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J Biol Chem, Vol. 273, Issue 26, 16104-16111, June 26, 1998

Characterization of the Human Transcobalamin II Promoter
A PROXIMAL GC/GT BOX IS A DOMINANT NEGATIVE ELEMENT

Ning Li, Shakuntla Seetharam, and Bellur Seetharam^§

From the Division of Gastroenterology and Hepatology, Departments of  Medicine and ^§ Biochemistry, Medical College of Wisconsin and Veterans Medical Center, Milwaukee, Wisconsin 53226

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.

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Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.

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