Multiple prolactin-responsive elements mediate G1 and S phase expression of the interferon regulatory factor-1 gene.
The interferon regulatory factor-1 (IRF-1) gene is both an immediate-early G1 phase gene and an S phase gene inducible by PRL in rat Nb2 T lymphocytes. To understand the mechanism by which PRL regulates the biphasic expression of IRF-1, we cloned the rat IRF-1 gene and functionally characterized the IRF-1 promoter. Upon transfection into Nb2 T cells, 1.7 kilobases (kb) of IRF-1 5'-flanking DNA linked to a chloramphenicol acetyl transferase (CAT) reporter gene mediated a 30-fold induction of CAT enzyme activity in response to 24 h of PRL stimulation. Deletion mutants containing 1.3, 0.6, and 0.2 kb 5'-flanking DNA were incrementally less transcriptionally active, although 0.2 kb still mediated a 12-fold induction by PRL. The sequence between -1.7 and -0.2 kb linked 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 course, we were able to assign G1 vs. S phase PRL responses of the IRF-1 gene 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 was 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 elements between 0.2 and 1.7 kb act in concert with the proximal 0.2-kb region during S phase progression.
Department of Microbiology and Immunology, Baylor College of Medicine, Houston, Texas 77030.
A M
AM
Stevens
L Y
LY
Yu-Lee
D000818:Animals; D001483:Base Sequence; D002453:Cell Cycle; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis / Q000378:metabolism; D004247:DNA / Q000032:analysis / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D016193:G1 Phase; D005786:Gene Expression Regulation; D017781:Genes, Immediate-Early; D017930:Genes, Reporter; D015752:Genes, env / Q000502:physiology*; D016762:Genes, fos / Q000235:genetics; D016259:Genes, myc / Q000235:genetics; D006801:Humans; D050856:Interferon Regulatory Factor-1; D051379:Mice; D008969:Molecular Sequence Data; D009955:Ornithine Decarboxylase / Q000235:genetics; D010750:Phosphoproteins / Q000235:genetics*; D011388:Prolactin / Q000235:genetics* / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000235:genetics; D051381:Rats; D016196:S Phase; D013601:T-Lymphocytes / Q000166:cytology; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection
D004268:DNA-Binding Proteins; C494257:IRF1 protein, human; D050856:Interferon Regulatory Factor-1; C494258:Irf1 protein, mouse; C494259:Irf1 protein, rat; D010750:Phosphoproteins; D014157:Transcription Factors; D011388:Prolactin; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D009955:Ornithine Decarboxylase