ETS1 AND ETS2 IN P53 REGULATION - SPATIAL SEPARATION OF ETS BINDING-SITES (EBS) MODULATE PROTEIN - DNA INTERACTION

p53 is an extensively studied tumor suppressor gene implicated in the genesis of a large number of varied tumours. However, the pathways of regulation for the wild-type p53 gene and its product are as yet unkno wn. In situ hybridization analyses of ETS1 and ETS2 expression during mouse embryogenesis, have shown a pattern similar to that of p53 gene expression. Significantly, we have identified several ETS-binding site s (EBS) in the promoter regions of the human and mouse p53 genes. In t he human promoter two of these EBS are present in the form of a palind rome, with the two EBS cores being separated by four nucleotides. This report shows that the EBS palindrome of the human p53 promoter has a high affinity for ETS1 and ETS2, and that such binding interaction int racellularly is able to activate the transcription of a CAT reporter g ene by 5-10-fold using COS cells. To investigate whether the spacing b etween the two EBS cores influences the DNA binding activity, we synth esized oligonucleotides with increasing distances (4,12,16 and 20 base s respectively) between the two EBS cores of the palindrome. We observ ed an inverse correlation between an increasing distance in the two EB S cores of the palindrome and the ETS1 and ETS2 DNA binding activity r espectively. Interestingly, optimal DNA binding activity was observed when the distance between the two EBS cores was four bases, identical to that which occurs in the natural promoter. Furthermore we show that the p53 mRNA is expressed at higher levels in NIH3T3 cells over-expre ssing ETS2 gene product, suggesting that the ETS2 transcription factor is a likely candidate for regulating the expression of p53 in vivo.