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J Biol Chem, Vol. 274, Issue 46, 32588-32595, November 12, 1999

Transcriptional Regulation of the MN/CA 9 Gene Coding for the Tumor-associated Carbonic Anhydrase IX
IDENTIFICATION AND CHARACTERIZATION OF A PROXIMAL SILENCER ELEMENT

Stefan Kaluz, Milota Kaluzová, René Opavský, Silvia Pastoreková, Adriana Gibadulinová, Franck Dequiedt^¶, Richard Kettmann^¶, and Jaromír Pastorek

From the  Institute of Virology, Slovak Academy of Sciences, 842 46 Bratislava, Slovak Republic and the ^¶ Department of Molecular Biology, Faculty of Agronomy, B-5030 Gembloux, Belgium

The MN/CA 9 (MN) gene encodes a tumor-associated isoenzyme of the carbonic anhydrase family. Functional characterization of the 3.5-kilobase pair MN 5' upstream region by deletion analysis led to the identification of the 173 to +31 fragment as the MN promoter. In vitro DNase I footprinting revealed the presence of five protected regions (PRs) within the MN promoter. Detailed deletion analysis of the promoter identified PR1 and PR2 (numbered from the transcription start) as the most critical for transcriptional activity. PR4 negatively affected transcription, since its deletion led to increased promoter activity and was confirmed to function as a promoter-, position-, and orientation-independent silencer element. Mutational analysis indicated that the direct repeat AGGGCacAGGGC is required for efficient repressor binding. Two components of the repressor complex (35 and 42 kDa) were found to be in direct contact with PR4 by UV cross-linking. Increased cell density, known to induce MN expression, did not affect levels of PR4 binding in HeLa cells. Significantly reduced repressor level seems to be responsible for MN up-regulation in the case of tumorigenic CGL3 as compared with nontumorigenic CGL1 HeLa × normal fibroblast hybrid cells.

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