CHARACTERIZATION OF THE HUMAN TRANSCOBALAMIN-II PROMOTER

Deletion and mutagenesis of the 5'-flanking region of the human transc obalamin II (TC II) transfected in hu man intestinal epithelial Caco-2 cells have revealed that TC II promoter activity is: (a) very weak; ( b) restricted to a core region (-29 to -163) that contained multiple t ranscription initiation sites; (c) not dependent on other potential el ements, such as a distally localized CCAAT box, a CF1, a HIP1 binding motif and a MED-1 element; (d) modulated weakly by a positive-acting G C box (-568-GAGGCGGTGC) and strongly by a proximal GC/GT overlapping b ox (-179 CCCCCGCCCCACCCC). Gel shift and immunosupershift analyses dem onstrated that both the positive-acting GC box and the negative-acting GC/GT box were recognized by Sp1 and Sp3. Co-transfection studies usi ng Sp1 and/or Sp3 expression plasmids revealed that while Sp1 stimulat ed, Sp3 repressed Sp1-mediated transactivation of TC II transcription. The proximal GC/GT box also acted as a negative element in human chro nic myelogenous leukemia K-562 and HeLa cells. These results suggest t hat tissue/cell specific expression of the TC II gene may be controlle d by the relative ratios of Sp1 and Sp3 that bind to the GC/GT box and the weak promoter activity of TC II is due to the transcriptional rep ression caused by the binding of Sp3 to the proximal GC/GT box.