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

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.

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.

Figure 2.

Native gel electrophoresis of fluorescein-labeled quadruplex. Lane 1 represents oligo T₂₅; lanes 2–4 represent quadruplex (2 µM) in absence and presence of 0.75 mM spermidine (Spd) and 50 µM spermine (Spm), respectively; lanes 5–7 represent duplex (2 µM) in absence and presence of 0.75 mM spermidine and 50 µM spermine, respectively.

Figure 3.

UV melting profile of c-MYC quadruplex (2 µM) in absence and presence of 0.75 mM spermidine and 50 µM spermine in 10 mM sodium cacodylate buffer, 100 mM NaCl, pH 7.4.

Figure 4.

Fluorescence-annealing curves obtained for equimolar mixture of quadruplex (0.15 µM) and its complementary strand in absence and presence of 0.75 mM spermidine and 50 µM spermine in 10 mM sodium cacodylate buffer, 100 mM NaCl, pH 7.4.

Figure 5.

Normalized fluorescence of quadruplex (0.15 µM) at 520 nm as a function of complementary strand concentrations in absence and presence of 0.75 mM spermidine and 50 µM of spermine in 10 mM sodium cacodylate buffer, 100 mM NaCl, pH 7.4 at 25°C.

Figure 6.

Binding data analysis for quadruplex–porphyrin (TMPyP4) interaction in absence (filled square) and presence (open circle) of 0.75 mM sperimidine and (open triangle) 50 µM spermine in10 mM sodium cacodylate buffer, 100 mM NaCl, pH 7.4 at 25°C. The y-axis represents r (moles of bound TMPyP4/1 mol of quadruplex) and x-axis represents C_f, which is concentration of free TMPyP4. Data analysis was performed using one-site and two-site hyperbolic equations. Fitting data and its residuals are shown for one-site (dash) and two-site binding (solid) for the quadruplex–porphyrin interaction.

Figure 7.

Fold change in the c-MYC transcripts assessed through real time PCR after 24 h of treatment of polyamines (0–15 mM spermidine and spermine) to HeLa cells. The fold change represents the log₂-fold change of c-MYC transcripts with respect to internal reference gene for treated samples versus control sample, which received no polyamine treatment. Expression level of B2m gene was taken as internal reference gene to normalize the real time PCR data. The data represent the mean values ± SDs from three separate experiments.