Abstract
Proliferating cell nuclear antigen (PCNA) synthesis is strictly regulated during the cell cycle. To investigate PCNA transcriptional regulation, we have analyzed protein-DNA interactions at the promoter region and in the first intron in quiescent fibroblasts and following serum stimulation. Twenty putative protein-binding sites, distributed in two divergent promoters at the PCNA locus, were identified in vivo by genomic footprinting. These elements bind transcription factors continuously throughout the cell cycle with the exception of one E2F consensus site, located in the first intron at position +583. This E2F site becomes strongly occupied 18 h after serum stimulation, implying that an E2F activator complex plays a role in activation of the PCNA gene at the onset of S phase. We detected a 500-600-base pair-long antisense transcript by Northern blot analysis. This RNA has no apparent coding capacity and is constitutively transcribed from a promoter located within the first intron. We suggest that silencing of the PCNA gene is accomplished through base pairing between sense pre-mRNA and antisense RNA. The binding of S phase-specific E2F complexes at the +583 element may help to overcome the negative effect of the antisense transcript, which results in up-regulation of PCNA expression in proliferating cells.
Highlights
Proliferating cell nuclear antigen (PCNA) synthesis is strictly regulated during the cell cycle
The induction of the human PCNA gene after serum stimulation was analyzed in synchronized fibroblasts by Northern blot analysis
Low amounts of PCNA mRNA were detected in serum-starved fibroblasts after a starvation time of either 2 or 14 days (Fig. 1A)
Summary
Proliferating cell nuclear antigen (PCNA) synthesis is strictly regulated during the cell cycle. Twenty putative protein-binding sites, distributed in two divergent promoters at the PCNA locus, were identified in vivo by genomic footprinting These elements bind transcription factors continuously throughout the cell cycle with the exception of one E2F consensus site, located in the first intron at position ؉583. This E2F site becomes strongly occupied 18 h after serum stimulation, implying that an E2F activator complex plays a role in activation of the PCNA gene at the onset of S phase. In vivo genomic footprinting has indicated that a key element for cell cycle regulation of the PCNA gene is located in the first intron We further characterized this regulatory element and suggest a dominant role of an E2F complex as an S phase-specific positive regulator of PCNA expression. The potential role of a newly discovered 500 – 600-bp-long antisense PCNA transcript originating from the first intron is discussed
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