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 -17 3 to +31 fragment as the MN promoter. lie vitro DNase I footprinting reveal ed the presence of five protected regions (PRs) within the MN promoter. Det ailed deletion analysis of the promoter identified PR1 and PR2 (numbered fr om the transcription start) as the most critical for transcriptional activi ty. PR4 negatively affected transcription, since its deletion led to increa sed promoter activity and was confirmed to function as a promoter-, positio n, and orientation-independent silencer element. Mutational analysis indica ted that the direct repeat AGGGCacAGGGC is required for efficient repressor binding. Two components of the repressor complex (35 and 42 kDa) were foun d to be in direct contact with PR4 by UV cross-linking. Increased cell dens ity, 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 nontu morigenic CGL1 HeLa x normal fibroblast hybrid cells.