ROLE OF THE CYCLIC ADENOSINE-3', 5'-MONOPHOSPHATE RESPONSE ELEMENT IN EFFICIENT EXPRESSION OF THE RAT THYROTROPIN RECEPTOR PROMOTER

The "minimal" promoter region of the TSH receptor gene, -195 to -39 basepairs (bp), exhibits basal promoter activity, thyroid specificity, and negative regulation by TSH via its cAMP signal. In FRT thyroid cells and by comparison to pTRCAT5'-199, 5'-deletion mutants of chloramphenicol acetyltransferase (CAT) constructs from -199 to -150 bp of the minimal promoter decrease basal CAT activity by 50%, whereas continued deletion to -146 bp increases activity more than 4-fold. Continued deletion to -131 bp results in basal activity less than that of the -199 bp construct. An octameric cAMP response element (CRE)-like sequence, TGAGGTCA, is within -146 to -131 bp and starts at -139 bp. Its mutation to a consensus CRE (TGACGTCA) or AP1 (TGAGTCA) site or mutation of several residues flanking its 3'-terminus can improve promoter activity as much as 8-fold compared to pTRCAT5'-199. A nonpalindromic mutation to CGAGGACA decreases basal promoter activity to the level of the 199-bp minimal promoter. The CRE-like sequence between -139 and -132 bp is a constitutive enhancer of promoter activity in FRT thyroid cells, since, ligated to a simian virus-40-promoter-driven CAT gene, it increases CAT activity in the absence of forskolin in proportion to copy number and independent of direction or position. It can, however, function as a cAMP-responsive CRE, as evidenced by the fact that forskolin increases the activity of the same simian virus-40-promoter-driven CAT gene constructs in Buffalo rat liver (BRL) cells. DNAase-I footprinting shows that the CRE region is protected by a purified binding region peptide of the CRE-binding protein, activating transcription factor-2, and recombinant AP1 (human c-jun) as well as by BRL, FRT, and FRTL-5 rat thyroid cell nuclear extracts. Gel mobility shift analyses show that multiple CRE-binding proteins in the BRL, FRT, and FRTL-5 cell nuclear extracts form complexes with the CRE-like site, that one of these is CRE-binding protein, and that all form complexes with mutant sequences of the CRE-like site in a manner that exactly parallels their effects on constitutive enhancer function in FRT thyroid cells. We show, therefore, that the CRE-like site in the minimal TSH receptor promoter functions as a constitutive enhancer of promoter activity in FRT thyroid cells yet is a cAMP-responsive CRE. We suggest that failure of the CRE-like site to act as a cAMP-inducible enhancer in FRT thyroid cells may relate to the ability of forskolin to cause negative regulation of the TSH receptor promoter in thyroid cells. Consistent with this, we show that sequences 3' to the CRE are important for cAMP-induced negative regulation of the TSH receptor minimal promoter. Thus, negative regulation by forskolin is evident in pTRCAT5'-131, which lacks the CRE site, and can be demonstrated with pTRCAT5'-146NP, the nonpalindromic mutant whose sequence loses enhancer activity and exhibits no specific interactions with CRE-binding nuclear proteins in FRTL-5, FRT, or BRL extracts. Activity is evident in FRT thyroid, but not BRL, cells and is expressed with a homologous, but not a heterologous, promoter, i.e. is thyroid and promoter specific in its expression. In addition to the sequences 3' to the CRE, which are important for cAMP-induced negative regulation of the TSH receptor minimal promoter, we show that a decanucleotide tandem repeat sequence immediately 5' to the CRE is a repressive element of constitutive CRE enhancer activity. Its repressive activity also appears to be thyroid specific, since it is expressed in FRT thyroid, but not BRL, cells.