A conserved quadruplex motif located in a transcription activation site of the human c-kit oncogene

Abstract

The c-kit gene encodes a receptor tyrosine kinase, whose engagement by its ligand triggers signals leading to cell proliferation. c-kit activity is elevated in gastrointestinal stromal tumors (GISTs), and its therapeutic inhibition by small molecules such as imatinib is clinically validated. We identified a putative quadruplex forming 21-nucleotide sequence upstream of the c-kit transcription initiation site (c-kit21), on the G-rich strand, which occupies a site required for core promoter activity. Here, we show by nuclear magnetic resonance (NMR), circular dichroism (CD), and ultraviolet (UV) spectroscopic methods that c-kit21 forms quadruplexes under physiological conditions. Mutational analysis of c-kit21 has provided insights into its structural polymorphism. In particular, one mutated form appears to form a single quadruplex species that adopts a parallel conformation. The quadruplex-forming sequence shows a high level of sequence conservation across human, mouse, rat, and chimpanzee. The small variation in sequence between the quadruplex in human/chimpanzee as compared to the rat/mouse was examined more closely by biophysical methods. Despite a variation in the sequence and length of loop 2, the quadruplexes showed both comparable CD spectra, indicative of parallel quadruplexes, and also similar thermal-stability profiles, suggesting conservation of biophysical characteristics. Collectively, the evidence suggests that this quadruplex is a serious target for a detailed functional investigation at the cell-biology level.

Figure 1

(A) Positions of the putative quadruplex sequence (c-kit21) and the Sp1-binding DNA sequences within the G-rich strand of the kit promoter. The numbering is relative to the transcription start site (+1). (B) Conservation of the DNA sequence in human, chimpanzee, mouse, and rat. (C) DNA sequences of the native putative quadruplex oligonucleotide c-kit21 and the mutated oligonucleotides c-kit18T and c-kit21T.

Figure 2

¹H NMR spectra of (A) c-kit21, (B) c-kit18T, and (C) c-kit21T sequences taken in 20 mM potassium phosphate buffer (pH 7.0) at 43 °C.

Figure 3

Normalized UV thermal difference spectra of (A) c-kit18T, c-kit21T, and c-kit21 sequences and (B) mouse/rat c-kit compared with human c-kit21. All spectra were taken in 10 mM Tris-HCl (pH 7.0) containing 100 mM KCl.

Figure 4

Normalized UV thermal melting curves of the c-kit21, c-kit21T, and c-kit18T sequences taken in 10 mM Tris-HCl (pH 7.0) containing 100 mM KCl.

Figure 5

(A) CD spectra of the c-kit18T, c-kit21T, and c-kit21 sequences. (B) Mouse/rat c-kit compared with human c-kit21. All spectra were taken in 10 mM Tris-HCl (pH 7.0) containing 100 mM KCl at 20 °C.