The promoter of the C1 inhibitor gene contains a polypurine.polypyrimidine segment that enhances transcriptional activity.

The C1 inhibitor (C1INH) promoter is unusual in two respects: 1) It contains no TATA sequence, but instead contains a TdT-like initiator element (Inr) at nucleotides -3 to +5; 2) it contains a polypurine.polypyrimidine tract between nucleotides -17 and -45. Disruption of the Inr by the introduction of point mutations reduced promoter activity by 40%. A TATA element inserted at nucleotide -30 in the wild-type promoter and in promoter constructs containing the mutated Inr led to a 2-fold increase in basal promoter activity. Previous studies suggested that the potential hinged DNA-forming polypurine.polypyrimidine tract might be important in the regulation of C1INH promoter activity. The present studies indicate that this region is capable of such intramolecular triple helix formation. Disruption of the polypurine.polypyrimidine sequence by substitution of 5 of the 23 cytosine residues with adenine prevented triple helix formation. Site-directed mutagenesis experiments demonstrate that the regulation of promoter activity is independent of hinged DNA-forming capacity but requires an intact AC box (ACCCTNNNNNACCCT) or the overlapping PuF binding site (GGGTGGG). The C1INH gene also contains a number of potential regulatory elements, including an Sp-1 and an hepatocyte nuclear factor-1 binding site and a CAAT box. The role of these elements in regulation of the C1INH promoter was examined. Elimination of the hepatocyte nuclear factor-1 site at nucleotides -94 to -81 by truncation reduced the activity of the promoter by approximately 50%. Similarly, site-directed mutations that disrupt this site reduce promoter activity by 70%.     

Structure and transcriptional regulation of the mouse ferrochelatase gene

Ferrochelatase (EC.4.99.1.1), the final step in the biosynthesis of heme, is widely expressed in various tissues and is induced in erythroid cells. We determined the structure of the mouse ferrochelatase gene after isolation and characterization of lambda phage clones mapping discrete regions of the cDNA. The gene spans about 25 kb and consists of 11 exons. The exon/intron boundary sequences conform to consensus acceptor (GTn)/donor (nAG) sequences, and exons in the gene encode functional protein domains. The promoter region contains multiple Sp1 sites, a CACCC box and GATA-1 binding sites. Function analysis of the promoter by transient transfection assay demonstrated that one Sp1 binding site located at -37/-32 is essential for basic expression of the ferrochelatase gene in both mouse erythroleukemia (MEL) and non-erythroid EL4 cells. In addition, the region (-66/-51) containing a CACCC box and the neighboring GC box partly contributes to the inducible activity of the reporter in MEL cells upon induction with dimethylsulfoxide. It appears that at least two promoter regions of the mouse ferrochelatase gene function in basic and inducible expression.     

Dual functions of transcription factors, transforming growth factor-beta-inducible early gene (TIEG)2 and Sp3, are mediated by CACCC element and Sp1 sites of human monoamine oxidase (MAO) B gene

Monoamine oxidases (MAO) A and B catalyze the oxidative deamination of many biogenic and dietary amines. Abnormal expression of MAO has been implicated in several psychiatric and neurodegenerative disorders. Human MAO B core promoter (-246 to -99 region) consists of CACCC element flanked by two clusters of overlapping Sp1 sites. Here, we show that cotransfection with transforming growth factor (TGF)-beta-inducible early gene (TIEG)2 increased MAO B gene expression at promoter, mRNA, protein, and catalytic activity levels in both SH-SY5Y and HepG2 cells. Mutation of the CACCC element increased the MAO B promoter activity, and cotransfection with TIEG2 further increased the promoter activity, suggesting that CACCC was a repressor element. This increase was reduced when the proximal Sp1 overlapping sites was mutated. Similar interactions were found with Sp3. These results showed that TIEG2 and Sp3 were repressors at the CACCC element but were activators at proximal Sp1 overlapping sites of MAO B. Gel-shift and chromatin immunoprecipitation assays showed that TIEG2 and Sp3 bound directly to CACCC element and the proximal Sp1 sites in both synthetic oligonucleotides and natural MAO B core promoter. TIEG2 had a higher affinity to Sp1 sites than CACCC element, whereas Sp3 had an equal affinity to both elements. Thus, TIEG2 was an activator, but Sp3 had no effect on MAO B gene expression. This study provides new insights into MAO B gene expression and illustrates the complexity of gene regulation.