Promoter activity of the proliferating-cell nuclear antigen gene is associated with inducible CRE-binding proteins in interleukin 2-stimulated T lymphocytes.
The proliferating-cell nuclear antigen (PCNA) gene encodes an auxiliary factor of DNA polymerase delta and functions in DNA replication during S phase. It is expressed at much higher levels in proliferating cells than in quiescent cells. We have studied the regulatory role of the 5'-flanking sequence of the murine PCNA gene in interleukin 2 (IL-2)-responsive cloned T cells (L2). Analysis of a set of deletion constructs in transient transfection assays measuring heterologous reporter gene (luciferase) activity demonstrated that the 182-bp 5'-flanking region provides full promoter activity in IL-2-stimulated L2 cells. While many elements contribute to PCNA promoter strength in IL-2-stimulated cells, the largest decrease in activity occurred with deletion of the tandem CRE (cyclic AMP response element) binding sites located at nucleotides -37 to -52. With a gel mobility shift assay, several IL-2-inducible DNA-protein complexes were detected, including CREB (CRE-binding) and ATF1 (activating transcription factor) proteins that are specific for the PCNA-CRE sequence. Methylation interference analysis confirmed specific binding of these proteins to the CRE sites. Mutation at the PCNA-CRE motif abolishes IL-2-inducible binding and reduces substantially PCNA promoter activity. These results indicate that IL-2-stimulated PCNA transcription may be partially mediated by these CRE-binding proteins.
Huang
D
D
Department of Pathology, School of Medicine, University of Pennsylvania, Philadelphia 19104.
Shipman-Appasamy
P M
PM
Orten
D J
DJ
Hinrichs
S H
SH
Prystowsky
M B
MB
D000818:Animals; D000951:Antigens, Neoplasm / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D002999:Clone Cells; D017362:Cyclic AMP Response Element-Binding Protein / Q000096:biosynthesis / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D017931:DNA Primers; D004261:DNA Replication; D007376:Interleukin-2 / Q000494:pharmacology*; D008156:Luciferases / Q000096:biosynthesis / Q000378:metabolism; D008745:Methylation; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D018809:Proliferating Cell Nuclear Antigen; D011401:Promoter Regions, Genetic; D016196:S Phase; D013601:T-Lymphocytes / Q000187:drug effects / Q000378:metabolism*; D006377:T-Lymphocytes, Helper-Inducer / Q000187:drug effects / Q000378:metabolism; D014162:Transfection
D000951:Antigens, Neoplasm; D017362:Cyclic AMP Response Element-Binding Protein; D017931:DNA Primers; D007376:Interleukin-2; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; D004247:DNA; D008156:Luciferases