Nuclear factor of activated T cells and YY1 combine to repress IL-5 expression in a human T-cell line Gretchen T.F. Schwenger, PhD, Régis Fournier, PhD,

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Nuclear factor of activated T cells and YY1 combine to repress IL-5 expression in a human T-cell line Gretchen T.F. Schwenger, PhD, Régis Fournier, PhD, Leanne M. Hall, Asc Dip, Colin J. Sanderson, PhD, ScD, Viatcheslav A. Mordvinov, PhD Journal of Allergy and Clinical Immunology Volume 104, Issue 4, Pages 820-827 (October 1999) DOI: 10.1016/S0091-6749(99)70293-9 Copyright © 1999 Mosby, Inc. Terms and Conditions

Fig. 1 Sequences protected by nuclear proteins of PER117 cells in –506 to –184 fragment of hIL-5 promoter. Protected sequence obtained by deoxyribonuclease I protection assays were visualized by labeling either coding strand (A) or noncoding strand (B). Digest of labeled probes was achieved either with no protein and 0.01 U of deoxyribonuclease I (naked DNA) or with 50 μg of nuclear protein and 1 U of deoxyribonuclease I. Protected areas were located at nucleotide level by enzymatic sequencing of probe. Protected sequences of each strand are displayed on A and B and on linear sequence on C. Journal of Allergy and Clinical Immunology 1999 104, 820-827DOI: (10.1016/S0091-6749(99)70293-9) Copyright © 1999 Mosby, Inc. Terms and Conditions

Fig. 2 EMSA with radiolabeled hPRE2o as probe and PER117 nuclear extracts. Free probe is shown at bottom of each lane. A, Unstimulated (Unstim) and PMA/CaI-stimulated (Stim) PER117 nuclear extracts with hPRE2o probe alone. B, Unstimulated PER117 nuclear extract used with hPRE2o probe and 50 or 100 times molar excess of unlabeled hPRE2o or AP2 oligonucleotides as competitor. C, Stimulated PER117 nuclear extract used with hPRE2o probe and 50 or 100 times molar excess of unlabelled hPRE2o or AP2 oligonucleotides as competitor. Journal of Allergy and Clinical Immunology 1999 104, 820-827DOI: (10.1016/S0091-6749(99)70293-9) Copyright © 1999 Mosby, Inc. Terms and Conditions

Fig. 3 A, EMSA with use of radiolabeled hPRE2o as probe and PMA/CaI-stimulated PER117 nuclear extracts. Free probe is shown at bottom of each lane. Unlabeled competitor oligonucleotides listed in Table I, hPRE2oA to hPRE2F (A to F ) are used at 100 times molar excess and are shown above each lane. B, hPRE2-IL5 region is shown with nucleotides critical for formation of K1 (underlined) and those critical for formation of K3 (highlighted). Journal of Allergy and Clinical Immunology 1999 104, 820-827DOI: (10.1016/S0091-6749(99)70293-9) Copyright © 1999 Mosby, Inc. Terms and Conditions

Fig. 4 Transfection of PER117 cells with wild-type and mutant hIL-5 constructs. Cells were either left unstimulated or stimulated with 10 ng/mL PMA and 0.25 μmol/L CaI for 16 hours. Stimulated results (n = 2) are presented as fold-induction ± SD compared with corresponding unstimulated data for each construct. Journal of Allergy and Clinical Immunology 1999 104, 820-827DOI: (10.1016/S0091-6749(99)70293-9) Copyright © 1999 Mosby, Inc. Terms and Conditions

Fig. 5 A, hPRE2-IL5 region is shown with comparison to consensus sequences for YY1 and NF-AT. Asterisk, Homology. B, EMSA with use of radiolabeled hPRE2o as probe and PMA/CaI-stimulated PER117 nuclear extracts. Free probe is shown at bottom of each lane. Unlabeled competitor oligonucleotides YY1 and NF-AT are listed in Table I and were used at 50 times molar excess. Supershift experiments were carried out with 2 μL of either YY1 or NF-AT antisera (2 μg protein). Journal of Allergy and Clinical Immunology 1999 104, 820-827DOI: (10.1016/S0091-6749(99)70293-9) Copyright © 1999 Mosby, Inc. Terms and Conditions

Fig. 6 Diagram of hIL-5 promoter showing characterized transcription factors active in gene regulation. Journal of Allergy and Clinical Immunology 1999 104, 820-827DOI: (10.1016/S0091-6749(99)70293-9) Copyright © 1999 Mosby, Inc. Terms and Conditions