2 DIFFERENT NEGATIVE REGULATORY ELEMENTS CONTROL THE TRANSCRIPTION OFT-CELL ACTIVATION GENE-3 IN ACTIVATED MAST-CELLS

T-cell activation gene 3 (TCA3) encodes a beta-chemokine that is trans criptionally regulated in mast cells; the gene has a functional NF-kap pa B 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), t his 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 acetyl transferase (CAT)-protein synthesis by a TCA3-CAT construct transfecte d into mast cells that were then activated. Only NRE-1 inhibited CAT-p rotein syn thesis 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 independen t of cell activation. Electrophoretic mobility shift assays showed DNA -protein complex formation with subsequences (CCCCCATTCT) of NRE-1 (NR E-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 alpha(IIb) integ rin gene. Site-directed mutagenesis demonstrated that both NRE-1a and NRE-2b are important in the negative regulation of TCA3 promoter activ ity. In vivo ligation-mediated PCR foot printing of the NRE-2 region r evealed protection between -1372 and -1354, which contains NRE-2b. The data thus demonstrate identity of a silencer motif, here termed NRE-2 b, in both the alpha(IIb), integrin gene and the TCA3, and that this s ilencer region in mast cells is functional both in vivo and in vitro. Further, evidence is presented that the promoter for TCA3 contains a n ovel silencer motif, termed NRE-1a, characterized by a CT-rich sequenc e.