Bisquinolinium compounds induce quadruplex-specific transcriptome changes in HeLa S3 cell lines
© Hartig et al; licensee BioMed Central Ltd. 2012
Received: 15 November 2011
Accepted: 13 March 2012
Published: 13 March 2012
Guanosine rich sequences capable of forming G-quadruplex (G4) motifs are enriched near the gene transcription start site (TSS) in the human genome. When probed at the single gene level, G-quadruplex motifs residing in promoter regions show substantial effects on gene transcription. Moreover, further changes in transcription levels are noticed when G4-motifs are targeted with G-quadruplex-specific small molecules.
Global studies concerning general changes of the transcriptome via targeting promoter-based G-quadruplex motifs are very limited and have so far only been carried out with compounds displaying weak selectivity for quadruplex sequences. Here we utilize two G-quadruplex-specific bisquinolinium derivatives PhenDC3 and 360A and investigate their effects on the expression of the HeLa S3 transcriptome. Our results show wide-spread changes in the transcriptome with specificity for genes with G-quadruplex motifs near their transcription start sites (TSS). Using real-time PCR we further confirmed the specificity of PhenDC3 and 360A as potent molecules to target G-quadruplex-regulated genes.
Specific effects on quadruplex-containing genes have been observed utilizing whole-transcriptome analysis upon treatment of cultured cells with quadruplex-selective bisquinolinium compounds.
G-quadruplex motifs are four-stranded DNA conformations adopted by certain guanosine-rich sequences  abundant in the human genome [2, 3]. Interestingly, these G-quadruplex motif-forming sequences show a pronounced positional bias and consequently a wide range of biological functions have been predicted for G-quadruplex motifs, including roles in transcription [4–6], translation [7, 8], replication , nucleosome positioning [10, 11], CpG methylation , recombination  and splicing . Due to their high over-representation near transcription start sites (TSS) , the role of G-quadruplex motifs as transcriptional regulators has been studied most intensively (reviewed in [16–19]). The first experimentally verified biological function for a G-quadruplex motif was the transcriptional regulation of the c-MYC gene [20, 21]. Similar modulatory effects in transcription have since been demonstrated for quadruplex motifs in the promoter regions of c-MYB, VEGF, c-KIT, KRAS, HRAS, PDGFR-β and BCL-2 expression. In several studies a small molecule specific to G-quadruplex motifs was used to demonstrate that quadruplex formation in vivo results in a perturbation of transcription (reviewed in [19, 29, 30]). However, since more than 55% of the genes in the human genome harbor at least one potential G-quadruplex motif 1 kb up- or downstream of the TSS , it is of interest to investigate whether addition of quadruplex-interacting compounds affects transcription of many or most of these genes.
Using the G-quadruplex-stabilizing molecule TMPyP4 , Verma et. al.  and Mikami-Terao et. al.  showed genome-wide effects on transcription in HeLa S3 and K562 cells, respectively. However, the specificity of the observed effects can be questioned because TMPyP4 is reported to show only poor selectivity for G-quadruplex motifs compared to e.g. duplex DNA . The two studies identified a rather small set of affected genes at prolonged (48 h) treatment using high (100 μM) concentrations of TMPyP4. In these studies, only 69  and 87  perturbations of mRNA levels were observed. Moreover, porphyrin molecules were reported to have cytotoxic effects starting at 50 μM [34, 35] and are photoactive [36, 37], potentially resulting in harmful side-effects. More recently, using a single-chain antibody specific to G-quadruplex motifs, Fernando et. al.  determined 1,767 significant differentially expressed genes in a HGC-27 cell line. It should be noted that the choice of the cell line has very likely a strong impact on the outcome of whole-transcriptome examinations as described here. For example, a direct comparison of differentially expressed genes upon compound incubation between two cell lines in our opinion makes only sense if the compared genes are indeed expressed in both cell lines. Hence the immediate comparison of transcripome datasets between different cell lines will result in many apparent contradictions that might result from different expression states of the genes in different cell lines. In a further study, G-quadruplex motifs were selectively pulled down from HT1080 cells using a biotinylated bisquinolinium derivative . However, in the latter study, an assignment of preferential genomic location for G4-motifs was not achieved, thereby limiting the insights gained regarding the in vivo-function of G-quadruplex formation. In conclusion, further genome-wide studies of transcriptional changes mediated by quadruplex-specific compounds are needed in order to judge the extent to which G-quadruplexes influence transcription.
Results and discussion
Transcriptome profiling of HeLa S3 cells treated with PhenDC3 or 360A
G-quadruplex motifs in differentially expressed genes
The distribution of G4 motifs in differentially expressed genes (Illumina IDs) after PhenDC3 and 360A treatment
No. of Genes (with G4 motif)
No. of G4 motifs
Average loop length
PhenDC3 - up-regulatedb
PhenDC3 - down-regulatedb
360A - up-regulatedb
360A - down-regulatedb
Quantitative Real Time PCR in HeLa S3 cells after treating with increasing concentrations of different molecules.
Effect of position and strand occupancy of G-quadruplex motifs
G-quadruplex motif loop length and composition in differentially expressed genes
In order to search for a consensus motif or common feature that might be responsible for the differing behavior in non-affected and differentially expressed genes in response to the compound treatment, we investigated the difference in sequence composition between the G-quadruplex motifs present in differentially expressed genes versus genes with unchanged expression. The loop length and its nucleotide composition has been extensively studied earlier and both are known to affect the thermodynamic stability of G-quadruplex motifs [49–51]. Hence, we first examined the average loop length of G-quadruplex motifs occurring in differentially expressed versus non-differentially expressed genes. As shown in Table 1 the average loop lengths of quadruplexes in the different sets turned out to be very similar.
Functional classification of the differentially expressed genes
Gene Ontology (GO) categories enriched/depleted (P < 0.01) of differentially expressed genes after treating HeLa S3 cells with PhenDC3
Enriched GO classes
cellular metabolic process
cellular biopolymer metabolic process
cellular macromolecule metabolic process
biopolymer metabolic process
macromolecule metabolic process
primary metabolic process
nucleobase, nucleoside, nucleotide and nucleic acid metabolic process
biopolymer biosynthetic process
cellular biopolymer biosynthetic process
RNA metabolic process
cellular macromolecule biosynthetic process
macromolecule biosynthetic process
regulation of transcription from RNA polymerase II promoter
cellular biosynthetic process
establishment of localization in cell
cellular protein metabolic process
Depleted GO classes
immune system process
G-protein coupled receptor protein signaling pathway
cell surface receptor linked signal transduction
multicellular organismal process
In summary, the present study shows that the bisquinolinium compounds PhenDC3 and 360A can induce G-quadruplex-motif-specific changes in transcription. In contrast to previous studies, our experiments employ more quadruplex-specific compounds, which is reflected in the over-representation of differentially regulated genes bearing quadruplex motifs in their promoter regions. In an earlier study using TMPyP4 as a more non-specific G-quadruplex binder 61% of the differentially expressed genes were connected to the occurrence of a G4 motif in their ± 1 kb region around TSS which is less than expected . In comparison, PhenDC3 and 360A showed enhanced specificity. The differences observed between the two compounds can result from slight variations in affinity for various quadruplex-forming sequences as shown recently . However, we were unable to detect obvious characteristics such as loop compositions and motifs common to quadruplexes that are associated with genes that showed changes. Such identifiers would also have been very interesting to observe also with respect to the therapeutic potential of quadruplex-targeting small molecules. On the other hand, given the rather late time point of 48 h post incubation with the quadruplex-inducing compound, we cannot rule out that some or many of the observed effects are secondary and therefore cannot be attributed to a direct effect of a quadruplex formation. Taking this into account, future studies of quadruplex-targeting compounds should also take into account earlier time points in order to ensure that indeed primary changes of gene expression are observed.
Interestingly, we found that more genes were up-regulated than down-regulated following compound treatment. Several studies reported quadruplexes as transcription suppressor elements when their effects on individual genes were studied [19, 28, 59]. However, positive effects on transcription levels via the formation of promoter G-quadruplexes have also been described [60, 61] and hence the influence of the formation of a promoter-based quadruplex on transcription is very likely dependent on the exact position and the promoter architecture. Similar up-regulation effects were noticed earlier in genome-wide microarray experiments following TMPyP4  or single-chain antibody  treatments. Similarly, in absence of WRN/BLM helicases which are believed to resolve G-quadruplex motifs, a strong association was observed between up-regulated genes and G-quadruplex motifs in promoter regions . It has been speculated that the changes could be mediated by an influence of the G4 motifs on the chromatin state [10, 12]. It is also possible that the absence of a helicase (or in our case stabilization of transient G-quadruplex motifs by small molecules) may result in formation of transcription factor binding sites or destruction of repressor binding sites. In addition, along with other non-canonical structures such as cruciform and triplexes, quadruplexes have been discussed as modulators of superhelical torsion. Stress-induced duplex destabilization (SIDD) is more prevalent in transcriptional regulatory sites  and quadruplex formation was shown to release supercoiling stress . The occurring denaturation of duplex DNA in regulatory regions near the transcription start site would be an easy way of interfering with the transcriptional machinery and its regulation. However, more detailed studies are required to further decipher the exact mechanisms underlying quadruplex-mediated changes in transcription. We also observed a positional bias in the distribution of G-quadruplex motifs in differentially expressed genes. G-quadruplexes are enriched on the reverse strand before the TSS or on the forward strand after the TSS when compared to the G4-motifs present in non-differentially expressed genes. Interestingly, G4 motifs present on the non-template strand have been shown to play a role in RNA pol II pausing , which provides a possible mechanism for how these may in turn regulate the expression of the downstream gene. Further, a similar distribution of the loop size and composition of G-quadruplexes between the perturbed and non-perturbed genes were found, suggesting the precise genomic position and the composition of the surrounding DNA are likely to play an important role in determining the effect a given quadruplex will have on gene-transcription.
RNA Isolation and microarray experiments
HeLa S3 cells (obtained from the DSMZ, the German Resource Center for Biological Materials, http://www.dsmz.de/) were cultured in DMEM high glucose medium with 10% Fetal Calf Serum (FCS). For PhenDC3, 360A and 8979A, 10 mM stock solutions were prepared in DMSO and diluted directly in the DMEM medium to 10 μM just before using. The HeLa S3 cells exhibited 92% and 100% cell viability at 10 μM PhenDC3 and 360A, respectively, after 48 hrs treatment. After 48 hours of treatment, the whole RNA was isolated using RNeasy plus mini kit (Qiagen). Microarray experiment was performed by Aros Applied Biotechnology (Denmark) as per the manufacturer's protocol. Briefly, RNA was amplified using Illumina TotalPrep RNA amplification kit and hybridized on Illumina HumanHT-12v4 expression BeadChips as recommended by the manufacturer. Hybridization data was obtained by iScan Bead Array scanner (Illumina) and preprocessed by quantile normalization (Compliance to MIAME guidelines).
Microarray data analysis
The intensity values after quantile normalization for all triplicate experiments were further analyzed using CLANS software http://bioinfoserver.rsbs.anu.edu.au/downloads/clans/. Briefly, all the Illumina IDs (probes) were assumed to be differentially expressed if their anova values were ≤ 0.5 and to be of interest if their correlation to a condition of interest (see above, table or figure listing the 8 conditions) was ≥ 0.9. All experiments were performed in triplicate and overall > 98% correlation was observed between replicates.
Data retrieval and G-quadruplex mapping
The Genbank (.gbk)files for the Homo sapiens reference genome build 36.2 were downloaded from the NCBI ftp site ftp://ftp.ncbi.nih.gov/genomes/H_sapiens/ARCHIVE/BUILD.36.2. The G-quadruplex motifs were identified using a custom PERL program. The motif searched for was 5'-(GGGN1-7)≥3GGG-3', wherein N = A, T, G or C. All G-quadruplex motifs were mapped to their nearest mRNA within ± 2 kb of Transcription Start Sites (TSS). To be certain the same sequence build was used for all analyses, we independently mapped the Illumina probe sequences (47,323) onto the available mRNA data using BLAT  and also the mRNA sequences (45,495; retrieved from the Genbank files) onto the Illumina IDs. One Illumina ID may map to multiple mRNAs and vice versa, due to the non-equivalence of the datasets. This resulted in 32,980 mRNAs mapping to 34,638 Illumina probe IDs. The 32,980 mRNAs could be matched to 24,651 NCBI genes. Next, we also mapped the G-quadruplexes present (53,397) in the ± 2 kb region of the TSS of the 17,384 mRNAs (16,607 genes). The terms "forward", "reverse", "before TSS", and "after TSS" used to classify quadruplexes refer to the position the G-rich quadruplex is found with respect to the coding (sense or template) strand of the associated gene. In this respect "forward" means the presence of a quadruplex on the coding (sense or template) strand.
Real time polymerase chain reaction
Two step real time PCR was performed to validate the microarray results. cDNA was prepared by using M-MuLV reverse transcriptase (Fermentas). Relative quantitative real time PCR was performed using LightCycler 480 SYBR Green I master (Roche) in a Roche LightCycler 480 system, for primer sequences see additional file 1, Figure S2. A negative control for each gene with no template was included. Reaction conditions consisted of 95°C for 1 min followed by 45 cycles of amplification (15 sec at 95°C, 15 sec at 60°C and 15 sec at 72°C). After amplification, melting analysis was performed to ensure a uniform amplification product.
Loop length and composition analysis
The different features (length and composition) of the G-quadruplex motifs were extracted via a PERL script and visualized using 'R' http://www.r-project.org/. For programmatic simplicity, the first three guanines from 5'-end were considered part of the stem and consecutive guanines, if any, were considered as part of the loop (length 1-7). To reduce redundancy in loop length and composition, each G-quadruplex motif was considered only once. All the statistical tests were performed in 'R' http://www.r-project.org/.
Gene Ontology (GO) classification
For functional classification all the differentially expressed gene names were converted to Uniprot standard gene name using gene merge (http://genemerge.cbcb.umd.edu/GeneMerge.php, ) and these converted gene names were used to obtain the GO category for biological function (http://genome.crg.es/GOToolBox/, ). Bonferroni correction was used to correct for multiple testing for PhenDC3. NOTE: The results for the 360A treated sample were not corrected for multiple testing as this removed all significant hits. These results should be interpreted with this in mind. Categories having p-value less than 0.01 was considered to be significantly enriched/depleted.
Availability of supplementary data
Further data sets including are available as Additional file 1 (containing Figure S1 and Tables S1, S2, and S3) and Additional File 2 (Table S4). The microarray data set supporting the results of this article is available online at the Gene Expression Omnibus repository (accession number GSE32170, http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=rnaxlqiuomkuips&acc=GSE3217)
We thank Kangkan Halder for helpful discussions. The work was funded by the VolkswagenStiftung via a Lichtenberg Professorship to JSH. JFR received funds from the "Ligue Nationale Contre le Cancer". MPTF and JFR thank the Agence Nationale pour la Recherche ANR (G4-TOOLBOX, ANR-Blan-09-355) for fundings. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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