Abstract
Protein disulfide isomerase (PDI) is a highly unusual multifunctional polypeptide that is identical to the beta-subunit of prolyl 4-hydroxylase, a cellular thyroid hormone-binding protein and a subunit of the microsomal triglyceride transfer protein complex, and very similar to a polypeptide functioning in vitro as a glycosylation site binding protein of oligosaccharyl transferase. The human PDI gene possesses several putative transcriptional control elements, including the highly unusual presence of six CCAAT boxes between -108 and -378 of the 5'-flanking region. We report here on a promoter analysis of this gene. Eleven PDI promoter elements recognized by DNA-binding proteins present in HeLa cell and HT-1080 cell nuclear extracts were identified by DNase I footprinting analysis within the first 630 nucleotides of the 5'-flanking region. Interestingly, these included all six CCAAT elements. Functional 5' deletion analysis suggested that only two or three of the CCAAT elements may contribute significantly to the promoter activity in HeLa cells. Mutations introduced into each of the CCAAT boxes separately indicated, however, that all six appear to contribute to the promoter strength, the largest decreases (approximately 50%) being seen with mutations in the second or fifth CCAAT box. These data thus suggested that efficient expression of the multifunctional PDI polypeptide is secured by multiple CCAAT elements, some of which appear to be functionally redundant. The 5' deletion analysis further suggested that a region between -623 and -518 may contain additional positively and negatively acting elements.
Highlights
Eleven Protein disulfide isomerase (PDI) promoter elements recognized by DNA-binding proteins present inHeLa cell andHT-1080cell nuclear extracts wereidentified by DNase I footprinting analysis within the first 630 nucleotide sequences were first reported for the rat enzyme [8]
Our results indicate that at least 11 PDI promoter elements may interact with DNA-binding proteins presentin HeLa cell
In order to study the transcriptional regulation of the human PDI gene, we prepared 11 promoter fragments of different length (Fig. I),four of these being used for DNase I footprinting experiments and all 11 for functional deletion analysis
Summary
Sequences Covered by the P D I Promoter Constructs-The 5’-flanking region of the human PDI gene contains a TATA box at position -21 relative to the transcription initiation site, two possible S p l binding sites a t -56 and -451, and six CCAAT boxes between -108 and -378, which are numbered from 1to 6 inrelation to thetranscription initiation site[21]. In order to study the transcriptional regulation of the human PDI gene, we prepared 11 promoter fragments of different length (Fig. I),four of these being used for DNase I footprinting experiments and all 11 for functional deletion analysis. These fragmentswere generated from a 3.3-kb genomicclone SGB-22, which covers 1.8 kb of the 5”flanking region and spans the first two exons of the human PDI gene [21]. Nucleotide sequences had previously been reported for 563 nt in 5’-flanking sequences [21], and additional upstreamsequences, extending to nt -840, were characterized here in order to allow interpretation of the results obtained with the three longest promoter constructs Two fragments, both containingall six CCAAT boxes, were isolated from the clone SGB-22. Binding site [42]. 1 GCCCTCTCGTCGCCCCCGCTGTCCCGCGGCCCCUCCGMGCGCCCCGCCTGATCCGTGTCCGACh~
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