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

Abstract

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.

Evidence That the Homeodomain Protein Gtx Is Involved in the Regulation of Oligodendrocyte Myelination

“…Several laboratories have demonstrated that relatively small regions of DNA sequence upstream of both the PL P and M BP genes are sufficient for developmental and tissue-specific activation of lacZ expression in oligodendrocytes in transgenic animals (Gow et al, 1992;Goujet-Z alc et al, 1993;Wright et al, 1993;Wrabetz et al, 1995), and transcription factors interacting with these regions have been identified (Berndt et al, 1992;K im and Hudson, 1992;Haas et al, 1993;Wrabetz et al, 1993). Only one of these factors, MyT1, a member of the zinc finger family of DNA-binding proteins, has been shown to be expressed in oligodendrocytes (Armstrong et al, 1995).…”

MEBA Derepresses the Proximal Myelin Basic Protein Promoter in Oligodendrocytes

“…In addition, we showed by transient transfection analysis in primary cultures of oligodendrocytes that 750, 420, or 149 nt of human MBP promoter was sufficient to activate a 5-10-fold increase in the expression of the CAT reporter gene in oligodendrocytes, but not in other cell types (12). We also showed that the 149-nt region contained two subregions with opposing functional activities: (a) the proximal 102-nt region activated the expression of CAT in most cell types; and (b) the more distal region, from Ϫ149 to Ϫ102 nt, silenced the expression of CAT in them.…”

“…Plasmids--All deletions of the proximal 149-nt MBP promoter were prepared by a modification of the exonuclease III/mung bean nuclease technique (Stratagene), as described previously (12). Transversion mutations spanning from 149 to 110 nt of the MBP promoter were prepared by polymerase chain reaction-mediated mutagenesis, as described previously (12).…”

“…Transversion mutations spanning from 149 to 110 nt of the MBP promoter were prepared by polymerase chain reaction-mediated mutagenesis, as described previously (12). All mutations were confirmed by sequence analysis.…”

“…This distal region contained a consensus nuclear factor 1 (NF1) site; a deletion that removed half of the NF1 site activated the expression of CAT in oligodendrocytes. Based on these results, we hypothesized that a specific alteration of NF1 may alleviate repression and contribute to the oligodendrocytespecific activation of MBP transcription (12).…”