TRANSCRIPTIONAL ACTIVATION OF THE P34(CDC2) GENE BY CDC2 PROMOTER-BINDING FACTOR NUCLEAR FACTOR-Y IN FETAL-RAT VENTRICULAR MYOCYTES

To determine how myocardial terminal differentiation is regulated by c ell cycle control genes, we studied cdc2 expression in rat cardiac mus cle and found that cdc2 mRNA and protein levels were reduced in neonat al compared with fetal ventricles and became undetectable in juvenile and adult ventricles. To further determine whether cdc2 downregulation is attributed to a decrease in transcription, transient expression as say was performed using the progressively truncated 6.2-, 1.8-, 1.1-, 0.7-, and 0.1-kb human cdc2 5' nanking regions. All five fragments act ivated reporter expression in fetal myocytes and were significantly le ss active in neonatal myocytes. The 0.1-kb fragment showed 65% of the activity of the 6.2-kb fragment. A protein binding site that contains an inverted CCAAT box was identified within the 0.1-kb fragment by DNa se I footprint assay and named the cdc2 promoter binding factor (CPBF) site. Point mutations within the CPBF site that abolish CPBF binding significantly decreased both 0.1- and 6.2-kb promoter activities. Comp etition and antibody supershift assays suggested that CPBF was identic al or related to the transcription factor, nuclear factor Y (NF-Y). Th e 0.1-kb promoter activity was suppressed by a dominant-negative NF-Y mutant in fetal myocytes. Taken together, our results demonstrate that cardiac cdc2 expression is downregulated after birth and turned off w hen the juvenile stage is attained. A 0.1-kb promoter fragment of cdc2 contains major information for both cdc2 transcriptional activation a nd suppression in fetal and neonatal myocytes, respectively. NF-Y or i ts related factor plays a critical role in activating the 0.1-kb cdc2 promoter.