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.
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.
Division of Gastroenterology and Hepatology, Department of Medicine, Medical College of Wisconsin and Veterans Medical Center, Milwaukee, Wisconsin 53226, USA.
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N
Li
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B
Seetharam
D001483:Base Sequence; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000378:metabolism; D017930:Genes, Reporter; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017384:Sequence Deletion; D016329:Sp1 Transcription Factor / Q000378:metabolism; D051705:Sp3 Transcription Factor; D016385:TATA Box; D014155:Transcobalamins / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D051720:Upstream Stimulatory Factors; D014805:Vitamin B 12
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; D014155:Transcobalamins; D014157:Transcription Factors; C497181:USF1 protein, human; C497184:USF2 protein, human; D051720:Upstream Stimulatory Factors; D051705:Sp3 Transcription Factor; D014805:Vitamin B 12