caa a22 4500 378858920 CHVBK 20180305123341.0 cr unu---uuuuu 161128e20031107xx s 000 0 eng 10.1515/BC.2003.157 doi (NATIONALLICENCE)gruyter-10.1515/BC.2003.157 Transcription of Cathepsin B in Glioma Cells: Regulation by an E-Box Adjacent to the Transcription Initiation Site [Elektronische Daten] [S. Yan, D. T. Jane, M. J. Dufresne, B. F. Sloane] We have previously isolated the human cathepsin B promoter and shown that Sp1 and Ets factors are involved in the regulation of cathepsin B expression. Using mutagenesis, transient transfection and electrophoretic mobility shift assays (EMSAs), we further identified regulatory factors that mediate cathepsin B transcription in U87 human glioblastoma cells. An Ebox element (CACGTG) adjacent to the transcription initiation site (at nucleotides -7 to -2) was found to be indispensable for cathepsin B promoter activity. Mutation of this E-box element in both pSCB2, a promoter construct with high promoter activity, and pSCB6, a construct with basal promoter activity, led to a 90% decrease in promoter activity in U87 cells. EMSAs demonstrated that upstream stimulatory factor 1 (USF-1) and upstream stimulatory factor 2 (USF-2) bound to the Ebox as a heterodimer. Chromatin immunoprecipitation assays revealed that both USF-1 and USF-2 were associated with the cathepsin B promoter. The roles of USF-1 and USF-2 in the regulation of cathepsin B expression were demonstrated by (i) cotransfection experiments showing that USF-1 or USF-2 increased promoter activity by 2.5-fold individually and by 3.4-fold together; (ii) cotransfection of pSCB6 with pUSF-2δN (a dominant negative USF-2 expression plasmid) resulting in an 80% decrease in promoter activity; and (iii) mutation of the Ebox element (from 5'-CACGTG to 5'-CGCGTT in the pSCB6 basal promoter construct) abolishing transactivation of cathepsin B by USF-1 and USF-2. These results collectively indicate that an Ebox at nucleotides 7 to 2 of the cathepsin B promoter is critical to the expression of cathepsin B and that binding of USF-1 and USF-2 to this E-box can regulate cathepsin B promoter activity. Copyright © 2003 by Walter de Gruyter GmbH & Co. KG Biochemistry nationallicence Molecular biology nationallicence Cellular biology nationallicence Yan S. aut Jane D. T. aut Dufresne M. J. aut Sloane B. F. aut Biological Chemistry Walter de Gruyter 384/10-11(2003-11-07), 1421-1427 1431-6730 384:10-11<1421 2003 384 bchm https://doi.org/10.1515/BC.2003.157 text/html Onlinezugriff via DOI 1 research article jats NATIONALLICENCE 856 40 https://doi.org/10.1515/BC.2003.157 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Yan S. aut NATIONALLICENCE 700 1- Jane D. T. aut NATIONALLICENCE 700 1- Dufresne M. J. aut NATIONALLICENCE 700 1- Sloane B. F. aut NATIONALLICENCE 773 0- Biological Chemistry Walter de Gruyter 384/10-11(2003-11-07), 1421-1427 1431-6730 384:10-11<1421 2003 384 bchm CC0 http://creativecommons.org/publicdomain/zero/1.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-gruyter