Molecular characterization of senescence marker protein-30 gene promoter: identification of repressor elements and functional nuclear factor binding sites.
BACKGROUND
Senescence marker protein-30 (SMP30), whose expression declines during aging in rat liver, has been proposed as an important aging marker. Besides apoptosis, SMP30 also protects cells against various other injuries by enhancement of membrane calcium-pump activity. The mechanism of this differential gene expression mechanism is not known. DNA-protein interactions, mutation analysis and luciferase reporter assay studies have been performed to elucidate the mechanism of transcriptional regulation of SMP30 gene.


RESULTS
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/EBPbeta (-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.


CONCLUSIONS
These studies provide novel insights into the mechanism that regulate SMP30 gene expression.
Rath
Bandita
B
Institute of Life Sciences, Nalco Square, Chandrasekharpur, Bhubaneswar, 751023, India. bandita27278@rediffmail.com
Pandey
Ravi S
RS
Debata
Priya R
PR
Maruyama
Naoki
N
Supakar
Prakash C
PC
D000818:Animals; D020029:Base Pairing; D001665:Binding Sites; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases; D018983:DNA Footprinting; D003850:Deoxyribonuclease I / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D050989:GATA2 Transcription Factor / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D009154:Mutation / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D051381:Rats; D012097:Repressor Proteins / Q000378:metabolism*; D020218:Response Elements / Q000235:genetics*; D051876:Sex-Determining Region Y Protein / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*
D022782:CCAAT-Enhancer-Binding Protein-beta; D002135:Calcium-Binding Proteins; D050989:GATA2 Transcription Factor; D047908:Intracellular Signaling Peptides and Proteins; D012097:Repressor Proteins; D051876:Sex-Determining Region Y Protein; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D002265:Carboxylic Ester Hydrolases; C513325:Rgn protein, rat; D003850:Deoxyribonuclease I