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A polymorphism affecting MYB binding within the promoter of the PDCD4 gene is associated with severe asthma in children.

Binia A, Van Stiphout N, Liang L, Michel S, Bhavsar PK, Fan Chung K, Brightling CE, Barnes PJ, Kabesch M, Bush A, Cookson WO, Moffatt MF - Hum. Mutat. (2013)

Bottom Line: In silico analysis of PDCD4 locus showed that rs6585018:G>A had the potential to affect MYB transcription factor binding, shown to act as a PDCD4-transcription inducer.Electromobility shift assays and reporter assays revealed that rs6585018:G>A alters MYB binding thereby influencing the expression of PDCD4.Our association between a variant MYB binding site in PDCD4 and the severest form of childhood asthma therefore suggests that PDCD4 is a novel molecule of importance to asthmatic inflammatory responses.

View Article: PubMed Central - PubMed

Affiliation: Molecular Genetics and Genomics Section, National Heart and Lung Institute, Imperial College London, London, United Kingdom. aristea.binia@rdls.nestle.com

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Reporter assay results comparing between Jurkat cell lines transfected with pGL3.rs6585018+A construct and pGL3.rs6585018+G construct. The means fold increase ±SD of five independent transient transfection experiments are shown. Paired t-test result was calculated comparing the mean luciferase activity of pGL3.rs6585018+A and pGL3.rs6585018+G in each experiment (*P < 0.02). Luciferase activity was normalized to Renilla and expressed relatively to empty expression vector. Mean luciferase activity relative to empty vector was 1.31 (SD 0.25) for pGL3.rs6585018+A and 1.14 (SD 0.19) for pGL3.rs6585018+G. The mean luminometer values of empty pGL3.promoter vector transfected alone into Jurkat cell line were 3030 light units. A representation of rs6585018+A and pGL3.rs6585018+G constructs is also provided.
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fig05: Reporter assay results comparing between Jurkat cell lines transfected with pGL3.rs6585018+A construct and pGL3.rs6585018+G construct. The means fold increase ±SD of five independent transient transfection experiments are shown. Paired t-test result was calculated comparing the mean luciferase activity of pGL3.rs6585018+A and pGL3.rs6585018+G in each experiment (*P < 0.02). Luciferase activity was normalized to Renilla and expressed relatively to empty expression vector. Mean luciferase activity relative to empty vector was 1.31 (SD 0.25) for pGL3.rs6585018+A and 1.14 (SD 0.19) for pGL3.rs6585018+G. The mean luminometer values of empty pGL3.promoter vector transfected alone into Jurkat cell line were 3030 light units. A representation of rs6585018+A and pGL3.rs6585018+G constructs is also provided.

Mentions: To confirm the allele-specific effects of rs6585018:G>A polymorphism on promoter activity, two luciferase reporter constructs were generated including the MYB binding sequence 5′ of PDCD4 and A/G at the rs6585018:G>A polymorphic site. After transient transfection into Jurkat cell lines, the relative luciferase activity of the pGL3.rs6585018+A transfected cells was found to be increased compared with pGL3.rs6585018+G transfected cells (P < 0.02) confirming the findings from the EMSA experiments (Fig. 5).


A polymorphism affecting MYB binding within the promoter of the PDCD4 gene is associated with severe asthma in children.

Binia A, Van Stiphout N, Liang L, Michel S, Bhavsar PK, Fan Chung K, Brightling CE, Barnes PJ, Kabesch M, Bush A, Cookson WO, Moffatt MF - Hum. Mutat. (2013)

Reporter assay results comparing between Jurkat cell lines transfected with pGL3.rs6585018+A construct and pGL3.rs6585018+G construct. The means fold increase ±SD of five independent transient transfection experiments are shown. Paired t-test result was calculated comparing the mean luciferase activity of pGL3.rs6585018+A and pGL3.rs6585018+G in each experiment (*P < 0.02). Luciferase activity was normalized to Renilla and expressed relatively to empty expression vector. Mean luciferase activity relative to empty vector was 1.31 (SD 0.25) for pGL3.rs6585018+A and 1.14 (SD 0.19) for pGL3.rs6585018+G. The mean luminometer values of empty pGL3.promoter vector transfected alone into Jurkat cell line were 3030 light units. A representation of rs6585018+A and pGL3.rs6585018+G constructs is also provided.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4296222&req=5

fig05: Reporter assay results comparing between Jurkat cell lines transfected with pGL3.rs6585018+A construct and pGL3.rs6585018+G construct. The means fold increase ±SD of five independent transient transfection experiments are shown. Paired t-test result was calculated comparing the mean luciferase activity of pGL3.rs6585018+A and pGL3.rs6585018+G in each experiment (*P < 0.02). Luciferase activity was normalized to Renilla and expressed relatively to empty expression vector. Mean luciferase activity relative to empty vector was 1.31 (SD 0.25) for pGL3.rs6585018+A and 1.14 (SD 0.19) for pGL3.rs6585018+G. The mean luminometer values of empty pGL3.promoter vector transfected alone into Jurkat cell line were 3030 light units. A representation of rs6585018+A and pGL3.rs6585018+G constructs is also provided.
Mentions: To confirm the allele-specific effects of rs6585018:G>A polymorphism on promoter activity, two luciferase reporter constructs were generated including the MYB binding sequence 5′ of PDCD4 and A/G at the rs6585018:G>A polymorphic site. After transient transfection into Jurkat cell lines, the relative luciferase activity of the pGL3.rs6585018+A transfected cells was found to be increased compared with pGL3.rs6585018+G transfected cells (P < 0.02) confirming the findings from the EMSA experiments (Fig. 5).

Bottom Line: In silico analysis of PDCD4 locus showed that rs6585018:G>A had the potential to affect MYB transcription factor binding, shown to act as a PDCD4-transcription inducer.Electromobility shift assays and reporter assays revealed that rs6585018:G>A alters MYB binding thereby influencing the expression of PDCD4.Our association between a variant MYB binding site in PDCD4 and the severest form of childhood asthma therefore suggests that PDCD4 is a novel molecule of importance to asthmatic inflammatory responses.

View Article: PubMed Central - PubMed

Affiliation: Molecular Genetics and Genomics Section, National Heart and Lung Institute, Imperial College London, London, United Kingdom. aristea.binia@rdls.nestle.com

Show MeSH
Related in: MedlinePlus