Presence and role of DNA G-quadruplex structures in the pathogenesis of XDP

XDP is a genetic movement disorder that human males can develop around 40 years of age. Genetic alterations located in the X chromosome are at the base of this disorder and the same haplotype is shared by all probands. An SVA retrotransposon antisense insertion within the intron 32 of TAF1 gene, which causes lowered TAF1 levels, is among the XDP characteristic mutations and is proposed to be crucial for the development of the disease. In fact, removal of the SVA brings TAF1 levels up to normal. Moreover, a truncated form of TAF1 comprising an intron which is retained, and truncated exactly at the site of SVA insertion, is present in XDP patients. When the SVA is removed, the truncated TAF1 levels drop down to healthy cell levels. It is not known how XDP SVA impairs TAF1 gene transcription. Our hypothesis was that G4s could fold within the SVA retrotransposon slowing down RNA polymerase. We started our investigation with in vitro experiments. We first identified putative G4 forming sequences with a G4 predicting tool, and we characterized the highest score sequences by circular dichroism, DMS footprinting and TaqPol STOP assay. Every tested sequence was proved to fold into highly stable, parallel topology G4s. We also studied the interaction of those sequences with different G4 ligands such as BRACO-19 and Quarfloxin. To assess if G4s can form in the double-stranded SVA sequence we set up a PCR STOP assay, in which we amplified the SVA from genome DNA extracted from XDP patient cells and healthy controls. In G4-inducing conditions, SVA amplification was totally impaired, suggesting that G4s were folded and able to block enzyme activity. To identify the SVA domains mainly responsible for this effect, we designed specific primers amplifying SVA domain regions and we proved that the VNTR and Hex domain are the domains that lead to amplification stop. This result was in complete accordance with the initial G4 prediction. To find out if those G4s were present within the SVA also in cells, we set up a BG4-ChIP-seq protocol on a difficult cell line, such as human fibroblasts, that displays 4 time less G4s than the model cell line K-562, that was one of the cell lines used in the development of the published protocol. We found a different G4 landscape between XDP affected cells and healthy control cells, that need to be further investigated. We did not reach a unique mapping alignment in the XDP SVA region, even when performing the more recent and efficient CUT&Tag protocol, that has very little background noise. However, we found coverage for every SVA family, indicating that SVAs (even those different from the SVA present in XDP) display folded G4s, a notion that has never been reported before. We finally proved that the hexameric domain of the XDP SVA displays folded G4s by BG4-ChIP-qPCR, designing specific Taqman primers that amplify the last part of the hexameric repeat. To assess the impact of the SVA G4s on TAF1 transcription, we treated XDP affected and healthy controls with increasing concentrations of G4 ligands for 24 hours. Strikingly, in SVA carrier cells there was an increase in the transcription of the first exons of TAF1 but not of the other exons. Our hypothesis is that when SVA G4s are stabilized by G4 ligands, the RNA Polymerase that is transcribing TAF1 stalls on the SVA until G4s are resolved. As a result, premature termination occurs, thus leading to increased truncated TAF1 with intron 32 retention, less full length transcripts that make the cell induce even more TAF1 transcription as a negative loop. For this reason, it is important to find a way to destabilize the SVA G4s. Our first attempt was using a new small molecule, PhPc, that was shown to destabilize G4s in vitro. Unfortunately, this compound was not able to destabilize the SVA G4s in cells. It did bind to them though, in such a way that led to an effect similar to the stabilizing G4 ligands. In conclusion we proved that G4s can fold within SVA in vitro

XDP è un malattia genetica che gli uomini possono sviluppare intorno ai 40 anni di età. Alterazioni genetiche localizzate nel cromosoma X sono alla base di questo disturbo e lo stesso aplotipo è condiviso da tutti i probandi. Un'inserzione antisenso del retrotrasposone SVA all'interno dell'introne 32 del gene TAF1, che causa livelli ridotti di TAF1, è tra le mutazioni caratteristiche XDP e si propone di essere cruciale per lo sviluppo della malattia. Infatti, la rimozione del SVA riporta i livelli di TAF1 alla normalità. Inoltre, nei pazienti XDP è presente una forma troncata di TAF1 comprendente un introne troncato esattamente nel sito di inserzione della SVA. Quando SVA viene rimosso, i livelli di TAF1 troncati scendono a livelli di cellule sane. Non è noto come XDP SVA comprometta la trascrizione del gene TAF1. La nostra ipotesi delle strutture G4 possano formarsi all'interno del retrotrasposone SVA rallentando l'RNA polimerasi