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, Jaromír Pastorek

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Abstract

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 x normal fibroblast hybrid cells.

Original languageEnglish (US)
Pages (from-to)32588-32595
Number of pages8
JournalJournal of Biological Chemistry
Volume274
Issue number46
DOIs
StatePublished - Nov 12 1999

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Transcriptional Silencer Elements
Carbonic Anhydrases
Hybrid Cells
Nucleic Acid Repetitive Sequences
Deoxyribonuclease I
Transcription
HeLa Cells
Isoenzymes
Tumors
Up-Regulation
Fibroblasts
Cell Count
Genes
Neoplasms
Carbonic Anhydrase IX
In Vitro Techniques

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Transcriptional regulation of the MN/CA 9 gene coding for the tumor- associated carbonic anhydrase IX. Identification and characterization of a proximal silencer element. / Kaluz, Stefan; Kaluzová, Milota; Opavský, René; Pastoreková, Silvia; Gibadulinová, Adriana; Dequiedt, Franck; Kettmann, Richard; Pastorek, Jaromír.

In: Journal of Biological Chemistry, Vol. 274, No. 46, 12.11.1999, p. 32588-32595.

Research output: Contribution to journalArticle

Kaluz, Stefan ; Kaluzová, Milota ; Opavský, René ; Pastoreková, Silvia ; Gibadulinová, Adriana ; Dequiedt, Franck ; Kettmann, Richard ; Pastorek, Jaromír. / Transcriptional regulation of the MN/CA 9 gene coding for the tumor- associated carbonic anhydrase IX. Identification and characterization of a proximal silencer element. In: Journal of Biological Chemistry. 1999 ; Vol. 274, No. 46. pp. 32588-32595.
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