Structural and functional characterisation of the mouse annexin A9 promoter

Annexin A9 is an atypical member of the annexin family of Ca(2+) and phospholipid-binding proteins, initially identified in EST data bases. Its amino acid sequences responsible for calcium coordination are mutated suggesting an atypical, Ca(2+)-independent cellular function in comparison to other family members. In line with a specialized function of annexin A9 is the restricted presence of its cDNA in EST libraries from different tissues. To identify elements mediating this regulation of annexin A9 transcription, we have cloned the mouse homolog of the human annexin A9 gene and characterised its promoter. By employing 2.5 kb of the most 5' flanking region of the gene, containing 5' non-coding sequence, exon I and intron I in luciferase reporter assays in annexin A9 positive HEPA 1-6 cells, we reveal the existence of a minimal promoter located at the 3' flank of intron I. The sequence covering this minimal promoter contains a binding site consensus for the transcription factor GATA-1 whose binding were verified by electrophoretic mobility shift assays (EMSA). Further mapping analysis also identified two elements in exon I with a negative regulatory function on gene transcription. This suggests that the entire region containing the non-protein coding exon I and the adjacent intron I is involved in the regulation of mouse annexin A9 transcription.