Limits...
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.

Show MeSH

Related in: MedlinePlus

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

License
getmorefigures.php?uid=PMC2691834&req=5

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.

Mentions: Next, we evaluated the effect of polyamines on the thermal stability of c-MYC quadruplex by monitoring the melting profile at 295 nm. The thermal melting was observed to be independent of quadruplex concentration both in absence and presence of polyamines, thereby suggesting formation of intramolecular structure (Supplementary Figure 2). The UV melting profile shows that this element forms a stable quadruplex with Tm of 62.5°C in 100 mM NaCl buffer. The increase in the concentration of spermidine (0–0.75 mM) and spermine (0–50 µM) resulted in increase in the stability of quadruplex structure. The maximum increase in thermal stability was obtained at 0.75 mM spermidine and 50 µM spermine with Tm of 71.5°C and 70°C, respectively (Figure 3 and Table 1). The enthalpy change associated with quadruplex melting was −24.3 kcal/mol in absence of polyamines, −54.0 kcal/mol and −67.0 kcal/mol in presence of 0.75 mM spermidine and 50 µM spermine, respectively. Similar experiments were also performed in potassium buffer. Quadruplex formed in potassium buffer is highly stable (Tm = 75°C, Supplementary Figure 3), but in presence of polyamines we did not obtain characteristic melting domain, as c-MYC quadruplex–K+ in presence of polyamines melts at temperatures >90°C (Supplementary Figure 3). Through UV melting study, it is difficult to dissect out the contributions from the participating populations: quadruplex, duplex and random coil. Therefore, we performed fluorescence-annealing experiments to understand the contribution of these populations and comprehend the fate of equilibrium (30,34). Annealing the system slowly from 95°C to 37°C containing both the G-rich and its complementary strand generates a competing environment that allows the G-rich oligonucleotide to either exist as quadruplex and/or hybridize to its complementary strand to form duplex depending on relative thermodynamic stabilities of these two secondary structures. We monitored the annealing profile of donor fluorescence intensity at 520 nm for c-MYC G-rich sequence with equimolar concentration of complementary strand in the absence and presence of polyamines (Figure 4). The annealing profile obtained for the equimolar mixture of G-rich and C-rich strand in absence of polyamines, initially showed a marginal decrease in fluorescence intensity upon decrease in temperatures from 95°C to 76°C. But at ∼76°C, a change in fluorescence profile was observed which showed increase in fluorescence with further decrease in temperature (76–37°C). The initial decrease and subsequent increase in fluorescence in the cooling profile corresponds to quadruplex and duplex formation, respectively. The fluorescence intensity change at different temperatures is reflective of these contributing populations. The fluorescence profile of donor intensity monitored for the equimolar mixture of G-rich and C-rich strand in presence of 0.75 mM spermidine and 50 µM spermine shows a prominent decrease in fluorescence intensity upon decrease in temperature from 95°C to 76°C and the profile shifts to higher temperature, as shown in Figure 4. Upon further decrease in temperature (76–37°C), the fluorescence intensity increased indicating duplex formation. The extent of increase in fluorescence intensity of donor is lesser in presence of polyamines, suggesting lesser amount duplex formation in comparison to absence of polyamines. The first derivative of these melting curves shows two peaks corresponding to quadruplex and duplex melting domains at higher and lower temperatures, respectively. The intensity and peak position of derivative curve reflect the amount of each participating population and their corresponding Tm values (34). We observed only a moderate shift in the duplex melting domain to higher temperatures in presence of polyamines. However, a prominent shift of quadruplex melting domain to higher temperatures was observed. This can be attributed to the competing secondary structures, quadruplex and duplex adopted by the G-rich sequence. Polyamine-induced stabilization of quadruplex facilitates greater quadruplex formation, and results in decrease in effective G-rich strands required for duplex formation, leading to a marginal increase in duplex stability in presence of polyamines. Polyamines show remarkable stabilizing effects on DNA–RNA hybrids, stem-loop structures and triplexes (41,42). Numerous studies have shown that polyamines interact with DNA anionic phosphate backbone, condense the DNA and induce B–Z transitions. More direct evidences have been obtained by NMR studies, which support the existence of nonspecific electrostatic interactions between DNA backbone and cationic polyamines. These studies also revealed that association of polyamines with B-DNA is weak, whereas their association with the folded quadruplex structure is stronger (43,44). Literature findings suggest that high affinity of polyamines toward non-B-DNA secondary structures is governed by both secondary structure and its base composition (45,46).Table 1.


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

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

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

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

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.
Mentions: Next, we evaluated the effect of polyamines on the thermal stability of c-MYC quadruplex by monitoring the melting profile at 295 nm. The thermal melting was observed to be independent of quadruplex concentration both in absence and presence of polyamines, thereby suggesting formation of intramolecular structure (Supplementary Figure 2). The UV melting profile shows that this element forms a stable quadruplex with Tm of 62.5°C in 100 mM NaCl buffer. The increase in the concentration of spermidine (0–0.75 mM) and spermine (0–50 µM) resulted in increase in the stability of quadruplex structure. The maximum increase in thermal stability was obtained at 0.75 mM spermidine and 50 µM spermine with Tm of 71.5°C and 70°C, respectively (Figure 3 and Table 1). The enthalpy change associated with quadruplex melting was −24.3 kcal/mol in absence of polyamines, −54.0 kcal/mol and −67.0 kcal/mol in presence of 0.75 mM spermidine and 50 µM spermine, respectively. Similar experiments were also performed in potassium buffer. Quadruplex formed in potassium buffer is highly stable (Tm = 75°C, Supplementary Figure 3), but in presence of polyamines we did not obtain characteristic melting domain, as c-MYC quadruplex–K+ in presence of polyamines melts at temperatures >90°C (Supplementary Figure 3). Through UV melting study, it is difficult to dissect out the contributions from the participating populations: quadruplex, duplex and random coil. Therefore, we performed fluorescence-annealing experiments to understand the contribution of these populations and comprehend the fate of equilibrium (30,34). Annealing the system slowly from 95°C to 37°C containing both the G-rich and its complementary strand generates a competing environment that allows the G-rich oligonucleotide to either exist as quadruplex and/or hybridize to its complementary strand to form duplex depending on relative thermodynamic stabilities of these two secondary structures. We monitored the annealing profile of donor fluorescence intensity at 520 nm for c-MYC G-rich sequence with equimolar concentration of complementary strand in the absence and presence of polyamines (Figure 4). The annealing profile obtained for the equimolar mixture of G-rich and C-rich strand in absence of polyamines, initially showed a marginal decrease in fluorescence intensity upon decrease in temperatures from 95°C to 76°C. But at ∼76°C, a change in fluorescence profile was observed which showed increase in fluorescence with further decrease in temperature (76–37°C). The initial decrease and subsequent increase in fluorescence in the cooling profile corresponds to quadruplex and duplex formation, respectively. The fluorescence intensity change at different temperatures is reflective of these contributing populations. The fluorescence profile of donor intensity monitored for the equimolar mixture of G-rich and C-rich strand in presence of 0.75 mM spermidine and 50 µM spermine shows a prominent decrease in fluorescence intensity upon decrease in temperature from 95°C to 76°C and the profile shifts to higher temperature, as shown in Figure 4. Upon further decrease in temperature (76–37°C), the fluorescence intensity increased indicating duplex formation. The extent of increase in fluorescence intensity of donor is lesser in presence of polyamines, suggesting lesser amount duplex formation in comparison to absence of polyamines. The first derivative of these melting curves shows two peaks corresponding to quadruplex and duplex melting domains at higher and lower temperatures, respectively. The intensity and peak position of derivative curve reflect the amount of each participating population and their corresponding Tm values (34). We observed only a moderate shift in the duplex melting domain to higher temperatures in presence of polyamines. However, a prominent shift of quadruplex melting domain to higher temperatures was observed. This can be attributed to the competing secondary structures, quadruplex and duplex adopted by the G-rich sequence. Polyamine-induced stabilization of quadruplex facilitates greater quadruplex formation, and results in decrease in effective G-rich strands required for duplex formation, leading to a marginal increase in duplex stability in presence of polyamines. Polyamines show remarkable stabilizing effects on DNA–RNA hybrids, stem-loop structures and triplexes (41,42). Numerous studies have shown that polyamines interact with DNA anionic phosphate backbone, condense the DNA and induce B–Z transitions. More direct evidences have been obtained by NMR studies, which support the existence of nonspecific electrostatic interactions between DNA backbone and cationic polyamines. These studies also revealed that association of polyamines with B-DNA is weak, whereas their association with the folded quadruplex structure is stronger (43,44). Literature findings suggest that high affinity of polyamines toward non-B-DNA secondary structures is governed by both secondary structure and its base composition (45,46).Table 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