Abstract
It has been >20 years since the formation of G-quadruplex (G4) secondary structures in gene promoters was first linked to the regulation of gene expression. Since then, the development of ...small molecules to selectively target G4s and their cellular application have contributed to an improved understanding of how G4s regulate transcription. One model that arose from this work placed these non-canonical DNA structures as repressors of transcription by preventing polymerase processivity. Although a considerable number of studies have recently provided sufficient evidence to reconsider this simplistic model, there is still a misrepresentation of G4s as transcriptional roadblocks. In this review, we will challenge this model depicting G4s as simple ‘off switches’ for gene expression by articulating how their formation has the potential to alter gene expression at many different levels, acting as a key regulatory element perturbing the nature of epigenetic marks and chromatin architecture.
Graphical Abstract
Graphical Abstract
DNA G-quadruplexes act as transcriptional hubs by means of diverse mechanisms, including: modulation of chromatin structure, regulatory protein recruitment and formation of DNA loops, stimulation of liquid-liquid phase separation and eliciting DNA damage and repair.
Substantial evidence now exists to support that formation of DNA G-quadruplexes (G4s) is coupled to altered gene expression. However, approaches that allow us to probe G4s in living cells without ...perturbing their folding dynamics are required to understand their biological roles in greater detail. Herein, we report a G4-specific fluorescent probe (SiR-PyPDS) that enables single-molecule and real-time detection of individual G4 structures in living cells. Live-cell single-molecule fluorescence imaging of G4s was carried out under conditions that use low concentrations of SiR-PyPDS (20 nM) to provide informative measurements representative of the population of G4s in living cells, without globally perturbing G4 formation and dynamics. Single-molecule fluorescence imaging and time-dependent chemical trapping of unfolded G4s in living cells reveal that G4s fluctuate between folded and unfolded states. We also demonstrate that G4 formation in live cells is cell-cycle-dependent and disrupted by chemical inhibition of transcription and replication. Our observations provide robust evidence in support of dynamic G4 formation in living cells.
G-quadruplexes (G4s) are nucleic acid secondary structures that form within guanine-rich DNA or RNA sequences. G4 formation can affect chromatin architecture and gene regulation and has been ...associated with genomic instability, genetic diseases and cancer progression. Here we present a high-resolution sequencing-based method to detect G4s in the human genome. We identified 716,310 distinct G4 structures, 451,646 of which were not predicted by computational methods. These included previously uncharacterized noncanonical long loop and bulged structures. We observed a high G4 density in functional regions, such as 5' untranslated regions and splicing sites, as well as in genes previously not predicted to contain these structures (such as BRCA2). G4 formation was significantly associated with oncogenes, tumor suppressors and somatic copy number alterations related to cancer development. The G4s identified in this study may therefore represent promising targets for cancer intervention.
Abstract
Genomic maps of DNA G-quadruplexes (G4s) can help elucidate the roles that these secondary structures play in various organisms. Herein, we employ an improved version of a G-quadruplex ...sequencing method (G4-seq) to generate whole genome G4 maps for 12 species that include widely studied model organisms and also pathogens of clinical relevance. We identify G4 structures that form under physiological K+ conditions and also G4s that are stabilized by the G4-targeting small molecule pyridostatin (PDS). We discuss the various structural features of the experimentally observed G-quadruplexes (OQs), highlighting differences in their prevalence and enrichment across species. Our study describes diversity in sequence composition and genomic location for the OQs in the different species and reveals that the enrichment of OQs in gene promoters is particular to mammals such as mouse and human, among the species studied. The multi-species maps have been made publicly available as a resource to the research community. The maps can serve as blueprints for biological experiments in those model organisms, where G4 structures may play a role.
Temperature is a major environmental cue affecting plant growth and development. Plants often experience higher temperatures in the context of a 24 h day-night cycle, with temperatures peaking in the ...middle of the day. Here, we find that the transcript encoding the bHLH transcription factor PIF7 undergoes a direct increase in translation in response to warmer temperature. Diurnal expression of PIF7 transcript gates this response, allowing PIF7 protein to quickly accumulate in response to warm daytime temperature. Enhanced PIF7 protein levels directly activate the thermomorphogenesis pathway by inducing the transcription of key genes such as the auxin biosynthetic gene YUCCA8, and are necessary for thermomorphogenesis to occur under warm cycling daytime temperatures. The temperature-dependent translational enhancement of PIF7 messenger RNA is mediated by the formation of an RNA hairpin within its 5' untranslated region, which adopts an alternative conformation at higher temperature, leading to increased protein synthesis. We identified similar hairpin sequences that control translation in additional transcripts including WRKY22 and the key heat shock regulator HSFA2, suggesting that this is a conserved mechanism enabling plants to respond and adapt rapidly to high temperatures.
This study argues that the Union maintains a double standard on working conditions in relation to the criteria established at international level, which allows to tolerate situations of labour ...exploitation as ‘unserious’, ‘proportionated’ or ‘normal’ phenomena. By analysing the gaps in the protection of the right to fair and just working conditions, the idea that an upward convergence of workers’ rights would be supported (and required) by the general duty to protect human dignity and to ensure fair competition in the Single Market is advanced. The relationship between these fundamental principles in the implementation of the right to fair and just working conditions is examined through the notion of ‘social market economy’. The aim is to illustrate what kind of measures could (or should) be taken to integrate the protection of human rights and market efficiency, and whether in practice there seems to be an articulation between these fundamental principles or, on the contrary, an insurmountable contradiction.
Following extensive evidence for the formation of four-stranded DNA G-quadruplex structures in vitro, DNA G-quadruplexes have been observed within human cells. Although chemically distinct, RNA can ...also fold in vitro into G-quadruplex structures that are highly stable because of the 2'-hydroxyl group. However, RNA G-quadruplexes have not yet been reported in cells. Here, we demonstrate the visualization of RNA G-quadruplex structures within the cytoplasm of human cells using a G-quadruplex structure-specific antibody. We also demonstrate that small molecules that bind to G-quadruplexes in vitro can trap endogenous RNA G-quadruplexes when applied to cells. Furthermore, a small molecule that exhibits a preference for RNA G-quadruplexes rather than DNA G-quadruplexes in biophysical experiments also shows the same selectivity within a cellular context. Our findings provide substantive evidence for RNA G-quadruplex formation in the human transcriptome, and corroborate the selectivity and application of stabilizing ligands that target G-quadruplexes within a cellular context.
Abstract
G-quadruplexes (G4s) are well known non-canonical DNA secondary structures that can form in human cells. Most of the tools available to investigate G4-biology rely on small molecule ligands ...that stabilise these structures. However, the development of probes that disrupt G4s is equally important to study their biology. In this study, we investigated the disruption of G4s using Locked Nucleic Acids (LNA) as invader probes. We demonstrated that strategic positioning of LNA-modifications within short oligonucleotides (10 nts.) can significantly accelerate the rate of G4-disruption. Single-molecule experiments revealed that short LNA-probes can promote disruption of G4s with mechanical stability sufficient to stall polymerases. We corroborated this using a single-step extension assay, revealing that short LNA-probes can relieve replication dependent polymerase-stalling at G4 sites. We further demonstrated the potential of such LNA-based probes to study G4-biology in cells. By using a dual-luciferase assay, we found that short LNA probes can enhance the expression of c-KIT to levels similar to those observed when the c-KIT promoter is mutated to prevent the formation of the c-KIT1 G4. Collectively, our data suggest a potential use of rationally designed LNA-modified oligonucleotides as an accessible chemical-biology tool for disrupting individual G4s and interrogating their biological functions in cells.
The promotion of the Olympic ideals is strongly correlated with the effective protection of human rights in the sporting context. However, countless practical examples of violations of athletes’ ...human rights have shown how the link between sport and human rights is continuously placed under strong pressure. Taking into consideration the thesis according to which human rights are an indirect legal source of the lex sportiva, this study analyses the gaps in the protection of the fundamental rights of athletes. By verifying the potential effects on the sporting juridical bodies’ competences and on the effective implications in sporting affairs for the European Court of Human Rights, the hypothesis of an emerging incorporation of human rights in sports legal orders will also be investigated. At the end of these pages, by identifying the limits and perspectives of the judicial protection of athletes’ human rights, we should also be able to evaluate if, and to what extent, a reform of sport justice bodies is urgently needed in order to guarantee the function of sport as an instrument of peace and sustainable development, vehicle of rights, and source of social inclusion.
Water is generally indicated as a public good that is essential for life. Within the Union water law, this emphatic qualification appears as a generic political declaration linked to the uncertain ...recognition of the right to water. By analysing the common private and public interests, this study argues that the restricted territorial sovereignty's theory, widely accepted for transboundary water resources, should be applied to all water resources. Water would be a shared European public good, subject to a sui generis community of property regime limiting the sovereignty of Member States over their water resources. By investigating the coherence of this community of property regime with the principle of neutrality enshrined in Article 345 TFEU, the essay suggests that the regulation of the water services would be drawn into the scope of the Treaties because of their instrumental nature in guaranteeing the exercise of fundamental rights, the social cohesion of the Union and the protection of a common public good. The idea is put forward that water services would be an emblematic case of European public services, understood as a specific development of the concept of service of general economic interest, instrumental to the implementation of the European model of society.
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