Upstream stimulatory factors are involved in the P1 promoter directed transcription of the A beta H-J-J locus.
BACKGROUND
Alternative splicing of the locus A beta H-J-J generates functionally distinct proteins: the enzyme aspartyl (asparaginyl) beta-hydroxylase (AAH), truncated homologs of AAH with a role in calcium homeostasis humbug and junctate and a structural protein of the sarcoplasmic reticulum membranes junctin. AAH and humbug are over expressed in a broad range of malignant neoplasms. We have previously reported that this locus contains two promoters, P1 and P2. While AAH and humbug are expressed in most tissues under the regulation of the P1 promoter, AAH, junctin and junctate are predominantly expressed in excitable tissues under the control of the P2 promoter. We previously demonstrated that Sp transcription factors positively regulate the P1 promoter.


RESULTS
In the present study, we extended the functional characterization of the P1 promoter of the A beta H-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.


CONCLUSIONS
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.
Finotti
Alessia
A
Department of Biochemistry and Molecular Biology, Molecular Biology Section, University of Ferrara, Via Fossato di Mortara 74, 44100 Ferrara, Italy. fntlss@unife.it
Treves
Susan
S
Zorzato
Francesco
F
Gambari
Roberto
R
Feriotto
Giordana
G
D001483:Base Sequence; D001665:Binding Sites; D045744:Cell Line, Tumor; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic; D051720:Upstream Stimulatory Factors / Q000235:genetics / Q000378:metabolism*
C497181:USF1 protein, human; C497184:USF2 protein, human; D051720:Upstream Stimulatory Factors; D006899:Mixed Function Oxygenases; C058291:aspartic acid 2-oxoglutarate-dependent dioxygenase