THE H19 TATA-LESS PROMOTER IS EFFICIENTLY REPRESSED BY WILD-TYPE TUMOR-SUPPRESSOR GENE-PRODUCT P53

The developmentally regulated H19 gene displays several remarkable pro perties: expression of an apparently non-translated mRNA, genomic impr inting (maternal allele only expressed), relaxation of the imprinting and/or epigenetic lesions demonstrated in some tumors. Despite several observations after relaxation of imprinting status of the gene, data on tr ans and cis-acting factors required for the human H19 gene expre ssion are still missing. As a first approach to address identification of factors involved in the regulation of the gene, we found that cell s from a p53 antisense-transfected HeLa clone displayed increased amou nts of H19 transcripts when compared to the non-transfected cells. Mor eover, a HeLa clone stably transfected with a temperature sensitive (t s) 143 Ala p53 mutant exhibited temperature-dependent regulation of H1 9 expression. This preliminary indication of the repressing effect of the p53 protein on H19 expression has been confirmed by transient cotr ansfection experiments in HeLa cells, using luciferase surrogate const ructs under the control of the 823 bp sequence immediately upstream of the transcription start point of the H19 gene, and different construc ts containing sense, antisense or a ts 143 Ala mutant p53 cDNA. We obs erved an increase of H19 promoter-driven activity in transient cotrans fections with the antisense p53 cDNA and the temperature sensitive mut ant p53 at the non-permissive temperature, but a decrease with sense w ild-type p53 cDNA. Furthermore, the cotransfection experiments were re peated in a cell line lacking endogenous p53, (Calu 6 cells) and the r esults provided additional evidence for a down regulation of the expre ssion of the H19 gene by the p53 protein.