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. 2010 Jul 19;5(7):e11647.
doi: 10.1371/journal.pone.0011647.

I-motif structures formed in the human c-MYC promoter are highly dynamic--insights into sequence redundancy and I-motif stability

Jixun Dai  1 , Emmanuel Hatzakis, Laurence H Hurley, Danzhou Yang
Affiliations

I-motif structures formed in the human c-MYC promoter are highly dynamic--insights into sequence redundancy and I-motif stability

Jixun Dai et al. PLoS One. .

Abstract

The GC-rich nuclease hypersensitivity element III1 (NHE III1) of the c-MYC promoter largely controls the transcriptional activity of the c-MYC oncogene. The C-rich strand in this region can form I-motif DNA secondary structures. We determined the folding pattern of the major I-motif formed in the NHE III1, which can be formed at near-neutral pH. While we find that the I-motif formed in the four 3' consecutive runs of cytosines appears to be the most favored, our results demonstrate that the C-rich strand of the c-MYC NHE III1 exhibits a high degree of dynamic equilibration. Using a trisubstituted oligomer of this region, we determined the formation of two equilibrating loop isomers, one of which contains a flipped-out cytosine. Our results indicate that the intercalative cytosine+-cytosine base pairs are not always necessary for an intramolecular I-motif. The dynamic character of the c-MYC I-motif is intrinsic to the NHE III1 sequence and appears to provide stability to the c-MYC I-motif.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The c-MYC NHE III1 sequences and I-motif structure.
(A) The promoter sequence of the NHE III1 element of the c-MYC gene and its modifications. mycPu27 is the wild-type 27-mer G-rich sequence of the c-MYC NHE III1; Pu22 is the modified G-rich sequence that adopts the single predominant c-MYC promoter G-quadruplex and was used for structure determination ; mycPy27 is the wild-type 27-mer C-rich sequence of the c-MYC NHE III1; Py27 is the wild-type C-rich promoter sequence with a 3′-AA; Py27(1245) is the modified Py27 that can only form the (1245) form of the c-MYC I-motif; Py22 is the truncated wild-type Py27 with a 3′-AA that can only form the (2345) form of the c-MYC I-motif; C11T, C20T, C11/20/23T, and C11/14/20/23T are the modified Py22 with single C-to-T substitutions. The numbering system is based on the G-rich strand and is shown above mycPy27. (B) A C+-C base pair (left), and a schematic drawing of a four-stranded I-motif structure (right). (C) CD melting curves of various C-rich c-MYC promoter sequences shown in (A) at pH 5.5. (D) Imino regions of 1D 1H NMR spectra of Py22, C11T, and C20T at 25°C, pH 5.5.
Figure 2
Figure 2. Variable temperature 1D 1H NMR of c-MYC I-motif sequences.
Imino proton regions of variable temperature 1D 1H NMR spectra of C11T, C20T, C11/20/23T, and C11/14/20/23T at pH 5.5.
Figure 3
Figure 3. Imino proton assignments of C11T and C20T c-MYC I-motif.
Imino proton assignments of C11T (A) and C20T (B) using 1D 15N-filtered experiments on site-specific 6% 15N-labeled oligonucleotides. Each site-specifically labeled cytosine is shown above its corresponding spectrum. The assignment of all cytosine imino protons is shown above the 1D spectra of the corresponding sequence. All samples are prepared at pH 5.5. NMR experiments were performed at 7°C except for the C7-labeled C20T which was performed at 1°C.
Figure 4
Figure 4. Folding structures of the c-MYC I-motifs.
Schematic drawing of the folding structures of the c-MYC I-motifs formed in the C11T sequence (A), the C20T sequence (B), and the two equilibrating conformations in the C11/20/23T sequence (C). The C+-C base pairs are shown in white boxes. The dashed boxes indicate the possible C+-C base pairs that are in dynamic equilibrium and thus show weaker and broader C+-C imino peaks. (cytosine  =  yellow sphere, adenine  =  green sphere, thymine  =  blue sphere.)
Figure 5
Figure 5. Imino proton assignment of C11/20/23T c-MYC I-motif.
Imino proton assignments of C11/20/23T using 1D 15N-filtered experiments on site-specific 15N-labeled oligonucleotides. Each site-specifically labeled cytosine is shown above its corresponding spectrum. The assignment of all cytosine imino protons is shown above the 1D 1H spectrum of C11/20/23T. All samples are at pH 5.5. NMR experiments were performed at 7°C.
Figure 6
Figure 6. Variable pH 1D 1H NMR of C11/14/20/23T c-MYC I-motif.
Cytosine- and thymine- imino regions of 1D 1H NMR spectra of C11/14/20/23T at various pHs at 7°C.
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