G-quadruplex formation within the promoter of the KRAS proto-oncogene and its effect on transcription

Abstract

In human and mouse, the promoter of the KRAS gene contains a nuclease hypersensitive polypurine-polypyrimidine element (NHPPE) that is essential for transcription. An interesting feature of the polypurine G-rich strand of NHPPE is its ability to assume an unusual DNA structure that, according to circular dichroism (CD) and DMS footprinting experiments, is attributed to an intramolecular parallel G-quadruplex, consisting of three G-tetrads and three loops. The human and mouse KRAS NHPPE G-rich strands display melting temperature of 64 and 73 degrees C, respectively, as well as a K+-dependent capacity to arrest DNA polymerase. Photocleavage and CD experiments showed that the cationic porphyrin TMPyP4 stacks to the external G-tetrads of the KRAS quadruplexes, increasing the T(m) by approximately 20 degrees C. These findings raise the intriguing question that the G-quadruplex formed within the NHPPE of KRAS may be involved in the regulation of transcription. Indeed, transfection experiments showed that the activity of the mouse KRAS promoter is reduced to 20% of control, in the presence of the quadruplex-stabilizing TMPyP4. In addition, we found that G-rich oligonucleotides mimicking the KRAS quadruplex, but not the corresponding 4-base mutant sequences or oligonucleotides forming quadruplexes with different structures, competed with the NHPPE duplex for binding to nuclear proteins. When vector pKRS-413, containing CAT driven by the mouse KRAS promoter, and KRAS quadruplex oligonucleotides were co-transfected in 293 cells, the expression of CAT was found to be downregulated to 40% of the control. On the basis of these data, we propose that the NHPPE of KRAS exists in equilibrium between a double-stranded form favouring transcription and a folded quadruplex form, which instead inhibits transcription. Such a mechanism, which is probably adopted by other growth-related genes, provides useful hints for the rational design of anticancer drugs against the KRAS oncogene.

Figure 1

(A) Sequences of the NHPPEs contained in the human and mouse KRAS genes. Positions relative to exon 0/intron 1 boundary are shown. (B) Human and mouse NHPPEs show a high sequence homology.

Figure 2

(A) CD spectra of the G-rich strands of the mouse (28Rm) and human (32Rh) NHPPEs and corresponding 4-base mutant sequences (28Rmut, 32Rmut). Spectra have been obtained in 50 mM Tris, pH 7.2, 100 mM KCl, oligonucleotide concentration 3 μM. (B) Native electrophoretic mobilities of the NHPPE duplexes, 28Rm, 32Rh, pyrimidine strands 28Ym and 32Yh, 28Rmut and 32Rmut under various experimental conditions. The oligonucleotides were radiolabelled, incubated for 5 h in the appropriate buffer and run at 25°C in a 20% polyacrylamide gel in TBE. Samples in lanes 6 and 7 (left panel) and 5 and 6 (right panel) were denatured with NaOH and renatured with HCl.

Figure 3

Polymerase stop assay showing primer elongation by the Klenow fragment. The single-stranded templates containing either 28Rm or 28Rmut are shown. These DNA substrates have been incubated overnight in buffers containing NaCl, KCl or KCl and TMPyP4. A primer elongation reaction was performed for 30 min at 37°C. Elongation products were separated in a 15% polyacrylamide, 8 M urea, denaturing gel. Sequencing reactions by the Sanger dideoxy method, using the same primer of the polymerase stop assay, were carried out. Klenow fragment elongation pauses in correspondence of the adenine preceding the first and second runs of guanines at the 3′ end of NHPPE, (positions are indicated by lines).

Figure 4

Polymerase stop assay performed with a double-stranded template (plasmid pKRS-413). In this experiment pKRS-413, containing the mouse KRAS promoter, has been incubated in 100 mM NaCl, 100 mM KCl or 100 mM KCl and 100 nM TMPyP4 at 45°C for 48 h. A primer elongation reaction was performed, using a primer located downstream from the G-rich strand. Reactions were performed as described in Figure 3. The adenines in the G-rich sequence are numbered.

Figure 5

(Top) Sequence of the mouse G-rich motif 28Rm subjected to DMS footprinting. The four guanines that are substituted with cytosines in 28Rmut are indicated (Bottom) DMS footprintings of the G-rich strand of NHPPE (28Rm) and 4-base mutant 28Rmut. DMS-induced cleavage of denatured 28Rm (lanes 1–3, F = formamide, Δ = heating); 28Rm after incubation in 100 mM KCl (lane 4) or 100 mM KCl plus 100 nM TMPyP4 (lane 5); 28Rmut in F (lane 6) and 100 mM KCl plus 100nM TMPyP4 (lane 7). The guanines cleaved by the DMS/piperidine treatment are labelled in the right side of the sequencing gel. (Bottom right) G-quadruplex structures proposed for 28Rm, based on DMS footprinting, CD, electrophoretic mobility and polymerase stop assays.

Figure 6

(Top) Sequence of the human G-rich motif 32Rh subjected to DMS footprinting. The guanines that are substituted with cytosines in 32Rmut are indicated. The parental 28Rm mouse sequence is also shown for comparison. Arrows indicate the cleavage sites (Bottom) DMS footprinting of the G-rich strand of the human NHPPE (32Rh) and of the corresponding 4-base mutant 32Rmut. DMS-induced cleavage of the denatured 32Rm (lanes 1-2); 32Rh after incubation in 50 mM KCl (lane 3) or 50 mM KCl plus 50 nM TMPyP4 (lane 4); 32Rh in 100 mM KCl (lane 5) or 100 mM KCl plus 100 nM TMPyP4 (lane 6); 32Rmut in formamide (lane 7), in 100 mM KCl (lane 8), in 100 mM KCl plus 100 nM TMPyP4 (lane 9). The guanines cleaved by the DMS/piperidine treatment are labelled in the right side of the sequencing gel.

Figure 7

(A) CD spectra of the mouse G-rich sequence 28Rm in the presence of 100 mM KCl with and without TMPyP4. The ellipticity arising from the 28Rm-porphyrin interaction is shown in the inset. DNA concentration is 3 μM, TMPyP4 increased from 3 to 18 μM. Cuvette pathlength 0.5 cm. (B) Photocleavage of the G-quadruplex formed by the mouse 28Rm sequence. DMS-induced cleavage of 28Rm in the denatured state (lane 1), under a quadruplex conformation (lane 2); photocleavage of 28Rm in the quadruplex conformation (lane 3); DMS-induced cleavage of 28Rmut (lane 4), Photocleavage of 28Rmut (lane 5). Right structure shows with arrows the guanines that are photocleaved.

Figure 8

Chloramphenicol acetyl transferase expression assay to determine the effect of TMPyP4 on the activity of KRAS promoter. Plasmid pKRS-413, containing CAT driven by the mouse KRAS promoter, has been transfected in 293 cells in the presence of increasing amounts of TMPyP4. As a control for transfection efficiency and aspecific transcription impairment plasmid pTK-βgal was cotrasfected with pKRS-413. The expression of CAT, with respect to that of β-gal, was determined by an ELISA assay, 48 h after transfection. The residual CAT expression, (CAT/β-gal) × 100, is reported in the histograms. The values are the average of three independent experiments in duplicate. Error bar are ±SE. These experiments showed that stabilization of G-quadruplex formed within NHPPE following cell treatment with TMPyP4, decreased the expression of a reporter gene driven by the KRAS promoter.

Figure 9

DNA–protein competition experiments. (A) EMSA showing that ³²P-labelled mouse NHPPE duplex (5 nM) binds to three nuclear proteins from a mouse NIH 3T3 cell extract (M1–M3) (lane 1). The competitors are: quadruplex 28Rm (lanes 2–4, 10-/50-/100-fold over the labelled duplex probe); quadruplex 32Rh (lanes 5–7, 10-/50-/100-fold), 4-base mutant 28Rmut (lanes 8–10, 10-/50-/100-fold), target duplex (10-/50-/100-fold) (lanes 11–13); quadruplex Q1 (for sequences see Table 1) (lane 14, 100-fold excess over probe) (lane 14). The autoradiography was analysed with a Gel Doc 2000 Imager System (Bio-Rad) and the percent OD in bands M1 and M2 respect to total OD in the lane was calculated. Set to 100 the value obtained in lane 1 (control), the values of M1+M2 in lanes 1–14 are 100, 92, 63, 29, 73, 46, 24, 88, 81, 99, 1, 0, 0 and 82, respectively. These values provide a quantitative estimate of the competition capacity of the tested oligonucleotides; (B) EMSA showing the ³²P-labelled mouse NHPPE duplex (5 nM) binds to two nuclear proteins from human Panc-1 cells (H1 and H2) (lane 1). The competitors are quadruplex 28Rm (lanes 2–5, 10-/20-/50-/100-fold over the probe); quadruplex 32Rh (lanes 9–10, 50-/100-fold); 4-base mutants 28Rmut (lanes 6–8, 10-/50-/100-fold); quadruplexes Q1 and Q2 (for sequences see Table 1) (lanes 11 and 12, 100-fold excess over probe); quadruplex CMYC (sequence in Table 1) (lanes 13, 14, 50-/100-fold); Percent OD in H1 respect to total OD, measured in the lanes 1–14 are 100, 94, 77, 60, 41, 79, 77, 77, 46, 27, 83, 87, 68 and 69; (C) Southwestern blots showing that 28Rm, but not the 4-base mutant 28Rmut, binds to nuclear proteins of 32, 75 and 115 kDa. The letters H and M on the top panels indicate human and mouse nuclear extracts, respectively. The mobilities of marker proteins are shown on the left.

Figure 10

(A) CAT assay to determine the effect of 28Rm and 32Rh on KRAS transcription. Cells 293 were co-transfected with plasmid pKRS-413, pTK-βgal and 1 μM quadruplex-forming oligonucleotide (28Rm, 32Rh) or 4-base mutant (28Rmut, 32Rmut). The expression of CAT with respect to that of β-gal, was determined by an ELISA assay, 48 h after transfection. The residual CAT expression, (CAT/β-gal) × 100, is reported in the histograms. The values are the average of three independent experiments in duplicate. Error bars are ±SE. (B) Model for regulation of transcription in the KRAS gene.