Presence, Location and Conservation of Putative G-Quadruplex Forming Sequences in Arboviruses Infecting Humans

Guanine quadruplexes (G4s) are non-canonical nucleic acid structures formed by guanine (G)-rich tracts that assemble into a core of stacked planar tetrads. G4s are found in the human genome and in the genomes of human pathogens, where they are involved in the regulation of gene expression and genome replication. G4s have been proposed as novel pharmacological targets in humans and their exploitation for antiviral therapy is an emerging research topic. Here, we report on the presence, conservation and localization of putative G4-forming sequences (PQSs) in human arboviruses. The prediction of PQSs was performed on more than twelve thousand viral genomes, belonging to forty different arboviruses that infect humans, and revealed that the abundance of PQSs in arboviruses is not related to the genomic GC content, but depends on the type of nucleic acid that constitutes the viral genome. Positive-strand ssRNA arboviruses, especially Flaviviruses, are significantly enriched in highly conserved PQSs, located in coding sequences (CDSs) or untranslated regions (UTRs). In contrast, negative-strand ssRNA and dsRNA arboviruses contain few conserved PQSs. Our analyses also revealed the presence of bulged PQSs, accounting for 17-26% of the total predicted PQSs. The data presented highlight the presence of highly conserved PQS in human arboviruses and present non-canonical nucleic acid-structures as promising therapeutic targets in arbovirus infections

Genome-wide mapping of i-motifs reveals their association with transcription regulation in live human cells

Lay Summary Among the secondary structures alternative to the DNA double helix, i-Motifs (iMs) and G-quadruplexes (G4s) are four-stranded non-canonical nucleic acid structures that form in cytosine- and guanine-rich regions, respectively. Because iMs fold in vitro under acidic conditions, they were long thought to form only in vitro. We now show that iMs, like G4s, form in live human cells mainly at gene promoters in open chromatin. iMs that are unstable in vitro still form in cells. iMs and G4s are cell-type specific and associated with increased transcription; however, transcript levels are remarkably different: low for iMs and high for G4s, indicating their distinct activity as regulators of the cell transcriptome. The iM/G4 interplay may represent a novel therapeutic target in disease.i-Motifs (iMs) are four-stranded DNA structures that form at cytosine (C)-rich sequences in acidic conditions in vitro. Their formation in cells is still under debate. We performed CUT & Tag sequencing using the anti-iM antibody iMab and showed that iMs form within the human genome in live cells. We mapped iMs in two human cell lines and recovered C-rich sequences that were confirmed to fold into iMs in vitro. We found that iMs in cells are mainly present at actively transcribing gene promoters, in open chromatin regions, they overlap with R-loops, and their abundance and distribution are specific to each cell type. iMs with both long and short C-tracts were recovered, further extending the relevance of iMs. By simultaneously mapping G-quadruplexes (G4s), which form at guanine-rich regions, and comparing the results with iMs, we proved that the two structures can form in independent regions; however, when both iMs and G4s are present in the same genomic tract, their formation is enhanced. iMs and G4s were mainly found at genes with low and high transcription rates, respectively. Our findings support the in vivo formation of iM structures and provide new insights into their interplay with G4s as new regulatory elements in the human genome

Caratterizzazione di domini G-rich vicinali nel promotore dell'oncogene c-KIT e loro interazione con ligandi selezionati

La formazione di strutture non canoniche del DNA, in particolare di G-quadruplex nelle regioni promotoriali di molti oncogeni, come c-KIT, e la loro stabilizzazione attraverso piccle molecole sta emergendo come una nuova strategia anticancro. In questa tesi si analizzano due potenziali ligandi rispetto a due regioni G-rich contenute nel promotore del gene c-KIT

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

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 polimerasiXDP 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 vitr

Profiling chicory sesquiterpene lactones by high resolution mass spectrometry

Sesquiterpene lactones (SLs) are the main determinants of radicchio bitterness and the control of their concentrations is a key point for the market value of this product. An innovative analytical approach based on two complementary mass spectrometers, Orbitrap-HRMS and MS/MS, was used for quantitative analysis of SLs in aerial part of four different varieties of chicory. Data highlighted the presence of eight SLs: 11\u3b2,13-dihydrolactucin, lactucin, 8-deoxy-lactucin, dihydro-8-deoxylactucin, dihydrolactucopicrin, lactucopicrin, lactuside C (jaquinellin glucoside) and dihydro-lactucopicrin oxalate. Significant varietal differences were found. The highest amount of SLs was found in the radicchio \u201cTreviso Precoce\u201d variety (189.71 \u3bcg/g), the lowest amount in \u201cTreviso Tardivo\u201d variety (45.78 \u3bcg/g). Lactucopicrin was the most abundant compound with concentration ranged between 99.36 in \u201cTreviso Precoce\u201d and 13.50 \u3bcg/g in \u201cTreviso Tardivo\u201d while dihydro-lactucopicrin oxalate was the less abundant in all analyzed varieties with an average concentration of about 1% on the total amount of SLs

G-Quadruplex Modulation of SP1 Functional Binding Sites at the KIT Proximal Promoter

The regulation of conformational arrangements of gene promoters is a physiological mechanism that has been associated with the fine control of gene expression. Indeed, it can drive the time and the location for the selective recruitment of proteins of the transcriptional machinery. Here, we address this issue at the KIT proximal promoter where three G-quadruplex forming sites are present (kit1, kit2 and kit*). On this model, we focused on the interplay between G-quadruplex (G4) formation and SP1 recruitment. By site directed mutagenesis, we prepared a library of plasmids containing mutated sequences of the WT KIT promoter that systematically exploited different G4 formation attitudes and SP1 binding properties. Our transfection data showed that the three different G4 sites of the KIT promoter impact on SP1 binding and protein expression at different levels. Notably, kit2 and kit* structural features represent an on-off system for KIT expression through the recruitment of transcription factors. The use of two G4 binders further helps to address kit2-kit* as a reliable target for pharmacological intervention

Photodynamic Therapy for ras-Driven Cancers: Targeting G-Quadruplex RNA Structures with Bifunctional Alkyl-Modified Porphyrins

Designing small molecules able to break down G4 structures in mRNA (RG4s) offers an interesting approach to cancer therapy. Here, we have studied cationic porphyrins (CPs) bearing an alkyl chain up to 12 carbons, as they bind to RG4s while generating reactive oxygen species upon photoirradiation. Fluorescence-activated cell sorting (FACS) and confocal microscopy showed that the designed alkyl CPs strongly penetrate cell membranes, binding to KRAS and NRAS mRNAs under low-abundance cell conditions. In Panc-1 cells, alkyl CPs at nanomolar concentrations promote a dramatic downregulation of KRAS and NRAS expression, but only if photoactivated. Alkyl CPs also reduce the metabolic activity of pancreatic cancer cells and the growth of a Panc-1 xenograft in SCID mice. Propidium iodide/annexin assays and caspase 3, caspase 7, and PARP-1 analyses show that these compounds activate apoptosis. All these data demonstrate that the designed alkyl CPs are efficient photosensitizers for the photodynamic therapy of ras-driven cancers

In Silico Identification of Piperidinyl-amine Derivatives as Novel Dual Binders of Oncogene c-myc/c-Kit G-quadruplexes

In the last years, it has been shown that the DNA secondary structure known as G-quadruplex is also involved in the regulation of oncogenes transcription, such as c-myc, c-Kit, KRAS, Bcl-2, VEGF, and PDGF. DNA G-quadruplexes, formed in the promoter region of these proto-oncogenes, are considered alternative anticancer targets since their stabilization causes a reduction of the related oncoprotein overexpression. In this study, a structure-based virtual screening toward the experimental DNA G-quadruplex structures of c-myc and c-Kit was performed by using Glide for the docking analysis of a commercial library of approximately 693 000 compounds. The best hits were submitted to thermodynamic and biophysical studies, highlighting the effective stabilization of both G-quadruplex oncogene promoter structures for three N-(4-piperidinylmethyl)amine derivatives, thus proposed as a new class of dual G-quadruplex binder