Articles Cite This: ACS Chem. Biol. 2017, 12, 2609-2618
pubs.acs.org/acschemicalbiology
Superhelicity Constrains a Localized and R‑Loop-Dependent Formation of G‑Quadruplexes at the Upstream Region of Transcription Ke-wei Zheng,†,§ Yi-de He,†,‡,§ Hong-he Liu,† Xin-min Li,† Yu-hua Hao,† and Zheng Tan*,† †
State Key Laboratory of Membrane Biology, Institute of Zoology, ‡University of Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing 100101, People’s Republic of China S Supporting Information *
ABSTRACT: Transcription induces formation of intramolecular G-quadruplex structures at the upstream region of a DNA duplex by an upward transmission of negative supercoiling through the DNA. Currently the regulation of such G-quadruplex formation remains unclear. Using plasmid as a model, we demonstrate that while it is the dynamic negative supercoiling generated by a moving RNA polymerase that triggers a formation of a G-quadruplex, the constitutional superhelicity determines the potential and range of the formation of a G-quadruplex by constraining the propagation of the negative supercoiling. G-quadruplex formation is maximal in negatively supercoiled and nearly abolished in relaxed plasmids while being moderate in nicked and linear ones. The formation of a G-quadruplex strongly correlates with the presence of an R-loop. Preventing R-loop formation virtually abolished G-quadruplex formation even in the negatively supercoiled plasmid. Enzymatic action and protein binding that manipulate supercoiling or its propagation all impact the formation of G-quadruplexes. Because chromosomes and plasmids in cells in their natural form are maintained in a supercoiled state, our findings reveal a physical basis that justifies the formation and regulation of G-quadruplexes in vivo. The structural features involved in G-quadruplex formation may all serve as potential targets in clinical and therapeutic applications.
G
-quadruplexes are four-stranded structures formed in guanine-rich nucleic acids. G-quadruplex forming motifs are significantly enriched around the transcription start site (TSS) in the genes of warm-blooded animals.1−4 Many studies suggested a regulatory role of G-quadruplexes in promoters in transcription, which was supported by the fact that ligands specifically bound with a G-quadruplex affect transcription efficiency.5−15 Our recent studies have shown that the formation of an intramolecular G-quadruplex can be induced by transcription in a linear DNA duplex around a TSS by two distinct mechanisms (Figure 1A). In the region downstream of TSS, a strand-biased formation of a G-quadruplex can be triggered, which occurs on the nontemplate, but not on the template DNA strand, by an approaching RNA polymerase (RNAP) via a destabilization of the base-pairing in the immediate front of the enzyme.16 When a G-quadruplex forming sequence is present in the upstream region of a TSS, G-quadruplex formation is induced without strand discrimination by the upward transmission of negative supercoiling generated by a transcribing RNAP.3 This G-quadruplex induction can take place in a linear DNA duplex in a distal locus thousands of base-pairs (bp) away from a TSS in the presence of PEG that was used to mimic a molecular crowding effect. According to their distribution in genes of mammalians, roughly one-third to half of G-quadruplex forming motifs reside at the upstream side of TSSs. This means that transcription generated negative supercoiling may potentially induce G© 2017 American Chemical Society
Figure 1. Transcription mediated (A) formation of an intramolecular DNA G-quadruplex and (B) structural changes in a DNA duplex. (A) The upward propagation of negative supercoiling induces formation of G-quadruplexes in the upstream region of TSS on both DNA strands. G-quadruplex formation downstream of TSS is triggered on the nontemplate, but not on the template strand by the destabilization of the DNA duplex in the immediate front of an approaching RNAP. (B) When tracking along a DNA, an RNA polymerase (RNAP) creates negative supercoiling behind it and positive supercoiling in front of it, which propagate in a divergent direction. The nascent RNA transcript may remain annealed or a released RNA transcript rehybridizes with the template DNA strand forming an R-loop structure.
Received: May 23, 2017 Accepted: August 28, 2017 Published: August 28, 2017 2609
DOI: 10.1021/acschembio.7b00435 ACS Chem. Biol. 2017, 12, 2609−2618
Articles
ACS Chemical Biology
Figure 2. Transcription mediated G-quadruplex formation in plasmids. (A) Detection by ligand-induced photocleavage. Transcribed plasmids were incubated with Zn-TTAPc, irradiated with UV light, cut at the Nhe I site, and labeled at the recessive 3′ end with a fluorescein-dUTP before being resolved on a polyacrylamide denaturing gel. G-quadruplex formation was indicated by the cleavage at the G-core. (B) G-quadruplex formation in the upstream region of transcription was K+-dependent and enhanced by a G-quadruplex-stabilizing ligand Zn-TTAPc. (C) G-quadruplex did not form on template strand at the downstream region of transcription. A C-MYC G-core was placed in the plasmid at the (B) upstream or (C) downstream region of a T7 promoter (T7P). “M” indicates a marker, and blue bars indicate terminators. G-cores were all on the template strand, in this and the rest of the figures. The plasmids in C were not transcribed (N) or transcribed (T) with T7 RNAP.
highly sensitive to its distance to the promoter in supercoiled plasmid and decreased sharply when the distance increased. This implied the driving force generated by a transcribing RNAP was constrained in a limited range to induce a localized destabilization of the DNA duplex and, as a result, facilitate a localized G-quadruplex formation. Additionally, we also found that the R-loop is essential for the formation of G-quadruplex, and preventing its formation virtually abolished G-quadruplex formation. On the other hand, R-loop formation also strongly depended on the superhelicity of plasmids, being maximal in supercoiled and minimal in relaxed ones. Collectively, these results suggested that the superhelicity and R-loop may play a cooperative role to warrant G-quadruplex formation. In support of the essential role of supercoiling, enzymatic manipulations and protein binding that affected supercoiling or its propagation all influenced G-quadruplex formation as expected.
quadruplex formation in a large fraction of the G-quadruplex forming motifs in these species. DNA in cells exists in its natural form in a supercoiled state. Under such a condition, a DNA double helix may be under- or overtwisted, resulting in negative or positive supercoiling, respectively, depending on the activities associated with the DNA, such as transcription and action of topoisomerases.17−19 Studies on the effect of superhelicity on G-quadruplex formation are rare. An early study reported that negative supercoiling facilitated the formation of a G-quadruplex in plasmid incubated in a physiological salt solution.20,21 A more recent work showed that DNA gyrase, an enzyme that introduces negative supercoiling into DNA, promoted the formation of a G-quadruplex in a circular DNA duplex.22 A most recent work studied G-quadruplex formation in a linear DNA duplex in which the supercoiling state was manipulated by magnetic tweezers. It was found that the probability of Gquadruplex formation increased with an increase in negative supercoiling.23 All these studies demonstrated the importance of DNA supercoiling in G-quadruplex formation. However, a study under dynamic conditions coupled with transcription has not been reported. Besides the static constitutional superhelicity, two major structural features are produced dynamically in a DNA during transcription. On one hand, a translocating RNAP generates a negative and positive supercoiling respectively behind and in front of the enzyme, leading to an under- and overtwisting of DNA strands in the two regions, respectively (Figure 1B).24−27 On the other hand, a transcribing RNAP may leave behind a non-B DNA structure called an R-loop in which an RNA transcript remains hybridized or a released RNA transcript rehybridizes with the template DNA strand.28,29 To seek mechanistic insight into the formation of an intramolecular DNA G-quadruplex in a more physiologically relevant situation, we studied how G-quadruplex formation triggered by downstream transcription activity was regulated by the structural features of a DNA duplex. We found that Gquadruplex formation was most efficient in negatively supercoiled plasmids and virtually diminished in relaxed ones. It was
■
RESULTS AND DISCUSSION G-Quadruplex Formation in Transcribed Plasmid. Several plasmids were constructed based on the pGL3-Basic vector (Figure S1). Transcriptions were carried out using plasmid containing a T7 promoter and a C-MYC G-core separated by 56 bp unless otherwise indicated. We used ligandinduced photocleavage to detect G-quadruplex formation with Zn-TTAPc that binds G-quadruplexes with high affinity and selectivity and cleaves the guanine residues in the G-quadruplex when irradiated by UV light.30,31 Analysis was first carried out in a supercoiled plasmid (Figure 2A) in which a G-core from the C-MYC gene was placed upstream of a T7 promoter (Figure 2B, top scheme). After transcription with T7 RNAP, the formation of G-quadruplex was revealed by the cleavages at the G-core (Figure 2B, lane 3), which were in sharp contrast to that in the two control samples where formation of Gquadruplex was suppressed by using Li+ in place of K+ (lane 2) or mutations in the G-core (lane 4). With a high specificity and affinity toward G-quadruplexes, the Zn-TTAPc also functions as a G-quadruplex stabilizer.32 When it was added during transcription, cleavage was significantly enhanced (lane 5), 2610
DOI: 10.1021/acschembio.7b00435 ACS Chem. Biol. 2017, 12, 2609−2618
Articles
ACS Chemical Biology
respectively, from the supercoiled one by appropriate enzymatic treatments (Figure 4A). Plasmid in bacteria is in a negatively
indicative of its G-quadruplex-dependency. We also placed the G-core downstream of the T7 promoter (Figure 2C, top scheme) and examined two situations in which the transcription was allowed to pass the G-core (lanes 1 and 2) or stop before the G-core (lanes 3 and 4). In either case, cleavage was barely detectable, indicating that G-quadruplex did not form in the downstream region when a G-core was placed on the template DNA strand as we previously saw in linear DNA duplexes.3,16 This result ensured that the formation of Gquadruplex upstream of T7P was not caused by such transcription events that might happen to pass the terminators. We further verified the formation of a G-quadruplex with a RNA polymerase arrest assay.31 In this experiment, Gquadruplex formation induced by the downstream transcription with T7 RNAP blocked the transcription initiated from an upstream SP6 promoter, generating prematurely terminated (PT) transcripts (Figure 3A). The PT band in Figure 3B, lane
Figure 4. Transcription mediated G-quadruplex formation dependent on the constitutional level of supercoiling in plasmid. (A) Plasmids resolved by agarose gel electrophoresis. (B) G-quadruplex formation in T7 RNAP transcribed plasmids. Supercoiled (S) plasmid (same as in Figure 3) was treated with topoisomorase I, Nt. BspQ I, and BspQ I enzyme to produce relaxed (R), nicked (N), and linear (L) plasmid, respectively. The plasmids were then transcribed, and G-quadruplex formation in the C-MYC G-core was detected by ligand-induced photocleavage as in Figure 2. The different cleavage pattern at the Gcore of the linear than that in the supercoiled and nicked plasmid was possibly caused by different folding topology.
supercoiled conformation which was well preserved in our isolated plasmid (lane 1). The four types of plasmids were all transcribed with a T7 promoter. As shown in Figure 4B, they displayed a formation of G-quadruplexes in an order of supercoiled > linear > nicked > relaxed. It was surprising that G-quadruplex formation was barely detectable in the relaxed plasmid (Figure 4B, lane 6). The positive and negative supercoilings generated by a translocating RNAP propagate in opposite directions. Because the plasmid was circular, the two opposite supercoilings, if allowed to travel freely, might soon meet with each other and become neutralized in the plasmid, leaving little chance for a Gquadruplex to form. A continuity of a DNA strand is required for the neutralization. In the nicked plasmid, the neutralization might be impeded by the nick in one strand while the other remained covalently circular. In the linear plasmid, the two strands were all open, which prevented the neutralization. These seem to explain the different degrees of G-quadruplex formation in the linear, nicked, and relaxed plasmids. The much more efficient formation of G-quadruplex in the supercoiled than in the rest plasmids suggests that the propagation and diffusion of the negative and positive supercoilings generated by an RNAP might possibly be constrained and isolated such that they stayed within a more localized region at higher magnitude to facilitate the formation of the G-quadruplex. If this was true, then we would expect a more localized formation of the Gquadruplex in the supercoiled plasmid. G-Quadruplex Induction in Supercoiled DNA Is Sensitive to the Distance from TSS. To test the aforementioned assumption, we examined G-quadruplex formation at different locations in supercoiled plasmids. We adjusted the distance between the T7 promoter and the CMYC G-core such that the two elements were separated by 56, 212, and 597 bp, respectively. As judged from the cleavages at the G-cores, the most efficient G-quadruplex formation was found with the 56 bp spacing (Figure 5A, lane 2) and a much
Figure 3. Transcription mediated G-quadruplex formation in plasmids detected by RNA polymerase arrest. (A) Plasmid contained a C-MYC G-core, T7, and SP6 promoter. The plasmid was first transcribed with T7 RNAP to induce a formation of G-quadruplex, and then SP6 RNAP was added together with a fluorescein-UTP to produce fluorescent SP6 RNA transcripts. An excess amount of competitive DNA was added at the same time to capture the T7 RNAPs to prevent them from further transcription. Fluorescent transcripts were resolved by denaturing polyacrylamide gel electrophoresis. (B) G-quadruplex formation in the G-core is indicated by the premature termination (PT) band below the full-length transcript (FT). Signal in lane 1 shows fluorescently labeled T7 transcripts.
4, its suppression by Li+ (lane 3) or mutation (lane 5), and enhancement by Zn-TTAPc (lane 6) are fully in agreement with the results in the photocleavage assays (Figure 2B, lanes 2−5). Besides the C-MYC G-core, formation of G-quadruplex induced by downstream transcription was also observed in Gcores from the CSTB, VEGFA gene, and human telomere DNA (Figure S2). On the other hand, we also conducted transcription employing two other prokaryotic promoters, TAC and LacUV5, and G-quadruplex formation was also detected in the G-core in the transcribed plasmids (Figure S3, lanes 4 and 6). These results suggested that the formation of a G-quadruplex in response to downstream transcription is a general phenomenon. Superhelicity Is Crucial for G-Quadruplex Formation in Plasmid. Superhelicity is an essential feature of genomic DNA in cells by which long chromosomes are packed into small volumes to be hosted in a nucleus. To examine how superhelicity affects the transcriptional formation of Gquadruplex, we prepared relaxed, nicked, and linear plasmid, 2611
DOI: 10.1021/acschembio.7b00435 ACS Chem. Biol. 2017, 12, 2609−2618
Articles
ACS Chemical Biology
correlation with the G-quadruplex formation in the plasmids, we found that the highest R-loop formation was in the supercoiled and lowest in the relaxed plasmid, whereas linear plasmid ranked between the two (Figures 6A and S4). These
Figure 5. Effect of distance between G-core and promoter on the formation of G-quadruplex in transcribed (A) supercoiled and (B) linear plasmids. A C-MYC G-core was placed 56, 212, and 597 bp, respectively, upstream of a T7 promoter in the plasmids. The plasmids were not transcribed (N) or transcribed (T) with T7 RNAP and subjected to the ligand-induced photocleavage as in Figure 2.
smaller one with the 212 bp in the supercoiled plasmid (lane 4). When the spacing increased to 597 bp, the cleavage was only slightly higher than the background (lane 6). We also tested the linear plasmid (Figure 5B). In this case, Gquadruplex formation did not show significant decay when the spacing increased from 56 to 212 bp (lane 4 versus 2), suggesting a weaker constraint in the linear than in the supercoiled plasmid. At 597 bp, faint cleavages were detected, indicating a large drop in G-quadruplex formation when the distance was further extended (lane 6). The weak formation of the G-quadruplex at a distal locus was in contrast to what we previously observed in linear dsDNA in which G-quadruplex formation only showed a small decay at a distance up to 2500 bp away from TSS when transcription was carried out in 40% (w/v) polyethylene glycol (PEG) 200. As a molecular crowding agent,3 PEG 200 brings changes to several environmental parameters, such as a decrease in dielectric constant and water activity, an increase in viscosity, and excluded volume,33 which could be responsible for the difference we observed. The fact that G-quadruplex formation was efficiently induced only in the very close vicinity upstream of the TSS (Figure 5A, lane 2) supported the assumption that the transcription generated supercoiling might be constrained within a limited region in a supercoiled plasmid. This constraint thus provided a most favorable environment for the formation of a G-quadruplex (Figure 4B, lane 5) which is compromised in the other three plasmids (Figure 4B, lanes 6−8). We did not examine the distance dependence for the relaxed plasmid since Gquadruplex formation was too weak even with a spacing of 56 bp (Figure 4B, lane 6). Upstream G-Quadruplex Formation Is Supported by R-Loop. During transcription, a DNA:RNA hybrid byproduct called R-loop is produced in the template DNA strand.28,29 We recently demonstrated that an R-loop forms along with a Gquadruplex during transcription.34 Previous work has shown that the transcription-generated negative supercoiling in a plasmid is dependent on the R-loop in the presence of gyrase,35,36 a topoisomerase that relaxes positive supercoiling. Given this role of the R-loop and the large variation in Gquadruplex formation in the plasmids of different superhelicity (Figure 4), we analyzed the formation of the R-loop in the plasmids using an R-loop footprinting method. 37,38 In
Figure 6. (A) Detection of R-loop formation in transcribed plasmids by nondenaturing bisulfate base conversion and DNA sequencing. Supercoiled (S), relaxed (R), and linear (L) plasmids (same as in Figure 4) were transcribed with T7 RNAP and then treated with sodium bisulfate. The DNA was then amplified, cloned, and sequenced. R-loops in plasmids were quantitated by the number of C to T conversions (numerator) over the clones (denominator), multiplied by a factor of 2, because the C to T conversion only occurred on one strand. (B) Prevention of R-loop formation by substitution of ITP for GTP inhibited G-quadruplex formation. (C) Hydrolysis of the R-loop with RNase H during transcription reduced the G-quadruplex amount. In both B and C, supercoiled (S) or linear (L) plasmid was not transcribed (N) or transcribed (T) with T7 RNAP using GTP or ITP (if indicated). Formation of G-quadruplex in the G-core was detected by ligand-induced photocleavage as in Figure 2.
results indicate that the R-loop was best preserved in the negatively supercoiled plasmid, which is what could be expected since negative supercoiling tends to separate the two DNA strands in a DNA helix. The formation of an R-loop can be prevented by substituting ITP for GTP in transcription.39−41 When we transcribed the supercoiled and linear plasmid with ITP, G-quadruplex formation was barely detectable in both plasmids (Figure 6B, lanes 3 and 6). The substitution also suppressed R-loop formation (Figure S5) but did not significantly alter the transcription activity as judged from the yields of RNA transcripts (Figure S6). The R-loop can be destroyed by RNase H that specifically digests the RNA strand in an Rloop.35,39,42 When we included RNase H during transcription, the amount of G-quadruplex in the supercoiled plasmid was dramatically reduced (Figure 6C, lane 3). The inhibition of Gquadruplex formation by ITP and RNase H, therefore, indicated an important role of R-loop in supporting Gquadruplex formation in transcription. Manipulation of Negative Supercoiling Affects GQuadruplex Formation. Supercoiling can be manipulated by 2612
DOI: 10.1021/acschembio.7b00435 ACS Chem. Biol. 2017, 12, 2609−2618
Articles
ACS Chemical Biology enzymes and DNA-binding proteins. For example, prokaryotic DNA topoisomerase I (Top1, a type IA topoisomerase) catalyzes the relaxation of negatively supercoiled DNA.43 In the presence of ATP, gyrase from prokaryotic cells can introduce negative supercoiling into plasmid by relaxing positive supercoiling.44,45 Binding of a protein, such as Lac I, to a plasmid is known to form a topological barrier to suppress supercoiling diffusion.46 To further explore the role of supercoiling, we looked at how G-quadruplex formation would be affected by such enzymes and protein. We first examined the effect of relaxation of negative supercoiling by Top1. This enzyme added in transcription reduced the formation of G-quadruplexes in a concentrationdependent manner (Figure 7A, lanes 3−5). A net reduction in
Figure 8. Restoration of the formation of a G-quadruplex in relaxed plasmid by negative supercoiling catalyzed by gyrase. (A) Gquadruplex formation detected by ligand-induced photocleavage. Supercoiled (S) or relaxed (R) plasmid containing a C-MYC G-core and a T7 promoter was transcribed by T7 RNAP in the absence and presence of 0.1 U/μL gyrase. (B) Supercoiling state of plasmids transcribed in the absence and presence of gyrase resolved by agarose gel electrophoresis after a digestion with RNase A and proteinase K.
Figure 7. Reduction of negative supercoiling by Top1 suppressed Gquadruplex formation. Supercoiled plasmid containing a C-MYC Gcore and a T7 promoter was transcribed by T7 RNAP. (A) Transcribed in the presence of 0, 0.015, 0.05, and 0.15 U/μL Top1 for 1 h. (B) Top1 (0.05 U/μL) added during a 1 h transcription (lane 4) or after transcription was terminated by competitive DNA and incubated for 0.5 or 1 h (lanes 7 and 8). G-quadruplex formation in the G-core (A and B bottom gel) was detected by ligand-induced photocleavage as in Figure 2. Supercoiling state (B top gel) was detected after a digestion with RNase A and proteinase K. S: supercoiled. R: relaxed.
by an increase in the negative supercoiling of the plasmid (Figure 8B, lane 5). Gyrase directly relaxes positive supercoiling by a transient cut-and-rejoining activity to introduce negative supercoiling.47 It was noted that the gyrase did not bring the negative supercoiling (Figure 8B, lane 5) to the original level (lane 1) of the plasmid, but the G-quadruplex formation was comparable to that in the supercoiled counterpart (Figure 8A, lane 8 versus 2). Similar to the effect of Top1, this result also supported that a dynamic change in local supercoiling might be more effective than a static global supercoiling in promoting Gquadruplex formation. The upstream formation of the G-quadruplex in response to downstream transcription activity relies on the upward transmission of negative supercoiling. A protein bound to DNA between a G-quadruplex forming site and promoter would hinder such a transmission of negative supercoiling.46 As a result, we expected this would reduce the formation of Gquadruplexes. To test this anticipation, we inserted one or two lac O1 operators between the C-MYC G-core and T7 promoter (Figure S1C) and performed transcription in the presence of Lac I protein that binds to lac O1. Indeed, decreased Gquadruplex formation was observed in the supercoiled plasmid (Figure 9A, lane 3). This effect was enhanced when two binding sites were arranged (lane 7) and defied by the Lac operon inducer IPTG that competes for Lac I (lanes 4 and 8). The suppression on G-quadruplex formation became more significant in the linear plasmid (Figure 9B), which suggested a more free transmission of negative supercoiling in the linear than in the closed supercoiled plasmid.
negative supercoiling was introduced in the plasmids when the Top1 was added either during or after the transcription (Figure 7B, lanes 4, 7, and 8, top gel). However, the reduction in Gquadruplex formation only occurred when Top1 was supplied during transcription (Figure 7B, lanes 4, bottom gel), indicating that the action of Top1 only affected the formation of Gquadruplex, but not when a G-quadruplex was already formed. At 0.05U/μL of Top1, G-quadruplex formation was barely detected (Figure 7A, lane 4) even though many plasmids were still supercoiled (Figure 7B, lane 4, top gel). This result seemed to suggest that the dynamic change in local supercoiling might be more effective than the static global supercoiling in promoting G-quadruplex formation. We then looked at the effect of relaxation of positive supercoiling by gyrase. When supercoiled plasmid was transcribed in the presence of gyrase, little effect was observed (Figure 8A, lane 3 versus 2). However, when relaxed plasmid was used in which original G-quadruplex formation was slightly detectable (lane 7), the addition of gyrase to the transcription dramatically enhanced G-quadruplex formation (lane 8). The formation of the G-quadruplex was transcription-dependent (lane 4) because gyrase alone was unable to induce formation of the G-quadruplex (lane 5). The enhancement in Gquadruplex formation (Figure 8A, lane 8) was accompanied 2613
DOI: 10.1021/acschembio.7b00435 ACS Chem. Biol. 2017, 12, 2609−2618
Articles
ACS Chemical Biology
Figure 9. Manipulation of supercoiling transmission by Lac Repressor (Lac I) affecting G-quadruplex formation. Supercoiled (A) or linear (B) plasmid contained one or two 21 bp Lac O1 operators between the G-core and T7 promoter. Lac I (0.5 μM) was incubated with plasmid at 37 °C for 20 min before transcription. IPTG (1 mM) was added with T7 RNAP when indicated. G-quadruplex formation in the G-core was detected by ligand-induced photocleavage as in Figure 2. Graphs at the bottom present a digital scan of the area within the two dotted lines in the gels to help judge the subtle changes in cleavage.
■
CONCLUSION The induction of G-quadruplex formation by downstream transcription offers a unique model to study G-quadruplex formation in a dynamic environment. The fact that the formation of the G-quadruplex was induced by T7 and endogenous bacteria RNAP suggests that G-quadruplex formation in the upstream region is a general phenomenon. Because the G-quadruplex forming motif does not overlap with the transcribed region, the two events do not directly interfere with each other. Therefore, the deduction of the cause−effect relationship is greatly simplified. On the basis of this model, our results revealed several characteristics of G-quadruplex formation in the upstream region in connection with transcription. Transcription-generated negative supercoiling propagates in a direction opposite that of transcription in a linear DNA duplex in vitro and in chromosomes in vivo.48,49 In a DNA duplex, the base-pairing of the two DNA strands needs to be disrupted for a G-quadruplex to form. Negative supercoiling provides such a chance by weakening the double helix, whereas positive supercoiling does the opposite. It is noted that the preexisting static superhelicity itself did not induce a formation of the G-quadruplex since it was not seen in the untranscribed plasmids. It was the additional negative supercoiling dynamically generated by a moving RNA polymerase that triggered the formation of a G-quadruplex. One important feature we observed was that the constitutional level of superhelicity was important for the formation of a G-quadruplex. It on one hand enhanced the formation of a Gquadruplex (Figure 4) and on the other limited the range in which the formation of a G-quadruplex is induced near the TSS (Figure 5). The enhanced G-quadruplex formation may have resulted from an additive effect of the overall global negative supercoiling and the local negative supercoiling generated by an
RNAP. In supercoiled plasmids, intrinsic bends can block the transport of torsional stress along DNA and the transcriptionally generated supercoiling is therefore maintained at higher magnitude in local topological domains.50−52 This behavior explains the preferable formation of a G-quadruplex near the TSS and the occurrence frequency of G-quadruplex forming motifs at the upstream side of TSS in warm-blooded animals, which is inversely related to their distance to the TSS.3,4 In contrast to the supercoiled plasmid, supercoiling was fully discharged prior to transcription in the relaxed, nicked, and linear plasmids. The compromised formation of the Gquadruplex in these plasmids (Figure 4B, lanes 6−8) might be attributed to their inability or deficiency to maintain and constrain the supercoiling generated during transcription. As a result, the dynamically generated supercoiling may transmit or diffuse more freely to become diluted over a larger range. Our data show that the R-loop is another structural feature that has a profound impact on the transcriptional formation of the G-quadruplex. Preventing R-loop formation virtually abolished G-quadruplex formation (Figure 6B). Digestion of the R-loop during transcription also resulted in a dramatic reduction in the amount of G-quadruplex (Figure 6C). According to previous studies, transcription led to a net gain of negative supercoiling in plasmids in an R-loop-dependent manner in the presence of gyrase.35,36 Positively supercoiled DNA is the preferred substrate to gyrase and the E. coli topoisomerase IV, a structural homolog to gyrase.53−55 When a transcribing RNAP generates local negative and positive supercoiling respectively behind and in front of it, gyrase relieves the positive supercoiling by cutting and rejoining a DNA strand.56 In this way, a plasmid gains a net increase in negative supercoiling. However, this did not occur when RNase H was included in the transcription to digest the RNA in an Rloop.35,36 This fact suggested that the R-loop was able to 2614
DOI: 10.1021/acschembio.7b00435 ACS Chem. Biol. 2017, 12, 2609−2618
Articles
ACS Chemical Biology
factors. An example of this effect is demonstrated in an earlier work where the Top1 inhibitor was found to potentiate the antitumor efficiency of a G-quadruplex-stabilizing drug.93 A recent work shows that topoisomerase 1 activity in human cells can be regulated by RNA polymerase II to favor efficient transcription.94 These findings imply that transcriptional machinery in cells may have an ability to manipulate Gquadruplex formation via topoisomerase, which in turn can regulate promoter output.
maintain the positive supercoiling to support the action of gyrase. The R-loop may therefore possibly maintain the negative supercoiling on the other side (Figure 1B) by the same mechanism. On the other hand, the R-loop has been shown to block or stall a transcribing RNAP,57 and such an RNAP might also prevent the merging of the two opposite supercoilings. For these reasons, we assume that the R-loop might function as an insulator to prevent the two opposite supercoilings from being neutralized with each other, such that G-quadruplex formation is facilitated by the negative supercoiling. Besides their effect, our data also show that R-loops are better preserved in supercoiled plasmid (Figure 6A). Therefore, the two structural forms were mutually supportive in their formation and stabilization. In our recent work, we have shown that the R-loop contributed an essential role to the transcriptional formation of the G-quadruplex in the downstream region of TSS.58 Our current work together with previous work demonstrated a more general importance of the R-loop in the formation of the G-quadruplex in transcription. G-quadruplex-forming sequences are prevalent in the genome of various organisms31,59−64 whose richness goes well beyond our previous anticipation.65 The formation of Gquadruplexes has been detected in the genome in both prokaryotic63 and eukaryotic66−70 cells. Their role in regulation of gene expression has been well documented.8,71−84 The role of the static superhelicity and the formation of a G-quadruplex driven by the dynamic negative supercoiling we observed provides a justification for the formation of G-quadruplexes in cells where DNA molecules are negatively supercoiled85 and dynamic negative supercoiling is generated in DNA tracking activities, for instance, replication and transcription. DNA in cells is packed in a condensed and dynamic architecture where topological changes are well constrained within defined regions. From our results, this feature may provide an essential environment for the formation of DNA G-quadruplexes in cells. The topological constraint may determine the range of dynamic supercoiling propagation and the magnitude of Gquadruplex formation. Besides the G-quadruplex, negative supercoiling also promotes the formation of another secondary structure, the imotif, on the C-rich DNA strand, through which additional modulation on gene expression can be inserted alongside the G-quadruplex.20,86−90 The formation of the i-motif has been shown to play a role in coordination with the G-quadruplex. Recent studies have demonstrated that the extent of negative superhelicity plays an important role in determining the formation and switching of structures, as well as transcriptional firing and binding of hnRNP K to the i-motif, suggesting that the transcriptionally induced negative superhelicity is also as important in controlling the access to the ON switch (i-motif) as the OFF switch (G-quadrulex).87,90 In a recent study, a high level of single-stranded DNA is observed in mammalian cells in regions upstream of promoters of active genes. In such regions, different noncanonical DNA structures, such as the G-quadruplexes, affect chromatin reorganization and transcriptome output.91 In the MYC gene, the degree of transcriptionally induced negative superhelicity regulates the transcriptional firing rate and the structure of the far upstream sequence element (FUSE), as well as its interactions with its binding proteins in the promoter.92 Our experiments with Top1, gyrase, and Lac I (Figures 7−9) illustrated how the control of G-quadruplex formation by DNA topology can be further regulated by additional physiological
■
MATERIALS AND METHODS
Plasmids and Linear DNA Duplex. Plasmids were constructed based on the pGL3-basic vector (Promega, USA) by insertion of the indicated elements (Table S1 and Figure S1). Supercoiled plasmid was purified from E. coli strain JM109 using the TIANprep Midi Plasmid Kit (Tiangen, China). Linear, nicked, and relaxed plasmids were prepared by treating supercoiled plasmid with BspQ I, Nt.BspQ I, and E. coli topoisomerase I (NEB), respectively, at 37 °C for 3 h followed by an incubation with 20 mM EDTA (pH 8.0) and 0.2 mg mL−1 proteinase K (Fermentas, Thermo Scientific) at 55 °C for 20 min. The plasmids were recovered using the Wizard DNA Clean-Up System (Promega, USA). In Vitro Transcription. Transcription using T7 RNAP was carried out essentially as described95 in the absence of PEG, with 3 μg of plasmid DNA in a total volume of 100 μL at 37 °C for 1 h in transcription buffer containing 33 mM Tris-acetate (pH 7.9), 10 mM magnesium acetate, 2 mM spermidine, 66 mM KCl or LiCl, 200 U T7 RNAP (Fermentas, Thermo Scientific), 0.1 U inorganic pyrophosphatase (Fermentas, Thermo Scientific), 1 U/μL RiboLock RNase inhibitor (Fermentas, Thermo Scientific), and 2 mM each NTP. Transcription using E. coli RNAP holoenzyme (NEB) was carried out with 0.5 μg of plasmid DNA in a total volume of 100 μL at 37 °C for 1 h in transcription buffer containing 40 mM Tris-HCl (pH 7.5), 0.15 M KCl, 10 mM MgCl2, 0.01% Triton X-100, 2 mM spermidine, 0.5 mM each NTP, 10 U E. coli RNAP holoenzyme, and 1 U/μL RiboLock RNase inhibitor (Fermentas, Thermo Scientific). Reaction was stopped with an addition of 1/25 vol of 0.5 M EDTA. When ITP was used in transcription, 0.4 mM GMP was supplied. RNA Polymerase Arrest Assay. Plasmids were transcribed by T7 and then by SP6 RNAP, and samples were processed as previously described.31 Ligand-Induced Photocleavage Footprinting. Photocleavage footprinting was conducted essentially as described previously.62 T7 RNAP transcribed samples were transferred into a 24-well microtiter plate and irradiated for 40 min with 365 nm UV light in a UVP CL1000 Ultraviolet Cross-linker (UVP, America) at a distance of 5 cm. Then, samples were digested with 0.4 mg mL−1 RNase A (Fermentas, Thermo Scientific) at 37 °C for 15 min and purified using the Wizard DNA Clean-Up System (Promega, USA). The plasmid was then digested with 0.03 U/μL Fastdigest Nhe I and labeled at the recessive 3′ end at 37 °C for 60 min with the klenow exo-polymerase (Fermentas, Thermo Scientific) and 1 μM fluorescein-12-dUTP (Fermentas, Thermo Scientific). The reaction was terminated by adding EDTA to 20 mM. For DNA precipitation, each 100 μL sample received 35 μL of 10 M ammonium acetate, 2 μL glycogen (10 mg mL−1), and 420 μL 100% ethanol; they were thoroughly mixed and left overnight at −20 °C. Precipitated DNA was dissolved, denatured, and resolved on a denaturing 12% polyacrylamide gel.30 The gel was scanned on a Typhoon 9400 (GE Healthcare, USA) imager and processed with the ImageQuant 5.2 software. Nondenaturing Bisulfate Base Conversion. DNA conversion was carried out using the EpiMark Bisulfite Conversion Kit (NEB) with modifications. Briefly, after transcription at 37 °C for 30 min, each 10 μL sample was mixed with 130 μL of bisulfate mix and incubated at 22 °C for 36 h. After completion of the conversion reaction, desulphonation and recovery of DNA were carried out according to the instructions of the kit. Putative R-loop regions were amplified using the EpiMark Hot Start Taq DNA Polymerase (NEB); 2615
DOI: 10.1021/acschembio.7b00435 ACS Chem. Biol. 2017, 12, 2609−2618
Articles
ACS Chemical Biology the resulting PCR products were purified using the Wizard DNA Clean-Up System (Promega, USA), cloned into pMD19-T simple vector, and transfected into E. coli strain JM109. At least 120 colonies were selected and sequenced for each sample. Plasmid with at least four neighboring C to T conversions in the transcription region was considered R-loop-positive.
■
TMPyP4 stabilization of promoter G-quadruplexes in leukemia cells. Tumor Biol. 37, 9967−9977. (9) Feng, Y., Yang, D., Chen, H., Cheng, W., Wang, L., Sun, H., and Tang, Y. (2016) Stabilization of G-quadruplex DNA and inhibition of Bcl-2 expression by a pyridostatin analog. Bioorg. Med. Chem. Lett. 26, 1660−1663. (10) Nagesh, N., Raju, G., Srinivas, R., Ramesh, P., Reddy, M. D., and Reddy, C. R. (2015) A dihydroindolizino indole derivative selectively stabilizes G-quadruplex DNA and down-regulates c-MYC expression in human cancer cells. Biochim. Biophys. Acta, Gen. Subj. 1850, 129− 140. (11) Taka, T., Joonlasak, K., Huang, L., Randall Lee, T., Chang, S. W., and Tuntiwechapikul, W. (2012) Down-regulation of the human VEGF gene expression by perylene monoimide derivatives. Bioorg. Med. Chem. Lett. 22, 518−522. (12) Gu, H. P., Lin, S., Xu, M., Yu, H. Y., Du, X. J., Zhang, Y. Y., Yuan, G., and Gao, W. (2012) Up-regulating relaxin expression by Gquadruplex interactive ligand to achieve antifibrotic action. Endocrinology 153, 3692−3700. (13) Agarwal, T., Roy, S., Chakraborty, T. K., and Maiti, S. (2010) Selective targeting of G-quadruplex using furan-based cyclic homooligopeptides: effect on c-MYC expression. Biochemistry 49, 8388−8397. (14) Waller, Z. A., Sewitz, S. A., Hsu, S. T., and Balasubramanian, S. (2009) A small molecule that disrupts G-quadruplex DNA structure and enhances gene expression. J. Am. Chem. Soc. 131, 12628−12633. (15) Bejugam, M., Sewitz, S., Shirude, P. S., Rodriguez, R., Shahid, R., and Balasubramanian, S. (2007) Trisubstituted isoalloxazines as a new class of G-quadruplex binding ligands: small molecule regulation of ckit oncogene expression. J. Am. Chem. Soc. 129, 12926−12927. (16) Liu, J. Q., Xiao, S., Hao, Y. H., and Tan, Z. (2015) Strand-Biased Formation of G-Quadruplexes in DNA Duplexes Transcribed with T7 RNA Polymerase. Angew. Chem., Int. Ed. 54, 8992−8996. (17) Kouzine, F., Gupta, A., Baranello, L., Wojtowicz, D., Ben-Aissa, K., Liu, J., Przytycka, T. M., and Levens, D. (2013) Transcriptiondependent dynamic supercoiling is a short-range genomic force. Nat. Struct. Mol. Biol. 20, 396−403. (18) Naughton, C., Avlonitis, N., Corless, S., Prendergast, J. G., Mati, I. K., Eijk, P. P., Cockroft, S. L., Bradley, M., Ylstra, B., and Gilbert, N. (2013) Transcription forms and remodels supercoiling domains unfolding large-scale chromatin structures. Nat. Struct. Mol. Biol. 20, 387−395. (19) Teves, S. S., and Henikoff, S. (2013) Transcription-generated torsional stress destabilizes nucleosomes. Nat. Struct. Mol. Biol. 21, 88− 94. (20) Sun, D., and Hurley, L. H. (2009) The importance of negative superhelicity in inducing the formation of G-quadruplex and i-motif structures in the c-Myc promoter: implications for drug targeting and control of gene expression. J. Med. Chem. 52, 2863−2874. (21) Sun, D. (2010) In vitro footprinting of promoter regions within supercoiled plasmid DNA. Methods Mol. Biol. 613, 223−233. (22) Lv, B., Li, D., Zhang, H., Lee, J. Y., and Li, T. (2013) DNA gyrase-driven generation of a G-quadruplex from plasmid DNA. Chem. Commun. (Cambridge, U. K.) 49, 8317−8319. (23) Selvam, S., Koirala, D., Yu, Z., and Mao, H. (2014) quantification of topological coupling between DNA superhelicity and G-quadruplex formation. J. Am. Chem. Soc. 136, 13967−13970. (24) Liu, L. F., and Wang, J. C. (1987) Supercoiling of the DNA template during transcription. Proc. Natl. Acad. Sci. U. S. A. 84, 7024− 7027. (25) Wu, H. Y., Shyy, S. H., Wang, J. C., and Liu, L. F. (1988) Transcription generates positively and negatively supercoiled domains in the template. Cell 53, 433−440. (26) Rahmouni, A. R., and Wells, R. D. (1992) Direct evidence for the effect of transcription on local DNA supercoiling in vivo. J. Mol. Biol. 223, 131−144. (27) Baranello, L., Levens, D., Gupta, A., and Kouzine, F. (2012) The importance of being supercoiled: how DNA mechanics regulate
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.7b00435. Additional Table S1, Figures S1−S6 (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*Tel.: +86 10-6480-7259. Fax: +86 (10) 6480-7099. E-mail: z.
[email protected]. ORCID
Zheng Tan: 0000-0003-0480-9686 Author Contributions §
These authors contributed equally to the work
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS This work was supported by the Ministry of Science and Technology of China, grant # 2013CB530802 and 2012CB720601, the National Natural Science Foundation of China, grant # 31470783 and 21432008, the China Postdoctoral Science Foundation 2013T60172, and the Young Elite Scientist Sponsorship Program of CAST.
■
REFERENCES
(1) Zhao, Y., Du, Z., and Li, N. (2007) Extensive selection for the enrichment of G4 DNA motifs in transcriptional regulatory regions of warm blooded animals. FEBS Lett. 581, 1951−1956. (2) Eddy, J., Vallur, A. C., Varma, S., Liu, H., Reinhold, W. C., Pommier, Y., and Maizels, N. (2011) G4 motifs correlate with promoter-proximal transcriptional pausing in human genes. Nucleic Acids Res. 39, 4975−4983. (3) Zhang, C., Liu, H. H., Zheng, K. W., Hao, Y. H., and Tan, Z. (2013) DNA G-quadruplex formation in response to remote downstream transcription activity: long-range sensing and signal transducing in DNA double helix. Nucleic Acids Res. 41, 7144−7152. (4) Xiao, S., Zhang, J. Y., Zheng, K. W., Hao, Y. H., and Tan, Z. (2013) Bioinformatic analysis reveals an evolutional selection for DNA:RNA hybrid G-quadruplex structures as putative transcription regulatory elements in warm-blooded animals. Nucleic Acids Res. 41, 10379−10390. (5) McLuckie, K. I., Waller, Z. A., Sanders, D. A., Alves, D., Rodriguez, R., Dash, J., McKenzie, G. J., Venkitaraman, A. R., and Balasubramanian, S. (2011) G-quadruplex-binding benzo[a]phenoxazines down-regulate c-KIT expression in human gastric carcinoma cells. J. Am. Chem. Soc. 133, 2658−2663. (6) Cogoi, S., and Xodo, L. E. (2006) G-quadruplex formation within the promoter of the KRAS proto-oncogene and its effect on transcription. Nucleic Acids Res. 34, 2536−2549. (7) Qin, Y., Rezler, E. M., Gokhale, V., Sun, D., and Hurley, L. H. (2007) Characterization of the G-quadruplexes in the duplex nuclease hypersensitive element of the PDGF-A promoter and modulation of PDGF-A promoter activity by TMPyP4. Nucleic Acids Res. 35, 7698− 7713. (8) Zidanloo, S. G., Hosseinzadeh Colagar, A., Ayatollahi, H., and Raoof, J. B. (2016) Downregulation of the WT1 gene expression via 2616
DOI: 10.1021/acschembio.7b00435 ACS Chem. Biol. 2017, 12, 2609−2618
Articles
ACS Chemical Biology dynamic processes. Biochim. Biophys. Acta, Gene Regul. Mech. 1819, 632−638. (28) Gowrishankar, J., Leela, J. K., and Anupama, K. (2013) R-loops in bacterial transcription: their causes and consequences. Transcription 4, 153−157. (29) Aguilera, A., and Garcia-Muse, T. (2012) R loops: from transcription byproducts to threats to genome stability. Mol. Cell 46, 115−124. (30) Zheng, K. W., Zhang, D., Zhang, L. X., Hao, Y. H., Zhou, X., and Tan, Z. (2011) Dissecting the strand folding orientation and formation of G-quadruplexes in single- and double-stranded nucleic acids by ligand-induced photocleavage footprinting. J. Am. Chem. Soc. 133, 1475−1483. (31) Zheng, K. W., Wu, R. Y., He, Y. D., Xiao, S., Zhang, J. Y., Liu, J. Q., Hao, Y. H., and Tan, Z. (2014) A competitive formation of DNA:RNA hybrid G-quadruplex is responsible to the mitochondrial transcription termination at the DNA replication priming site. Nucleic Acids Res. 42, 10832−10844. (32) Ren, L., Zhang, A., Huang, J., Wang, P., Weng, X., Zhang, L., Liang, F., Tan, Z., and Zhou, X. (2007) Quaternary ammonium zinc phthalocyanine: inhibiting telomerase by stabilizing G quadruplexes and inducing G-quadruplex structure transition and formation. ChemBioChem 8, 775−780. (33) Chen, Z., Zheng, K. W., Hao, Y. H., and Tan, Z. (2009) Reduced or diminished stabilization of the telomere G-quadruplex and inhibition of telomerase by small chemical ligands under molecular crowding condition. J. Am. Chem. Soc. 131, 10430−10438. (34) Zhang, J. Y., Zheng, K. W., Xiao, S., Hao, Y. H., and Tan, Z. (2014) Mechanism and manipulation of DNA:RNA hybrid Gquadruplex formation in transcription of G-rich DNA. J. Am. Chem. Soc. 136, 1381−1390. (35) Leng, F., Amado, L., and McMacken, R. (2004) Coupling DNA supercoiling to transcription in defined protein systems. J. Biol. Chem. 279, 47564−47571. (36) Drolet, M., Bi, X., and Liu, L. F. (1994) Hypernegative supercoiling of the DNA template during transcription elongation in vitro. J. Biol. Chem. 269, 2068−2074. (37) Yu, K., Chedin, F., Hsieh, C. L., Wilson, T. E., and Lieber, M. R. (2003) R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B cells. Nat. Immunol. 4, 442−451. (38) Ginno, P. A., Lott, P. L., Christensen, H. C., Korf, I., and Chedin, F. (2012) R-loop formation is a distinctive characteristic of unmethylated human CpG island promoters. Mol. Cell 45, 814−825. (39) Roy, D., Yu, K., and Lieber, M. R. (2008) Mechanism of R-loop formation at immunoglobulin class switch sequences. Mol. Cell. Biol. 28, 50−60. (40) Mizuta, R., Mizuta, M., and Kitamura, D. (2005) Guanine is indispensable for immunoglobulin switch region RNA-DNA hybrid formation. J. Electron Microsc. 54, 403−408. (41) Duquette, M. L., Handa, P., Vincent, J. A., Taylor, A. F., and Maizels, N. (2004) Intracellular transcription of G-rich DNAs induces formation of G-loops, novel structures containing G4 DNA. Genes Dev. 18, 1618−1629. (42) Tracy, R. B., and Lieber, M. R. (2000) Transcription-dependent R-loop formation at mammalian class switch sequences. EMBO J. 19, 1055−1067. (43) Masse, E., and Drolet, M. (1999) Escherichia coli DNA topoisomerase I inhibits R-loop formation by relaxing transcriptioninduced negative supercoiling. J. Biol. Chem. 274, 16659−16664. (44) Reece, R. J., and Maxwell, A. (1991) DNA gyrase: structure and function. Crit. Rev. Biochem. Mol. Biol. 26, 335−375. (45) Gellert, M., Mizuuchi, K., O’Dea, M. H., and Nash, H. A. (1976) DNA gyrase: an enzyme that introduces superhelical turns into DNA. Proc. Natl. Acad. Sci. U. S. A. 73, 3872−3876. (46) Leng, F., Chen, B., and Dunlap, D. D. (2011) Dividing a supercoiled DNA molecule into two independent topological domains. Proc. Natl. Acad. Sci. U. S. A. 108, 19973−19978. (47) Cozzarelli, N. R. (1980) DNA gyrase and the supercoiling of DNA. Science 207, 953−960.
(48) Kouzine, F., Liu, J., Sanford, S., Chung, H. J., and Levens, D. (2004) The dynamic response of upstream DNA to transcriptiongenerated torsional stress. Nat. Struct. Mol. Biol. 11, 1092−1100. (49) Kouzine, F., Sanford, S., Elisha-Feil, Z., and Levens, D. (2008) The functional response of upstream DNA to dynamic supercoiling in vivo. Nat. Struct. Mol. Biol. 15, 146−154. (50) Droge, P., and Nordheim, A. (1991) Transcription-induced conformational change in a topologically closed DNA domain. Nucleic Acids Res. 19, 2941−2946. (51) Nelson, P. (1999) Transport of torsional stress in DNA. Proc. Natl. Acad. Sci. U. S. A. 96, 14342−14347. (52) Wang, Z., and Droge, P. (1997) Long-range effects in a supercoiled DNA domain generated by transcription in vitro. J. Mol. Biol. 271, 499−510. (53) Crisona, N. J., Strick, T. R., Bensimon, D., Croquette, V., and Cozzarelli, N. R. (2000) Preferential relaxation of positively supercoiled DNA by E. coli topoisomerase IV in single-molecule and ensemble measurements. Genes Dev. 14, 2881−2892. (54) Kozyavkin, S. A., Slesarev, A. I., Malkhosyan, S. R., and Panyutin, I. G. (1990) DNA linking potential generated by gyrase. Eur. J. Biochem. 191, 105−113. (55) Westerhoff, H. V., O’Dea, M. H., Maxwell, A., and Gellert, M. (1988) DNA supercoiling by DNA gyrase. A static head analysis. Cell Biophys. 12, 157−181. (56) Gubaev, A., and Klostermeier, D. (2014) The mechanism of negative DNA supercoiling: a cascade of DNA-induced conformational changes prepares gyrase for strand passage. DNA Repair 16, 23−34. (57) El Hage, A., French, S. L., Beyer, A. L., and Tollervey, D. (2010) Loss of Topoisomerase I leads to R-loop-mediated transcriptional blocks during ribosomal RNA synthesis. Genes Dev. 24, 1546−1558. (58) Zhao, Y., Zhang, J. Y., Zhang, Z. Y., Tong, T. J., Hao, Y. H., and Tan, Z. (2017) Real-time detection reveals responsive cotranscriptional formation of persistent intramolecular DNA and intermolecular DNA:RNA hybrid G-quadruplexes stabilized by R-loop. Anal. Chem. 89, 6036. (59) Huppert, J. L., and Balasubramanian, S. (2005) Prevalence of quadruplexes in the human genome. Nucleic Acids Res. 33, 2908−2916. (60) Chambers, V. S., Marsico, G., Boutell, J. M., Di Antonio, M., Smith, G. P., and Balasubramanian, S. (2015) High-throughput sequencing of DNA G-quadruplex structures in the human genome. Nat. Biotechnol. 33, 877−881. (61) Li, X. M., Zheng, K. W., Zhang, J. Y., Liu, H. H., He, Y. D., Yuan, B. F., Hao, Y. H., and Tan, Z. (2015) Guanine-vacancy-bearing Gquadruplexes responsive to guanine derivatives. Proc. Natl. Acad. Sci. U. S. A. 112, 14581−14586. (62) Zheng, K. W., Xiao, S., Liu, J. Q., Zhang, J. Y., Hao, Y. H., and Tan, Z. (2013) Co-transcriptional formation of DNA:RNA hybrid Gquadruplex and potential function as constitutional cis element for transcription control. Nucleic Acids Res. 41, 5533−5541. (63) Wu, R. Y., Zheng, K. W., Zhang, J. Y., Hao, Y. H., and Tan, Z. (2015) Formation of DNA:RNA hybrid G-quadruplex in bacterial cells and its dominance over the intramolecular DNA G-quadruplex in mediating transcription termination. Angew. Chem., Int. Ed. 54, 2447− 2451. (64) Xiao, S., Zhang, J. Y., Wu, J., Wu, R. Y., Xia, Y., Zheng, K. W., Hao, Y. H., Zhou, X., and Tan, Z. (2014) Formation of DNA:RNA hybrid G-quadruplexes of two G-quartet layers in transcription: expansion of the prevalence and diversity of G-quadruplexes in genomes. Angew. Chem., Int. Ed. 53, 13110−13114. (65) Bedrat, A., Lacroix, L., and Mergny, J. L. (2016) Re-evaluation of G-quadruplex propensity with G4Hunter. Nucleic Acids Res. 44, 1746−1759. (66) Laguerre, A., Hukezalie, K., Winckler, P., Katranji, F., Chanteloup, G., Pirrotta, M., Perrier-Cornet, J. M., Wong, J. M., and Monchaud, D. (2015) Visualization of RNA-Quadruplexes in Live Cells. J. Am. Chem. Soc. 137, 8521−8525. (67) Biffi, G., Di Antonio, M., Tannahill, D., and Balasubramanian, S. (2013) Visualization and selective chemical targeting of RNA G2617
DOI: 10.1021/acschembio.7b00435 ACS Chem. Biol. 2017, 12, 2609−2618
Articles
ACS Chemical Biology quadruplex structures in the cytoplasm of human cells. Nat. Chem. 6, 75−80. (68) Biffi, G., Tannahill, D., McCafferty, J., and Balasubramanian, S. (2013) Quantitative visualization of DNA G-quadruplex structures in human cells. Nat. Chem. 5, 182−186. (69) Schaffitzel, C., Berger, I., Postberg, J., Hanes, J., Lipps, H. J., and Pluckthun, A. (2001) In vitro generated antibodies specific for telomeric guanine-quadruplex DNA react with Stylonychia lemnae macronuclei. Proc. Natl. Acad. Sci. U. S. A. 98, 8572−8577. (70) Yuan, L., Tian, T., Chen, Y., Yan, S., Xing, X., Zhang, Z., Zhai, Q., Xu, L., Wang, S., Weng, X., Yuan, B., Feng, Y., and Zhou, X. (2013) Existence of G-quadruplex structures in promoter region of oncogenes confirmed by G-quadruplex DNA cross-linking strategy. Sci. Rep. 3, 1811. (71) Li, Y., Syed, J., Suzuki, Y., Asamitsu, S., Shioda, N., Wada, T., and Sugiyama, H. (2016) Effect of ATRX and G-Quadruplex Formation by the VNTR Sequence on alpha-Globin Gene Expression. ChemBioChem 17, 928−935. (72) Sun, H., Xiang, J., Shi, Y., Yang, Q., Guan, A., Li, Q., Yu, L., Shang, Q., Zhang, H., Tang, Y., and Xu, G. (2014) A newly identified G-quadruplex as a potential target regulating Bcl-2 expression. Biochim. Biophys. Acta, Gen. Subj. 1840, 3052−3057. (73) Nguyen, G. H., Tang, W., Robles, A. I., Beyer, R. P., Gray, L. T., Welsh, J. A., Schetter, A. J., Kumamoto, K., Wang, X. W., Hickson, I. D., Maizels, N., Monnat, R. J., Jr., and Harris, C. C. (2014) Regulation of gene expression by the BLM helicase correlates with the presence of G-quadruplex DNA motifs. Proc. Natl. Acad. Sci. U. S. A. 111, 9905− 9910. (74) He, Q., Zeng, P., Tan, J. H., Ou, T. M., Gu, L. Q., Huang, Z. S., and Li, D. (2014) G-quadruplex-mediated regulation of telomere binding protein POT1 gene expression. Biochim. Biophys. Acta, Gen. Subj. 1840, 2222−2233. (75) Dhayan, H., Baydoun, A. R., and Kukol, A. (2014) G-quadruplex formation of FXYD1 pre-mRNA indicates the possibility of regulating expression of its protein product. Arch. Biochem. Biophys. 560, 52−58. (76) Endoh, T., Kawasaki, Y., and Sugimoto, N. (2013) Suppression of gene expression by G-quadruplexes in open reading frames depends on G-quadruplex stability. Angew. Chem., Int. Ed. 52, 5522−5526. (77) Huang, W., Smaldino, P. J., Zhang, Q., Miller, L. D., Cao, P., Stadelman, K., Wan, M., Giri, B., Lei, M., Nagamine, Y., Vaughn, J. P., Akman, S. A., and Sui, G. (2012) Yin Yang 1 contains G-quadruplex structures in its promoter and 5′-UTR and its expression is modulated by G4 resolvase 1. Nucleic Acids Res. 40, 1033−1049. (78) Shklover, J., Weisman-Shomer, P., Yafe, A., and Fry, M. (2010) Quadruplex structures of muscle gene promoter sequences enhance in vivo MyoD-dependent gene expression. Nucleic Acids Res. 38, 2369− 2377. (79) Shahid, R., Bugaut, A., and Balasubramanian, S. (2010) The BCL-2 5′ untranslated region contains an RNA G-quadruplex-forming motif that modulates protein expression. Biochemistry 49, 8300−8306. (80) Brooks, T. A., and Hurley, L. H. (2010) Targeting MYC Expression through G-Quadruplexes. Genes Cancer 1, 641−649. (81) Verma, A., Yadav, V. K., Basundra, R., Kumar, A., and Chowdhury, S. (2009) Evidence of genome-wide G4 DNA-mediated gene expression in human cancer cells. Nucleic Acids Res. 37, 4194− 4204. (82) Halder, K., Wieland, M., and Hartig, J. S. (2009) Predictable suppression of gene expression by 5′-UTR-based RNA quadruplexes. Nucleic Acids Res. 37, 6811−6817. (83) Verma, A., Halder, K., Halder, R., Yadav, V. K., Rawal, P., Thakur, R. K., Mohd, F., Sharma, A., and Chowdhury, S. (2008) Genome-wide computational and expression analyses reveal Gquadruplex DNA motifs as conserved cis-regulatory elements in human and related species. J. Med. Chem. 51, 5641−5649. (84) Wieland, M., and Hartig, J. S. (2007) RNA quadruplex-based modulation of gene expression. Chem. Biol. 14, 757−763. (85) Bowater, R. P. (2001) Supercoiled DNA: Structure, In eLS, John Wiley & Sons, Ltd.
(86) Brooks, T. A., and Hurley, L. H. (2009) The role of supercoiling in transcriptional control of MYC and its importance in molecular therapeutics. Nat. Rev. Cancer 9, 849−861. (87) Sutherland, C., Cui, Y., Mao, H., and Hurley, L. H. (2016) A Mechanosensor Mechanism Controls the G-Quadruplex/i-Motif Molecular Switch in the MYC Promoter NHE III1. J. Am. Chem. Soc. 138, 14138−14151. (88) Kendrick, S., Kang, H. J., Alam, M. P., Madathil, M. M., Agrawal, P., Gokhale, V., Yang, D., Hecht, S. M., and Hurley, L. H. (2014) The dynamic character of the BCL2 promoter i-motif provides a mechanism for modulation of gene expression by compounds that bind selectively to the alternative DNA hairpin structure. J. Am. Chem. Soc. 136, 4161−4171. (89) Kang, H. J., Kendrick, S., Hecht, S. M., and Hurley, L. H. (2014) The transcriptional complex between the BCL2 i-motif and hnRNP LL is a molecular switch for control of gene expression that can be modulated by small molecules. J. Am. Chem. Soc. 136, 4172−4185. (90) Kaiser, C. E., Van Ert, N. A., Agrawal, P., Chawla, R., Yang, D., and Hurley, L. H. (2017) Insight into the Complexity of the i-Motif and G-Quadruplex DNA Structures Formed in the KRAS Promoter and Subsequent Drug-Induced Gene Repression. J. Am. Chem. Soc. 139, 8522−8536. (91) Kouzine, F., Wojtowicz, D., Baranello, L., Yamane, A., Nelson, S., Resch, W., Kieffer-Kwon, K. R., Benham, C. J., Casellas, R., Przytycka, T. M., and Levens, D. (2017) Permanganate/S1 Nuclease Footprinting Reveals Non-B DNA Structures with Regulatory Potential across a Mammalian Genome. Cell Syst. 4, 344. (92) Michelotti, G. A., Michelotti, E. F., Pullner, A., Duncan, R. C., Eick, D., and Levens, D. (1996) Multiple single-stranded cis elements are associated with activated chromatin of the human c-myc gene in vivo. Mol. Cell. Biol. 16, 2656−2669. (93) Leonetti, C., Scarsella, M., Riggio, G., Rizzo, A., Salvati, E., D’Incalci, M., Staszewsky, L., Frapolli, R., Stevens, M. F., Stoppacciaro, A., Mottolese, M., Antoniani, B., Gilson, E., Zupi, G., and Biroccio, A. (2008) G-quadruplex ligand RHPS4 potentiates the antitumor activity of camptothecins in preclinical models of solid tumors. Clin. Cancer Res. 14, 7284−7291. (94) Baranello, L., Wojtowicz, D., Cui, K., Devaiah, B. N., Chung, H. J., Chan-Salis, K. Y., Guha, R., Wilson, K., Zhang, X., Zhang, H., Piotrowski, J., Thomas, C. J., Singer, D. S., Pugh, B. F., Pommier, Y., Przytycka, T. M., Kouzine, F., Lewis, B. A., Zhao, K., and Levens, D. (2016) RNA Polymerase II Regulates Topoisomerase 1 Activity to Favor Efficient Transcription. Cell 165, 357−371. (95) Zheng, K. W., Chen, Z., Hao, Y. H., and Tan, Z. (2010) Molecular crowding creates an essential environment for the formation of stable G-quadruplexes in long double-stranded DNA. Nucleic Acids Res. 38, 327−338.
2618
DOI: 10.1021/acschembio.7b00435 ACS Chem. Biol. 2017, 12, 2609−2618
