%0 Journal Article
%T Upstream stimulatory factors are involved in the P1 promoter directed transcription of the AbetaH-J-J locus
%A Alessia Finotti
%A Susan Treves
%A Francesco Zorzato
%A Roberto Gambari
%A Giordana Feriotto
%J BMC Molecular Biology
%D 2008
%I BioMed Central
%R 10.1186/1471-2199-9-110
%X In the present study, we extended the functional characterization of the P1 promoter of the A¦ÂH-J-J locus. We demonstrated by quantitative Real-time RT-PCR that mRNAs from the P1 promoter are actively transcribed in all the human cell lines analysed. To investigate the transcription mechanism we transiently transfected HeLa cells with sequentially deleted reporter constructs containing different regions of the -661/+81 P1 nucleotide sequence. Our results showed that (i) this promoter fragment is a powerful activator of the reporter gene in HeLa cell line, (ii) the region spanning 512 bp upstream of the transcription start site exhibits maximal level of transcriptional activity, (iii) progressive deletions from -512 gradually reduce reporter expression.The region responsible for maximal transcription contains an E-box site; we characterized the molecular interactions between USF1/2 with this E-box element by electrophoretic mobility shift assay and supershift analysis. In addition, our USF1 and USF2 chromatin immunoprecipitation results demonstrate that these transcription factors bind the P1 promoter in vivo.A functional role of USF1/USF2 in upregulating P1-directed transcription was demonstrated by analysis of the effects of (i) in vitro mutagenesis of the P1/E-box binding site, (ii) RNA interference targeting USF1 transcripts.Our results suggest that USF factors positively regulate the core of P1 promoter, and, together with our previously data, we can conclude that both Sp and USF DNA interaction and transcription activity are involved in the P1 promoter dependent expression of AAH and humbug.We have previously characterized the human A¦ÂH-J-J locus, a genomic sequence which generates functionally distinct proteins [1], including the enzyme aspartyl (asparaginyl) ¦Â-hydroxylase (AAH), junctin, a structural protein of sarcoplasmic reticulum, humbug and junctate, the truncated homologs of AAH calcium binding proteins [1,2]. AAH catalyzes posttranslational hydroxylati
%U http://www.biomedcentral.com/1471-2199/9/110