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Licensed Unlicensed Requires Authentication Published by De Gruyter June 1, 2005

Transcription of Cathepsin B in Glioma Cells: Regulation by an E-Box Adjacent to the Transcription Initiation Site

  • S. Yan , D. T. Jane , M. J. Dufresne and B. F. Sloane
From the journal Biological Chemistry

Abstract

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.

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Published Online: 2005-06-01
Published in Print: 2003-11-07

Copyright © 2003 by Walter de Gruyter GmbH & Co. KG

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