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 (CREbinding) and ATFI (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.The activation of antigen-specific T lymphocytes from Go to S phase usually requires several signals. Initially, antigen interacts with the T-cell antigen receptor, which leads to the transition from Go to GI and the expression of interleukin 2 (IL-2) receptor and lymphokines including IL-2. Further GI progression requires additional signals provided by the interaction of IL-2 with its high-affinity receptor, which results in the expression of genes related to G, activation, and binding of prolactin to its receptor, which results in entry into S phase and DNA replication (11,14,35,38,67).One of the genes expressed during IL-2-driven G, progression is proliferating-cell nuclear antigen (PCNA) (49, 69). PCNA was originally described as a proliferation-associated nuclear antigen that was thought to be expressed in a cellcycle-specific manner (8,47). While PCNA is an auxiliary protein for DNA polymerase delta (leading-strand polymerase) (9, 59) and PCNA-associated immunofluorescence closely parallels [3H]thymidine incorporation during cell cycle progression (42), synthesis and expression of PCNA can be detected in all phases of the cell cycle (G11SG2/M) in proliferating cells, suggesting that the protein is easily extractable when it is not associated with the polymerase (10, 49). Beach and coworkers showed recently that D-type cyclins can directly interact with PCNA and suggested this interaction as one possible mechanism by which the cell cycle machinery could * Corresponding author. Present address:

Proliferating cell nuclear antigen (PCNA) RNA levels are regulated by transcription as well as changes in stability, in growing cells. We have cloned the murine PCNA cDNA and a fragment of the murine PCNA gene flanking the transcription initiation site. Comparison of the murine deduced amino acid sequence with the PCNA sequence from rat, human, Drosophila, Saccharomyces cerevisiae, and higher plants, reveals extensive homology between species. The homology is likely to be related to the fundamental role of PCNA as an auxiliary protein for DNA replication. Consensus sequences for transcriptional regulatory factors identified within 520 bp 5' of the cap site of the murine PCNA gene include: an inverted CCAAT site, an enhancer core element (EBP-1), three cAMP-response elements (CRE-BP), one AP-2 site, three Sp1 sites, and two octamer sequences. The first 20 bp of the transcriptional unit are homologous to an initiator element, which may direct transcription from RNA polymerase II in the absence of a TATAA box. The consensus elements in the murine PCNA gene are similar in sequence and/or location to elements identified in the genes for human, Drosophilia, and yeast PCNA.

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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.