Allele-specific transcription of the asthma-associated PHD finger protein 11 gene (PHF11) modulated by octamer-binding transcription factor 1 (Oct-1)

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Allele-specific transcription of the asthma-associated PHD finger protein 11 gene (PHF11) modulated by octamer-binding transcription factor 1 (Oct-1) Richard J. Holt, DPhil, Youming Zhang, DPhil, Aristea Binia, PhD, Anna L. Dixon, DPhil, Claire Vandiedonck, DPhil, William O. Cookson, DPhil, Julian C. Knight, DPhil, Miriam F. Moffatt, DPhil Journal of Allergy and Clinical Immunology Volume 127, Issue 4, Pages 1054-1062.e2 (April 2011) DOI: 10.1016/j.jaci.2010.12.015 Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Gene structure of PHF11. Figure drawn approximately to scale. The locations of the genes according to the Human March 2006 assembly are as follows: SETDB2: chr13:48,916,511-48,964,299; PHF11: chr13:48,967,802-49,001,118; RCBTB1: chr13:49,004,083-49,057,720. The detailed structure of PHF11 is shown with exons numbered. The positions of SNPs rs3765526, rs9526569, and rs1046295 are indicated, together with their names and P values from the study by Zhang et al.13 Journal of Allergy and Clinical Immunology 2011 127, 1054-1062.e2DOI: (10.1016/j.jaci.2010.12.015) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Allele-specific protein-DNA interactions at PHF11 involving rs1046295. A, EMSAs corresponding to the A or G alleles of rs1046295 with BEAS-2B, Calu-3, and Daudi nuclear extracts. B, Competition EMSAs using Daudi nuclear extract. Rs1046295 allele A (lanes 1-10) and G (lanes 11-20). Molar excesses of unlabeled competitor oligonucleotides for allele A, allele G, and an unrelated probe, rs71307668 (unrel.), indicated. C, Supershift EMSA experiments for rs1046295 using Daudi nuclear extract. Supershift indicated by S. Journal of Allergy and Clinical Immunology 2011 127, 1054-1062.e2DOI: (10.1016/j.jaci.2010.12.015) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Allele-specific protein-DNA interactions at PHF11 confirming the ability of only Oct-1 to bind at rs1046295. A, Competition EMSAs using Daudi nuclear extract. Rs1046295 allele A (lanes 1-8) and G (lanes 9-16). Molar excesses of unlabeled competitor oligonucleotides for the Oct-1 and SRY consensus sequences, and an unrelated probe (Unrel.), are indicated. B, EMSAs performed for rs1046295 allele A using Daudi and CCL-159 nuclear extracts and Oct-1 (POU2F1) and SRY recombinant proteins. Addition of antibodies for SRY or YY1 (negative control) is indicated (lanes 7-9). Journal of Allergy and Clinical Immunology 2011 127, 1054-1062.e2DOI: (10.1016/j.jaci.2010.12.015) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Boxplots showing the allelotyping results. Combined genomic DNA results (blue), combined means of cDNA ratios of all individuals (red), with and without sample outliers, and replicate cDNA results for each individual (brown). Peak area ratio of allele A:allele G used for 8 PCR replicates. Moderate outliers (outside 1.5∗interquartile range) are displayed as a circle, extreme outliers (outside 2∗interquartile range) as a circle in the box-whiskers. Samples 8198-4 (ratio is infinite), 8033-3, and 8103-4 (outlying results) not shown. Significance of any difference between cDNA and genomic DNA peak ratios indicated (∗P < .05; ∗∗P < .01; ∗∗∗P < .001). Journal of Allergy and Clinical Immunology 2011 127, 1054-1062.e2DOI: (10.1016/j.jaci.2010.12.015) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Summary of MALDI-TOF mass spectrometry allelotyping Summary of MALDI-TOF mass spectrometry allelotyping. A, MALDI-TOF performed on genomic DNA for a heterozygote. B, MALDI-TOF performed on cDNA from the same individual. i, cDNA peak ratio identical to genomic DNA peak ratio, indicating no preferential allele expression. ii, Ratio of cDNA peaks differs from the genomic DNA, indicating preferential A allele expression. iii, Ratio of cDNA peaks differs from the genomic DNA, indicating preferential G allele expression. Journal of Allergy and Clinical Immunology 2011 127, 1054-1062.e2DOI: (10.1016/j.jaci.2010.12.015) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Protein-DNA interactions at PHF11 involving rs1046295 using 5 cell lines. EMSAs corresponding to the A or G alleles of rs1046295 with CCL-114, CCL-159, Calu-3, BEAS-2B, and Daudi nuclear extracts. Multiple reactions with CCL-114 and CCL-159 nuclear extracts represent protein aliquots prepared on separate dates. Journal of Allergy and Clinical Immunology 2011 127, 1054-1062.e2DOI: (10.1016/j.jaci.2010.12.015) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions