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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.

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Quad-SNP affects promoter activity of RPS3. (A) Scheme showing part of RPS3 promoter with sequence of the PG4 motif given in bold; Quad-SNP is underlined. (B) CD spectra of PG4 motif sequences S3A and S3B, melting temperature (Tm) in right frame. (C) Scheme showing promoter reporter systems inserted upstream of the firefly luciferase gene. Luciferase reporter activity of reporter clones with either S3A or S3B relative to no insert clone is shown below; activity in case of S3B in A549 cells was not detectable (asterisks). Experiments were done in triplicate; Renilla luciferase activity was used to normalize transfection efficiency.
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gkr1258-F3: Quad-SNP affects promoter activity of RPS3. (A) Scheme showing part of RPS3 promoter with sequence of the PG4 motif given in bold; Quad-SNP is underlined. (B) CD spectra of PG4 motif sequences S3A and S3B, melting temperature (Tm) in right frame. (C) Scheme showing promoter reporter systems inserted upstream of the firefly luciferase gene. Luciferase reporter activity of reporter clones with either S3A or S3B relative to no insert clone is shown below; activity in case of S3B in A549 cells was not detectable (asterisks). Experiments were done in triplicate; Renilla luciferase activity was used to normalize transfection efficiency.

Mentions: Next, to test above findings we sought to study a PG4 motif/SNP combination that was independent of the data sets analysed above and asked: (i) whether the specific nucleotide substitution resulted in disruption of the G-quadruplex structure and (ii) if the disruption caused any alteration in expression of the gene. For this the SNP (rs17880356, G to C) found in the promoter of the ribosomal protein S3 (RPS3) (Figure 3A), which plays a critical role in initiation of translation, was selected. In order to determine whether this sequence adopted the quadruplex motif, and if the substitution significantly disrupted the structure, we first synthesized two oligonucleotides, S3A and S3B comprising the PG4 motif representing both the alleles of the Quad-SNP found in RPS3, where S3A had the G-base while S3B had the substitution (G to C). G-quadruplex forming potential was determined using CD spectroscopy—S3A gave a well formed parallel quadruplex whereas S3B showed decrease in peak height at 260 nm suggesting loss of structural stability (Figure 3B). We also found that the Tm of the G-quadruplex motif was 62.1°C whereas that of the S3B motif was substantially decreased to 48°C, consistent with CD results (Figure 3B, right panel).Figure 3.


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)

Quad-SNP affects promoter activity of RPS3. (A) Scheme showing part of RPS3 promoter with sequence of the PG4 motif given in bold; Quad-SNP is underlined. (B) CD spectra of PG4 motif sequences S3A and S3B, melting temperature (Tm) in right frame. (C) Scheme showing promoter reporter systems inserted upstream of the firefly luciferase gene. Luciferase reporter activity of reporter clones with either S3A or S3B relative to no insert clone is shown below; activity in case of S3B in A549 cells was not detectable (asterisks). Experiments were done in triplicate; Renilla luciferase activity was used to normalize transfection efficiency.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC3351168&req=5

gkr1258-F3: Quad-SNP affects promoter activity of RPS3. (A) Scheme showing part of RPS3 promoter with sequence of the PG4 motif given in bold; Quad-SNP is underlined. (B) CD spectra of PG4 motif sequences S3A and S3B, melting temperature (Tm) in right frame. (C) Scheme showing promoter reporter systems inserted upstream of the firefly luciferase gene. Luciferase reporter activity of reporter clones with either S3A or S3B relative to no insert clone is shown below; activity in case of S3B in A549 cells was not detectable (asterisks). Experiments were done in triplicate; Renilla luciferase activity was used to normalize transfection efficiency.
Mentions: Next, to test above findings we sought to study a PG4 motif/SNP combination that was independent of the data sets analysed above and asked: (i) whether the specific nucleotide substitution resulted in disruption of the G-quadruplex structure and (ii) if the disruption caused any alteration in expression of the gene. For this the SNP (rs17880356, G to C) found in the promoter of the ribosomal protein S3 (RPS3) (Figure 3A), which plays a critical role in initiation of translation, was selected. In order to determine whether this sequence adopted the quadruplex motif, and if the substitution significantly disrupted the structure, we first synthesized two oligonucleotides, S3A and S3B comprising the PG4 motif representing both the alleles of the Quad-SNP found in RPS3, where S3A had the G-base while S3B had the substitution (G to C). G-quadruplex forming potential was determined using CD spectroscopy—S3A gave a well formed parallel quadruplex whereas S3B showed decrease in peak height at 260 nm suggesting loss of structural stability (Figure 3B). We also found that the Tm of the G-quadruplex motif was 62.1°C whereas that of the S3B motif was substantially decreased to 48°C, consistent with CD results (Figure 3B, right panel).Figure 3.

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