Non-duplex G-Quadruplex DNA Structure: A Developing Story from Predicted Sequences to DNA Structure-Dependent Epigenetics and Beyond

Abstract

The story of the non-duplex DNA form known as the G-quadruplex (G4) has traversed a winding path. From initial skepticism followed by debate to a surge in interest, the G4 story intertwines many threads. Starting with computational predictions of a gene regulatory role, which now include epigenetic functions, our group was involved in many of these advances along with many other laboratories. Following a brief background, set in the latter half of the last century when the concept of the G4 as a structure took ground, here we account the developments. This is through a lens that though focused on our groups' research presents work from many other groups that played significant roles. Together these provide a broad perspective to the G4 story. Initially we were intrigued on seeing potential G4 (pG4)-forming sequences, then known to be found primarily at the telomeres and immunoglobin switch regions, occurring throughout the genome and being particularly prevalent in promoters of bacteria. We further observed that pG4s were not only prevalent but also conserved through evolution in promoters of human, chimpanzee, mouse and rat genomes. This was between 2005 and 2007. Encouraged by these partly and partly in response to the view held by many that genome-wide presence of G4s were genomic "accidents", the focus shifted to seeking experimental evidence.In the next year, 2008, two independent findings showed promise. First, on treating human cancer cells with G4-binding ligands, we observed widespread change in gene expression. Second, our search for the missing G4-specific transcription factor, without which, importantly, G4s in promoters posed only half the story, yielded results. We determined how NM23-H2 (also known as NME2 or NDPK-B) interacts with G4s and how interaction of NM23-H2 with a G4 in the promoter of the oncogene c-myc was important for regulation of c-myc transcription. NM23-H2, and subsequently many other similar factors discovered by multiple groups, is possibly giving shape to what might be the "G4-transcriptome". Later, a close look at NM23-H2-G4 interaction in regulation of the human reverse transcriptase gene (hTERT) revealed the role of G4s in local epigenetic modifications. Meanwhile work from others showed how G4s impact histone modifications following replication. Together these show the intrinsic role of DNA sequence, through formation of DNA structure, in epigenetics.More recent work, however, was waiting to reveal aspects that tend to bring forth a completely new understanding of G4s. We observed that the telomere-repeat-binding-factor-2 (TRF2), known canonically to be telomere-associated, binds extensively outside telomeres throughout the genome. Moreover, a large fraction of the non-telomeric TRF2 sites comprise G4s. Second, the extent of non-telomeric TRF2 binding at promoters was dependent on telomere length. Thereby TRF2-induced epigenetic gene regulation was telomere-dependent. Together these implicate underlying connections that show signs of addressing an intriguing unanswered question that takes us back to the beginning: Why are G4s prevalent in two distinct regions, the telomeres and gene promoters?

Figure 1. (A) G-quadruplex formation by stacking of G-tetrads stabilized by Hoogsteen base pairing showing stem and loop configurations. (B) Sequence patterns with varying loop length that adopt the G4 fold readily. GGG trimers are connected through sequences of varying lengths comprising all four bases. Algorithms for G-quadruplex mining are typically based on this formalism. G-quadruplexes with interruption within the GGG trimers (bulge formation) and loop lengths of more than 15 bases have also been reported. (C) Basic topology of intra- and intermolecular G-quadruplexes; many variations of this based on the orientation and directionality of the loop sequence have been reported; the three most common intramolecular forms are shown.

Figure 2. (A) Potential G4 (pG4) sequences are predominant near transcription start sites (TSSs) of human, chimpanzee, mouse, and rat genomewide. Each row represents a chromosome; TSSs of all genes in a chromosome are centered in each row. (B) Human promoter pG4s conserved within “orthologous” mouse and rat promoters appear in clusters when plotted relative to distance from the TSS; each row represents one promoter; heat map indicates presence of pG4 with respect to distance from TSS in 773 human promoters (plotted in a 100 bp window; lower panel). Frequency of pG4 motifs near TSS shown in upper panel. Adapted with permission from ref . Copyright 2008 American Chemical Society.

Figure 3. Interaction of NM23-H2 with the parallel type of c-myc promoter G4 transcriptionally activates c-myc expression. Disruption of the G4 results in loss of NM23-H2 binding and reduced transcription of c-myc.

Figure 4. NM23-H2-G4 interaction recruits the REST-co-REST— LSD1 repressor complex for transcriptional repression of genes.

Figure 5. (A) Extensive non-telomeric TRF2 association genome-wide with G-quadruplexes. (B) TRF2-G4 interaction induces recruitment of epigenetic modifier factors for transcription regulation.

Figure 6. Non-telomeric TRF2 binding is telomere-sensitive. Sequestered telomeric TRF2 decreases as telomeres shorten resulting in increased non-telomeric TRF2 binding. This leads to TRF2-induced epigenetic regulation in a telomere-dependent way.