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Elevated polyamines induce c-MYC overexpression by perturbing quadruplex-WC duplex equilibrium.

Kumar N, Basundra R, Maiti S - Nucleic Acids Res. (2009)

Bottom Line: The relative free energy difference (DeltaDeltaG degrees) between the duplex and quadruplex structure indicate that polyamines stabilize and favor c-MYC quadruplex over duplex.Our results suggest that polyamines induce structural transition of c-MYC quadruplex to a transcriptionally active motif with distinctive molecular recognition property, which drives c-MYC expression.These findings may allow exploiting quadruplex-polyamines interaction for developing antiproliferative strategies to combat aberrant gene expression.

View Article: PubMed Central - PubMed

Affiliation: Proteomics and Structural Biology Unit, Institute of Genomics and Integrative Biology, CSIR, Mall Road, Delhi 110 007, India.

ABSTRACT
The biological role of quadruplexes and polyamines has been independently associated with cancer. However, quadruplex-polyamine mediated transcriptional regulation remain unaddressed. Herein, using c-MYC quadruplex model, we have attempted to understand quadruplex-polyamine interaction and its role in transcriptional regulation. We initially employed biophysical approach involving CD, UV and FRET to understand the role of polyamines (spermidine and spermine) on conformation, stability, molecular recognition of quadruplex and to investigate the effect of polyamines on quadruplex-Watson Crick duplex transition. Our study demonstrates that polyamines affect the c-MYC quadruplex conformation, perturb its recognition properties and delays duplex formation. The relative free energy difference (DeltaDeltaG degrees) between the duplex and quadruplex structure indicate that polyamines stabilize and favor c-MYC quadruplex over duplex. Further, we investigated the influence of polyamine mediated perturbation of this equilibrium on c-MYC expression. Our results suggest that polyamines induce structural transition of c-MYC quadruplex to a transcriptionally active motif with distinctive molecular recognition property, which drives c-MYC expression. These findings may allow exploiting quadruplex-polyamines interaction for developing antiproliferative strategies to combat aberrant gene expression.

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CD spectra recorded for c-MYC quadruplex (5 µM) in absence and presence of (a) spermidine and (b) spermine in 10 mM sodium cacodylate buffer, 100 mM NaCl, pH 7.4 at 25°C.
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Figure 1: CD spectra recorded for c-MYC quadruplex (5 µM) in absence and presence of (a) spermidine and (b) spermine in 10 mM sodium cacodylate buffer, 100 mM NaCl, pH 7.4 at 25°C.

Mentions: The CD spectrum recorded for 22-mer purine-rich sequence of NHE III of c-MYC promoter in 100 mM NaCl buffer depicts an intense positive peak at 265 nm, a small positive peak at 295 nm and a negative peak at 240 nm, thus suggesting the existence of a predominant parallel quadruplex population along with a small antiparallel population (Figure 1). The increasing concentrations of polyamines (0–0.75 mM spermidine and 0–50 µM spermine) lead to a prominent increase in the magnitude of positive peak at 265 nm and a moderate increase in the negative peak at 240 nm with a concomitant decrease in the magnitude of positive peak at 295 nm. This suggests that polyamines induce structural transitions which result in decrease in antiparallel population and a simultaneous increase in parallel population. Similar experiments were performed in KCl buffer, and only a moderate effect for the structural transition from antiparallel to parallel conformation was observed (Supplementary Figure 1). In KCl buffer, c-MYC quadruplex displays very high thermal stability (Tm = 75°C, Supplementary Figure 3) and this structure might resist the induction of any further structural transitions by polyamines. A further increase in concentration of spermidine (>0.75 mM) and spermine (>50 µM) resulted in precipitation of DNA both in NaCl and KCl buffers. Therefore, the working concentration was restricted to 0–0.75 mM spermidine and 0–50 µM spermine in our subsequent experiments. Since, this 22-mer G-rich sequence has potential to adopt more than one conformation, the difference in their mobility would allow resolving these populations through native gel electrophoresis. We performed native gel electrophoresis to observe the structural transitions induced by polyamines, as shown in Figure 2 (lanes 2–4). The presence of two discrete bands for quadruplex signifies existence of mixed conformers in NaCl buffer, and is in concordance with our CD observations. However, in presence of both 0.75 mM spermidine and 50 µM spermine, we observed decrease in the intensity of lower band and an increase in the intensity of upper band suggesting the shift towards one type of conformer, probably the parallel population as observed in the CD study.Figure 1.


Elevated polyamines induce c-MYC overexpression by perturbing quadruplex-WC duplex equilibrium.

Kumar N, Basundra R, Maiti S - Nucleic Acids Res. (2009)

CD spectra recorded for c-MYC quadruplex (5 µM) in absence and presence of (a) spermidine and (b) spermine in 10 mM sodium cacodylate buffer, 100 mM NaCl, pH 7.4 at 25°C.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2691834&req=5

Figure 1: CD spectra recorded for c-MYC quadruplex (5 µM) in absence and presence of (a) spermidine and (b) spermine in 10 mM sodium cacodylate buffer, 100 mM NaCl, pH 7.4 at 25°C.
Mentions: The CD spectrum recorded for 22-mer purine-rich sequence of NHE III of c-MYC promoter in 100 mM NaCl buffer depicts an intense positive peak at 265 nm, a small positive peak at 295 nm and a negative peak at 240 nm, thus suggesting the existence of a predominant parallel quadruplex population along with a small antiparallel population (Figure 1). The increasing concentrations of polyamines (0–0.75 mM spermidine and 0–50 µM spermine) lead to a prominent increase in the magnitude of positive peak at 265 nm and a moderate increase in the negative peak at 240 nm with a concomitant decrease in the magnitude of positive peak at 295 nm. This suggests that polyamines induce structural transitions which result in decrease in antiparallel population and a simultaneous increase in parallel population. Similar experiments were performed in KCl buffer, and only a moderate effect for the structural transition from antiparallel to parallel conformation was observed (Supplementary Figure 1). In KCl buffer, c-MYC quadruplex displays very high thermal stability (Tm = 75°C, Supplementary Figure 3) and this structure might resist the induction of any further structural transitions by polyamines. A further increase in concentration of spermidine (>0.75 mM) and spermine (>50 µM) resulted in precipitation of DNA both in NaCl and KCl buffers. Therefore, the working concentration was restricted to 0–0.75 mM spermidine and 0–50 µM spermine in our subsequent experiments. Since, this 22-mer G-rich sequence has potential to adopt more than one conformation, the difference in their mobility would allow resolving these populations through native gel electrophoresis. We performed native gel electrophoresis to observe the structural transitions induced by polyamines, as shown in Figure 2 (lanes 2–4). The presence of two discrete bands for quadruplex signifies existence of mixed conformers in NaCl buffer, and is in concordance with our CD observations. However, in presence of both 0.75 mM spermidine and 50 µM spermine, we observed decrease in the intensity of lower band and an increase in the intensity of upper band suggesting the shift towards one type of conformer, probably the parallel population as observed in the CD study.Figure 1.

Bottom Line: The relative free energy difference (DeltaDeltaG degrees) between the duplex and quadruplex structure indicate that polyamines stabilize and favor c-MYC quadruplex over duplex.Our results suggest that polyamines induce structural transition of c-MYC quadruplex to a transcriptionally active motif with distinctive molecular recognition property, which drives c-MYC expression.These findings may allow exploiting quadruplex-polyamines interaction for developing antiproliferative strategies to combat aberrant gene expression.

View Article: PubMed Central - PubMed

Affiliation: Proteomics and Structural Biology Unit, Institute of Genomics and Integrative Biology, CSIR, Mall Road, Delhi 110 007, India.

ABSTRACT
The biological role of quadruplexes and polyamines has been independently associated with cancer. However, quadruplex-polyamine mediated transcriptional regulation remain unaddressed. Herein, using c-MYC quadruplex model, we have attempted to understand quadruplex-polyamine interaction and its role in transcriptional regulation. We initially employed biophysical approach involving CD, UV and FRET to understand the role of polyamines (spermidine and spermine) on conformation, stability, molecular recognition of quadruplex and to investigate the effect of polyamines on quadruplex-Watson Crick duplex transition. Our study demonstrates that polyamines affect the c-MYC quadruplex conformation, perturb its recognition properties and delays duplex formation. The relative free energy difference (DeltaDeltaG degrees) between the duplex and quadruplex structure indicate that polyamines stabilize and favor c-MYC quadruplex over duplex. Further, we investigated the influence of polyamine mediated perturbation of this equilibrium on c-MYC expression. Our results suggest that polyamines induce structural transition of c-MYC quadruplex to a transcriptionally active motif with distinctive molecular recognition property, which drives c-MYC expression. These findings may allow exploiting quadruplex-polyamines interaction for developing antiproliferative strategies to combat aberrant gene expression.

Show MeSH
Related in: MedlinePlus