Introduction {#s1}
============

The landscape of genomic DNA has shown a myriad of alternate DNA structures such as cruciform ([@B9]), G-quadruplexes (G4s) ([@B37]), triplexes ([@B18]), and i-motifs ([@B1]). These structures can form within genomic DNA (B-DNA), as seen in the case of left-handed Z-DNA ([@B33]), or require the opening of base pairs leading to generation of single-stranded regions within genomic DNA as seen in cruciform DNA ([@B9]) and G4s ([@B35]). Genome-wide prediction of secondary structure-forming regions in various genomes is possible because of their propensity to favor specific sequence patterns. It has been proven that Z-DNA favors purine--pyrimidine repeats flanked by specific sequences for B-DNA/Z-DNA junction formation ([@B7]; [@B32]), whereas cruciform structures can be formed in palindromic regions ([@B40]). The abundance of secondary structures has led to attempts to identify the probable roles of these structures in replication, gene regulation, and DNA damage/repair. These structures have been implicated in several diseases, such as amyotrophic lateral sclerosis, frontotemporal dementia, Fanconi anemia, Bloom's syndrome, and fragile X disease ([@B61]; [@B57]).

G4s are among the most widely studied DNA secondary structures formed from consecutive blocks of two or more guanines separated by a single-stranded region called a loop. Four consecutive G-runs form G-stacks with Hoogsteen bonds \[(C2)NH2:N7 and O6:N1H\], which are stabilized by several monovalent and divalent cations such as K^+^, Na^+^, Ca^2+^, and Sr^2+^, which have been reviewed elsewhere ([@B55]; [@B4]). K^+^ is the best stabilizer of the G4 due to its favorable ionic radius and Gibbs free energy of solvation ([@B65]). Computational tools such as Quadparser, PQSFinder, G4Hunter, and QGRS Mapper have been developed to predict putative G4-forming sequences ([@B50]). They are based on pattern matching and scoring algorithms using the schema G~x~N~y~G~x~N~y~G~x~N~y~G~x~, where N is any nucleotide, x is ≥2, and y is ≥1. However, y is usually considered as 1--7 as longer loops are flexible and destabilize the G4 ([@B26]). Several putative G4-forming sequences were predicted in the genomes of prokaryotes and eukaryotes and accessible through web servers such as QuadBase and NonBDB ([@B62]; [@B11]). In addition, high-throughput sequencing has also been used to experimentally verify G4 formation in the genomes of many organisms ([@B41]) and construct whole-genome experimental G4 maps.

The enrichment of G4s throughout several genomes especially in the *cis*-regulatory regions ([@B12]; [@B41]) has led to the development of several small molecules that can bind and stabilize G4s, including porphyrins, benzoquinolines, and perylene diimide ([@B58]). Some of the most widely used G4 stabilizers are TMPyP4, NMM-IX, pyridostatin (PDS), and BRACO-19. To study the effects of these chemicals on G4-mediated gene regulation, individual reporter assays are performed by cloning specific regulatory regions in reporter vectors and analyzing reporter gene expression in the presence of a G4-stabilizing ligand ([@B21]) ([**Figure 1**](#f1){ref-type="fig"}). G4-mediated *cis*-regulatory activity has been confirmed by reporter assays in various genes such as *CMYC*, *C-KIT*, and *BCL2* ([@B56]; [@B2]; [@B39]).

![Schematic showing the comparison between conventional reporter assays and Genome-Wide Association Studies (GWAS). **(A)** Conventional reporter-based study of G-quadruplexes (G4s) in the specific gene promoters, in this case, Gene A. **(B)** GWAS expression analysis showing binding of G4 ligand to G4s formed in untargeted regions (Gene B) of the genome. **(C)** Protein A is normally expressed and performs its regulatory activity on Gene C in the absence of G4 ligand, whereas G4-mediated cis-regulation in the presence of G4 ligand leads abnormal regulation of Gene C by Protein A.](fgene-10-01002-g001){#f1}

Although these ligands display considerable selectivity for G4 structures over single-stranded and double-stranded DNA, it is essential to study the effect of ligand binding to untargeted G4-forming regions in the genome ([**Figure 1**](#f1){ref-type="fig"}). A key factor influencing specificity and the *cis*-regulatory impact is to identify the conditions that affect G4 formation. Apart from intracellular K^+^, Na^+^, and Mg^2+^, there are several other conditions that can affect G4 formation within both prokaryotic and eukaryotic cells. Proteins that can interact with G4s have been described ([@B8]; [@B23]). Some studies have shown that G4 formation is influenced by the chromatin status and that euchromatin shows more G4-forming sequences than heterochromatin ([@B24]), corroborating the idea that actively transcribed genes show higher propensity to form G4s. Several universal transcription factors, such as MYC, SP1, and VEGF, are also regulated by G4s. Changes in expression levels of such transcription factors may affect the expression of genes regulated by them ([**Figure 1**](#f1){ref-type="fig"}).

The cross-reactivity between G4 ligands and i-motifs has also been reported since i-motifs are present on the opposite strand of the G4. Ligands such as TMPyP4 and berberine were shown to bind to i-motifs, although their ability to stabilize them was lower than that of G4s ([@B17]; [@B42]; [@B49]). The prevalence of G4s in the RNA has also been shown recently ([@B36]; [@B64]), and the functional effects of RNA G4 stabilization have also been reviewed before ([@B16]). DNA G4-binding ligands such as TMPyP4 have also been shown to destabilize RNA G4s ([@B48]; [@B46]; [@B66]). However, the functional impact of the cross-reactivity of G4 ligands to DNA and RNA has not been well studied. A combination of high-throughput studies along with individual RNA analysis can also be utilized to understand the impact of cross-reactivity of G4 ligands. In addition, since RNA G4s have been extensively discussed in previous reviews ([@B16]), we do not include the studies on RNA G4s in this review.

Therefore, the addition of a G4-stabilizing ligand can be expected to impact multiple regions in the genome and affect transcription of multiple genes at the same time. High-throughput studies do not provide a fine-grained analysis of the dynamics of individual genes regulated by G4s. Therefore, individual reporter assays are required to analyze the effect of G4s on specific target genes ([**Figure 1**](#f1){ref-type="fig"}). Concomitantly, individual reporter assays on all genes of an organism combined with genome or transcriptome-wide studies can provide a better understanding of how individual G4s can regulate gene expression ([**Figures 1A, B**](#f1){ref-type="fig"}). In this review, we discuss the importance of combining high-throughput experiments with studies on individual G4s in humans, bacteria, and viruses to obtain a better picture of G4-mediated *cis* regulation.

Current Status of Studies on *cis*-Regulation by G4s {#s2}
====================================================

Studies on *Cis*-Regulatory G4s in Humans {#s2_1}
-----------------------------------------

Quadparser-based computational analysis of the human genome for the prediction of G4-forming sequences based on the schema G~3+~N~1--7~G~3+~N~1--7~G~3+~N~1--7~G~3+~ initially revealed 370,000 G4 sequences ([@B28]). Regulatory regions of the human genome were enriched in G4s ([@B30]; [@B59]), and the distribution of G4-forming sequences was also dependent on the function of the gene; for example, tumor suppressors contained lower G4-forming sequences than did proto-oncogenes ([@B15]). Later studies showed that promoter G4 regions overlapped with DNAse hypersensitive sites in over 40% of human genes ([@B29]). Experimental confirmation of the impact of G4 formation on transcription and translation was carried out initially in specific genes such as *CMYC* ([@B63]), *KRAS* ([@B13]), *HRAS* ([@B43]), and *BCL2* ([@B47]). However, experimental evidence of G4 formation to corroborate the computational analysis was still pending. Later, high-throughput sequencing studies *in vitro* showed that over 700,000 G4s can be formed in the genome in the presence of KCl and PDS ([@B12]). These studies proposed that G4 formation in the regulatory regions may have an impact on gene regulation. However, they could not elucidate the impact of nonspecific binding of G4 stabilizers or the downstream impact of G4-mediated gene regulation on other genes. These limitations may be overcome by high-throughput transcriptome-wide differential expression studies.

Several studies have involved treating cells for specific periods of time with G4-stabilizing ligands and analyzing changes in gene expression for changes before and after treatment ([**Table 1**](#T1){ref-type="table"}). In most studies, TMPyP4 was used as the G4-stabilizing ligand. Initial studies using the HeLa S3 cell line showed that the G4-binding ligand could cause changes in gene expression ([@B20]). They observed that proto-oncogenes, such as *CMYC*, *CMYB*, and *CFOS*, were downregulated under TMPyP4 treatment, but not by TMPyP2. Another study on the same cell line showed similar results and found that the promoter regions of differentially expressed genes, including *CMYC*, *CMYB*, and *CFOS*, contained G4-forming sequences ([@B59]). Interestingly, this study also found that there was no statistically significant correlation between the presence of G4s and the expression fold change. The same group performed a subsequent study with TMPyP4 on the A549 cell line and compared the results with BMVC and a TMPyP4 analog TyPy ([@B60]). This study also observed 863 significantly upregulated and 298 significantly downregulated genes similar to their previous study on Hela S3 cells. Therefore, they shortlisted 12 genes containing G4s in their promoters from the microarray results and analyzed them individually by quantitative Real-Time PCR (qRT-PCR), demonstrating that the genes were indeed affected by G4-stabilizing ligands.

###### 

Compilation of studies on *cis*-regulatory G4s in humans, bacteria, and viruses.

  List of high-throughput G4 cis-regulatory studies in *Homo sapiens*                                                                                                                                                                                                                                                             
  --------------------------------------------------------------------- -------------------------- --------------------------------------------------------------------------------------------------------- -------------------------------- ------------------------------------------------ ------------------ --------------- ----------
  *H. sapiens*                                                          X\*                        --                                                                                                        TMPyP4                           100 µM                                           24, 36, and 48 h   HeLa S3         ([@B20])
  *H. sapiens*                                                          V\*                        Custom algorithm, G~3~N~1--7~G~3~N~1--7~G~3~N~1--7~G~3~                                                   TMPyP4                           100 µM                                           24 and 48 h        HeLa S3         ([@B59])
  *H. sapiens*                                                          X                          --                                                                                                        TMPyP4                           100 µM                                           48 h               K562            ([@B44])
  *H. sapiens*                                                          V                          Custom algorithm, G~3~N~1--7~G~3~N~1--7~G~3~N~1--7~G~3~                                                   TMPyP4                           100 µM                                           48 h               HeLa S3, A549   ([@B60])
  *H. sapiens*                                                          V                          Custom algorithm, G~3~N~1--7~G~3~N~1--7~G~3~N~1--7~G~3~                                                   PhenDC3, 360A                    10 µM                                            48 h               HeLa S3         ([@B22])
  List of G4 cis regulatory studies in bacteria                                                                                                                                                                                                                                                                                   
  Organism                                                              Computational prediction   Prediction algorithm                                                                                      Individual gene reporter assay   Transcriptome/differential expression analysis   Reference                          
  *Escherichia coli*                                                    V                          Custom algorithm, G~2--5~N~1--5~G~2--5~N~1--5~G~2--5~N~1--5~G~2--5~                                       X                                X                                                ([@B53])                           
  *Deinococcus radiodurans*                                             V                          Custom algorithm and QGRS Mapper, G~2--5~N~1--5~G~2--5~N~1--5~G~2--5~N~1--5~G~2--5~                       V                                X                                                ([@B34])                           
  *Mycobacterium tuberculosis*                                          V                          Custom algorithm, ~G2--5~N~7/11/15~G~2--5~N~7/11/15~G~2--5~N~7/11/15~G~2--5~                              X                                X                                                ([@B51])                           
  *Xanthomonas* sp., and *Nostoc* sp.                                   V                          ProQuad Database                                                                                          X                                X                                                ([@B54])                           
  Domain bacteria (∼1500 genomes)                                       V                          G4Hunter; Default parameters                                                                              X                                X                                                ([@B3])                            
  *E. coli*                                                             V                          Quadparser, G~≥3~N~1--7~ G~≥3~N~1--7~ G~≥3~N~1--7~G~≥3~                                                   X                                X                                                ([@B31])                           
  *E. coli*                                                             V                          ProQuad, G~2--5~N~1--5~G~2--5~N~1--5~G~2--5~N~1--5~G~2--5~                                                V                                X                                                ([@B27])                           
  *Streptococcus pneumoniae*                                            V                          Custom algorithm, QGRS Mapper, PQSFinder, G~≥3~N~0--10~G~≥3~N~0--10~ G~≥3~N~0--10~G~≥3~                   V                                X                                                ([@B45])                           
  *Rhodobacter*, *Trypanosoma*, *Plasmodium*, *E. coli*, *Leishmania*   V                          Custom algorithm, G~3~N~1--12~G~3~N~1--12~G~3~N~1--12~G~3~ and G~2~N~1--12~G~2~N~1--12~G~2~N~1--12~G~2~   X                                X                                                ([@B41])                           
  Deinococcales and Thermales                                           V                          Quadparser, G~3~N~1--12~G~3~N~1--12~G~3~N~1--12~G~3~ and G~2~N~1--12~G~2~N~1--12~G~2~N~1--12~G~2~         X                                X                                                ([@B14])                           
  List of G4 cis-regulatory studies in viruses                                                                                                                                                                                                                                                                                    
  Organism                                                              Computational prediction   Prediction algorithm                                                                                      Individual gene reporter assay   Transcriptome/differential expression analysis   Reference                          
  Human herpesvirus                                                     V                          Quadparser, G~3~N~1--7~ G~3~N~1--7~ G~3~N~1--7~G~3~                                                       V                                X                                                ([@B5])                            
  Hepatitis B virus                                                     V                          Quadparser, G~3~N~1--7~ G~3~N~1--7~ G~3~N~1--7~G~3~                                                       V                                X                                                ([@B6])                            
  Human cytomegalovirus                                                 V                          Custom script, G~3--6~N~1--7~G~3--6~N~1--7~G~3--6~N~1--7~G~3--6~                                          V                                X                                                ([@B52])                           
  Alphaherpesviruses                                                    V                          QGRS Mapper, Quadbase G~2~N~1--7~G~2~N~1--7~G~2~N~1--7~G~2~, G~3~N~1--12~G~3~N~1--12~G~3~N~1--12~G~3~     V                                X                                                ([@B19])                           

"X" indicates "absent," and "V" indicates "present."

A similar study on the effect of gene expression by TMPyP4 in the K562 cell line showed that only 33 genes were upregulated and 54 genes were downregulated and proposed that TMPyP4 might act by repressing *CMYC* and activating MAPK family kinases ([@B44]). The same group observed similar effects using retinoblastoma cell lines in response to TMPyP4 and demonstrated that the induction of p53 and activation of MAPK kinases could contribute to the antitumor effects of TMPyP4. However, in both studies, they could only speculate that the G4 stabilization by TMPyP4 could affect the regulation of differentially expressed genes. In addition, they also observed telomere shortening in both K562 and retinoblastoma cell lines where G4 stabilization prevents telomerase from binding to the 3′ end of the telomere and maintaining telomere length. So, it is difficult to predict whether the effect of G4 is by gene regulation or telomere shortening. TMPyP4 was developed to target telomeric G4s ([@B25]), but the transcriptome-wide study showed nonspecific activities, underscoring the need for further genome-wide studies in cells.

Another study compared the effect of bisquinolinium drugs 360A and PhenDC3 on gene expression in Hela S3 cell lines and showed that 1157 genes were downregulated and 1529 upregulated in PhenDC3. In the case of 360A, only 249 downregulated and 401 upregulated genes were observed ([@B22]). This clearly indicates that although the small molecules were developed as G4-binding ligands, they show significant nonspecific effects, which need to be explored further. In addition, the mechanisms that dynamically control G4 formation are yet to be understood, so combined genome and transcriptome-wide mechanistic and functional analysis is required to unravel the mysteries of gene regulation by G4 stabilization.

Studies on *Cis*-Regulatory G4s in Bacteria {#s2_2}
-------------------------------------------

The bacterial genome is considerably simpler than the eukaryotic genome due to the absence of the complex organization that is found in the human genome. However, various computational studies and individual promoter region analysis have shown that the genomes of several bacteria contain G4-forming sequences ([**Table 1**](#T1){ref-type="table"}). Genome-wide prediction has identified G4-forming sequences in the genomes of *Escherichia coli* ([@B53]), *Deinococcus radiodurans* ([@B34]), *Mycobacterium tuberculosis* ([@B51]), *Xanthomonas* sp., and *Nostoc* sp. ([@B54]). These studies also showed that the G4-forming sequences were predominantly restricted to regulatory regions such as promoters ([@B53]). In each study, individual luciferase assays carried out on selected promoter regions showed variable responses to the addition of G4 ligands. For example, in the case of *D.* radiodurans, some promoters showed higher activity when the bacterium was treated with NMM-IX, whereas others showed diminished activity, although all promoters contained G4-forming sequences ([@B34]). This lack of correlation exhibited in promoter luciferase assays and whole transcriptome studies indicates that the landscape of gene regulation by G4 is more complex than expected even in prokaryotes, despite the simple organization of their genome.

In the case of *E. coli*, a systematic study on the effect of the location of G4 relative to the transcription start site (TSS) was performed in the genome using reporter assays where the G4-forming sequences were cloned according to their genomic locations into pQE luciferase reporter plasmids ([@B27]). The results revealed that G4 formation in the 5′ UTR significantly affected reporter gene expression, but the 3′ UTR G4s had a negligible effect on gene expression. It was also observed that the G4 sequences within 20 bp downstream of the TSS showed maximum upregulation or downregulation depending on whether the G4 was formed on the antisense or sense strand, respectively. Interestingly, there was no effect of NMM-IX or other G4-stabilizing ligands on G4-mediated gene regulation ([@B27]), indicating that more studies are required to explain gene regulation by G4s in *E. coli*.

In a recent study on G4 sequences in the *E. coli* genome, the predicted G4-forming regions were aligned using ClustalW to identify repetitive sequence motifs ([@B31]). This was based on the idea that similar sequences will have similar regulatory roles. In this analysis, only 52 sequences matched their stringent schema with G-tract length 1 to 3 and loop length 1 to 7. They further classified these into two groups of well-aligned sequence and performed reporter assays using the representative sequence. Interestingly, all the sequences were within the regulatory regions flanking the open reading frame, and the group was able to identify two sequence motifs conserved in several bacteria. However, the functional impact of these sequences needs to be investigated.

The recent interest in studies on bacterial G4s has necessitated the formulation of a streamlined approach to facilitate interpretation of the roles of G4s in gene regulation. Since the bacterial system has been extensively studied as a model organism, it will be interesting to combine both high-throughput and individualistic approaches to gain a comprehensive picture of bacterial G4s.

Studies on *Cis*-Regulatory G4s in Viruses {#s2_3}
------------------------------------------

Viruses provide an exciting platform for studying the impact of G4s at the genomic and transcriptomic level due to their small genome size. Some viral DNA can be chromatinized as in eukaryotes, and the genetic materials of some DNA and RNA viruses can be integrated into the human genome and affected by the same parameters as human genomic DNA. Therefore, they present an ideal platform for studies that especially focus on the holistic effects of G4-binding ligands especially to understand G4-mediated gene regulation.

Genome-wide computational analyses of G4s in several viral genomes have revealed that DNA viruses had a higher number of G4s per 1 kb compared to RNA viruses ([@B38]). Many computational and individual reporter studies were performed in a number of viruses to evaluate the *cis*-regulatory effects of G4s ([**Table 1**](#T1){ref-type="table"}). One of the first systematic genome-wide studies on G4s in viruses was performed in human herpesvirus genomes ([@B5]). Preliminary computational analysis of G4s present in regulatory regions of the herpesvirus genome revealed their prevalence in regulatory and long terminal repeat regions. Among regulatory regions, immediate-early genes showed higher densities of G4-forming sequences when compared with early or late gene promoters. Overall, alpha-herpesviruses such as herpes simplex virus-1 (HSV-1) and varicella-zoster virus genomes had a higher G4-density than did human and mouse genomes, indicating that G4 formation in these viruses would have more impact on gene regulation. In this study, the authors considered G4-forming sequences found in only three genes, namely, UL2 and UL24 of HSV-1 and K15 of Kaposi's sarcoma-associated herpesvirus, for further experimental study. It was observed that G4s could suppress gene expression in the presence of the G4-stabilizing ligands BRACO19 and TMPyP4. However, since several G4-forming sequences were predicted, the questions of how many G4-forming sequences actually form G4s and how many G4s regulate viral gene expression still need to be explored.

In lieu of this important question, a recent study systematically checked the effect of G4s on gene regulation in all genes of the human cytomegalovirus, which belongs to the beta-herpesvirus subfamily ([@B52]). Unlike previous reports that only tested a few G4s, the genome-wide analysis for all conventional, long-loop, and bulged-G4 schema identified 36 G4-forming sequences associated with 20 viral genes including all immediate-early, early, and late genes. Most of these sequences formed G4s *in vitro*, and their stability could be further increased by NMM-IX treatment. The cell-based assays using reporter constructs with promoters containing G4s indicated that out of 20 genes only 9 were suppressed effectively by G4-stabilizing ligand NMM-IX. This is interesting because while all tested genes contained G4-forming sequences (evidenced by *in vitro* assays) in their promoter regions, only half of these genes were affected by the ligand. Therefore, it was proposed that there exists a context-dependent mechanism by which G4s influence viral genes. It is also possible that other factors that are involved in controlling gene expression, such as the binding of human transcription factors, are implicated in G4 activity as shown earlier for HPV ([@B10]). This proves to be an exciting field for further study and can facilitate the construction of G4-mediated regulatory networks. This also shows that although many promoters might contain G4s that can affect gene expression when tested individually, genome-wide studies are also important in studying the collective impact of G4 stabilization.

Future Perspectives {#s3}
===================

The role of G4s in the genomes of prokaryotes and eukaryotes is an exciting area of research because of the plethora of reports showing the influence of G4s on specific gene expression. However, there is an absence of reconciliation between studies on individual G4s and high-throughput genome and transcriptome-wide studies, especially for *cis*-regulatory G4s. With the advent of next-generation sequencing technologies and high-throughput reporter assays, it should be possible to construct complex G4 networks with the ability to incorporate computational and experimental analysis and present a combined view of G4-mediated regulation. For example, the computational G4 prediction and individual gene reporter assays can be compared with high-throughput differential expression studies to identify candidates that show the maximum effect. The regions can be compared with Chromatin Immuno Precipitation (ChIP) studies to correlate with transcription factor binding sites or chromatin binding sites. Construction of a central repository to store the results of functional analysis from various publications will also facilitate the comparison of data and provide a holistic picture of gene regulation by G4s.

Author Contributions {#s4}
====================

SR, J-HA, and KK wrote the manuscript.

Funding {#s5}
=======

This work was supported by National Research Foundation of Korea funded by the Ministry of Science and ICT (2019R1A2C2006676 to J-HA and 2019R1A2C2089148 to KK).

Conflict of Interest {#s6}
====================

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

[^1]: Edited by: Daniel Yero, Autonomous University of Barcelona, Spain

[^2]: Reviewed by: Mahdi Zeraati, University of New South Wales, Australia; Luca Pandolfini, University of Cambridge, United Kingdom; Aaron John Stevens, University of Otago, Christchurch New Zealand

[^3]: This article was submitted to Evolutionary and Genomic Microbiology, a section of the journal Frontiers in Genetics
