Abstract
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.
Highlights
From the Division of Gastroenterology and Hepatology, Department of ‡Medicine and §Biochemistry, Medical College of Wisconsin and Veterans Medical Center, Milwaukee, Wisconsin 53226
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, respectively, were required for the full transcriptional activity of this fragment
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 Upstream stimulatory factor (USF) 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
Summary
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. We have identified a 69-bp sequence (Ϫ581/Ϫ513) from the distal region of the TC II promoter [25] that did not contain an obvious TATA box or a known Inr element but possessed a high promoter activity in transfected cells in an orientation-independent manner. This finding suggests that TATAϪ/InrϪ promoters may use an E box as a core element to direct basal transcription
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