DNA G-quadruplex formation in response to remote downstream transcription activity: long-range sensing and signal transducing in DNA double helix

Abstract

G-quadruplexes, four-stranded structures formed by Guanine-rich nucleic acids, are implicated in many physiological and pathological processes. G-quadruplex-forming sequences are abundant in genomic DNA, and G-quadruplexes have recently been shown to exist in the genome of mammalian cells. However, how G-quadruplexes are formed in the genomes remains largely unclear. Here, we show that G-quadruplex formation can be remotely induced by downstream transcription events that are thousands of base pairs away. The induced G-quadruplexes alter protein recognition and cause transcription termination at the local region. These results suggest that a G-quadruplex-forming sequence can serve as a sensor or receiver to sense remote DNA tracking activity in response to the propagation of mechanical torsion in a DNA double helix. We propose that the G-quadruplex formation may provide a mean for long-range sensing and communication between distal genomic locations to coordinate regulatory transactions in genomic DNA.

Figure 1.

G-quadruplex formation induced by downstream transcription. (A) Schemes illustrating the structure of the DNAs in which a G₃(TG₃)₃ motif (Q) or its mutant (M) was placed upstream of one or between two divergently oriented T7 promoters (T7), which was flanked by 45 bp at its 3′ side so that transcription can proceed for 45 nucleotides (nt). (B) G-quadruplex formation in transcribed DNA detected by native gel electrophoresis. DNAs with a FAM dye at the 5′-end of the nontemplate strand were subjected to transcription followed by digestion with RNase A and H or heat denaturation/renaturation. The structure of the DNAs and cation in the transcription buffer is indicated above the gel. N, H, T indicates non-transcribed, heated (21,22) and transcribed DNA, respectively. The schemes at the left illustrate the structures of the corresponding DNA bands. (C) Verification of G-quadruplex formation by DMS footprinting. Cleavage fragments were resolved on a denaturing gel (top) and digitized (bottom) for comparison.

Figure 2.

Characterization of G-quadruplex formation induced by downstream transcription. DNA carrying a G₃(TG₃)₃ was transcribed with T7 polymerase and resolved on a native gel. G-quadruplex-bearing DNA was expressed as percentage of total DNA (% qDNA). (A) G-quadruplex formation as a function of the distance a polymerase could translocate. One group of DNAs carried a G₃(TG₃)₃ between two divergently oriented T7 promoters (filled circle). Another group of DNAs was derived from this group by mutating the T7 promoter in the upstream region (open circle). The size of the sequences flanking the 3′ side of the T7 promoter(s) varied from 45–205 bp. (B) G-quadruplex formation as a function of NTP concentration. DNA bearing a G₃(TG₃)₃ upstream of a T7 promoter (Figure 1A, scheme at left) was transcribed at various concentrations of NTP and resolved on a native gel. Data show the mean and standard deviation of three independent experiments. (C) G-quadruplex formation in long dsDNA. The DNAs contained a G₃(TG₃)₃ upstream of a T7 promoter, which were separated by various length of base pairs (left panel). DNA of 600 bps or longer was cut at a restriction site 230 bp downstream of the G₃(TG₃)₃ after transcription, producing a FAM-labeled 333-bp fragment that could be resolved by native gel electrophoresis. The remaining samples of shorter DNA were treated under the same condition, but in the absence of restriction enzyme. Data (right panel) show the mean and standard deviation of three independent experiments. D_1/2 denotes the distance required for G-quadruplex formation to drop to the midvalue between the minimum and maximum.

Figure 3.

Distribution of PQS occurrence in the 5000 bp region flanking the 5′ and 3′ end of genes. Frequency was normalized to the number of sequences and expressed as the number of occurrences in 100 sequences within a 100 nt window. A gene here denotes the transcribed region, including 5′ untranslated region (UTR), exons, introns and 3′ UTR. TSS and TES indicate transcription start and end site, respectively. Inserts in the left panels show PQS mapping at 20 nt resolution within the 1000 nt region downstream of TES.

Figure 4.

Statistics of genes carrying PQS motifs in different species. (A) Percentage genes having PQS in the upstream region of their TSS. The value at a given position gives the percentage of genes that have at least one PQS in the range from −1 to the given position. (B) Percentage genes having PQS within the −1 to −5000 bp upstream region of their TSS. The species is ordered according to the species tree provided on the Ensembl website. PQS on both the template and non-template strand was counted in (A) and (B).

Figure 5.

Model of downstream transcription activity sensing by G-quadruplex formation and its direct effect on DNA. The fast propagation of negative supercoiling generated by a proximal or distal downstream transcription or DNA tracking event induces a G-quadruplex formation at the PQS site and subsequently affects protein recognition and hinders protein translocation along the DNA.