C) by RT-qPCR analysis. The transcript levels of each exon were normalized to the housekeeping gene β-actin and to the untreated sample (n = 3). G4 ligand treatment decreased TAF1 transcription of the exons flanking and downstream of the SVA and increased transcription of the most upstream exons in XDP cells compared with WT cells.

Figure 5.

TAF1intron 32 retention levels in XDP and WT NPCs. (A) TAF1 RNA-seq profile of G4 ligand treated XDP cell line with B19 (left panel) or Q (right panel). Untreated (NT) nXDP NPC cell line (pink). RNA-seq profile presented is the mean profile of n = 3 biological experiments. (B) TAF1 RNA-seq profile of G4 ligand treated nWT cell line with B19 (left panel) or Q (right panel). Non-treated (NT) nWT NPC cell line (grey). RNA-seq profile presented is the mean profile of n = 3 biological experiments. (C) RNA-seq TAF1 intron 32 coverage in NPCs upon G4 ligand treatment. Intron 32 coverage is present only in nXDP cells. Statistical analysis was performed with the non-parametric simple t-test (n = 3).

Figure 6.

PhpC destabilises G4 Hex. (A) Molecular structure of PhpC. (B) CD analysis of Hex G4 alone (red) or with the G4 destabiliser PhpC (purple). CD spectra were recorded at 25°C: they show a parallel G4 topology with a positive peak at 265 nm. PhpC spectra show a decrease in molar ellipticity, indicating destabilisation of the G4 structure. (C) Representative agarose gel of a nested PCR stop assay for the HEX domain in 100 mM KCl (left panel). Amplification increases at lower compound concentrations. Quantification of the gel bands of the nested PCR stop assay at different KCl concentrations (right panel). Only at 100 mM KCl PhpC shows an increase in amplification at low concentration. No such effect is observed at 0–50 mM KCl. Error bars represent the standard deviation (n = 2). (D) RT-qPCR of exons flanking and following SVA insertion in XDP and WT NPCs treated for 24 h with 0.01 μM PhpC (n = 3). A small but significant increase in transcription of these exons is observed, but only in nXDP cell lines. nWT cells were unaffected. Statistical analysis was performed using the non-parametric sample t-test (ns = P ≥ 0.05, *P < 0.05, **P < 0.01). Error bars represent the standard deviation. Results in XDP cells are shown in pink and control cells in grey. Treated samples are depicted in purple and non-treated samples in light blue.