T-cell activation gene 3 (TCA3) encodes a β-chemokine that is transcriptionally regulated in mast cells; the gene has a functional NF-κB element at positions -194 to -185. The 5´-flanking region of this gene is also known to have a negative regulatory region between -2057 and -1342. To characterize the negative regulatory elements (NREs), this region was sequenced and then digested by HindIII enzyme into two fragments, NRE-1 (-2057 to -1493) and NRE-2 (-1492 to -1342). Both NRE-1 and NRE-2 in the 5´–3´ orientation inhibited chloramphenicol acetyltransferase (CAT)-protein synthesis by a TCA3–CAT construct transfected into mast cells that were then activated. Only NRE-1 inhibited CAT-protein synthesis in the 3´–5´ orientation. Further deletion of the 5´ region of NRE-1 partially abolished the inhibitory activity. Both NRE-1 and NRE-2 inhibited the activity of a CD20–CAT construct independent of cell activation. Electrophoretic mobility shift assays showed DNA–protein complex formation with subsequences (CCCCCATTCT) of NRE-1 (NRE-1a) and (CCATGA) of NRE-2 (NRE-2b). NRE-1a appears to be novel. NRE-2b is identical with a putative silencer motif in the αIIb integrin gene. Site-directed mutagenesis demonstrated that both NRE-1a and NRE-2b are important in the negative regulation of TCA3 promoter activity. In vivo ligation-mediated PCR footprinting of the NRE-2 region revealed protection between -1372 and -1354, which contains NRE-2b. The data thus demonstrate identity of a silencer motif, here termed NRE-2b, in both the αIIb integrin gene and the TCA3, and that this silencer region in mast cells is functional both in vivoand in vitro. Further, evidence is presented that the promoter for TCA3 contains a novel silencer motif, termed NRE-1a, characterized by a CT-rich sequence.
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April 1997
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Research Article|
April 15 1997
Two different negative regulatory elements control the transcription of T-cell activation gene 3 in activated mast cells Available to Purchase
Chad K. OH;
Chad K. OH
*Department of Pediatrics, Harbor-UCLA Medical Center, Torrance, CA 90509, U.S.A.
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Markus NEURATH;
Markus NEURATH
†The Mucosal Immunity Section, Laboratory of Clinical Investigation, NIAID, NIH, Bethesda, MD 20892, U.S.A.
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Jeong-Je CHO;
Jeong-Je CHO
*Department of Pediatrics, Harbor-UCLA Medical Center, Torrance, CA 90509, U.S.A.
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Tekli SEMERE;
Tekli SEMERE
‡Laboratory of Allergic Diseases, NIAID, NIH, Bethesda, MD 20892, U.S.A.
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Dean D. METCALFE
Dean D. METCALFE
§
‡Laboratory of Allergic Diseases, NIAID, NIH, Bethesda, MD 20892, U.S.A.
§To whom correspondence should be addressed.
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Publisher: Portland Press Ltd
Received:
August 22 1996
Revision Received:
November 28 1996
Accepted:
December 12 1996
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1997
1997
Biochem J (1997) 323 (2): 511–519.
Article history
Received:
August 22 1996
Revision Received:
November 28 1996
Accepted:
December 12 1996
Citation
Chad K. OH, Markus NEURATH, Jeong-Je CHO, Tekli SEMERE, Dean D. METCALFE; Two different negative regulatory elements control the transcription of T-cell activation gene 3 in activated mast cells. Biochem J 15 April 1997; 323 (2): 511–519. doi: https://doi.org/10.1042/bj3230511
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