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Quadruplex-single nucleotide polymorphisms (Quad-SNP) influence gene expression difference among individuals.

Baral A, Kumar P, Halder R, Mani P, Yadav VK, Singh A, Das SK, Chowdhury S - Nucleic Acids Res. (2012)

Bottom Line: Findings reveal significant association between quadruplex-SNPs and expression of the corresponding gene in individuals (P < 0.0001).The quadruplex motif, and not the disrupted-motif, enhanced transcription in human cell lines of different origin.Together, these findings build direct support for quadruplex-mediated transcription and suggest quadruplex-SNPs may play significant role in mechanistically understanding variations in gene expression among individuals.

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
Non-canonical guanine quadruplex structures are not only predominant but also conserved among bacterial and mammalian promoters. Moreover recent findings directly implicate quadruplex structures in transcription. These argue for an intrinsic role of the structural motif and thereby posit that single nucleotide polymorphisms (SNP) that compromise the quadruplex architecture could influence function. To test this, we analysed SNPs within quadruplex motifs (Quad-SNP) and gene expression in 270 individuals across four populations (HapMap) representing more than 14,500 genotypes. Findings reveal significant association between quadruplex-SNPs and expression of the corresponding gene in individuals (P < 0.0001). Furthermore, analysis of Quad-SNPs obtained from population-scale sequencing of 1000 human genomes showed relative selection bias against alteration of the structural motif. To directly test the quadruplex-SNP-transcription connection, we constructed a reporter system using the RPS3 promoter-remarkable difference in promoter activity in the 'quadruplex-destabilized' versus 'quadruplex-intact' promoter was noticed. As a further test, we incorporated a quadruplex motif or its disrupted counterpart within a synthetic promoter reporter construct. The quadruplex motif, and not the disrupted-motif, enhanced transcription in human cell lines of different origin. Together, these findings build direct support for quadruplex-mediated transcription and suggest quadruplex-SNPs may play significant role in mechanistically understanding variations in gene expression among individuals.

Show MeSH
Incorporation of the G-quadruplex motif and not sequence per se induces promoter activity. (A) Scheme showing the constructs made to insert either a G-quadruplex-forming (G4) or disrupted G4 (disG4) as control sequence upstream of SV40 promoter in a luciferase reporter vector. (B) CD spectra of oligonucleotide used for G4 motif and disG4 showing disruption of the quadruplex motif in case of disG4. (C) Luciferase reporter activity of clones harbouring G4 or disG4 in human cell lines with respect to the no-insert construct. All experiments were done in triplicate using Renilla luciferase activity as transfection control.
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gkr1258-F4: Incorporation of the G-quadruplex motif and not sequence per se induces promoter activity. (A) Scheme showing the constructs made to insert either a G-quadruplex-forming (G4) or disrupted G4 (disG4) as control sequence upstream of SV40 promoter in a luciferase reporter vector. (B) CD spectra of oligonucleotide used for G4 motif and disG4 showing disruption of the quadruplex motif in case of disG4. (C) Luciferase reporter activity of clones harbouring G4 or disG4 in human cell lines with respect to the no-insert construct. All experiments were done in triplicate using Renilla luciferase activity as transfection control.

Mentions: To test quadruplex-mediated transcription in a more direct fashion we made a synthetic G-quadruplex motif and incorporated this upstream of an exogenous promoter reporter system constituting the SV40 promoter upstream of the firefly luciferase gene (Figure 4A). An analogous system was made by introducing a similar sequence wherein the quadruplex motif was disrupted by specific nucleotide changes to constitute a negative control that did not adopt the quadruplex form. We confirmed that the substitutions led to disruption of the structure using CD (Figure 4B) and DNA melting experiments (data not shown). Following this luciferase activity was checked in two cell lines and reporter activity from firefly luciferase was normalized using Renilla luciferase counts to control for transfection efficiency. Promoter activity increased on quadruplex insertion by ∼1.9- and 2.3-folds in HT1080 and A549 cells, respectively (Figure 4C). In contrast, reporter activity when the quadruplex motif was disrupted was similar to the inherent SV40 promoter activity. Together these results showed that incorporation of the quadruplex motif results in altered promoter activity due to the presence of the structural motif and is lost when the structure is specifically disrupted.Figure 4.


Quadruplex-single nucleotide polymorphisms (Quad-SNP) influence gene expression difference among individuals.

Baral A, Kumar P, Halder R, Mani P, Yadav VK, Singh A, Das SK, Chowdhury S - Nucleic Acids Res. (2012)

Incorporation of the G-quadruplex motif and not sequence per se induces promoter activity. (A) Scheme showing the constructs made to insert either a G-quadruplex-forming (G4) or disrupted G4 (disG4) as control sequence upstream of SV40 promoter in a luciferase reporter vector. (B) CD spectra of oligonucleotide used for G4 motif and disG4 showing disruption of the quadruplex motif in case of disG4. (C) Luciferase reporter activity of clones harbouring G4 or disG4 in human cell lines with respect to the no-insert construct. All experiments were done in triplicate using Renilla luciferase activity as transfection control.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351168&req=5

gkr1258-F4: Incorporation of the G-quadruplex motif and not sequence per se induces promoter activity. (A) Scheme showing the constructs made to insert either a G-quadruplex-forming (G4) or disrupted G4 (disG4) as control sequence upstream of SV40 promoter in a luciferase reporter vector. (B) CD spectra of oligonucleotide used for G4 motif and disG4 showing disruption of the quadruplex motif in case of disG4. (C) Luciferase reporter activity of clones harbouring G4 or disG4 in human cell lines with respect to the no-insert construct. All experiments were done in triplicate using Renilla luciferase activity as transfection control.
Mentions: To test quadruplex-mediated transcription in a more direct fashion we made a synthetic G-quadruplex motif and incorporated this upstream of an exogenous promoter reporter system constituting the SV40 promoter upstream of the firefly luciferase gene (Figure 4A). An analogous system was made by introducing a similar sequence wherein the quadruplex motif was disrupted by specific nucleotide changes to constitute a negative control that did not adopt the quadruplex form. We confirmed that the substitutions led to disruption of the structure using CD (Figure 4B) and DNA melting experiments (data not shown). Following this luciferase activity was checked in two cell lines and reporter activity from firefly luciferase was normalized using Renilla luciferase counts to control for transfection efficiency. Promoter activity increased on quadruplex insertion by ∼1.9- and 2.3-folds in HT1080 and A549 cells, respectively (Figure 4C). In contrast, reporter activity when the quadruplex motif was disrupted was similar to the inherent SV40 promoter activity. Together these results showed that incorporation of the quadruplex motif results in altered promoter activity due to the presence of the structural motif and is lost when the structure is specifically disrupted.Figure 4.

Bottom Line: Findings reveal significant association between quadruplex-SNPs and expression of the corresponding gene in individuals (P < 0.0001).The quadruplex motif, and not the disrupted-motif, enhanced transcription in human cell lines of different origin.Together, these findings build direct support for quadruplex-mediated transcription and suggest quadruplex-SNPs may play significant role in mechanistically understanding variations in gene expression among individuals.

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
Non-canonical guanine quadruplex structures are not only predominant but also conserved among bacterial and mammalian promoters. Moreover recent findings directly implicate quadruplex structures in transcription. These argue for an intrinsic role of the structural motif and thereby posit that single nucleotide polymorphisms (SNP) that compromise the quadruplex architecture could influence function. To test this, we analysed SNPs within quadruplex motifs (Quad-SNP) and gene expression in 270 individuals across four populations (HapMap) representing more than 14,500 genotypes. Findings reveal significant association between quadruplex-SNPs and expression of the corresponding gene in individuals (P < 0.0001). Furthermore, analysis of Quad-SNPs obtained from population-scale sequencing of 1000 human genomes showed relative selection bias against alteration of the structural motif. To directly test the quadruplex-SNP-transcription connection, we constructed a reporter system using the RPS3 promoter-remarkable difference in promoter activity in the 'quadruplex-destabilized' versus 'quadruplex-intact' promoter was noticed. As a further test, we incorporated a quadruplex motif or its disrupted counterpart within a synthetic promoter reporter construct. The quadruplex motif, and not the disrupted-motif, enhanced transcription in human cell lines of different origin. Together, these findings build direct support for quadruplex-mediated transcription and suggest quadruplex-SNPs may play significant role in mechanistically understanding variations in gene expression among individuals.

Show MeSH