THE HUMAN ARF CELL-CYCLE REGULATORY GENE PROMOTER IS A CPG ISLAND WHICH CAN BE SILENCED BY DNA METHYLATION AND DOWN-REGULATED BY WILD-TYPE P53

The INK4a/ARF locus encodes two proteins involved in tumor suppression in a manner virtually unique in mammalian cells. Distinct first exons , driven from separate promoters, splice onto a common exon 2 and 3 bu t utilize different reading frames to produce two completely distinct proteins, both of which play roles in cell cycle control. INK4a, a cri tical element of the retinoblastoma gene pathway, binds to and inhibit s the activities of CDK4 and CDK6, while ARF, a critical element of th e p53 pathway, increases the level of functional p53 via interaction w ith MDM2, Here we clone and characterize the promoter of the human ARF gene and show that it is a CpG island characteristic of a housekeepin g gene which contains numerous Spl sites. Both ARF and INK4a are coord inately expressed in tells except when their promoter regions become d e novo methylated. In one of these situations, ARF transcription could be reactivated by treatment,vith the DNA methylation inhibitor 5-aza- 2'-deoxycytidine, and the reactivation kinetics of ARF and INK4a were found to differ slightly in a cell line in which both genes were silen ced by methylation, The ARF promoter was also found to be highly respo nsive to E2F1 expression, in keeping with previous results at the RNA level. Lastly, transcription from the ARF promoter was down-regulated by wild-type p53 expression, and the magnitude of the effect correlate d with the status of the endogenous p53 gene, This finding points to t he existence of an autoregulatory feedback loop between p53, MDM2, and ARF, aimed at keeping p53 levels in check.