Transcriptional activity and Sp 1/3 transcription factor binding to the P1 promoter sequences of the human AbetaH-J-J locus

Alternative splicing of the locus AbetaH-J-J generates functionally distinct proteins: the enzyme aspartyl (asparaginyl) beta-hydroxylase, humbug and junctate (truncated homologs of aspartyl (asparaginyl) beta-hydroxylase with a role in calcium regulation), and junctin (a structural protein of the sarcoplasmic reticulum membrane). Aspartyl (asparaginyl) beta-hydroxylase and humbug are overexpressed in a broad range of malignant neoplasms. We have previously reported the gene structure of this locus, showing the presence of two putative promoters, P1 and P2, and characterized the P2 sequences, directing tissue-specific transcription of junctin, aspartyl (asparaginyl) beta-hydroxylase and junctate. In addition, aspartyl (asparaginyl) beta-hydroxylase and humbug are expressed from exon 1 by the P1 promoter. The present study identifies and functionally characterizes the P1 promoter activity of the AbetaH-J-J locus. We demonstrate that mRNAs from the P1 promoter are actively transcribed in all the human tissues and cell lines analyzed, and define the transcription start point in HeLa and RD cells. To investigate the transcription mechanism we cloned 1.7 kb upstream of exon 1 from a human BAC clone, and produced progressively deleted reporter constructs. Our results showed that: (a) the 1.7 kb fragment was a powerful activator of the reporter gene in human hepatoblastoma (HepG2) and human embryonic rhabdomyosarcoma (RD) cell lines; (b) 512 bp upstream of the transcription start site were essential for maximal promoter activity; and (c) progressive deletions from -512 resulted in gradually decreased reporter expression. The region responsible for maximal transcription contains at least 12 GC boxes homologous to binding sequences of specific transcription factor 1 (Sp1); by electrophoretic mobility shift assay and supershift analysis, we identified three GC-rich elements that bind Sp transcription factor family nuclear factors with very high efficiency. A functional role of Sp transcription factors in upregulating P1-directed transcription was demonstrated by analysis of the effects of: (a) in vitro mutagenesis of the Sp1 transcription factor binding sites; (b) transfection with Sp transcription factor 1/3 expression vectors; and (c) treatment with decoy oligonucleotides targeting Sp transcription factors. In addition, Sp1 and Sp3 transcription factor chromatin immunoprecipitation demonstrated in vivo binding of these proteins to P1 promoter. Our results suggest that Sp transcription factors positively regulate the core of the P1 promoter, and the comparison of the two promoters of the AbetaH-J-J locus demonstrates that they are very different with regard to transcriptional efficiency and ability to direct tissue-specific transcription.