MULTIPLE PROLACTIN-RESPONSIVE ELEMENTS MEDIATE G1 AND S-PHASE EXPRESSION OF THE INTERFERON REGULATORY FACTOR-I GENE

The interferon regulatory factor-1 (IRF-1) gene is both an immediate-e arly G1 phase gene and an S phase gene inducible by PRL in rat Nb2 T l ymphocytes. To understand the mechanism by which PRL regulates the bip hasic expression of IRF-1, we cloned the rat IRF-1 gene and functional ly characterized the IRF-1 promoter. Upon transfection into Nb2 T cell s, 1.7 kilobases (kb) of IRF-1 5'-flanking DNA linked to a chloramphen icol acetyl transferase (CAT) reporter gene mediated a 30-fold inducti on of CAT enzyme activity in response to 24 h of PRL stimulation. Dele tion mutants containing 1.3, 0.6, and 0.2 kb 5'-flanking DNA were incr ementally less transcriptionally active, although 0.2 kb still mediate d a 12-fold induction by PRL. The sequence between -1.7 and -0.2 kb li nked to a heterologous thymidine kinase promoter failed to respond to PRL stimulation, suggesting that the activity of upstream PRL response elements may require an interaction with promoter-proximal elements. By assaying CAT enzyme activity across a 24-h PRL induction time cours e, we were able to assign G1 vs. S phase PRL responses of the IRF-1 ge ne to different regions of the IRF-1 5'-flanking and promoter DNA. The 0.2-kb IRF-CAT construct was induced by PRL stimulation during the G1 phase of the cell cycle. In contrast, the 1.7-kb IRF-CAT construct wa s inducible by PRL during both G1 and S phase of the cell cycle. Hence , the PRL-induced biphasic expression of the IRF-1 gene appears to be controlled by separate PRL-responsive elements: elements in the first 0.2 kb of the IRF-1 promoter region act during early activation, and e lements between 0.2 and 1.7 kb act in concert with the proximal 0.2 kb region during S phase progression.