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An IgE-associated polymorphism in STAT6 alters NF-κB binding, STAT6 promoter activity, and mRNA expression  Michaela Schedel, PhD, Remo Frei, PhD, Christian.

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Presentation on theme: "An IgE-associated polymorphism in STAT6 alters NF-κB binding, STAT6 promoter activity, and mRNA expression  Michaela Schedel, PhD, Remo Frei, PhD, Christian."— Presentation transcript:

1 An IgE-associated polymorphism in STAT6 alters NF-κB binding, STAT6 promoter activity, and mRNA expression  Michaela Schedel, PhD, Remo Frei, PhD, Christian Bieli, MD, Lisa Cameron, PhD, Jerzy Adamski, PhD, Roger Lauener, MD, Michael Kabesch, MD  Journal of Allergy and Clinical Immunology  Volume 124, Issue 3, Pages e6 (September 2009) DOI: /j.jaci Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 In vitro STAT6 promoter activity is silenced depending on rs Luciferase assays were performed using Jurkat T cells with the STAT6 promoter vector pBP86 (1.1 kb) (A) or pBP78 (5.6 kb) (B) and further constructs in addition carrying STAT6 intron 2 either expressing the wild-type C (pBP86_WT, pBP78_WT) or the polymorphic T allele (pBP86_PO, pBP78_PO) of rs (N = 10).∗P < .001; ∗∗P < .0001; P < P/I = PMA/ionomycin. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Polymorphic allele of rs in STAT6 intron 2 induces a novel binding site for NF-κB in a T-cell line. EMSA analysis with a STAT6 probe either carrying the wild-type C (rs324011_WT) or the polymorphic T (rs324011_PO) allele of SNP rs was performed with nuclear extract from Jurkat T cells (5 μg). ♣, Competition experiment using an unrelated oligo; §, supershift experiment using an unrelated antibody. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 Polymorphic allele of rs in STAT6 intron 2 induces a novel binding site for NF-κB in primary T cells. EMSA analysis with a STAT6 probe either carrying the WT or the PO of rs was performed by using nuclear extract from freshly isolated primary CD4+ T cells stimulated with PMA/ionomycin (50 ng/mL; 1 μmol/L). Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 Ex vivo expression of STAT6 splice variants are influenced by rs alleles. Association between gene expression of 3 STAT6 splice variants (non-STAT6d/STAT6e, STAT6d, STAT6e) and SNP rs in intron 2 of the STAT6 gene is displayed in GMRs with the 95% CI. ∗.05 > P ≥ .01; ∗∗P < .01. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Localization of STAT6 on chromosome 12q13 and linkage disequilibrium patterns (r2 plots). A, Position of the STAT6 gene on chromosome 12q13 in relation to adjacent genes Tachykinin 3, Myosin IA, Nerve growth factor-induced clone IA-binding protein 2 and Low density lipoprotein receptor-related protein 1 and position of frequent STAT6 SNPs (minor allele frequency > 0.03) and linkage disequilibrium (r2 plot) from the HapMap database. B, Gene structure of STAT6 indicating the location of SNPs in linkage disequilibrium with rs C, Linkage disequilibrium (r2 plot) between SNPs rs and rs recently associated with elevated total IgE levels by the genome-wide analysis study.E1 Colors in the linkage disequilibrium (LD) plot from Haploview: white (r2 = 0), shades of gray (0 < r2 < 1), and black (r2 = 1). Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Generation of the STAT6 luciferase constructs
Generation of the STAT6 luciferase constructs. A, STAT6 promoter constructs pBP78 (5.6 kb), pBP88 (2.8 kb), and pBP86 (1.1 kb). B, STAT6 intron 2 was cloned 5′3′ into the STAT6 pBP86 and pBP78 promoter constructs downstream of the luciferase gene carrying either the wild-type C or the polymorphic T allele. C, STAT6 intron 2 carrying either the wild-type C or the polymorphic T allele was cloned into the pBP78 promoter construct in the 3′5′ orientation. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 STAT6 promoter activity in human cells
STAT6 promoter activity in human cells. Jurkat T cells were transiently transfected with either the pGL3-basic vector or the STAT6 promoter constructs pBP78, pBP88, and pBP86. Cells were left in medium or stimulated with PMA/ionomycin (P/I = 12.5 ng/mL; 0.25 μmol/L) 3 hours after transfection and harvested 24 hours after transfection (N = 10). Luciferase activity was normalized for transfection efficiency by using the control plasmid pRL-TK. Relative luciferase activity in relative light units (RLU). Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 EMSA analysis with a NF-κB consensus sequenceE2 using nuclear extract (5 μg) from Jurkat T cells either left in medium or stimulated with PMA/ionomycin (50 ng/mL; 1 μmol/L). The competitors (100-fold molar excess) and supershift antibodies (4 μg) are noted below the respective lanes. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 Quantitative real-time PCR of STAT6 splice variants (non-STAT6d/STAT6e, STAT6d, STAT6e). A, Quantitative mRNA analyses were first performed by using a forward primer at the STAT6 exon 16/17 border and a reverse primer in exon 19. B, Three PCR products were observed containing the predicted exonic regions of STAT6 (further referred to as non-STAT6d/STAT6e) and 2 novel splice variants, STAT6d and STAT6e. C, Isoform-specific primers were used for all further analyses. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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