G4 DNA Motifs Research Articles

G4-Ligand-Conjugated Oligonucleotides Mediate Selective Binding and Stabilization of Individual G4 DNA Structures.

G-quadruplex (G4) DNA structures are prevalent secondary DNA structures implicated in fundamental cellular functions, such as replication and transcription. Furthermore, G4 structures are directly correlated to human diseases such as cancer and have been highlighted as promising therapeutic targets for their ability to regulate disease-causing genes, e.g., oncogenes. Small molecules that bind and stabilize these structures are thus valuable from a therapeutic perspective and helpful in studying the biological functions of the G4 structures. However, there are hundreds of thousands of G4 DNA motifs in the human genome, and a long-standing problem in the field is how to achieve specificity among these different G4 structures. Here, we developed a strategy to selectively target an individual G4 DNA structure. The strategy is based on a ligand that binds and stabilizes G4s without selectivity, conjugated to a guide oligonucleotide, that specifically directs the G4-Ligand-conjugated oligo (GL-O) to the single target G4 structure. By employing various biophysical and biochemical techniques, we show that the developed method enables the targeting of a unique, specific G4 structure without impacting other off-target G4 formations. Considering the vast amount of G4s in the human genome, this represents a promising strategy to study the presence and functions of individual G4s but may also hold potential as a future therapeutic modality.

A transposon-introduced G-quadruplex motif is selectively retained and constrained to downregulate CYP321A1.

Insects utilize xenobiotic compounds to up- and downregulate cytochrome P450 monooxygenases (P450s) involved in detoxification of toxic xenobiotics including phytochemicals and pesticides. G-quadruplexes (G4)-forming DNA motifs are enriched in the promoter regions of transcription factors and function as cis-acting elements to regulate these genes. Whether and how P450s gain and keep G4 DNA motifs to regulate their expression still remain unexplored. Here, we show that CYP321A1, a xenobiotic-metabolizing P450 from Helicoverpa zea, a polyphagous insect of economic importance, has acquired and preserved a G4 DNA motif by selectively retaining a transposon known as HzIS1-3 that carries this G4 DNA motif in its promoter region. The HzIS1-3 G4 DNA motif acts as a silencer to suppress the constitutive and induced expression of CYP321A1 by plant allelochemicals flavone and xanthotoxin through folding into an intramolecular parallel or hybrid-1 conformation in the absence or presence of K+ . The G4 ligand N-methylmesoporphyrin IX (NMM) strengthens the silencing effect of HzIS1-3 G4 DNA motif by switching its structure from hybrid-1 to hybrid-2. The enrichment of transposons in P450s and other environment-adaptation genes implies that selective retention of G4 DNA motif-carrying transposons may be the main evolutionary route for these genes to obtain G4 DNA motifs.

Characterization of G4 DNA formation in mitochondrial DNA and their potential role in mitochondrial genome instability.

Mitochondria possess their own genome which can be replicated independently of nuclear DNA. Mitochondria being the powerhouse of the cell produce reactive oxygen species, due to which the mitochondrial genome is frequently exposed to oxidative damage. Previous studies have demonstrated an association of mitochondrial deletions to aging and human disorders. Many of these deletions were present adjacent to non-B DNA structures. Thus, we investigate noncanonical structures associated with instability in mitochondrial genome. Insilico studies revealed the presence of >100 G-quadruplex motifs (of which 5 have the potential to form 3-plate G4 DNA), 23 inverted repeats, and 3 mirror repeats in the mitochondrial DNA (mtDNA). Further analysis revealed that among the deletion breakpoints from patients with mitochondrial disorders, majority are located at G4 DNA motifs. Interestingly, ~50% of the deletions were at base-pair positions 8271-8281, ~35% were due to deletion at 12362-12384, and ~12% due to deletion at 15516-15545. Formation of 3-plate G-quadruplex DNA structures at mitochondrial fragile regions was characterized using electromobility shift assay, circular dichroism (CD), and Taq polymerase stop assay. All 5 regions could fold into both intramolecular and intermolecular G-quadruplex structures in a KCl-dependent manner. G4 DNA formation was in parallel orientation, which was abolished in the presence of LiCl. The formation of G4 DNA affected both replication and transcription. Finally, immunolocalization of BG4 with MitoTracker confirmed the formation of G-quadruplex in mitochondrial genome. Thus, we characterize the formation of 5 different G-quadruplex structures in human mitochondrial region, which may contribute toward formation of mitochondrial deletions.

The highly conserved proteins Nucleolin and Sub1 play critical roles in regulating G4 DNA‐induced genome instability.

Sequences containing multiple guanine‐runs can form the four‐stranded G‐quadruplex (G4) DNA. The significance of G4 DNA in etiology of blood cancers is underscored by the manifest enrichment of G4 sequence motifs proximal to the leukemia/lymphoma‐associated translocation breakpoints. But how G4 DNA‐motifs instigate chromosome abnormalities leading to malignant transformation is yet to be resolved. We developed quantitative assays to measure and characterize multiple types of G4 DNA‐induced genome instability events in the context of the yeast genome. This system allowed us to demonstrate that the level and direction of transcription are important determinants of the stability of G4 sequences. Using this approach, we demonstrated that the topological change ensuing in the absence of Topoisomerase I (Top1) can severely endanger genome instability at actively transcribed G4 motifs. The G4‐associated instability in top1Δ yeasts was suppressed partially by the bacterial topA (ecTopA), which removes negative supercoils and not at all by the gyrases (ecGyrase), which removes positive supercoils or the RNase H1 overexpression, which removes RNA:DNA hybrids or R‐loops. Taking advantage of the G4 DNA‐conducive cellular environment in the top1Δ yeasts, we also demonstrated that, upon disruption of Sub1, a general transcription co‐activator, genome instability linked to co‐transcriptionally formed G4 DNA is significantly elevated and that the highly conserved DNA binding domain of Sub1 and its human homolog PC4 is sufficient in preventing genome instability at actively transcribed G4 motifs. Consistent with its role in suppressing G4‐mediated instability, Sub1 interacts specifically with co‐transcriptionally formed G4 DNA in vivo and yeast cells become highly sensitivity to G4‐stabilizing chemical ligands by the loss of Sub1. We demonstrated that Sub1 forms a complex with the G4‐resolving helicase Pif1 suggesting a possible mechanism by which Sub1 suppresses G4‐associated genome instability. Additionally, we discuss our most recent findings regarding the role of the highly conserved nucleolar protein Nucleolin (Nsr1 in yeast) in G4 DNA‐associated genome instability. Deletion of Nsr1, a known G4 DNA‐binding protein, surprisingly led to a significant decrease of G4‐induced recombination. We present a model where interaction with Nsr1 shift the equilibrium between B DNA and G4 DNA toward stable G4 DNA and thereby create strong genome‐destabilizing replication block.Support or Funding InformationWork in the NK lab was supported by grants from the Welch Foundation (AU1875) and the National Institutes of Health (GM116007).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A G-quadruplex DNA structure resolvase, RHAU, is essential for spermatogonia differentiation.

G-quadruplex (G4) DNA and G4 DNA resolvase are involved in a variety of biological processes. To understand the biological function of G4 DNA structures and their resolvases in spermatogenesis, we investigated the distribution of G4 structures in mouse testis and identified their alterations during spermatogenesis. Meanwhile, we studied the function of RNA helicase associated with AU-rich element (RHAU), a G4 DNA resolvase, in spermatogenesis with a germ-cell-specific knockout mouse model. The results showed that the ablation of RHAU in germ cells caused the increase of G4 structures and thus resulted in the decrease of spermatogonial differentiation. c-kit, a spermatogonia differentiation-related gene, contains two G4 DNA motifs on its promoter. We found its expression was significantly downregulated in RHAU conditional knockout testis. A further analysis demonstrated that RHAU directly bound to the G4 structures to activate c-kit expression. We concluded that RHAU regulates spermatogonia differentiation by promoting c-kit expression via directly binding to the G4 DNA motifs c-kit promoter.

Discovery of novel rules for G-quadruplex-forming sequences in plants by using bioinformatics methods

The G-quadruplex is one of the most frequently studied secondary DNA structures and consists of 4 guanine residues that interact through Watson-Crick and Hoogsteen pairing. The G-quadruplex formation is thought to be a molecular switch for gene expression. Genome-wide analyses of G-quadruplexes have been published for many species; however, only one genome-wide analysis of G-quadruplexes in plants has been reported. Here, we propose a new approach involving a two-step procedure for identifying G-quadruplex-forming sequences (potential G4 DNA motif regions: G4MRs) and classifying positional relationships between G4MRs and genes. By using this approach, we exhaustively searched for G4MRs in the whole genomes of 8 species: Arabidopsis thaliana, Oryza sativa subsp. japonica, Populus trichocarpa, Vitis vinifera, Homo sapiens, Danio rerio, Drosophila melanogaster, and Caenorhabditis elegans. We classified genes on the basis of their positional relationships to their proximal G4MRs. We identified novel rules for G4MRs in plants, such as G4MR-enrichment in the template strands at transcription start sites (TSSs). Next, we focused on the template strands of TSSs and conducted gene ontology (GO) analysis of genes proximal to G4MRs. We identified GO terms such as chloroplast and nucleosome (or histone) in O. sativa. Although these terms were strongly associated in O. sativa, weak associations were identified in other plants. These results will be helpful for elucidating the functional roles of G4 DNA.

FANCJ coordinates two pathways that maintain epigenetic stability at G-quadruplex DNA

We have previously reported that DT40 cells deficient in the Y-family polymerase REV1 are defective in replicating G-quadruplex DNA. In vivo this leads to uncoupling of DNA synthesis from redeposition of histones displaced ahead of the replication fork, which in turn leads to loss of transcriptional repression due to failure to recycle pre-existing repressive histone post-translational modifications. Here we report that a similar process can also affect transcriptionally active genes, leading to their deactivation. We use this finding to develop an assay based on loss of expression of a cell surface marker to monitor epigenetic instability at the level of single cells. This assay allows us to demonstrate G4 DNA motif-associated epigenetic instability in mutants of three helicases previously implicated in the unwinding of G-quadruplex structures, FANCJ, WRN and BLM. Transcriptional profiling of DT40 mutants reveals that FANCJ coordinates two independent mechanisms to maintain epigenetic stability near G4 DNA motifs that are dependent on either REV1 or on the WRN and BLM helicases, suggesting a model in which efficient in vivo replication of G-quadruplexes often requires the established 5′–3′-helicase activity of FANCJ acting in concert with either a specialized polymerase or helicase operating in the opposite polarity.

G-Quadruplex DNA Sequences Are Evolutionarily Conserved and Associated with Distinct Genomic Features in Saccharomyces cerevisiae

G-quadruplex DNA is a four-stranded DNA structure formed by non-Watson-Crick base pairing between stacked sets of four guanines. Many possible functions have been proposed for this structure, but its in vivo role in the cell is still largely unresolved. We carried out a genome-wide survey of the evolutionary conservation of regions with the potential to form G-quadruplex DNA structures (G4 DNA motifs) across seven yeast species. We found that G4 DNA motifs were significantly more conserved than expected by chance, and the nucleotide-level conservation patterns suggested that the motif conservation was the result of the formation of G4 DNA structures. We characterized the association of conserved and non-conserved G4 DNA motifs in Saccharomyces cerevisiae with more than 40 known genome features and gene classes. Our comprehensive, integrated evolutionary and functional analysis confirmed the previously observed associations of G4 DNA motifs with promoter regions and the rDNA, and it identified several previously unrecognized associations of G4 DNA motifs with genomic features, such as mitotic and meiotic double-strand break sites (DSBs). Conserved G4 DNA motifs maintained strong associations with promoters and the rDNA, but not with DSBs. We also performed the first analysis of G4 DNA motifs in the mitochondria, and surprisingly found a tenfold higher concentration of the motifs in the AT-rich yeast mitochondrial DNA than in nuclear DNA. The evolutionary conservation of the G4 DNA motif and its association with specific genome features supports the hypothesis that G4 DNA has in vivo functions that are under evolutionary constraint.

Genome-wide colonization of gene regulatory elements by G4 DNA motifs

G-quadruplex (or G4 DNA), a stable four-stranded structure found in guanine-rich regions, is implicated in the transcriptional regulation of genes involved in growth and development. Previous studies on the role of G4 DNA in gene regulation mostly focused on genomic regions proximal to transcription start sites (TSSs). To gain a more comprehensive understanding of the regulatory role of G4 DNA, we examined the landscape of potential G4 DNA (PG4Ms) motifs in the human genome and found that G4 motifs, not restricted to those found in the TSS-proximal regions, are bias toward gene-associated regions. Significantly, analyses of G4 motifs in seven types of well-known gene regulatory elements revealed a constitutive enrichment pattern and the clusters of G4 motifs tend to be colocalized with regulatory elements. Considering our analysis from a genome evolutionary perspective, we found evidence that the occurrence and accumulation of certain progenitors and canonical G4 DNA motifs within regulatory regions were progressively favored by natural selection. Our results suggest that G4 DNA motifs are ‘colonized’ in regulatory regions, supporting a likely genome-wide role of G4 DNA in gene regulation. We hypothesize that G4 DNA is a regulatory apparatus situated in regulatory elements, acting as a molecular switch that can modulate the role of the host functional regions, by transition in DNA structure.

Selection for the G4 DNA motif at the 5′ end of human genes

Formation of G4 DNA may occur in the course of replication and transcription, and contribute to genomic instability. We have quantitated abundance of G4 motifs and potential for G4 DNA formation of the nontemplate strand of 5' exons and introns of transcripts of human genes. We find that, for all human genes, G4 motifs are enriched in 5' regions of transcripts relative to downstream regions; and in 5' regulatory regions relative to coding regions. Notably, although tumor suppressor genes are depleted and proto-oncogenes enriched in G4 motifs, abundance of G4 motifs in the 5' regions of transcripts of genes in these categories does not differ. These results support the hypothesis that G4 motifs are under selection in the human genome. They further show that for tumor suppressor genes and proto-oncogenes, independent selection determines potential for G4 DNA formation of 5' regulatory regions of transcripts and downstream coding regions.

Genome-wide analysis reveals regulatory role of G4 DNA in gene transcription

G-quadruplex or G4 DNA, a four-stranded DNA structure formed in G-rich sequences, has been hypothesized to be a structural motif involved in gene regulation. In this study, we examined the regulatory role of potential G4 DNA motifs (PG4Ms) located in the putative transcriptional regulatory region (TRR, -500 to +500) of genes across the human genome. We found that PG4Ms in the 500-bp region downstream of the annotated transcription start site (TSS; PG4M(D500)) are associated with gene expression. Generally, PG4M(D500)-positive genes are expressed at higher levels than PG4M(D500)-negative genes, and an increased number of PG4M(D500) provides a cumulative effect. This observation was validated by controlling for attributes, including gene family, function, and promoter similarity. We also observed an asymmetric pattern of PG4M(D500) distribution between strands, whereby the frequency of PG4M(D500) in the coding strand is generally higher than that in the template strand. Further analysis showed that the presence of PG4M(D500) and its strand asymmetry are associated with significant enrichment of RNAP II at the putative TRR. On the basis of these results, we propose a model of G4 DNA-mediated stimulation of transcription with the hypothesis that PG4M(D500) contributes to gene transcription by maintaining the DNA in an open conformation, while the asymmetric distribution of PG4M(D500) considerably reduces the probability of blocking the progression of the RNA polymerase complex on the template strand. Our findings provide a comprehensive view of the regulatory function of G4 DNA in gene transcription.

Quadruplex-Coupled Kinetics Distinguishes Ligand Binding between G4 DNA Motifs

G-quadruplex (or G4 DNA) specific ligands are important potential anticancer molecules as telomerase inhibitors. On the other hand, emerging evidence implicates G4 DNA in regulation of several oncogenes making telomerase inhibitors amenable to undesired effects (Borman, S. (2007) Chem. Eng. News 85 (22), 12-17). Therefore molecules which can discriminate between G4 DNA are of interest, both as telomerase inhibitors and for selective intervention of gene expression. Design of selective molecules requires resolution of the coupled equilibria between intramolecular quadruplex-formation and bimolecular ligand-binding. Several previous studies have reported G4-ligand binding kinetics, however the primary equilibrium of intramolecular G4 DNA folding/unfolding was not considered. Here, we quantitatively assess the linked equilibrium in G4-ligand complexes using a novel real time surface plasmon resonance-based technique. Kinetic constants for G4 folding/unfolding and ligand binding were simultaneously determined, for the first time, from a single reaction by resolving the coupled equilibrium. We demonstrate the coupled model by showing that affinity of TMPyP4 (a well-established anticancer telomerase inhibitor) for the human telomere quadruplex is only 3-fold more than the c-MYC promoter G4, which is known to repress c-MYC. This provides quantitative rationale to poor selectivity of TMPyP4 in recently observed cell-based assays. In the light of recent advances indicating G4's regulatory potential in several important genes, quantitative evaluation of selectivity vis-à-vis affinity as presented here will augment design and preliminary screening of new molecules.

Extensive selection for the enrichment of G4 DNA motifs in transcriptional regulatory regions of warm blooded animals

A comprehensive analysis of potential G4 DNA motifs (G4Ms) in genomic regions flanking transcription start sites (TSS) was performed across 13 animal species. We found that G4Ms are significantly enriched in the transcriptional regulatory regions (TRRs) of warm-blooded animals. Further analysis of human genes in different temporal groups reveals that the enrichment is not specific to genes found only in warm-blooded species but instead exist in a wide range of genes. Our findings therefore suggest that the high prevalence of G4Ms in TRRs is extensively selected in warm-blooded animals, supporting the hypothesis that G4Ms are involved in the regulation of gene transcription.

Enrichment of G4 DNA motif in transcriptional regulatory region of chicken genome

G-quadruplex or G4 DNA is a stable, four-stranded DNA structure formed from guanine-rich regions. Based on the hypothesis that G4 DNA participated in the regulation of transcription, we analyzed G4 DNA in 5kb 5′ flanking regions of 2892 chicken RefSeq genes with annotated transcription start sites (TSS). In total, 4769 distinct putative G4 DNA motifs (G4M) were identified in 1880 (65%) genes. The pattern of distribution of the G4M showed a gradient along the 5′ flanking regions; from −5 to −4kb, to −1kb to the TSS, the frequency (number of G4M per kilobase) increased significantly from 0.192 to 0.768, and 62.56% of the G4M in the 1kb upstream regions were located in the region −400 to the TSS, where a core promoter is always present. Thus, 38.24% of the analyzed genes contained at least one G4M in the 400bp upstream region. Our findings support the hypothesis that G4M are involved in gene transcription.

Genome-wide prediction of G4 DNA as regulatory motifs: Role inEscherichia coliglobal regulation

The role of nonlinear DNA in replication, recombination, and transcription has become evident in recent years. Although several studies have predicted and characterized regulatory elements at the sequence level, very few have investigated DNA structure as regulatory motifs. Here, using G-quadruplex or G4 DNA motifs as a model, we have researched the role of DNA structure in transcription on a genome-wide scale. Analyses of >61,000 open reading frames (ORFs) across 18 prokaryotes show enrichment of G4 motifs in regulatory regions and indicate its predominance within promoters of genes pertaining to transcription, secondary metabolite biosynthesis, and signal transduction. Based on this, we predict that G4 DNA may present regulatory signals. This is supported by conserved G4 motifs in promoters of orthologous genes across phylogenetically distant organisms. We hypothesized a regulatory role of G4 DNA during supercoiling stress, when duplex destabilization may result in G4 formation. This is in line with our observations from target site analysis for 55 DNA-binding proteins in Escherichia coli, which reveals significant (P<0.001) association of G4 motifs with target sites of global regulators FIS and Lrp and the sigma factor RpoD (sigma70). These factors together control >1000 genes in the early growth phase and are believed to be induced by supercoiled DNA. We also predict G4 motif-induced supercoiling sensitivity for >30 operons in E. coli, and our findings implicate G4 DNA in DNA-topology-mediated global gene regulation in E. coli.

The Yeast STM1 Gene Encodes a Purine Motif Triple Helical DNA-binding Protein

The formation of triple helical DNA has been evoked in several cellular processes including transcription, replication, and recombination. Using conventional and affinity chromatography, we purified from Saccharomyces cerevisiae whole-cell extract a 35-kDa protein that avidly and specifically bound a purine motif triplex (with a K(d) of 61 pM) but not a pyrimidine motif triplex or duplex DNA. Peptide microsequencing identified this protein as the product of the STM1 gene. Confirmation that Stm1p is a purine motif triplex-binding protein was obtained by electrophoretic mobility shift assays using either bacterially expressed, recombinant Stm1p or whole-cell extracts from stm1Delta yeast. Stm1p has previously been identified as G4p2, a G-quartet nucleic acid-binding protein. This suggests that some proteins actually recognize features shared by G4 DNA and purine motif triplexes, e.g. Hoogsteen hydrogen-bonded guanines. Genetically, the STM1 gene has been identified as a multicopy suppressor of mutations in several genes involved in mitosis (e.g. TOM1, MPT5, and POP2). A possible role for multiplex DNA and its binding proteins in mitosis is discussed.