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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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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.
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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.

Mentions: The interesting biophysical observations prompted us to perform molecular assay to extrapolate the findings of polyamines-induced structural transition, stabilization and modulation of molecular recognition properties of c-MYC quadruplex structure to c-MYC transcription. Literature cites independent observations of both quadruplex and increased polyamine concentrations being involved in uncontrolled cellular proliferation. This hints toward the existence of an underlying phenomenon, which may associate physical interaction between quadruplex and intracellular polyamines to ensue cancer progression. The aberrant gene expression observed during cancer progression can be attributed to modulation of molecular recognition, physical interaction and function of nucleic acid structures upon interaction with polyamines. c-MYC is an important proto-oncogene which is involved in growth differentiation of cells and regulation of other proto-oncogenes (15,17). The aberrant expression of c-MYC is associated with broad range of cancers. Further, the quadruplex element in the promoter region of c-MYC proto-oncogene is structurally and functionally well characterized, and this motif is believed to play a role in c-MYC expression (18,49). We have previously established that quadruplex–duplex equilibrium has important role in modulating gene expression (50). As quadruplex in c-MYC promoter serves as a structural target for gene regulation, any perturbation in quadruplex–duplex equilibrium induced by polyamines would affect downstream c-MYC expression. To ascertain this belief, we assessed c-MYC transcript levels in absence and presence of polyamines in cell culture system. We performed real time PCR experiment to analyze the relative changes in c-MYC gene expression with respect to the internal reference gene. c-MYC transcript levels were assessed after treating the cells with increasing concentrations (0–15 mM) of spermidine and spermine and data were analyzed by mathematical model for relative quantification of target (c-MYC) versus internal reference gene (B2m) expression (51). We obtained a concentration-dependent increase in c-MYC transcript levels upon treating the cells with increasing concentrations (0–15 mM) of spermidine and spermine (Figure 7). These experiments were also performed using β-actin gene as internal reference gene. It is noteworthy to mention that β-actin harbors the quadruplex forming sequence potential in its promoter region, whereas B2m lacks this potential in its promoter. Interestingly, polyamine does not significantly affect the gene expression of both B2m and β-actin genes, thereby resulting in similar trend in c-MYC activation (Figure 7 and Supplementary Figure 10). Polyamine interaction with this c-MYC quadruplex motif affects its stability, molecular recognition and induces structural transition into a transcriptionally active conformer, which drives c-MYC activation. This implies that the quadruplex–polyamine interaction will translate into molecular consequences, where the quadruplex in the promoter region serves as functional regulatory motif and controls the expression of the downstream gene.Figure 7.


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

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

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

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.
Mentions: The interesting biophysical observations prompted us to perform molecular assay to extrapolate the findings of polyamines-induced structural transition, stabilization and modulation of molecular recognition properties of c-MYC quadruplex structure to c-MYC transcription. Literature cites independent observations of both quadruplex and increased polyamine concentrations being involved in uncontrolled cellular proliferation. This hints toward the existence of an underlying phenomenon, which may associate physical interaction between quadruplex and intracellular polyamines to ensue cancer progression. The aberrant gene expression observed during cancer progression can be attributed to modulation of molecular recognition, physical interaction and function of nucleic acid structures upon interaction with polyamines. c-MYC is an important proto-oncogene which is involved in growth differentiation of cells and regulation of other proto-oncogenes (15,17). The aberrant expression of c-MYC is associated with broad range of cancers. Further, the quadruplex element in the promoter region of c-MYC proto-oncogene is structurally and functionally well characterized, and this motif is believed to play a role in c-MYC expression (18,49). We have previously established that quadruplex–duplex equilibrium has important role in modulating gene expression (50). As quadruplex in c-MYC promoter serves as a structural target for gene regulation, any perturbation in quadruplex–duplex equilibrium induced by polyamines would affect downstream c-MYC expression. To ascertain this belief, we assessed c-MYC transcript levels in absence and presence of polyamines in cell culture system. We performed real time PCR experiment to analyze the relative changes in c-MYC gene expression with respect to the internal reference gene. c-MYC transcript levels were assessed after treating the cells with increasing concentrations (0–15 mM) of spermidine and spermine and data were analyzed by mathematical model for relative quantification of target (c-MYC) versus internal reference gene (B2m) expression (51). We obtained a concentration-dependent increase in c-MYC transcript levels upon treating the cells with increasing concentrations (0–15 mM) of spermidine and spermine (Figure 7). These experiments were also performed using β-actin gene as internal reference gene. It is noteworthy to mention that β-actin harbors the quadruplex forming sequence potential in its promoter region, whereas B2m lacks this potential in its promoter. Interestingly, polyamine does not significantly affect the gene expression of both B2m and β-actin genes, thereby resulting in similar trend in c-MYC activation (Figure 7 and Supplementary Figure 10). Polyamine interaction with this c-MYC quadruplex motif affects its stability, molecular recognition and induces structural transition into a transcriptionally active conformer, which drives c-MYC activation. This implies that the quadruplex–polyamine interaction will translate into molecular consequences, where the quadruplex in the promoter region serves as functional regulatory motif and controls the expression of the downstream gene.Figure 7.

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