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J Biol Chem, Vol. 273, Issue 43, 28170-28177, October 23, 1998

A 69-Base Pair Fragment Derived from Human Transcobalamin II Promoter Is Sufficient for High Bidirectional Activity in the Absence of a TATA Box and an Initiator Element in Transfected Cells
ROLE OF AN E BOX IN TRANSCRIPTIONAL ACTIVITY

Ning Li and Bellur Seetharam^§

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

A 69-base pair (bp) (581/513) fragment derived from human transcobalamin II distal promoter constructed upstream of a chloramphenicol acetyltransferase reporter gene demonstrated high bidirectional promoter activity in transfected epithelial Caco-2 cells. DNase I footprinting, gel mobility shift, supershift, and mutagenesis studies with the 69-bp fragment demonstrated that a GC box (568/559) and an E box (523/528), which interacted with Sp1/Sp3 and USF1/USF2 (where USF is upstream stimulatory factor), respectively, were required for the full transcriptional activity of this fragment. Whereas mutations in the GC box reduced the promoter activity by 50%, mutations in the E box alone or in both the E box and GC box resulted in 90% loss of transcriptional activity. The essential role of the E box in the bidirectional promoter activity was further demonstrated by transient transfection in Caco-2, K-562, and HeLa cells using a 29-bp (541/513) fragment that contained only the E box. Based on these results we suggest that 1) the E box is essential for both the GC box-dependent and -independent promoter activity of the 69-bp fragment, 2) cooperative interactions between Sp1/Sp3 and USFs are required for the full activation of the 69-bp promoter activity, and 3) the single E box is able to mediate bidirectional transcription in transfected cells in the absence of an obvious TATA box or a known initiator element.

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