MOLECULAR-CLONING AND CHARACTERIZATION OF THE MOUSE RB1 PROMOTER

We report the isolation and characterization of the mouse RB1 promoter and surrounding DNA sequences, and the identification of elements req uired for basal transcriptional activity. The mouse RB1 promoter, like the human homologue, has a high G + C content, constitutes a CpG isla nd and is devoid of typical TATA and CAAT boxes. The first 235 base-pa irs upstream of the translation initiation codon in the mouse promoter exhibit 80% sequence homology with the human sequence. This homology includes a region which contains putative binding sites for the transc ription factors Sp1, ATF and E2F/DRTF1. Four major transcription initi ation sites were identified downstream of this conserved region. Mutat ional analysis revealed that the Sp1 and ATF binding sites, but not th e putative E2F/DRTF1 binding site, are critical for promoter activity. Complete disruption of the putative Sp1 and ATF sites abrogated trans cription, whereas the introduction of point mutations, previously iden tified in the Sp1 and ATF sites in two low penetrance retinoblastoma f amilies, reduced promoter activity in a cell type specific manner. Les s reduction in activity occured in retinoic acid induced differentiate d P19 cells and NIH3T3 mouse fibroblasts than in undifferentiated embr yonal carcinoma P19 cells. Activity of the RB1 promoter was found to b e stimulated in retinoic-acid induced differentiated P19 cells compare d to undifferentiated P19; this stimulation required intact Sp1 and AT F sites but not the putative E2F/DRTF1 binding site. Our results indic ate that basal level of RB1 expression is governed by Sp1 and ATF.