A polymorphism affecting MYB binding within the promoter of the PDCD4 gene is associated with severe asthma in children.
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.
Affiliation: Molecular Genetics and Genomics Section, National Heart and Lung Institute, Imperial College London, London, United Kingdom. email@example.comShow MeSH
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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.
Affiliation: Molecular Genetics and Genomics Section, National Heart and Lung Institute, Imperial College London, London, United Kingdom. firstname.lastname@example.org