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. 2002 Sep 3;99(18):11593-8.
doi: 10.1073/pnas.182256799. Epub 2002 Aug 23.

Direct evidence for a G-quadruplex in a promoter region and its targeting with a small molecule to repress c-MYC transcription

Adam Siddiqui-Jain  1 , Cory L Grand, David J Bearss, Laurence H Hurley
Affiliations

Direct evidence for a G-quadruplex in a promoter region and its targeting with a small molecule to repress c-MYC transcription

Adam Siddiqui-Jain et al. Proc Natl Acad Sci U S A. .

Abstract

The nuclease hypersensitivity element III(1) upstream of the P1 promoter of c-MYC controls 85-90% of the transcriptional activation of this gene. We have demonstrated that the purine-rich strand of the DNA in this region can form two different intramolecular G-quadruplex structures, only one of which seems to be biologically relevant. This biologically relevant structure is the kinetically favored chair-form G-quadruplex, which is destabilized when mutated with a single G --> A transition, resulting in a 3-fold increase in basal transcriptional activity of the c-MYC promoter. The cationic porphyrin TMPyP4, which has been shown to stabilize this G-quadruplex structure, is able to suppress further c-MYC transcriptional activation. These results provide compelling evidence that a specific G-quadruplex structure formed in the c-MYC promoter region functions as a transcriptional repressor element. Furthermore, we establish the principle that c-MYC transcription can be controlled by ligand-mediated G-quadruplex stabilization.

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Figures

Figure 1
Figure 1
Determination of the structures of the unimolecular G-quadruplexes formed after incubation of the Pu 27 strand (Pu27) in 100 mM KCl for 48 h at 37°C. (A) Promoter structure of the c-MYC gene; shown in Inset is the 27-mer sequence of the purine-rich strand upstream of the P1 promoter (3). (B) Nondenaturing gel analysis (15% polyacrylamide/12.5 mM KCl/NaCl, 4°C, 16 h) of Pu 27 preincubated under the conditions specified in the figure at a strand concentration of ≈25 μM. (C) DMS footprinting of band 1 in B. (Left) DMS treatment of the denatured Pu27 (lanes 1 and 2) and the isolated band (lanes 3 and 4). The Pu27 base sequence is shown to the left. (Right) Proposed structure based upon the footprinting pattern. Guanines showing DMS-induced cleavage are labeled in both Left and Right. Base colors: red, guanine; green, thymine; and orange, adenine. (D) As in C, but for band 2 in B.
Scheme 1
Scheme 1
Structures of the aptamers T30695 (24) and TBA (23) in comparison to that proposed for the chair G-quadruplex from Pu27 (Center).
Figure 2
Figure 2
Effect of mutations of Pu27 on transcriptional activity and stability of the G-quadruplexes. Site-directed mutagenesis was performed as described in Methods. (A) Cartoon showing the mutations that destabilize the chair (I), basket (III), and chair and basket (II) alongside the wild-type (Pu27) and null-mutant (IV) sequences. The positions of the mutants in the chair and basket G-quadruplex regions are shown by the arrows. The arrows from the asterisks point to the corresponding positions in the non-G-quadruplex regions of the alternative basket or chair structure (the mutant bases in I–IV are shown in bold) in which mutations occur. (B) Diagram of the c-MYC-luciferase reporter vector (modified from ref. 46). The Del-4 plasmid, a gift from Bert Vogelstein, places ≈850 bp of c-myc sequence, including the two major promoters (P1 and P2), in control of a luciferase expression cassette. (C) Expression of luciferase normalized to the wild-type and various mutant sequences shown in A. Transfection and luciferase assays were performed as described in Methods. Each data point is the average of three experiments. (D) Polymerase stop assay (29) for determination of the effect of mutations on the stability of the G-quadruplex structure at 45°C in the presence of 10 mM KCl. Each data point is the average of three experiments. The cartoon above the gel illustrates the principle of the assay. The G-quadruplex structure is shown in proximity to the pause site for Taq polymerase. (E) Quantitation of the results from the polymerase stop assay shown in D.
Figure 3
Figure 3
Photocleavage and stabilization of the basket and chair G-quadruplexes formed from Pu27 as a result of interaction with TMPyP4 and TMPyP2. (A) Structures of TMPyP4 and TMPyP2. The overlapping arcs shown in the TMPyP2 structure demonstrate the steric clash between the 2-methylpyridyl groups and the 3-pyrrol hydrogens. (B) Photo-induced cleavage of the denatured (lanes 1–6) and isolated (lanes 7–12) basket G-quadruplex structures with TMPyP4 (lanes 1–3 and 7–9) and TMPyP2 (lanes 4–6 and 10–12). Photocleavage was carried out for the times shown at the top of the gel. Photocleavage of bands 1 and 2 (see Fig. 1B), each with TMPyP4 and TMPyP2, was conducted essentially as outlined in ref. , except that salmon sperm DNA was used instead of calf thymus DNA in the stop buffer. Guanines cleaved by TMPyP4 and TMPyP2 are shown to the right of the gel and are identified with arrows on the cartoon on the right. Base colors: red = guanine; green = thymine; orange = adenine. (C) As for B except that TMPyP4 and TMPyP2 were evaluated on separate gels, and the chair G-quadruplex was used in place of the basket. Blue arrows show the implied positions for TMPyP4 end-stacking to the two intact G-tetrads. (D Left) The polymerase stop assay was used to compare the stabilization of the G-quadruplex structure by TMPyP2 (lanes 4–8) and TMPyP4 (lanes 9–12) by using increasing concentrations of TMPyP2 (0.5–20 μM) and TMPyP4 (0.5–10 μM) at 60°C (10 mM KCl). “A” refers to an adenine sequencing reaction. (Right) The ratio of the full-length product to the major arrest product was plotted against concentrations. Each data point is the average of three experiments.
Figure 4
Figure 4
Effect of cationic porphyrins on c-MYC mRNA synthesis in two different Burkitt's lymphoma cell lines and on the stability of mutant G-quadruplex structures and luciferase expression mediated by wild-type and mutant promoters. (a) Diagram of the rearrangements involved in the Ramos and CA46 Burkitt's lymphoma cell lines (modified from ref. 2). Vertical arrows indicate the breakage and rejoining points between chromosomes 14 and 8 for each translocation. (b) RT-PCR for c-MYC and β-actin in Ramos (lanes 1–3) and CA46 (lanes 4–6) cell lines after no treatment (lanes 1 and 4) and treatment with 100 μM TMPyP2 (lanes 2 and 5) and TMPyP4 (lanes 3 and 6) for 48 h (for experimental details, see ref. 33). The experiment was repeated, with comparable results. (c) Polymerase stop assay (29) for determination of the effect of TMPyP4 on the stabilization of the G-quadruplex in the wild-type (lanes 1–5) and various mutant (see Fig. 2A) sequences (lanes 6–25). TMPyP4 was added at the concentrations shown, and the experiment was carried out under the same conditions as in Fig. 2D. Above the gel are the approximate IC50 values for polymerase arrest by TMPyP4 determined in this experiment. (d) Luciferase expression assays to determine the effect of TMPyP4 on promoter activity of the wild-type and various mutants (see Fig. 2A). HeLa S3 cells were transiently transfected as described earlier. Twenty-four hours after transfection, cells were exposed to 100 μM TMPyP2 or TMPyP4 or an equivalent volume of water as a control added to the growth medium. Treatments lasted for 24 h, after which the cells were lysed, and the lysates were tested for luciferase activity, as in Fig. 2B. Experiments were performed in duplicate. Error bars represent 1 SD above and below the mean % luciferase activity. The numbers are given relative to treatment with TMPyP2, which, like TMPyP4, shows about a 20% nonspecific inhibition of luciferase activity in both these plasmids, as well as in an unrelated vector (pGL3 control).
Scheme 2
Scheme 2
Model for the activation and repression of gene transcription involving the accessory role of NM23-H2 in interconversion of the unstructured purine and pyrimidine single-stranded DNA forms to the paranemic secondary DNA structures. Interaction of the G-quadruplex structure with TMPyP4 stabilizes the gene-off form by inhibition of conversion to the single-stranded gene-on forms (see text for further details).
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