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GA-Binding Protein Factors, in Concert with the Coactivator CREB Binding Protein/P300, Control the Induction of the Interleukin 16 Promoter in T Lymphocytes

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GA-Binding Protein Factors, in Concert with the Coactivator CREB Binding Protein/P300, Control the Induction of the Interleukin 16 Promoter in T Lymphocytes Proc. Natl. Acad. Sci. USA Vol. 96, pp. 1541–1546, February 1999 Immunology GA-binding protein factors, in concert with the coactivator CREB binding protein/p300, control the induction of the interleukin 16 promoter in T lymphocytes NORBERT BANNERT*†,ANDRIS AVOTS†‡,MICHAEL BAIER*§,EDGAR SERFLING‡, AND REINHARD KURTH* *Paul-Ehrlich-Institute, Paul-Ehrlich-Strasse 51-59, D-63225 Langen, Germany; and ‡Department of Molecular Pathology, Institute of Pathology, University of Wu¨rzburg, Josef-Schneider-Strasse 2, D-97080 Wu¨rzburg, Germany Communicated by Maurice R. Hilleman, Merck and Co., Inc, West Point, PA, December 17, 1998 (received for review October 28, 1998) ABSTRACT Interleukin 16 (IL-16) is a chemotactic cy- induces the expression of certain activation markers, such as tokine that binds to the CD4 receptor and affects the activa- IL-2 receptor subunits (13). The proliferative response of tion of T cells and replication of HIV. It is expressed as a large CD41 T cells cotreated with IL-16 and IL-2 for long periods 67-kDa precursor protein (pro-IL-16) in lymphocytes, mac- is therefore selectively enhanced (14), suggesting the possible rophages, and mast cells, as well as in airway epithelial cells use of IL-16 as an immune therapy to counteract conditions of from asthmatics after challenge with allergen. This pro-IL-16 CD41 T cell deficiency such as that associated with AIDS. is subsequently processed to the mature cytokine of 13 kDa. To Moreover, IL-16 inhibits the replication of T cell and mono- study the expression of IL-16 at the transcriptional level, we cyte tropic HIV strains in vitro (15–17). HIV patients receiving cloned the human chromosomal IL-16 gene and analyzed its anti-retroviral therapy display rapidly increasing IL-16 serum promoter. The human IL-16 gene consists of seven exons and levels, possibly reflecting the recovery of the immune system six introns. The 5* sequences up to nucleotide 2120 of the as a consequence of the reduced viral load (18). human and murine IL-16 genes share >84% sequence homol- To learn more about the expression of IL-16, we have ogy and harbor promoter elements for constitutive and in- determined the genomic organization of the human IL-16 ducible transcription in T cells. Although both promoters lack gene, including the structure and function of its promoter. A any TATA box, they contain two CAAT box-like motifs and DNA fragment spanning the nucleotides from 2408 to 188 of three binding sites of GA-binding protein (GABP) transcrip- the IL-16 gene was found to exert constitutive promoter tion factors. Two of these motifs are part of a highly conserved activity in lymphoid and nonlymphoid cells. A strong inducible and inducible dyad symmetry element shown previously to promoter activity in T cells was identified within the immediate control a remote IL-2 enhancer and the CD18 promoter. In 59 upstream DNA of approximately 90 bp. Although this concert with the coactivator CREB binding proteinyp300, promoter fragment lacks any TATA box-like sequence ele- which interacts with GABPa, the binding of GABPa and -b to ment, it contains three binding sites for Ets-like proteins. One the dyad symmetry element controls the induction of IL-16 of these fragments overlaps with the previously reported promoter in T cells. Supplementing the data on the processing transcriptional start sites (4), whereas the other two sites of pro-IL-16, our results indicate the complexity of IL-16 generate a dyad symmetry element (DSE). We show here that expression, which is tightly controlled at the transcriptional these sites are bound by GA-binding protein (GABP) factors and posttranslational levels in T lymphocytes. which, in concert with the coactivators CREB binding protein (CBP)yp300, appear to play an important role in the control y a Interleukin 16 (IL-16), originally identified in supernatants of of the IL-16 promoter. CBP p300 binds to GABP and mitogen-activated peripheral blood mononuclear cells enhances strongly the GABP mediated IL-16 promoter induc- (PBMCs) (1), is synthesized as a large 67-kDa precursor tion. protein (2–4). This pro-IL-16 is almost exclusively expressed in lymphatic tissues (4) and contains three postsynaptic density MATERIALS AND METHODS protein, disc-large, zonulin-1 (PDZ) domains, i.e., modules involved in protein-protein interactions (5, 6). The maturation Cell Culture, DNA Transfections, and Reporter Gene As- of pro-IL-16 appears to be mediated by caspase 3 leading to the says. Human Jurkat T cells were cultured in RPMI 1640 bioactive IL-16, which consists of the C-terminal 121 amino medium, and HeLa cells and 293 embryonic kidney cells were acids of the precursor (4, 7). Activated CD81 and CD41 T cells cultured in DMEM medium. Both media were supplemented synthesize identical levels of IL-16 transcripts, but mature with 10% fetal calf serum. DNA transfections were performed IL-16 is predominantly secreted by CD81 T lymphocytes in by using the SuperFect transfection reagent (Qiagen, Chats- response to antigens, histamine, or serotonin (8, 9). In addi- worth, CA) according to the manufacturers protocol. 293 cells tion, human mast cells also synthesize pro-IL-16, and treat- were transfected by using a conventional calcium phosphate ment with anaphylatoxin, C5a, or 12-O-tetradecanoylphorbol- coprecipitation technique. Luciferase assays were performed 13-acetate (TPA) increases the IL-16 mRNA level as much as by using the Luciferase Assay Reagent (Promega). As trans- 10-fold (10). fection controls, the pSEAP2-control plasmid (CLONTECH) IL-16 binds to the CD4 receptor and is therefore selectively active on CD41 T lymphocytes, monocytes, and eosinophils Abbreviations: Ab, antibody; CBP, CREB binding protein; EMSA, (11, 12). Like other cytokines, IL-16 exerts numerous and electrophoretic mobility-shift assay; PBMC, peripheral blood mono- partially contradictory effects in vitro. Although it is able to nuclear cells; PDZ, postsynaptic density protein, disc-large, zonulin-1; 1 y TPA, 12-O-tetradecanoylphorbol-13-acetate; DSE, dyad symmetry render CD4 T cells unresponsive to TCR CD3 stimuli, it element; ERE, Ets-related element; IL, interleukin. Data deposition: The sequences reported in this paper have been The publication costs of this article were defrayed in part by page charge submitted to the GenBank database [accession nos. AF077011 (human IL-16 gene and promoter) and AF077012 (murine IL-16 promoter)]. payment. This article must therefore be hereby marked ‘‘advertisement’’ in †N.B. and A.A. contributed equally to this work. accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. e-mail: baierm@ PNAS is available online at www.pnas.org. rki.de. 1541 Downloaded by guest on October 1, 2021 1542 Immunology: Bannert et al. Proc. Natl. Acad. Sci. USA 96 (1999) was cotransfected and assays were performed by using the as blocker). After rotating for2hat4°Candextensive washings Great EscAPe SEAP chemiluminescence detection kit with the binding buffer, bound proteins were eluted by boiling (CLONTECH). in Laemmli sample buffer, fractionated on a 12.5% polyacryl- Recombinant DNA Work and Sequence Analysis. Overlap- amide gel, and immunodetected by using GABPa1b-specific ping human IL-16 gene fragments were amplified by using Abs. human PBMC DNA, the Tth-Polymerase Mix (CLONTECH), and the following primer pairs: (59-CTGCTGCTACCACAG- GAAGACACAGCAG-39 and 59-GAGCTGATTCTCTGC- RESULTS CTGATGGA-39;59-GAGCACCTAGGATCACACATC-39 Organization of the Human IL-16 Gene. For the analysis of and 59-CTGACCAGACTGAAGGGAACTGTGGTC-39;59- the chromosomal IL-16 gene, overlapping genomic DNA CAGAGCTGAGAGAATATACAGAGGGTC-39 and 59- fragments from human PBMCs were amplified by using IL-16 9 9 TTCTAGATCTGCTCCTCCTGCCAAGCT-3 ;5-CATGT- cDNA-specific primer pairs and sequenced. The human IL-16 9 9 CACCATCTTACACAAGGAG-3 and 5 -CATCACTGGC- gene spans approximately 12.8 kb and consists of seven exons 9 9 TGCAGAGGCTGAGGCTGCAG-3 ;5 -CACAGAGTGTT- and six introns (Fig. 1). Exon I contains the largest part of the 9 9 TCCAAATGGGCTGGCC-3 and 5 -AGCTGTGTGT- 59-untranslated mRNA region. The first ATG, which corre- 9 9 GTTATTGGCTTTGGCTTC-3 ;5-ACCACAGCTGCTG- sponds to the translational start codon (4), starts on nucleotide GAGACTCCTAG-39 and 59-GTGGTAAGTAGTAGCAAT- 9 position 51 of exon II. This exon is unusually long (1096 bp), TTTATTTGATAC-3 ) The amplification products were encoding 348 aa harboring the proline-rich part and the cloned in the pGEM-T-vector (Promega) and sequenced. To N-terminal region of the first PDZ domain (19). Exon III and minimize sequencing errors, multiple clones were sequenced the first nucleotides of exon IV code for the remainder of the for every fragment. first PDZ domain. PDZ-2 is encoded by the exons IV and V. For the cloning of the chromosomal DNA upstream of the Exons VI and VII contain the sequence for PDZ-3 (found in human IL-16 cDNA-sequence two cDNA based reverse- mature IL-16). The cleavage site for the putative processing complementary primers, hGSP1: 59-TCTTCTCCACCCTG- enzyme caspase 3 is encoded in exon V. GCTCGCCCTCTGT-39 and hGSP2: 59-ACTTGGTCTGT- Characterization of the IL-16 Promoter. Fig. 2 shows a GGGCCGATACCAGGT-39, were used in a nested PCR with sequence comparison between the 59-flanking regions of hu- parts of the human PromoterFinder DNA Walking kit man and murine IL-16 genes spanning the nucleotides from (CLONTECH) according to the manufacturer’s protocol. position 2440 to 1130. The 59-untranslated mRNA regions Fragments of 1.7 kb (DraI library) and 1.9 kb (PvuII library) 2 were amplified and cloned in pGEM-T. Sequencing revealed and the immediate upstream regions (up to 120) from both the expected 62 bp of the 59 end of the IL-16 cDNA sequence genes share a high sequence homology, suggesting their func- tional importance.
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