Characterization of a brain-specific Sp1-like activity interacting with an unusual binding site within the myelin proteolipid protein promoter.
The proteolipid protein (PLP) gene encodes the main integral protein of the myelin membrane of the central nervous system. The expression of the gene is regulated in a cell- and development-specific manner. Comparison of approximately 1.5 kb of the upstream noncoding region from man, mouse, and rat gene revealed an extensive sequence identity of about 95% between -250 and +100 (the most upstream transcription start site is defined as +1) but only about 50% identity further upstream. To define potential cis-acting elements in the promoter of the mouse PLP gene the upstream region was studied by transfection of C6 glioblastoma cells and CHO fibroblasts with various 5' deletion constructs fused to the reporter gene luciferase. We localized a promoter at position -184 to +90, which is active in both cell lines. Analysis of this region by DNase I foot-printing experiments and band shift analysis with nuclear extracts from myelinating brain, liver, C6, and CHO cells shows the binding of several different proteins to the promoter region. One brain-specific and two ubiquitous factors bound to the sequence AAGGGGAGGAG (DR1/2 box). This motif is also present in the upstream region of other myelin-specific genes and in some variants of the glia cell-specific virus JC. The factors bound with similar affinity to a Sp1-binding site. Therefore one of the ubiquitous factors seems to be Sp1 suggesting that Sp1 may play a role in the transcriptional regulation of the PLP gene. It has been shown that the DR1/2 box-binding factors are Zn(2+)-dependent. By Southwestern blotting it has been demonstrated that the DR1/2 box binds a protein of about 66 kDa that is enriched in brain.
Janz
R
R
Institute of Biochemistry, Medical Faculty, University of Cologne.
Stoffel
W
W
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D001923:Brain Chemistry; D016466:CHO Cells; D006224:Cricetinae; D003850:Deoxyribonuclease I / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009185:Myelin Proteins / Q000378:metabolism*; D018991:Myelin Proteolipid Protein; D015345:Oligonucleotide Probes; D010957:Plasmids; D011401:Promoter Regions, Genetic; D051381:Rats; D014162:Transfection; D014407:Tumor Cells, Cultured
D009185:Myelin Proteins; D018991:Myelin Proteolipid Protein; D015345:Oligonucleotide Probes; D003850:Deoxyribonuclease I