No abstract

Regulation of myelin proteolipid protein (Plp) gene expression is tightly controlled, both spatially and temporally. Previously, we have shown with transgenic mice that a Plp-lacZ fusion gene (which includes the entire sequence for Plp intron 1 DNA) is regulated in a similar manner to endogenous Plp gene expression. Furthermore, by deletion-transfection analyses using assorted Plp-lacZ constructs with partial deletion of Plp intron 1 sequences, we have shown that the first intron possesses an antisilencer region that is capable of overcoming repression mediated by two distinct regions located elsewhere within intron 1 DNA. Here, we report the ability of various fragments encompassing the antisilencer region to restore ␤-galactosidase activity when inserted into Plp-lacZ constructs, which originally exhibited low levels of ␤-galactosidase activity. Additional constructs were generated to test the effects of these antisilencer-containing fragments in constructs that are missing either one or both of the negative regulatory regions that are overridden during antisilencing. Transfection analyses, in conjunction with protein-DNA binding assays, suggest that several nuclear factors are necessary for derepression of Plp gene activity in an oligodendroglial cell line. Moreover, either the "core" or complete antisilencing region can act in an additive or synergistic fashion when multiple copies are inserted into the Plp-lacZ constructs.

The myelin proteolipid protein (Plp) gene is expressed in oligodendrocytes and encodes the most abundant protein found in mature CNS myelin. Expression of the gene is dynamic and peaks during the active myelination period of CNS development. The surge in Plp gene activity during this period has been purported to be mediated by a positive regulatory region located within the first intron. This region, designated ASE for antisilencer/enhancer, is located approximately 1 kb downstream of exon 1 sequences and encompasses nearly 100 bp. However, neither the critical nucleotides within this region, nor the associated DNA-binding proteins have been identified. In the present study, DNase I footprinting analysis demonstrated widespread protection of the region on both the coding and non-coding strands suggesting that multiple transcription factors are likely involved. Targeting of putative DNAprotein binding sites contained within the ASE by gel shift, transfection and mutagenesis studies revealed the importance of several AP-1-like binding sites in governing high levels of Plp gene expression in oligodendrocytes. Our results suggest that factors, which bind to these sites, form the core of a multiprotein complex that assembles on the ASE and ultimately affects the temporal regulation of the gene in oligodendrocytes.

A unique combined luminescence assay for firefly (Photinus pyralis) luciferase and beta-galactosidase (beta-gal) reporter gene products is described. Luciferase and beta-gal activities are determined with the same aliquot of cell lysate prepared from cells contransfected with both reporter genes, thereby reducing manual labor and increasing experimental accuracy. With the Dual-Light assay system, luciferase activity is measured first with an enhanced luciferase assay, followed by quantitation of beta-gal with Galacton-Plus chemiluminescent substrate and Sapphire-II enhancer. Highly sensitive detection of luciferase (2 fg) and beta-gal (8 fg) is achieved with a dynamic range over seven orders of magnitude of enzyme concentration. Comparative analysis of both independent and combined (Dual-Light) detection methods for cells contransfected with luciferase and beta-gal reporter genes is also described.

The myelin proteolipid protein (Plp) gene encodes the most abundant protein found in mature CNS myelin. Expression of the gene is regulated spatiotemporally, with maximal expression occurring in oligodendrocytes during the myelination period of CNS development. Plp gene expression is tightly controlled. Misregulation of the gene in humans can result in the dysmyelinating disorder Pelizaeus-Merzbacher disease, and in transgenic mice carrying a null mutation or extra copies of the gene can result in a variety of conditions, from late onset demyelination and axonopathy, to severe early onset dysmyelination. In this study we have examined the effects of Plp intron 1 DNA in mediating proper developmental expression of Plp-lacZ fusion genes in transgenic mice. Our results reveal the importance of Plp intron 1 sequences in instigating the expected surge in Plp-lacZ gene activity during (and following) the active myelination period of brain development. Transgene expression was also detected in the testis (Leydig cells), however, the presence or absence of Plp intron 1 sequences had no effect on the temporal profile in the testis. Surprisingly, expression of the transgene missing Plp intron 1 DNA was always higher in the testis, as compared to the brain, in all of the transgenic lines generated.