Analysis of the human MBP promoter in primary cultures of oligodendrocytes: positive and negative cis-acting elements in the proximal MBP promoter mediate oligodendrocyte-specific expression of MBP.
Since the regulation of myelin basic protein expression depends primarily on the initiation of transcription, we analyzed the 5' flanking region of the human myelin basic protein gene in transient transfection studies in primary cultures of developing oligodendrocytes. We demonstrated that 149 base pairs 5' of the initiation of transcription was sufficient to direct oligodendrocyte-specific expression of myelin basic protein. The capsite of the fusion transcript was identical with that of the endogenous myelin basic protein transcript, and chloramphenicol acetyl transferase reporter gene expression was restricted to oligodendrocytes in these cultures. Within this 149 base pair region, one distal, negative cis-acting segment, containing a consensus nuclear factor I site, and one proximal, positive cis-acting segment were identified. The distal segment behaved more negatively in Cos-7 cells than in oligodendrocytes, reducing expression to background levels. Furthermore, these functionally important cis-acting segments bound oligodendrocyte nuclear proteins in a pattern differing from other cells, including Cos-7 cells. Interestingly, the distal segment increased heterologous SV40 promoter activity in oligodendrocytes but had no effect on the SV40 promoter in Cos-7 cells. We conclude that the functionally negative distal segment may mediate oligodendrocyte-specific expression of MBP by restricting its expression in other cells. These experiments strongly support using primary cultures of oligodendrocytes for analyzing the myelin-specific promoters.
Wrabetz
L
L
Department of Neurology, University of Pennsylvania School of Medicine, Philadelphia.
Shumas
S
S
Grinspan
J
J
Feltri
M L
ML
Bozyczko
D
D
McMorris
F A
FA
Pleasure
D
D
Kamholz
J
J
D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D002460:Cell Line; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D005910:Glioma; D006367:HeLa Cells; D006801:Humans; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D004676:Myelin Basic Protein / Q000235:genetics*; D009457:Neuroglia / Q000378:metabolism; D009836:Oligodendroglia / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins; D014162:Transfection
D004268:DNA-Binding Proteins; D004676:Myelin Basic Protein; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase