Differentiation-responsive elements in the 5' region of the mouse tissue plasminogen activator gene confer two-stage regulation by retinoic acid and cyclic AMP in teratocarcinoma cells.

F9 cells induced to differentiate with retinoic acid (RA) increase transcription of the tissue plasminogen activator (t-PA) gene. Further treatment of these cells with cyclic AMP (cAMP) results in an additional stimulation of t-PA gene transcription. To investigate the mechanism of this two-stage regulation, 4 kilobase pairs (kbp) of 5'-flanking sequence from the murine t-PA gene was isolated. Two major start sites for transcription were found, neither of which depended on a classical TATA motif for correct initiation. By using transient transfection assays, it was determined that 4-kbp of flanking sequence could confer on reporter genes the same two-stage differentiation-specific expression as was observed for the endogenous t-PA gene. Deletion analyses of this 4-kbp fragment showed that 190 bp of flanking sequence was sufficient to bestow the same degree of two-stage regulation on reporter gene constructs. Within this region of DNA, sequence analysis revealed a possible cAMP regulatory element, a CTF/NF-1 recognition sequence, two potential Sp1 sites, and five potential binding sites for transcription factor AP-2. The deletion experiments, coupled with the positions of these potential cis-acting elements, suggest that multiple transcription factors, including those that bind to cAMP regulatory element, CTF/NF-1, Sp1, and AP-2 sites, may be involved in regulation of the t-PA gene during F9 cell differentiation.