Transcriptional regulation of the MN/CA 9 gene coding for the tumor-associated carbonic anhydrase IX. Identification and characterization of a proximal silencer element.
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.
Kaluz
S
S
Institute of Virology, Slovak Academy of Sciences, 842 46 Bratislava, Slovak Republic.
Kaluzová
M
M
Opavský
R
R
Pastoreková
S
S
Gibadulinová
A
A
Dequiedt
F
F
Kettmann
R
R
Pastorek
J
J
D000951:Antigens, Neoplasm / Q000235:genetics; D001483:Base Sequence; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases / Q000235:genetics*; D003432:Cross-Linking Reagents; D018983:DNA Footprinting; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000032:analysis; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D020868:Gene Silencing; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics; D014162:Transfection; D014466:Ultraviolet Rays
D000951:Antigens, Neoplasm; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D012097:Repressor Proteins; C089226:CA9 protein, human; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases