REPRESSION OF ADENOVIRUS-E1A ENHANCER ACTIVITY BY A NOVEL ZINC FINGER-CONTAINING DNA-BINDING PROTEIN RELATED TO THE GLI-KRUPPEL PROTEIN

We have previously shown that expression of the E1A oncogene is negati vely regulated in rodent fibroblast cells by a nuclear factor (phiAP3) that binds to the E1A promoter region just upstream of the canonical enhancer element. To understand how phiAP3 can regulate E1A gene trans cription by inactivation of the enhancer function, we have used an oli gonucleotide probe containing a binding site for this protein to clone the mouse phiAP3 gene. DNA sequence analysis of the 2.3 kb cDNA revea led the presence of six well-conserved zinc ringer DNA-binding motifs, which were highly related to those found in the GLI-Kruppel family of human zinc ringer proteins. Analysis of the tissue distribution of th e phiAP3 mRNA suggested that its expression was ubiquitous but at vari able levels, most likely as a result of post-transcriptional regulatio n of mRNA stability. The phiAP3 factor is a nuclear phosphoprotein; th e extent of its phosphorylation is regulated during the cell cycle. Pr eferential binding of the hyperphosphorylated form of this protein to DNA was observed. Co-expression of the phiAP3 cDNA and a luciferase re porter gene under the control of the E1A promoter/enhancer in several human cell lines resulted in repression of E1A enhancer activity. In c ontrast, when the phiAP3 binding site upstream of the enhancer was mut ated, no inhibition of enhancer function was observed. Based on these observations we conclude that we have cloned the cellular phiAP3 gene, and that the DNA-binding activity of this protein is regulated during the cell cycle.