Do DNA Triple Helices or Quadruplexes Have a Role in Transcription?

Certain DNA sequences preferentially adopt multistranded, non-B-form structures under physiological conditions. These include three-stranded DNA triplexes and four-stranded DNA quadruplexes. Several lines of evidence suggest that multiplex structures can form in vivo, either from the addition of oligonucleotides or through the transient formation of single-stranded regions. The consequences of multiplex structures on many DNA-dependent biological processes have been described. In this chapter we will review the effects of different DNA multiplexes on the process of transcription. The influence of parameters such as multiplex type and multiplex formation conditions on different transcription mechanistic steps in organisms spanning from prokaryotes to Xenopus oocytes and mammalian cells will be discussed.

Introduction

Oligopurine/oligopyrimidine-rich DNA sequences have long been known to preferentially adopt multistranded structures quite different from the familiar Watson-Crick base-paired, right-handed, antiparallel-stranded, B-form double-helical structure.^1,2 Examples include triple helical DNA (triplexes) and G-quartet-containing quadruplexes (G₄), both of which can form under physiological conditions, and once formed, are extremely stable.^3,6

Although a considerable amount of information is available about the properties of DNA multiplex structures in vitro, little is known about their existence and biological roles in vivo.^7,10 Sequences capable of forming these structures abound in all eukaryotic organisms.¹¹ Examples include the G-rich 3' overhangs on the ends of chromosomes and long oligopurine tracts within the promoter regions of several genes. DNA multiplexes have been invoked as necessary intermediates in many biological processes, including chromosome condensation, recombination, replication, telomere function, and transcriptional control.^12,18 Potentially deleterious multiplex structures could also form as a consequence of essential biological processes that use the DNA as a template (e.g., replication and transcription) with removal of these structures then being necessary for viability.^19,20 In this chapter, we review the literature to address these questions: (1) can DNA multiplexes such as triplexes and quadruplexes affect the process of transcription, and (2) do DNA multiplexes play a role in transcription regulation in vivo?

DNA Triplexes

As has been well known, certain nucleic acid sequences preferentially adopt a triple-helical structure under the proper conditions.^3,4 Triplex structures are characterized by a single polynucleotide strand residing in the former major groove of a homopurine-homopyrimidine duplex (fig. 1A), which are reviewed in Chapter 1 of this book. Two triplex motifs are known. The parallel- or pyrimidine-motif (Py) has a C- or T-rich third strand bound in a parallel orientation with respect to the duplex homopurine strand, while the antiparallel- or purine-motif (Pu) has the opposite orientation and a primarily A- or G-rich third strand. Both types of triplexes utilize Hoogsteen hydrogen bonding between their third strands and purines in their duplex acceptors. The primary base triplets of Py triplexes are T•A•T and C•G•C⁺, while the base triplets of Pu triplexes are T•A•A, T•A•T, and C•G•G (fig. 1B,C). Py triplexes can occur with RNA being present as any of the three strands, while Pu triplexes only occur with DNA.^21,22 Both inter- and intramolecular triplexes have been observed. The former involves a third DNA strand that originates from either a second DNA molecule or from a distal site on the same molecule, while the latter involves homopurine-homopyrimidine sequences immediately adjacent to the duplex acceptor (fig. 2A). Four isomers of intramolecular triplexes can exist dependent on the half-element strand that serves as the third strand (fig. 2B). Intramolecular triplexes are also known as H-DNA or H'-DNA, depending on whether they contain Py or Pu triplexes, respectively.

In theory, a homopurine-homopyrimidine duplex should be capable of forming triplexes of either motif. However, under physiological conditions, cytosine protonation is not favored, and C•G•G is the most stable base triplet in the purine motif. T-rich nucleic acids would be expected, therefore, to form Py triplexes, while G-rich DNAs would form Pu triplexes. The same is true for intramolecular triplexes, with the additional condition that the different isomers are not isoenergetic.^23,24 In both intermolecular and intramolecular triplexes, contiguous homopurine-homopyrimidine runs of at least 10 base pairs are required for the duplex acceptor, since shorter triplexes are not very stable under physiological conditions, and even single base interruptions are known to greatly destabilize triplexes.^25,27 Triplex formation is kinetically slow compared to duplex annealing.^25,28 However, once formed, triplex RNA and DNA are very stable, exhibiting half-lives on the order of days.^25,29

DNA Quadruplexes

DNAs (and RNAs) containing guanine tracts will associate in vitro to form four-stranded, right-handed helices known as quadruplexes or tetraplexes.^5,6 These G₄ nucleic acids are characterized by stacked G-quartet structures, square planar arrays of four guanines, each serving as the donor and acceptor of two Hoogsteen hydrogen bonds. Electronegative carbonyl oxygens line the center of the G-ring, where they interact with a suitably sized monovalent cation, typically Na⁺ or K⁺ (fig. 3A). Several isoforms of DNA and RNA quadruplexes have been described by NMR and X-ray crystallographic studies.^30,31 The isoforms are characterized by either parallel or cis or trans antiparallel strand orientations and may be composed of either intermolecular or intramolecular or both types of hydrogen bonding (fig. 3B). G-rich nucleic acids can be highly polymorphic, adoption of the exact G₄ structure depending on several factors including nucleotide sequence, strand concentration, and the types and concentrations of monovalent, divalent, and polyvalent cations present. Formation of G₄ nucleic acids requires one or more polynucleotide strands, each containing one or more runs of two or more contiguous guanosine nucleotides. Four parallel-stranded intermolecular G4 nucleic acids (Fig. 3B, structure 1) require only a single G-tract. However, their strand stoichiometry and very slow formation kinetics lessen the likelihood that this form of G₄ nucleic acid often occurs in vivo. More likely in vivo are G₄ multiplex species formed from polynucleotides containing multiple G-runs, which have the ability to form intramolecular Hoogsteen hydrogen bonds. These species include purely intramolecular G4' nucleic acids that require only a single DNA or RNA molecule (fig. 3B,2), and G'2 hairpin dimer species that can link two separate polynucleotides (fig. 3B,3,4). Formation of intermolecular species may be rather slow under physiological conditions, though intermediates containing intramolecular G•G Hoogsteen base pairs (e.g., G') form quite rapidly.³² Once formed, each of the G₄ species is quite stable, with measured enthalpies approaching -25 kcal/mole of G-quartet.³³ Thus, equilibration between different G₄ species is glacially slow under physiological conditions, and the thermodynamically favored structure is not necessarily the species that occurs in vivo.

Multiplexes and Transcription

A considerable body of evidence indicates that multiplex nucleic acids may affect transcription. Briefly, transcription requires a start site (+1), usually indicated by an arrow in most schematic representation, to define where transcription begins and in which direction it proceeds. In addition, transcription requires an RNA polymerase, which is the enzyme that catalyzes the template-directed sequential condensation of ribonucleotides to generate a product RNA. In many cases, the RNA polymerase itself does not directly recognize the +1 site but relies on auxiliary proteins for this purpose. In addition, these proteins and/or RNA polymerase do not typically interact directly with the +1 site but rather recognize nearby sequences known as the promoter. Once these proteins and RNA polymerase have assembled on a promoter, addition of ribonucleotides will allow transcription to begin. The process of transcription initiation is shown schematically in Figure 4A. Afterward, the transcribing RNA polymerase can proceed downstream of the +1 site and generate an RNA transcript (wavy line) in a process known as elongation (fig. 4B). Note that transcription is a multistep process: promoter recognition, initiation, elongation, and that the overall rates of transcription depend on the efficiencies of these different steps. These steps are often affected by a class of nucleic acid binding proteins (specific transcription factors), which can greatly modulate transcription.

Multiplex structures are believed to interfere with transcription primarily through two different mechanisms: promoter occlusion and elongation arrest.³⁴ In promoter occlusion (fig. 4C), a DNA multiplex interferes with the binding of a transcription factor to a gene promoter. Note that, for occlusion to occur, the sites of transcription factor binding and multiplex formation need to overlap, and the extent of overlap necessary depending on the transcription factor and multiplex structure used. As shown in this example (fig. 4C), the typical occluded protein is a specific transcription factor that normally stimulates transcription initiation or elongation. However, it is also possible to inhibit transcription of a targeted promoter by occluding a DNA-binding basic transcription factor (e.g., TFIID). Likewise, it is possible to stimulate transcription through protein occlusion, if the occluded protein is a transcriptional repressor. In elongation arrest (fig. 4D), a post-initiation RNA polymerase II has its progress impeded by a downstream-situated multiplex. Note that a multiplex alone usually cannot effectively impede elongation by an RNA polymerase, especially eukaryotic RNA polymerases that normally function in a chromatin environment. Thus, unless the multiplex is located immediately downstream of a transcription pause or termination site, it is usually necessary for the multiplex to direct a subsequent covalent modification of the template (e.g., a cross-link or strand break) that renders it unsuitable for elongation.

Conceivably, there are several other mechanisms by which a multiplex structure might affect transcription. Some are shown schematically in Figure 5. For example, multiplex-forming oligonucleotides could themselves adopt structures that bind proteins involved in transcription (fig. 5A). Note that these could include proteins directly involved in RNA synthesis (e.g., transcription factors) as well as proteins that ultimately modulate their activity (e.g., signal transduction proteins). Given the appropriate sequence homology, multiplex-forming oligonucleotides could bind to RNA transcripts through conventional Watson-Crick base pairing, thereby leading to transcript degradation (and apparent loss) through endogenous RNase H activity (fig. 5B). Alternatively, multiplexes could inhibit transcription through the delivery of nonspecific and specific inhibitors of transcription, instead of through the direct occlusion of stimulatory transcription factors (Fig. 5C). Discerning these possible mechanisms relies on the use of adequate and sufficient controls, including mutagenesis of multiplex-forming sequences, order-of-addition experiments, and physical verification of multiplex structures.

Intermolecular Triplexes and Transcription

Most studies on the modulation of transcription by multiplexes have been done with oligonucleotides and intermolecular triplexes, because of the ease of forming such structures, the variety of controls that can be performed, and the flexibility possible through use of chemically modified triplex-forming oligonucleotides (TFOs). Studies on intermolecular triplexes have been performed both in vitro and in vivo, “in vivo” referring to any living organism, including cultured cells.

Intermolecular triplex effects on transcription have been investigated in vitro for a number of model systems, including prokaryotic, eukaryotic, and various hybrid systems. A list of representative studies is presented in Table 1. Both triplex motifs, purine and pyrimidine, have been explored, as have binding modes that are less well defined. Occlusion of specific transcription factors or general transcription factor/RNA polymerase binding has been proposed and/or reported in many studies.^{35,39,42,45,47,51,54,56,71} Typical observed results have been in the range of 50% to 105% transcription inhibition when 0.2 to 50 μM TFO was present. Control reactions usually involved oligonucleotides (ODN) that were not capable of triplex formation or templates that lacked TFO binding sites. Some unusual findings include the demonstration that a TFO targeting an upstream stimulatory transcription factor could apparently inhibit transcript appearance through partial hybridization to these transcripts and RNase H-mediated RNA degradation, and that transcription was inhibited when triplexes were located distal to transcription factor binding sites.39,88 Promotion of transcription has also been described in vitro, through the direct delivery of transcription activators by hybrid TFOs.⁸⁵ Inhibition of transcription elongation has been observed in vitro as well.^{38,40,49,52,58,60,61,68,72} Typical effects range from 60% to 95% transcription inhibition, depending on several factors including the location of the intermolecular triplex relative to the start site of transcription, the type of RNA polymerase investigated, and whether the TFO was noncovalently bound or whether it directed a covalent modification of the DNA template. Taken together, these data demonstrated that many types of intermolecular triple helices can specifically and effectively inhibit several types of transcription through multiple mechanisms in vitro.

Given the observed successes with intermolecular triplexes in vitro, several research groups have investigated the effects of intermolecular triplexes on transcription in vivo (see Table 1). Both transcription factor occlusion and polymerase elongation mechanisms of triplex action have been investigated in vivo, with reports of efficiencies in excess of -90% reported in some circumstances, depending on oligonucleotide type, delivery method, and target site. Substantial transcription stimulation in vivo mediated by an activation domain peptide/triplex-forming oligonucleotide hybrid has also been reported.⁸⁵ These findings suggest that intermolecular triplexes appear to be an efficient means of inhibiting specific gene transcription in vivo.

In experiments performed on in vivo targets, researchers encounter complications not found with in vitro experiments, including maintaining oligonucleotide stability in the presence of serum and cellular nucleases, delivering adequate concentrations of TFO to the proper cellular compartment (nucleus), and ensuring that triplex-formation actually occurs. Each of these difficulties has been addressed by a variety of means. Stability questions have been addressed by chemical modifications of the TFO termini and/or its phosphodiester backbone.^{36,41,42,44,46,47,53,55,62,66,69,70,75,82,84} Delivery difficulties have been surmounted by transfection with cationic lipids, electroporation, microinjection, or synthesis in situ.^{48,53,55,57,65,67,70,76,80,81,83,85,86} Even triplex formation, which can be highly problematic in an intracellular milieu with its high protein concentrations and surfeit of nonspecific nucleic acid targets, has been overcome by first preforming triplexes on their plasmid targets in vitro and then introducing the entire complex into cells.^{41,44,46,50,63,64,66,69,71,73,75,78,79,82,87} Note that these ex vivo experiments, although successful at addressing particular aspects of triplex-mediated transcription modulation, do not completely address the overall feasibility of triplexes in vivo. In addition, since very few investigators have actually demonstrated triplex formation in vivo, and oligonucleotides can affect cells through multiple specific and nonspecific mechanisms, most studies supporting triplex effects in vivo are not as compelling as they could be.

Intramolecular Triplexes and Transcription

Oligopurine•oligopyrimidine sequences have long been understood to play an instrumental role in the regulation of transcription for many genes.¹ It has also been well known that certain oligopurine•oligopyrimidine sequences, especially those possessing mirror repeats, can form intramolecular triplexes in vitro under conditions of low pH or increased negative superhelicity.³ Thus it has been tempting to speculate that intramolecular triplexes are responsible for the transcriptional regulation observed at these sites. There is some evidence that intramolecular triplexes can form in vivo, albeit under less than physiological conditions in prokaryotic systems.^89,90 Additionally, in triplex-specific antibody studies, cross-reactive structures have been identified near the centromeres of chromosomes.^12,91 However, most reports in the literature regarding the involvement of H-DNA (or its purine-motif counterpart, H'-DNA) on transcription are only suppositions; few researchers have tested whether these sequences actually form intramolecular triplexes, and most physical studies have been performed in vitro. Nonetheless, a few exemplary studies have been done to investigate the possible role of intramolecular triplex structures on transcriptional regulation. Some are presented in Table 2.

Intramolecular triplexes may affect transcription from two locales: either proximally upstream the transcription start site or at any distance downstream. In the former case, intramolecular triplexes located within gene promoters are believed to arise in response to increased negative superhelical tension, which can result from nearby transcription. Such triplexes could then inhibit subsequent transcription events by displacing necessary transactivating proteins (fig. 4C) or by recruiting repressive proteins (fig. 5C). An alternative view is that the single-stranded region resulting from intramolecular triplex formation could serve as an entry point for RNA polymerase and thus serve as an activator of transcription.¹⁰⁵ In the latter case, downstream intramolecular triplexes could arise as a result of processes that locally denature the DNA template (e.g., replication, transcription). These downstream triplexes would then either impede subsequent transcription elongation (fig. 4D) or inhibit transcription elongation by sequestering essential proteins (fig. 5A) or by delivering repressive proteins (fig. 5C).

Promoter-based intramolecular triplex effects on transcription have been reported to be quite variable, with magnitudes ranging from highly stimulatory to no effect to moderately inhibitory.^{15,92,94,96,97,102,104} More telling have been the results of the corresponding control experiments, which in the majority of studies showed no correlation between intermolecular triplex formation and transcriptional strength.^{92,94,97,102,104} For downstream intramolecular triplexes, significant inhibitory effects have been consistently reported both in vitro and in vivo, though their exact correlation with a specific triplex structure has been somewhat weak.^98,101 All in all, these studies suggested that the transcriptional effects ascribed to relatively short polypurine•polypyrimidine sequences located upstream of many genes is most likely not the result of intramolecular triplex formation, whereas the transcriptional effects observed with very long downstream polypurine•polypyrimidine sequences may well involve some form of intramolecular triplex, especially of the H' variety.

Quadruplexes and Transcription

While quadruplexes, especially of the G-quartet variety, have primarily been invoked as playing a role in the biogenesis of chromosome telomeres, recent studies have suggested that they may also have a role in the transcriptional regulation of certain genes.^9,10 G-rich sequences capable of forming quadruplex structures in vitro have been identified in the immunoglobulin switch region, the c-myc promoter, and upstream of the insulin gene.^32,106,107 Use of a single-chain antibody fragment probe specific for guanine quadruplexes has led to identification of cross-reactive species in the macronucleus but not the micronucleus of Stylonychia lemnae, suggesting that quadruplexes do exist in vivo.¹⁷ Less clear is how such quadruplex structures arise, although arguments concerning the formation of intramolecular triplexes, including local negative superhelical tension in the promoter region, chromatin remodeling, and the consequence of transcription and/or replication events may also apply here.^9,19 At present only a few studies directly describe quadruplex effects on transcription (see Table 3). Effects are believed to occur at the level of transcription factor occlusion and/or transcription factor recruitment, and significant effects, both stimulatory and repressive, have been observed.^108,111 One major weakness of all these studies is the lack of in vivo characterization of G₄ structures, which makes ascribing transcriptional effects to bona fide quadruplexes a bit tenuous.

Conclusions

Do DNA multiplexes affect transcription? From the aforementioned studies, the following conclusions can be made: (1) Some intermolecular triplexes can significantly repress transcription in vitro. However, their effectiveness in vivo often requires triplex preassembly in vitro. (2) Intramolecular triplexes may be responsible for impeding transcription on long, repeated sequences. (3) G-quadruplexes may affect transcription.

Acknowledgments

This work was supported by a grant from the Robert A. Welch Foundation (G-1199), and is dedicated to the memory of Claude Hélène (1938-2003).

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