Characterization of the human transcobalamin II promoter. A proximal GC/GT box is a dominant negative element.
Deletion and mutagenesis of the 5'-flanking region of the human transcobalamin II (TC II) transfected in human 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 transcription initiation sites; (c) not dependent on other potential elements, 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 GC box (-568-GAGGCGGTGC) and strongly by a proximal GC/GT overlapping box (-179 CCCCCGCCCCACCCC). Gel shift and immunosupershift analyses demonstrated that both the positive-acting GC box and the negative-acting GC/GT box were recognized by Sp1 and Sp3. Co-transfection studies using Sp1 and/or Sp3 expression plasmids revealed that while Sp1 stimulated, Sp3 repressed Sp1-mediated transactivation of TC II transcription. The proximal GC/GT box also acted as a negative element in human chronic myelogenous leukemia K-562 and HeLa cells. These results suggest that tissue/cell specific expression of the TC II gene may be controlled 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 repression caused by the binding of Sp3 to the proximal GC/GT box.
Li
N
N
Division of Gastroenterology and Hepatology, Medical College of Wisconsin and Veterans Medical Center, Milwaukee, Wisconsin 53226, USA.
Seetharam
S
S
Seetharam
B
B
D001483:Base Sequence; D018938:Caco-2 Cells; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D017384:Sequence Deletion; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism; D051705:Sp3 Transcription Factor; D014155:Transcobalamins / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D004268:DNA-Binding Proteins; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; D014155:Transcobalamins; D014157:Transcription Factors; D051705:Sp3 Transcription Factor