Molecular characterization of senescence marker protein-30 gene promoter: Identification of repressor elements and functional nuclear factor binding sites

We have characterized up to -2750 bp of the promoter by DNA-protein interactions studies. Twenty eight transcription factor binding sites have been identified by DNase I footprinting and electrophoretic mobility shift assay (EMSA). Transient transfection of 5' and 3' -deleted promoter-reporter constructs and luciferase assay illustrated the region between -128/+157 bp is sufficient to drive promoter activity. We have mapped an essential regulatory region between -513 to -352 bp which causes a drastic decline of reporter activity. This region contains CdxA, GATA2 and SRY transcription factor binding sites. Individual mutation of these three sites showed increase in reporter activity. Mutation in SRY site (-403/-368) showed maximum increase in reporter activity among these three sites. Therefore, we suggest that SRY like protein may be acting as a strong repressor of SMP30 gene along with CdxA and GATA-2. We also report that mutation of both Sp1 (172/-148 bp) and a C/EBPβ (-190/-177 bp) transcription binding site located adjacent to each other on SMP30 gene promoter, causes a significant enhancement in reporter activity than individual mutation, thus may be causing the repression of SMP30 promoter activity.These studies provide novel insights into the mechanism that regulate SMP30 gene expression.Senescence marker protein-30 (SMP30), a 34 kDa protein, is preferentially expressed in hepatocytes and renal tubular epithelia. SMP30 is unique in that, its expression is maintained at a high level throughout the tissue maturation process, then decreases in an androgen-independent manner during senescent stages in both sexes [1,2]. Analysis of murine genomic clone revealed that SMP30 is organized into seven exons and six introns spanning approximately 17.5 kb. The full length cDNA fragment (1.6 kb) contains an open reading frame of 897 bp encoding 299 amino acids. Cloned SMP30 promoter is approximately 3 kb in length and up to -1.5 kb of upstream promoter region has been sequ