Molecular and functional analysis of the XPBC/ERCC-3 promoter: transcription activity is dependent on the integrity of an Sp1-binding site.
The human XPBC/ERCC-3 gene, which corrects the excision-repair defect in xeroderma pigmentosum group B cells and the UV-sensitive CHO mutant 27-1 cells, appears to be expressed constitutively in various cell types and tissues. We have analysed the structure and functionality of the XPBC/ERCC-3 promoter. Transcription of the XPBC/ERCC-3 gene is initiated from heterogeneous sites, with a major startpoint mapped at position -54 (relative to the translation start codon ATG). The promoter region does not possess classical TATA and CAAT elements, but it is GC-rich and contains three putative Sp1-binding sites. In addition, there are two elements related to the cyclic AMP (cAMP)-response element (CRE) and the 12-O-tetradecanoyl phorbol-13-acetate-response element (TRE) in the 5'-flanking region. Transient expression analysis of XPBC/ERCC-3 promoter-CAT chimeric plasmids revealed that a 127-bp fragment, spanning position -129 to -3, is minimally required for the promoter activity. Transcription of the XPBC/ERCC-3 promoter depends on the integrity of a putative Sp1-binding site in close proximity to the major cap site. Band shift assays showed that this putative Sp1-binding site can interact specifically with a nuclear factor, most likely transcription factor Sp1 (or an Sp1-like factor) in vitro.
Ma
L
L
Sylvius Laboratories, University of Leiden, The Netherlands.
Weeda
G
G
Jochemsen
A G
AG
Bootsma
D
D
Hoeijmakers
J H
JH
van der Eb
A J
AJ
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D016466:CHO Cells; D006224:Cricetinae; D004260:DNA Repair / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D014983:Xeroderma Pigmentosum / Q000201:enzymology / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor