Structure and characterization of rat cyclin D3 promoter

To investigate the transcription initiation mechanism of the gene encoding cyclin D3, one of the major G1 cyclins to promote the G1/S transition, we cloned the 5' portion of rat cyclin D3 gene and analyzed the promoter region. The major transcription start point of the gene was identified by the primer extension experiment to be 207 bp upstream from the ATG start codon and its promoter region was found to have no canonical TATA box. The DNA mobility shift assay and DNase I footprinting analysis using nuclear extracts from Nb2 cells stimulated by prolactin (PRL) have clearly revealed that the promoter possesses two binding sites for the PRL-induced transcription factors. One is located between -1 and -59 bp (Region I) and the other between -328 and -380 bp (Region II). The induction of transcription factors by PRL was blocked by simultaneous addition of cycloheximide, suggesting that protein synthesis was necessary for the release of these PRL-responsive transcription factors. Region I contained putative binding elements for ATF/CREB, SP1 and AP2, and Region II contained elements for a MCBF/MGF and a novel factor. These results suggest that the cyclin D3 gene is a TATA-less gene whose transcription is regulated by multiple mitogen-inducible transcription factors including an unknown factor.