Effects of chromosomal integration site upon p53 interactions with DNA consensus sequence homologies

In the present study, we report that, despite the presence of one perfect p 53 consensus sequence homology (designated SCL CS) and four half-sites with in the 3'-untranslated region of the stem cell leukemia (SCL) gene, the nat ive endogenous gene is not regulated by p53, We employ a tet-repressible sy stem to show that, under conditions in which the WAF1 mRNA steady-state lev el is upregulated fourfold by p53, the SCL mRNA level is not altered, In a previous report, ne demonstrated that p53 interactions with the SCL CS can upregulate downstream reporter gene activity 43-fold in transient reporter assays, This disparity prompted us to explore the differences between p53 r egulation of SCL CS activity in organized (chromosomally integrated) and di sorganized (non-replicating episomal plasmid) chromatin. We show that p53 c an increase (between 3-80-fold), decrease (between 5-33-fold) or have no ef fect upon transactivation of an SCL CS/reporter fusion gene depending upon chromosomal integration site. Most studies used to characterize p53 binding sites employ transient transfection assays. Our results suggest that chara cterization of consensus sequence homologies by assay of transiently transf ected cells mag be inaccurate.