Functional analysis of the promoter of the mitochondrial phosphate carrier human gene: identification of activator and repressor elements and their transcription factors.
The phosphate carrier (PiC) catalyses the import of phosphate into mitochondria where it is needed for ATP synthesis. We have analysed the 5'-flanking region of the human PiC gene and found that it has a single transcriptional initiation site and lacks a TATA box. Through deletion analysis of the -1213/-25 nt region, we identified an activation domain (-223/-25) and an inhibition domain (-1017/-814). The most effective promoter activity in transfected HeLa cells corresponded to the region containing putative binding sites for Sp1 (-163/-142; where Sp1 stands for stimulating protein-1) and CREB (-138/-116; where CREB stands for cAMP-response-element-binding protein). These DNA sequences were active in gel-shift assays in the presence of HeLa cell nuclear extracts or recombinant Sp1 and CREB respectively. Forskolin increased PiC promoter activity via the CREB site. Both footprinting and transfection of deletion constructs of the inhibition region (-1017/-814) showed that PiC silencer activity extends over 25 nt (-943/-919), which specifically binds two proteins present in HeLa cell nuclear extracts. These transcription factors were purified by DNA affinity, analysed by MS and identified as p54(nrb)/NonO (nuclear RNA binding protein) and PSF (protein-associated splicing factor). The PiC silencer region cloned in front of the ferritin promoter conferred a strong inhibition to the heterologous promoter. These findings may provide insight into control of PiC gene expression in different cell types and under different growth conditions. To our knowledge, this is the first study to analyse the regulation of the PiC gene expression in any cell.
Iacobazzi
Vito
V
Laboratory of Biochemistry and Molecular Biology, Department of Pharmaco-Biology, University of Bari, Via Orabona 4, 70125 Bari, Italy.
Infantino
Vittoria
V
Costanzo
Paola
P
Izzo
Paola
P
Palmieri
Ferdinando
F
D001483:Base Sequence; D005576:Colforsin / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics*; D020868:Gene Silencing; D006367:HeLa Cells; D006801:Humans; D024101:Mitochondrial Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D028061:Phosphate Transport Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D017384:Sequence Deletion / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism*; D024363:Transcription Initiation Site; D015533:Transcriptional Activation / Q000187:drug effects
D024101:Mitochondrial Proteins; D028061:Phosphate Transport Proteins; C422067:SLC25A3 protein, human; D014157:Transcription Factors; D005576:Colforsin