MD simulations and FRET reveal an environment-sensitive conformational plasticity of importin-ß.

The nuclear pore complex mediates nucleocytoplasmic transport of macromolecules in eukaryotic cells. Transport through the pore is restricted by a hydrophobic selectivity filter comprising disordered phenylalanine-glycine-rich repeats of nuclear pore proteins. Exchange through the pore requires specialized transport receptors, called exportins and importins, that interact with cargo proteins in a RanGTP-dependent manner. These receptors are highly flexible superhelical structures composed of HEAT-repeat motifs that adopt various degrees of extension in crystal structures. Here, we performed molecular-dynamics simulations using crystal structures of Importin-ß in its free form or in complex with nuclear localization signal peptides as the starting conformation. Our simulations predicted that initially compact structures would adopt extended conformations in hydrophilic buffers, while contracted conformations would dominate in more hydrophobic solutions, mimicking the environment of the nuclear pore. We confirmed this experimentally by Förster resonance energy transfer experiments using dual-fluorophore-labeled Importin-ß. These observations explain seemingly contradictory crystal structures and suggest a possible mechanism for cargo protection during passage of the nuclear pore. Such hydrophobic switching may be a general principle for environmental control of protein function.

Reporter assays for studying quadruplex nucleic acids.

DNA and RNA G-quadruplexes have gained increasing attention due to their potential role in a wide range of biological functions. The majority of functional studies characterize the influence of quadruplexes in gene expression including transcription and translation. Many of these studies have used reporter assays to elucidate the effect of quadruplexes at certain positions in promoters and untranslated mRNA regions (UTRs). Reporter assays are the preferred method to ascertain the biological function of DNA or RNA G-quadruplexes intracellularly due to their ready availability, fast cloning and experimental setup and reproducibility. Moreover, these reporter assays are also helpful to compare or screen for selectivity and efficacy of small molecules that target DNA and RNA G-quadruplexes in the cellular context. Here we briefly discuss various aspects of reporter assays followed by a review of available studies using reporter assays to understand the role and functions of DNA and RNA quadruplexes in gene expression.

Predictable suppression of gene expression by 5'-UTR-based RNA quadruplexes.

Four-stranded DNA and RNA quadruplexes or G4 motifs are non-B DNA conformations that are presumed to form in vivo, although only few explicit evidence has been reported. Using bioinformatics the presence of putative DNA G-quadruplexes within critical promoter regions has been demonstrated and a regulatory role in transcription has been suspected. However, in genomic DNA the presence of the complementary strand interferes with the potential to form a quadruplex motif. Contrarily RNA G4 motifs have no such limitation and consequently strong interference with gene expression is suspected. Nevertheless, experimental evidence is scarce. Here we show a well-defined structure-function relationship of synthetic quadruplex sequences in 5'-UTRs in multiple mammalian cell-lines. We establish a universal 'translational suppressor' effect of these motifs on gene expression at the translational level and show for the first time that specific features such as loop-length and the number of 'GGG'-repeats further determine the suppressive impact. Moreover, a consistent and predictable repression of gene expression is observed for naturally occurring RNA G4 motifs, augmenting the functional relevance of these unusual nucleic acid structures.

Metastases suppressor NM23-H2 interaction with G-quadruplex DNA within c-MYC promoter nuclease hypersensitive element induces c-MYC expression.

Regulatory influence of the G-quadruplex or G4 motif present within the nuclease hypersensitive element (NHE) in the promoter of c-MYC has been noted. On the other hand, association of NM23-H2 to the NHE leads to c-MYC activation. Therefore, NM23-H2 interaction with the G4 motif within the c-MYC NHE presents an interesting mechanistic possibility. Herein, using luciferase reporter assay and chromatin immunoprecipitation we show NM23-H2 mediated c-MYC activation involves NM23-H2-G4 motif binding within the c-MYC NHE. G4 motif complex formation with recombinant NM23-H2 was independently confirmed using fluorescence energy transfer, which also indicated that the G4 motif was resolved to an unfolded state within the protein-bound complex. Taken together, this supports transcriptional role of NM23-H2 via a G4 motif.

Kinetic resolution of bimolecular hybridization versus intramolecular folding in nucleic acids by surface plasmon resonance: application to G-quadruplex/duplex competition in human c-myc promoter.

The human oncogene c-myc is regulated by G-quadruplex formation within the nuclease hypersensitive element (NHE III(I)) in the c-myc promoter, making the quadruplex a strong anti-cancer target. With respect to this, the competing equilibrium between intramolecular quadruplex folding and bimolecular duplex formation is poorly understood and very few techniques have addressed this problem. We present a method for simultaneously determining the kinetic constants for G-quadruplex folding/unfolding and hybridization in the presence of the complementary strand from a single reaction using an optical biosensor based on surface plasmon resonance (SPR). Using this technique, we demonstrate for the first time that quadruplex formation in the c-myc promoter is favored at low strand concentrations. Our results indicate favorable quadruplex folding (equilibrium folding constant K(F) of 2.09 calculated from the kinetic parameters: folding rate constant, k(f) = 1.65 x 10(-2) s(-1) and unfolding rate constant, k(u) = 7.90 x 10(-3) s(-1)) in 150 mM K+. The hybridization rate constants detected concurrently gave a bimolecular association constant, k(a) = 1.37 x 10(5) M(-1) s(-1) and dissociation constant, k(d) = 4.94 x 10(-5) s(-1). Interestingly, in the presence of Na+ we observed that G-quadruplex folding was unfavorable (K(F) = 0.54). Implication of our results on the c-myc transcription activation model is discussed in light of aberrant c-myc expression observed on destabilization of the G-quadruplex.

RNA quadruplexes.

Opposed to DNA quadruplex sequences, RNA quadruplexes are still less well characterized. On the other hand, RNA quadruplexes are found to be at least as stable as their DNA counterparts. They show the same dependence on metal ions but seem to be much more restricted with respect to the adopted conformations. Other than DNA, which is mostly found to be double-stranded inside cells, RNAs are produced during transcription without its complementary sequence. The absence of a second strand that is able to hybridize and form a duplex makes the folding of RNA quadruplexes a likely event of intramolecular structure formation. Consequently, the formation of RNA quadruplexes in cellular RNAs has recently been suggested and the study of their influence and potential roles in cellular processes has just started. Here we give an overview of the RNA quadruplex field, summarizing issues such as structures, stabilities, and anticipated roles of these interesting four-stranded, guanosine-rich sequences.

Small molecule-triggered assembly of DNA nanoarchitectures.

The utilization of toehold-containing DNA strands allows for the assembly of complex nanostructures via kinetically driven hybridization reactions. Here, we have rendered this strategy ligand-dependent, resulting in small-molecule-inducible DNA nanoarchitectures.

Guanine quadruplex DNA structure restricts methylation of CpG dinucleotides genome-wide.

Cytosine methylation in mammals is important for epigenetic control of the transcriptome. Although altered methylation is frequently encountered in disease situations, particularly cancer, the relationship between genome-wide methylation and DNA structure is poorly understood. It is now evident that alternative DNA forms are functionally relevant in replication, recombination and transcription. Herein, we researched the role of alternative DNA structure in cytosine methylation using quadruplex DNA as a case study. Our findings from analysis of 2.1 million CpGs in humans, across 12 tissues from the Human Epigenome Project (HEP), revealed a striking correlation within each tissue: CpGs with low methylation were enriched (P = 5.24E(-20)) whereas CpGs with high methylation were relatively depleted (P = 9.28E(-15)), within quadruplex-forming regions. This was further substantiated on considering 1.07E(8) methylcytosines from genome-wide sequencing within embryonic stem cells and differentiated fibroblasts. To further test the predictions we experimentally determined methylation in >600,000 CpGs across 18 individuals using bisulfite mapping and found significantly low methylation of CpGs within quadruplex-forming regions (P = 1.36E(-08)). Together, these suggest the role of guanine-quadruplexes in CpG methylation and directly impact our understanding of the inter-relationship between DNA conformation and global cytosine methylation.

Genome-wide analysis predicts DNA structural motifs as nucleosome exclusion signals.

Several factors are known to determine chromatin organization. However, the role of non-canonical DNA structure has not been studied in this context. Our recent observations indicated a widespread role of a particular non-canonical DNA structure, the G-quadruplex, or G4 motifs, in gene regulation. Herein, we first analyzed potential G4 (PG4) motif occurrence vis-à-vis nucleosome occupancy signals. Genome-wide analysis using reported nucleosome positions in Saccharomyces cerevisiae and human (ENCODE regions and 3239 promoters) showed exclusion of nucleosomes by regions that have a relatively high density of PG4 motifs (P < 0.0001). This was supported by the enrichment of PG4 motifs within DNase I hypersensitive sites, which generally exclude nucleosomes. Based on these, we asked whether PG4 motifs had any distinct regulatory function. Two non-overlapping gene-sets in humans were tested-with PG4-enriched (Set I) or nucleosome-enriched (Set II) promoters. Gene-sets I and II were enriched in distinct functions: apoptosis and cellular signaling vs. development and immunity-related, respectively (P < 0.01). Sets I and II also showed different tissue-specific expression in 35 normal human tissues. In S. cerevisiae, we noted significant enrichment of PG4 motif regulated genes in cellular response to heat-shock, while genes with nucleosome-enriched promoters were not significantly represented. Our results show a structural motif as a possible nucleosome exclusion signal for the first time, and predict an alternate/additional regulatory role of G4 motifs, which could be distinct from gene regulation by remodeling of nucleosomes.

Genome-wide computational and expression analyses reveal G-quadruplex DNA motifs as conserved cis-regulatory elements in human and related species.

Using a combination of in silico and experimental approaches, we present evidence that the G-quadruplex (G4) motif (an alternative higher-order DNA conformation) has regulatory potential. Genome-wide analyses of 99980 human, chimpanzee, mouse, and rat promoters showed enrichment of sequence with potential to adopt G4 (potential G4 or PG4) motifs near transcription start sites (TSS; P < 0.0001), supporting earlier findings. Interestingly, we found >700 orthologously related promoters in human, mouse, and rat conserve PG4 motif(s). The corresponding genes have enriched (z score > 4.0) tissue-specific expression in 75 of 79 human tissues and are significantly overrepresented in signaling and regulation of cell-cycle (P < 10(-05)). This is supported by results from whole genome expression experiments in human HeLa S3 cells following treatment with TMPyP4 [5,10,15,20-tetra(N-methyl-4-pyridyl) porphine chloride], which is known to bind the G4 motif inside cells. Our results implicate G4-motif mediated regulation as a more general mode of transcription control than currently appreciated.

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.

Genome-wide prediction of G4 DNA as regulatory motifs: role in Escherichia coli global 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.

Quadruplex-duplex competition in the nuclease hypersensitive element of human c-myc promoter: C to T mutation in C-rich strand enhances duplex association.

The nuclease hypersensitive element NHE III(I) is an important anti-cancer target as the transcription of oncogene c-myc is largely regulated by it. It has been postulated that regulatory control is mediated by G-quadruplex formation in the NHE anti-sense strand through a competition between the duplex and the quadruplex states. A mutation in the NHE has been implicated in cancer. In this study, the reported mutation has been characterized vis-a-vis the kinetics of i-tetraplex formation (in the sense strand) and its effect on duplex formation. We found that i-tetraplex formation was destabilized by approximately 1.4 kcal/mol (DeltaDeltaG at 20 degrees C, pH 5.8). Observed hysteresis allowed us to analyze the kinetics of folding for the mutant (M3). Though we observed higher association (DeltaEon approximately -23.4 kcal/mol) and dissociation (DeltaEoff approximately 22.1 kcal/mol) activation energies (at pH 5.3) for the wild-type (P1) tetraplex folding, the kinetics of folding and unfolding for M3 was somewhat faster at pH 5.3 and 5.8. Interestingly, Surface plasmon resonance (BIAcore) analysis of hybridization at pH 6.6 indicated a higher association constant for M3 (approximately 22.5 x 10(4)M(-1)s(-1)) than P1 (approximately 3.2 x 10(4)M(-1)s(-1)). The equilibrium dissociation constants also indicated favorable duplex association for M3 (approximately 22.2 and approximately 190.6 nM for M3 and P1, respectively). We envisage that the increased affinity for the duplex state due to the mutation could play a functional role in the aberrant regulation of c-myc.