Julian Leon Huppert

G-quadruplexes in promoters throughout the human genome

Certain G-rich DNA sequences readily form four-stranded structures called G-quadruplexes. These sequence motifs are located in telomeres as a repeated unit, and elsewhere in the genome, where their function is currently unknown. It has been proposed that G-quadruplexes may be directly involved in gene regulation at the level of transcription. In support of this hypothesis, we show that the promoter regions (1 kb upstream of the transcription start site TSS) of genes are significantly enriched in quadruplex motifs relative to the rest of the genome, with >40% of human gene promoters containing one or more quadruplex motif. Furthermore, these promoter quadruplexes strongly associate with nuclease hypersensitive sites identified throughout the genome via biochemical measurement. Regions of the human genome that are both nuclease hypersensitive and within promoters show a remarkable (230-fold) enrichment of quadruplex elements, compared to the rest of the genome. These quadruplex motifs identified in promoter regions also show an interesting structural bias towards more stable forms. These observations support the proposal that promoter G-quadruplexes are directly involved in the regulation of gene expression.

G-quadruplexes: the beginning and end of UTRs

Molecular mechanisms that regulate gene expression can occur either before or after transcription. The information for post-transcriptional regulation can lie within the sequence or structure of the RNA transcript and it has been proposed that G-quadruplex nucleic acid sequence motifs may regulate translation as well as transcription. Here, we have explored the incidence of G-quadruplex motifs in and around the untranslated regions (UTRs) of mRNA. We observed a significant strand asymmetry, consistent with a general depletion of G-quadruplex-forming RNA. We also observed a positional bias in two distinct regions, each suggestive of a specific function. We observed an excess of G-quadruplex motifs towards the 5′-ends of 5′-UTRs, supportive of a hypothesis linking 5′-UTR RNA G-quadruplexes to translational control. We then analysed the vicinity of 3′-UTRs and observed an over-representation of G-quadruplex motifs immediately after the 3′-end of genes, especially in those cases where another gene is in close proximity, suggesting that G-quadruplexes may be involved in the termination of gene transcription.

Structure, location and interactions of G‐quadruplexes

Four‐stranded G‐rich DNA structures called G‐quadruplexes have been the subject of increasing interest recently. Experimental and computational techniques have been used to implicate them in important biological processes such as transcription and translation. In this minireview, I discuss how they form, what structures they adopt and with what stability. I then discuss the computational approaches used to predict them on a genomic scale and how the information derived can be combined with experiments to understand their biological functions. Other minireviews in this series deal with G‐quadruplex nucleic acids and human disease [Wu Y & Brosh RM Jr (2010) FEBS J] and making sense of G‐quadruplex and i‐motif function in oncogene promoters [Brooks TA et al. (2010) FEBS J].

Hunting G-quadruplexes

Whilst DNA spends much of its time in the double-stranded form, frequently in storage, wrapped around histones and packaged as chromatin, it can also form other complex structures, which may play a role in natural regulation and gene control. These alternative structures therefore also present an interesting novel series of targets for artificial intervention, and so may lead to novel therapeutics. In this review, I describe the current understanding of how genomic and bioinformatics studies may be used to understand the roles that one such structure, the four-stranded guanine-rich G-quadruplex, may play in the genome, and outline how these may be considered as targets for intervention. I will also describe recent work looking at RNA G-quadruplexes, and the biological roles they may play.

Four-stranded DNA: cancer, gene regulation and drug development.

DNA can form many structures other than the famous double helix. In particular, guanine-rich DNA of particular sequences can form four-stranded structures, called G-quadruplexes. This article describes the structural form of these sequences, techniques for predicting which sequences can fold up in this manner and efforts towards stability prediction. It then discusses the biological significance of these structures, focusing on their importance in telomeric regions at the end of chromosomes, and their existence in gene promoters and mRNA, where they may be involved with regulating transcription and translation, respectively. Ligands that are capable of selectively binding to these structures are introduced and described, as are DNA aptamers that form G-quadruplex structures; both of these classes of compound have been investigated as anticancer agents in clinical trials. The growing use of G-quadruplexes in the nanotechnology field is also outlined. The article concludes with an analysis of future directions the field may take, with some proposals for further important studies.

Genome-wide analysis of a G-quadruplex-specific single-chain antibody that regulates gene expression

G-quadruplex nucleic acids have been proposed to play a role in a number of fundamental biological processes that include transcription and translation. We have developed a single-chain antibody that is selective for G-quadruplex DNA over double-stranded DNA, and here show that when it is expressed in human cells, it significantly affects the expression of a wide variety of genes, in a manner that correlates with the presence of predicted G-quadruplexes. We observe cases where gene expression is increased or decreased, and that there are apparent interactions with G-quadruplex motifs at the beginning and end of the genes, and on either strand. The outcomes of this genome-wide study demonstrate that G-quadruplex recognition by the antibody has physiological consequences, and provides insights into some of the complexity associated with G-quadruplex-based regulation.

Direct visualization of G-quadruplexes in DNA using atomic force microscopy

The formation of G-quadruplexes in G-rich regions of DNA is believed to affect DNA transcription and replication. However, it is currently unclear how this formation occurs in the presence of a complementary strand. We have used atomic force microscopy (AFM) to image stable RNA/DNA hybrid loops generated by transcription of the plasmid pPH600, which contains a 604-bp fragment of the murine immunoglobulin Sγ3 switch region. We show that the non-RNA-containing portion folds into G-quadruplexes, consistent with computational predictions. We also show that hybrid formation prevents further transcription from occurring, implying a regulatory role. After in vitro transcription, almost all (93%) of the plasmids had an asymmetric loop, a large asymmetric blob or a spur-like projection at the appropriate position on the DNA contour. The loops disappeared following treatment of the transcribed plasmid with RNase H, which removes mRNA hybridized with the template strand. Replacement of K+ in the transcription buffer with either Na+ or Li+ caused a reduction in the percentage of plasmids containing loops, blobs or spurs, consistent with the known effects of monovalent cations on G-quadruplex stability. The minimal sample preparation required for AFM imaging has permitted direct observation of the structural changes resulting from G-quadruplex formation.

A Toolbox for Predicting G-Quadruplex Formation and Stability

G-quadruplexes are four stranded nucleic acid structures formed around a core of guanines, arranged in squares with mutual hydrogen bonding. Many of these structures are highly thermally stable, especially in the presence of monovalent cations, such as those found under physiological conditions. Understanding of their physiological roles is expanding rapidly, and they have been implicated in regulating gene transcription and translation among other functions. We have built a community-focused website to act as a repository for the information that is now being developed. At its core, this site has a detailed database (QuadDB) of predicted G-quadruplexes in the human and other genomes, together with the predictive algorithm used to identify them. We also provide a QuadPredict server, which predicts thermal stability and acts as a repository for experimental data from all researchers. There are also a number of other data sources with computational predictions. We anticipate that the wide availability of this information will be of use both to researchers already active in this exciting field and to those who wish to investigate a particular gene hypothesis.

Seven essential questions on G-quadruplexes

Abstract The helical duplex architecture of DNA was discovered by Francis Crick and James Watson in 1951 and is well known and understood. However, nucleic acids can also adopt alternative structural conformations that are less familiar, although no less biologically relevant, such as the G-quadruplex. G-quadruplexes continue to be the subject of a rapidly expanding area of research, owing to their significant potential as therapeutic targets and their unique biophysical properties. This review begins by focusing on G-quadruplex structure, elucidating the intermolecular and intramolecular interactions underlying its formation and highlighting several substructural variants. A variety of methods used to characterize these structures are also outlined. The current state of G-quadruplex research is then addressed by proffering seven pertinent questions for discussion. This review concludes with an overview of possible directions for future research trajectories in this exciting and relevant field.

Distance-dependent duplex DNA destabilization proximal to G-quadruplex/i-motif sequences

G-quadruplexes and i-motifs are complementary examples of non-canonical nucleic acid substructure conformations. G-quadruplex thermodynamic stability has been extensively studied for a variety of base sequences, but the degree of duplex destabilization that adjacent quadruplex structure formation can cause has yet to be fully addressed. Stable in vivo formation of these alternative nucleic acid structures is likely to be highly dependent on whether sufficient spacing exists between neighbouring duplex- and quadruplex-/i-motif-forming regions to accommodate quadruplexes or i-motifs without disrupting duplex stability. Prediction of putative G-quadruplex-forming regions is likely to be assisted by further understanding of what distance (number of base pairs) is required for duplexes to remain stable as quadruplexes or i-motifs form. Using oligonucleotide constructs derived from precedented G-quadruplexes and i-motif-forming bcl-2 P1 promoter region, initial biophysical stability studies indicate that the formation of G-quadruplex and i-motif conformations do destabilize proximal duplex regions. The undermining effect that quadruplex formation can have on duplex stability is mitigated with increased distance from the duplex region: a spacing of five base pairs or more is sufficient to maintain duplex stability proximal to predicted quadruplex/i-motif-forming regions.