Aurore Guédin

The Yeast Pif1 Helicase Prevents Genomic Instability Caused by G-Quadruplex-Forming CEB1 Sequences In Vivo

In budding yeast, the Pif1 DNA helicase is involved in the maintenance of both nuclear and mitochondrial genomes, but its role in these processes is still poorly understood. Here, we provide evidence for a new Pif1 function by demonstrating that its absence promotes genetic instability of alleles of the G-rich human minisatellite CEB1 inserted in the Saccharomyces cerevisiae genome, but not of other tandem repeats. Inactivation of other DNA helicases, including Sgs1, had no effect on CEB1 stability. In vitro, we show that CEB1 repeats formed stable G-quadruplex (G4) secondary structures and the Pif1 protein unwinds these structures more efficiently than regular B-DNA. Finally, synthetic CEB1 arrays in which we mutated the potential G4-forming sequences were no longer destabilized in pif1Δ cells. Hence, we conclude that CEB1 instability in pif1Δ cells depends on the potential to form G-quadruplex structures, suggesting that Pif1 could play a role in the metabolism of G4-forming sequences.

A G-quadruplex structure within the 5′-UTR of TRF2 mRNA represses translation in human cells

Telomeres protect chromosome ends from being recognized as double-stranded breaks. Telomeric function is ensured by the shelterin complex in which TRF2 protein is an essential player. The G-rich strand of telomere DNA can fold into G-quadruplex (G4) structure. Small molecules stabilizing G4 structures, named G4 ligands, have been shown to alter telomeric functions in human cells. In this study, we show that a guanine-rich RNA sequence located in the 5′-UTR region of the TRF2 mRNA (hereafter 91TRF2G) is capable of forming a stable quadruplex that causes a 2.8-fold decrease in the translation of a reporter gene in human cells, as compared to a mutant 5′-UTR unable to fold into G4. We also demonstrate that several highly selective G4 ligands, the pyridine dicarboxamide derivative 360A and bisquinolinium compounds Phen-DC(3) and Phen-DC(6), are able to bind the 91TRF2G:RNA sequence and to modulate TRF2 protein translation in vitro. Since the naturally occurring 5′-UTR TRF2:RNA G4 element was used here, which is conserved in several vertebrate orthologs, the present data substantiate a potential translational mechanism mediated by a G4 RNA motif for the downregulation of TRF2 expression.

Thioflavin T as a fluorescence light-up probe for G4 formation

Thioflavin T (ThT) becomes fluorescent in the presence of the G-quadruplex structure such as that formed by the human telomeric motif. In this report, we extend and generalize these observations and show that this dye may be used as a convenient and specific quadruplex probe. In the presence of most, but not all, G4-forming sequences, we observed a large increase in ThT fluorescence emission, whereas the presence of control duplexes and single strands had a more limited effect on emission. This differential behavior allowed us to design a high-throughput assay to detect G4 formation. Hundreds of different oligonucleotides may be tested in parallel for G4 formation with a simple fluorescence plate reader. We applied this technique to a family of aptamers not previously recognized as G4-forming sequences and demonstrated that ThT fluorescence signal may be used to predict G4 formation.

Sequence effects in single-base loops for quadruplexes

Intramolecular G-quadruplexes formed by a single DNA strand have attracted much interest due to the possibility that they may form in telomeres, oncogene promoter sequences and other biologically relevant regions of the genome. Therefore, it is important to understand the rules that govern the formation of these intramolecular structures and to determine their stabilities. We compared here 27 different sequences containing four tracts of three guanines with a medium (3) or relatively long (6-9 bases) central loop and two loops composed of a single nucleotide H (A, T or C) corresponding to the GGGHGGGN3-9GGGHGGG motif. These sequences are similar to sequence motifs that can be found in repeated and promoter sequences. Several conclusions were reached: (i) all sequences are prone to form very stable quadruplexes in potassium (Tm between 55 degrees C and 83 degrees C); (ii) these quadruplexes also form in sodium but with a strongly decreased Tm, with a 21 to 36 degrees C difference in melting temperature between Na+ and K+; (iii) a long (9 bases) central loop had a minimal deleterious impact on the stability of the quadruplex; (iv) pyrimidines are preferred over adenine in both single-base loops; (v) the folding topology is relatively robust in potassium: the CD spectra of all oligonucleotides matched the one of all-parallel stranded reference quadruplexes; (vi) conversely, in sodium the folding is diverse and sequence-dependent, as judged from CD and electrophoresis results.

Stability of intramolecular quadruplexes: sequence effects in the central loop

Hundreds of thousands of putative quadruplex sequences have been found in the human genome. It is important to understand the rules that govern the stability of these intramolecular structures. In this report, we analysed sequence effects in a 3-base-long central loop, keeping the rest of the quadruplex unchanged. A first series of 36 different sequences were compared; they correspond to the general formula GGGTTTGGGHNHGGGTTTGGG. One clear rule emerged from the comparison of all sequence motifs: the presence of an adenine at the first position of the loop was significantly detrimental to stability. In contrast, adenines have no detrimental effect when present at the second or third position of the loop. Cytosines may either have a stabilizing or destabilizing effect depending on their position. In general, the correlation between the Tm or ΔG° in sodium and potassium was weak. To determine if these sequence effects could be generalized to different quadruplexes, specific loops were tested in different sequence contexts. Analysis of 26 extra sequences confirmed the general destabilizing effect of adenine as the first base of the loop(s). Finally, analysis of some of the sequences by microcalorimetry (DSC) confirmed the differences found between the sequence motifs.

Proteasomal degradation of retinoid X receptor α reprograms transcriptional activity of PPARγ in obese mice and humans

Obese patients have chronic, low-grade inflammation that predisposes to type 2 diabetes and results, in part, from dysregulated visceral white adipose tissue (WAT) functions. The specific signaling pathways underlying WAT dysregulation, however, remain unclear. Here we report that the PPARgamma signaling pathway operates differently in the visceral WAT of lean and obese mice. PPARgamma in visceral, but not subcutaneous, WAT from obese mice displayed increased sensitivity to activation by its agonist rosiglitazone. This increased sensitivity correlated with increased expression of the gene encoding the ubiquitin hydrolase/ligase ubiquitin carboxyterminal esterase L1 (UCH-L1) and with increased degradation of the PPARgamma heterodimerization partner retinoid X receptor alpha (RXRalpha), but not RXRbeta, in visceral WAT from obese humans and mice. Interestingly, increased UCH-L1 expression and RXRalpha proteasomal degradation was induced in vitro by conditions mimicking hypoxia, a condition that occurs in obese visceral WAT. Finally, PPARgamma-RXRbeta heterodimers, but not PPARgamma-RXRalpha complexes, were able to efficiently dismiss the transcriptional corepressor silencing mediator for retinoid and thyroid hormone receptors (SMRT) upon agonist binding. Increasing the RXRalpha/RXRbeta ratio resulted in increased PPARgamma responsiveness following agonist stimulation. Thus, the selective proteasomal degradation of RXRalpha initiated by UCH-L1 upregulation modulates the relative affinity of PPARgamma heterodimers for SMRT and their responsiveness to PPARgamma agonists, ultimately activating the PPARgamma-controlled gene network in visceral WAT of obese animals and humans.

G-Quadruplex Formation Interferes with P1 Helix Formation in the RNA Component of Telomerase hTERC

Guanine-rich nucleic acids can adopt unusual structures called guanine quadruplexes (G4) that are based on stacked guanine quartets (Figure 1A, bottom right). DNA sequences that are prone to adopt such structures are found in telomeric repeats, in several promoters, in ribosomal DNA, and in the immunoglobulin switch region; these structures appear to be biologically relevant. Formation of G4 has been demonstrated in vivo in ciliate telomeres, and during G-rich sequence transcription (G-loop). More recently, G4 formation in RNA sequences has been investigated. Telomerase, the nucleoprotein complex that is responsible for telomere maintenance and homeostasis, is a key component of cell proliferation and its reactivation is often associated with tumorigenesis. Inhibitors of telomerase function, including antisense oligonucleotides that target the telomerase RNA component (hTERC) or G-quadruplex ligands that target the telomeric 3’-overhang substrate for telomerase elongation and binding, offer therapeutic promise. The telomerase RNA is structurally conserved among many organisms, but its primary sequence and size are not. In most vertebrates, the predicted structures possess a helical domain called the P1 helix that is located 5’ to the template sequence. This helix is thought to serve as a template boundary element to limit reverse transcription to the 6-nucleotide template. In many mammals, a 5’ tail precedes this helix, but no precise function has been assigned to this tail. Analysis of these sequences shows a skewed base composition with several well-conserved runs of guanines in this region (Figure 1B). Such G-rich RNA sequences have the potential to form G quadruplexes that may interfere with P1 helix formation and its template boundary function (Figure 1A). In this work, we demonstrate that RNA fragments with the sequence of the 41 5’-most nucleotides of hTERC form a quadruplex. This G4 formation hinders the formation of a helix that corresponds to P1. We further demonstrate that this guanine quadruplex interacts with a specific guanine-quadruplex ligand, 360A ; the presence of 360A enhanced inhibition of P1 helix assembly. We synthesized an oligoribonucleotide with the sequence of the first 41 nucleotides of hTERC, 41R (Table 1). We also designed and synthesized the 41m control sequence (changes are underlined in Table 1), in which guanine runs have been disrupted by G-to-A changes. UV absorbance as a function of temperature offers a convenient method for characterizing ACHTUNGTRENNUNGnucleic acids structures. With 41R, we observed a typical cooperative reversible transition around 295 nm; the temperature of mid-transition (Tm) depended upon the nature and the concentration of the monovalent cation present in the solution. Tm values ranged from 38 8C in 100 mm LiCl to more than 80 8C in 100 mm KCl (Figure 2A). Results are presented in detail in Table 2. The structure that is formed by this sequence was surprisingly stable even in the absence of added potassium and sodium (Tm =39 8C, see Table 2). The addition of as little as 0.1 mm KCl in the absence of any other added monovalent cation was sufficient to stabilize the structure. The temperature range in which the transition occurred was independent of the concentration of oligonucleotide in the 0.5 to 25 mm range; this demonstrates that folding was intramolecular (data not shown). No transition was observed at 295 nm for the 41m control. These finding suggest that 41R adopts an intramolecular G-quadruplex structure. Thermal difference spectra (TDS) for 41R in Li , Na or K conditions were also typical of G quadruplexes, with a negative peak around 297 nm and two positives peaks around 274 nm and 242 nm (Figure 2B). Circular dichroism (CD) spectra of 41R had a positive peak at 263 nm and a negative peak at 240 nm (Figure 2C), as is typical of G4 formation. Although CD spectra with these characteristics have been attributed to parallel G quadruplexes in the light of recent publications, 16] we prefer to describe these as type I spectra. These spectra are characteristic of quadruplexes with all stacked quartets with the same polarity, likely to be in the present case with all guanine glyocosidic bonds in the anti conformation and thus with parallel strands. CD and TDS spectra for 41m do not correspond to those typical of G quadruplexes (not shown). Finally, enzymatic probing with RNase T1 confirmed quadruplex formation. In the wild-type 41R sequence, several Gs were protected in buffer that contained K , but not in buffer that contained only Li as a cation (see Figure S1, left in the Supporting Information). These results confirm the formation of a cation-dependent folded structure by 41R : In K , five protected clusters of G were observed; this suggests either a complex intramolecular folding or the coexistence of at least two foldings that share three runs of Gs. In contrast, digestion of 41m showed only a slight protection of the central part of the ACHTUNGTRENNUNGsequence (Figure S1, right). Having demonstrated that the 5’ fragment of hTERC 5’ forms a stable quadruplex, we then investigated whether this structure could interfere with secondary structure formation. Some of the guanines that are involved in the intramolecular quadruplex are base-paired in the P1 helix, thus making these [a] J. Gros, A. Gu din, Dr. J.-L. Mergny, Dr. L. Lacroix INSERM, U565, Mus um National d’Histoire Naturelle (MNHN) USM 503-CNRS UMR 5153 “Acides Nucleiques: Dynamique, Ciblage et Fonctions Biologiques” 57 rue Cuvier, Case Postale 26, 75231 Paris cedex 05 (France) Fax: (+33)1-40-79-36-89 E-mail : lacroix@mnhn.fr Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.

Thermal Melting Studies of Ligand DNA Interactions

A simple thermal melting experiment may be used to demonstrate the stabilization of a given structure by a ligand (usually a small molecule, sometimes a peptide). Preparation of the sample is straightforward, and the experiment itself requires an inexpensive apparatus. Furthermore, reasonably low amounts of sample are required. A qualitative analysis of the data is simple: An increase in the melting temperature (T(m)) indicates preferential binding to the folded form as compared to the unfolded form. However, it is perilous to derive an affinity constant from an increase in T(m) as other factors play a role.

“One Ring to Bind Them All”—Part I: The Efficiency of the Macrocyclic Scaffold for G-Quadruplex DNA Recognition

Macrocyclic scaffolds are particularly attractive for designing selective G-quadruplex ligands essentially because, on one hand, they show a poor affinity for the “standard” B-DNA conformation and, on the other hand, they fit nicely with the external G-quartets of quadruplexes. Stimulated by the pioneering studies on the cationic porphyrin TMPyP4 and the natural product telomestatin, follow-up studies have developed, rapidly leading to a large diversity of macrocyclic structures with remarkable-quadruplex binding properties and biological activities. In this review we summarize the current state of the art in detailing the three main categories of quadruplex-binding macrocycles described so far (telomestatin-like polyheteroarenes, porphyrins and derivatives, polyammonium cyclophanes), and in addressing both synthetic issues and biological aspects.

Recognition of G-Quadruplex DNA by Triangular Star-Shaped Compounds: With or Without Side Chains?

We report the synthesis of two new series of triangular aromatic platforms, either with three aminoalkyl side chains (triazatrinaphthylene series, TrisK: six compounds), or without side chains (triazoniatrinaphthylene, TrisQ). The quadruplex-DNA binding behavior of the two series, which differ essentially by the localization of the cationic charges, was evaluated by means of FRET-melting and G4-FID assays. For the trisubstituted triazatrinaphthylenes (TrisK), the length of the substituents and the presence of terminal hydrogen-bond-donor groups (NH(2)) were shown to be crucial for ensuring a high quadruplex affinity (ΔT(1/2) values of up to 20 °C at 1 μM for the best candidate, TrisK3-NH) and selectivity versus duplex DNA. Subsequently, comparison of data collected on both the telomeric- and c-myc-quadruplex showed that the nonsubstituted TrisQ is even more efficient than TrisK3-NH, both in terms of quadruplex affinity (ΔT(1/2)=26 °C in K(+) buffer) and selectivity versus duplex DNA. Structural considerations conducted with the c-myc quadruplex indicate that both TrisK3-NH and TrisQ stack well onto the G-quartet but in an offset position, which might be influenced by the formation of multiple hydrogen bonds with the target in the former case. Finally, the nonsubstituted TrisQ displays a binding profile very similar to some of the best quadruplex binders, BRACO-19 and bisquinolinium 360A, used herein as references, and thereby represents a highly promising novel molecular design for quadruplex recognition.