Characterization of the MN/CA 9 promoter proximal region: a role for specificity protein (SP) and activator protein 1 (AP1) factors

Abstract

MN/CA IX (MN) is a tumour-associated isoenzyme of the carbonic anhydrase family. Previous deletion analysis of the MN promoter established that protected regions (PRs) 1 and 2 are crucial for its transcriptional activity. Computer-assisted searching indicated putative binding sites for activator protein (AP) 2 and specificity protein (SP) 1 transcription factors, plus a CACCC box in PR1 and an AP1 site in PR2. PR1 produced four complexes in electrophoretic mobility-shift assay (EMSA) with HeLa nuclear extracts. Of these, three were completely competed with the SP1 and transforming growth factor-beta retinoblastoma control-element CACCC box (RCE) probes, whereas the AP2 probe competed against the same three complexes partially. Supershift EMSA identified SP1 in the complex 1 and SP3 in the complexes 2 and 4. Point mutations in the SP1 site abrogated the PR1 function, while mutations affecting the overlapping CACCC box/AP2 site in PR1 had minor effect on MN promoter activity. Block-replaced MN promoter mutants that had a consensus binding site (SP1 or AP2) or the RCE in place of PR1 demonstrated the stringent selectivity of the PR1 position as only the SP1 mutant reconstituted the MN promoter activity. The consensus SP1 probe generated the same SP1 and SP3 complexes as PR1 in EMSA; therefore we conclude that SP activity is both necessary and sufficient in the PR1 position. The critical role of AP1 in the PR2 position was confirmed by supershift of the PR2 complex with c-Fos antibody and markedly decreased activity of the construct with a mutated AP1 site. Detailed deletion analysis proved that PR1+PR2 account for 90% of the MN promoter activity, while neither PR1 nor PR2 on their own are sufficient for transactivation. Thus, synergistic co-operation between SP and AP1 factors bound to the adjacent PR1 and PR2, respectively, is necessary for MN transcriptional activity. The PR1+PR2 module also stimulated transcription from a heterologous promoter. The modulation of AP1 activity with PMA stimulated MN expression and activated the MN promoter, whereas inhibition of protein kinase C activity had no effect on MN expression in HeLa cells.

DNA Damage Is a Prerequisite for p53-Mediated Proteasomal Degradation of HIF-1α in Hypoxic Cells and Downregulation of the Hypoxia Marker Carbonic Anhydrase IX

“…SP3-binding PR1 domain (31). Transient-transfection experiments confirmed induction of CA9 promoter activity by hypoxia (0.5% O 2 ) and DFO in both MCF-7 and Saos-2 cells.…”

Strict regulation of CAIXG250/MN by HIF-1α in clear cell renal cell carcinoma

“…CAIX promoter studies have established the involvement of HIF, and several critical promoter regions have been identified in HeLa cells, harboring AP1-and Sp1-binding sites (Kaluz et al, 1999;Kaluzova et al, 2001). The Sp1/HRE (HRE: hypoxia-responsive element) containing portion of the CAIX promoter has been suggested as a novel type of HRE (Kaluz et al, 2003).…”

Diagnostic, prognostic and therapeutic implications of carbonic anhydrases in cancer

“…The strong inducibility in hypoxia that is conferred by the minimal CA9 promoter may be of use in targeting gene therapy vectors to areas of tumour hypoxia (Dachs et al, 1997). Other factors may be of significance in producing the strong upregulation of CAIX in cancer, since p53 mutation modulates expression, the promoter is less methylated in cancer (Cho et al, 2001) and contains binding sites for activator protein 1 and specificity protein transcription factors 1 and 3 (Kaluzova et al, 2001).…”