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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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Supershift assays for PDCD4 SNP rs6585018:G>A using 10 μg of either A549, Daudi or Jurkat nuclear extracts. Lanes 1 and 8 contain only the hot probes rs6585018_A and rs6585018_G, respectively. Lanes 2–7 include the rs6585018_A hot probe and lanes 9–14 the rs6585018_G hot probe. A549 extracts are included in lanes 2 and 9 without the antibody and in lanes 3 and 10 with 0.3 ng of the MYB antibody. Daudi extracts are included in lanes 4 and 11 without the antibody and 5 and 12 with 0.3 ng of the MYB antibody. Jurkat extracts are included in lanes 6 and 13 without the antibody and in lanes 7 and 14 with 0.3 ng of the MYB antibody. As expected the specific protein–DNA complex is still observed in all cell lines tested. The addition of a MYB antibody (lanes 3, 5, 7, 10, 12, 14) results in the loss of the protein–DNA complexes.
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fig04: Supershift assays for PDCD4 SNP rs6585018:G>A using 10 μg of either A549, Daudi or Jurkat nuclear extracts. Lanes 1 and 8 contain only the hot probes rs6585018_A and rs6585018_G, respectively. Lanes 2–7 include the rs6585018_A hot probe and lanes 9–14 the rs6585018_G hot probe. A549 extracts are included in lanes 2 and 9 without the antibody and in lanes 3 and 10 with 0.3 ng of the MYB antibody. Daudi extracts are included in lanes 4 and 11 without the antibody and 5 and 12 with 0.3 ng of the MYB antibody. Jurkat extracts are included in lanes 6 and 13 without the antibody and in lanes 7 and 14 with 0.3 ng of the MYB antibody. As expected the specific protein–DNA complex is still observed in all cell lines tested. The addition of a MYB antibody (lanes 3, 5, 7, 10, 12, 14) results in the loss of the protein–DNA complexes.

Mentions: Examining the ENCODE data for the region chr10:112,630,451–112,631,970 (GRCh37/hg19 assembly), the area including rs6585018:G>A has been reported to be rich in active regulatory elements, such as strong active enhancer regions, DNase I hypersensitivity sites and histone modifications as predicted by integrating chromatin immunoprecipitation sequencing (ChIP-seq) data [Dunham et al., 2012]. Transcription factor binding analysis revealed that one out of five PDCD4 SNPs, rs6585018:G>A, had the potential to disrupt the binding of the transcription factor MYB (v-myb myeloblastosis viral oncogene homolog) (Supp. Table S4). An allele-specific band formation was found by EMSA using nuclear extracts from Jurkat and A549 cell lines. Competition assays in Jurkat (Fig. 3) and A459 cells (Supp. Fig. S3) revealed the formation of a protein–DNA complex specific for the A allele of rs6585018:G>A and identical results were obtained using a probe containing the MYB-consensus binding site (Supp. Fig. S4). This was further confirmed by supershift assays using anti-MYB in the reaction (Fig. 4) as well as assays in which a nonspecific antibody was included in the reaction (anti-SRY) (Supp. Fig. S5). Repetition of both the nuclear extractions and EMSA reactions gave identical results.


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)

Supershift assays for PDCD4 SNP rs6585018:G>A using 10 μg of either A549, Daudi or Jurkat nuclear extracts. Lanes 1 and 8 contain only the hot probes rs6585018_A and rs6585018_G, respectively. Lanes 2–7 include the rs6585018_A hot probe and lanes 9–14 the rs6585018_G hot probe. A549 extracts are included in lanes 2 and 9 without the antibody and in lanes 3 and 10 with 0.3 ng of the MYB antibody. Daudi extracts are included in lanes 4 and 11 without the antibody and 5 and 12 with 0.3 ng of the MYB antibody. Jurkat extracts are included in lanes 6 and 13 without the antibody and in lanes 7 and 14 with 0.3 ng of the MYB antibody. As expected the specific protein–DNA complex is still observed in all cell lines tested. The addition of a MYB antibody (lanes 3, 5, 7, 10, 12, 14) results in the loss of the protein–DNA complexes.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4296222&req=5

fig04: Supershift assays for PDCD4 SNP rs6585018:G>A using 10 μg of either A549, Daudi or Jurkat nuclear extracts. Lanes 1 and 8 contain only the hot probes rs6585018_A and rs6585018_G, respectively. Lanes 2–7 include the rs6585018_A hot probe and lanes 9–14 the rs6585018_G hot probe. A549 extracts are included in lanes 2 and 9 without the antibody and in lanes 3 and 10 with 0.3 ng of the MYB antibody. Daudi extracts are included in lanes 4 and 11 without the antibody and 5 and 12 with 0.3 ng of the MYB antibody. Jurkat extracts are included in lanes 6 and 13 without the antibody and in lanes 7 and 14 with 0.3 ng of the MYB antibody. As expected the specific protein–DNA complex is still observed in all cell lines tested. The addition of a MYB antibody (lanes 3, 5, 7, 10, 12, 14) results in the loss of the protein–DNA complexes.
Mentions: Examining the ENCODE data for the region chr10:112,630,451–112,631,970 (GRCh37/hg19 assembly), the area including rs6585018:G>A has been reported to be rich in active regulatory elements, such as strong active enhancer regions, DNase I hypersensitivity sites and histone modifications as predicted by integrating chromatin immunoprecipitation sequencing (ChIP-seq) data [Dunham et al., 2012]. Transcription factor binding analysis revealed that one out of five PDCD4 SNPs, rs6585018:G>A, had the potential to disrupt the binding of the transcription factor MYB (v-myb myeloblastosis viral oncogene homolog) (Supp. Table S4). An allele-specific band formation was found by EMSA using nuclear extracts from Jurkat and A549 cell lines. Competition assays in Jurkat (Fig. 3) and A459 cells (Supp. Fig. S3) revealed the formation of a protein–DNA complex specific for the A allele of rs6585018:G>A and identical results were obtained using a probe containing the MYB-consensus binding site (Supp. Fig. S4). This was further confirmed by supershift assays using anti-MYB in the reaction (Fig. 4) as well as assays in which a nonspecific antibody was included in the reaction (anti-SRY) (Supp. Fig. S5). Repetition of both the nuclear extractions and EMSA reactions gave identical results.

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