Patricia A. Wight

Mutations in the Myelin Proteolipid Protein Gene Alter Oligodendrocyte Gene Expression in Jimpy and Jimpymsd Mice

Abstract: The mouse myelin proteolipid protein (PLP) gene has been studied in normal and jimpymsd mice. Potential upstream regulatory regions of the normal gene have been cloned and mapped, but when these regions were studied in jimpymsd mice by Southern blots, no alterations were observed, relative to the normal gene. To assess whether the low ratio of PLP to DM20 proteins in this mutant reflected an altered PLP/DM20 ratio mRNAs, S1 nuclease analyses were undertaken, which demonstrated that at all ages studied in both jimpy and jimpymsd mice, PLP mRNA was elevated above DM20 mRNA. When exon 3 (the site of the alternative splice signal for DM20 mRNA) of the jimpymsd PLP gene was sequenced, no mutation was identified. The transcription of the PLP gene in normal and mutant animals was studied. The transcription rate increases in normal animals with development, and in very young jimpymsd or jimpy mice, the transcription rate of the PLP gene was close to that of agematched normal animals. However, by 10 days of age, the transcription rate of this gene in both mutants was significantly below that of age‐matched controls. The transcription rate of the myelin basic protein (MBP) gene was also reduced, indicating that expression of both genes is affected by this mutation. In contrast, the transcription rate of the glycerol phosphate dehydrogenase (GPDH) gene, an early marker of oligodendrocytes, is equal to or greater than normal in both mutants. We have confirmed an earlier report of a point mutation in exon 6 of the jimpymsd PLP gene, which converts an alanine to a valine. This mutation apparently alters oligodendrocyte metabolism such that the cell can differentiate to express early oligodendrocyte genes such as GPDH, but it cannot differentiate to a stage where it expresses the PLP and MBP genes at normal high levels.

The first intron of the myelin proteolipid protein gene confers cell type-specific expression by a transcriptional repression mechanism in non-expressing cell types

Chimeric genes containing portions of the mouse myelin proteolipid protein (PLP) gene fused to the lacZ reporter gene were used to detect the effect of PLP intron 1 sequences on cell type-specific expression. A transfected fusion gene containing PLP intron 1 sequences was expressed in an oligodendrocyte cell line but not in a liver cell line, consistent with endogenous PLP gene expression. However, an analogous fusion gene missing the first intron was expressed in either oligodendrocyte or liver transfected cells. These studies suggest that transcriptional repressor element(s) located in PLP intron 1 are important in extinguishing expression in non-glial cell types and that the promoter alone functions in an indiscriminate manner. This moderately large intron (>8 kb) was sequenced to aid in future fine mapping of these cell-specific regulatory element(s).

Where, when and how much: regulation of myelin proteolipid protein gene expression

The myelin proteolipid protein (PLP) gene (Plp) encodes the most abundant protein found in myelin from the central nervous system (CNS). Expression of the gene is regulated in a spatiotemporal manner with maximal levels of expression occurring in oligodendrocytes during the active myelination period of CNS development, although other cell types in the CNS as well as in the periphery can express the gene to a much lower degree. In oligodendrocytes, Plp gene expression is tightly regulated. Underexpression or overexpression of the gene has been shown to have adverse effects in humans and other vertebrates. In light of this strict control, this review provides an overview of the current knowledge of Plp gene regulation.

Antisilencing: myelin proteolipid protein gene expression in oligodendrocytes is regulated via derepression.

Abstract: Antisilencer or antirepressor elements have been described, thus far, for only a few eukaryotic genes and were identified by their ability not to augment gene expression per se but to override repression mediated via negative transcription regulatory elements. Here we report the first case of antisilencing for a neural‐specific gene, the myelin proteolipid protein (PLP) gene (Plp). PLP is the most abundant protein found in CNS myelin. The protein is synthesized in oligodendrocytes, and its expression is regulated developmentally. Previously we have shown that a PLP‐lacZ transgene (which includes the entire sequence for Plp intron 1) is regulated in mice, in a manner consistent with the spatial and temporal expression of the endogenous Plp gene. In the present report, we demonstrate by transfection analyses, using various PLP‐lacZ deletion constructs, that Plp intron 1 DNA contains multiple elements that collectively regulate Plp gene expression in oligodendrocytes. One of these regulatory elements functions as an antisilencer element, which acts to override repression mediated by at least two negative regulatory elements located elsewhere within Plp intron 1 DNA. The mechanism for antisilencing appears to be complex as the intragenic region that mediates this function binds multiple nuclear factors specifically.

Characterization of an intronic enhancer that regulates myelin proteolipid protein (Plp) gene expression in oligodendrocytes

The myelin proteolipid protein (Plp) gene is expressed in oligodendrocytes and encodes the most abundant protein (∼50%) present in mature myelin from the central nervous system (CNS). Plp gene activity is low to nonexistent early in development but sharply increases, concurrently with the active myelination period of CNS development. Work from our laboratory suggests that the temporal regulation of Plp gene expression in mice is mediated by a positive regulatory element located within Plp intron 1 DNA. We have termed this regulatory element/region ASE (for antisilencer/enhancer). The ASE is situated approximately 1 kb downstream of exon 1 DNA and encompasses nearly 100 bp. To understand the mechanisms by which the ASE augments Plp gene expression in oligodendrocytes, Plp‐lacZ constructs were generated and transfected into a mouse oligodendroglial cell line (N20.1). Results presented here demonstrate that upstream regulatory elements in the Plp promoter/5′‐flanking DNA are not required for ASE activity; the ASE worked perfectly well when the thymidine kinase (TK) promoter was substituted for the Plp promoter. However, the relative location of the ASE appears to be important. When placed upstream of 2.4 kb of Plp 5′‐flanking DNA, or downstream of the lacZ expression cassette, the ASE was no longer effective. Thus, the ASE might have to be in the context of the intron in order to function. To begin to identify the crucial nucleotides within the ASE, orthologous sequences from rat, human, cow, and pig Plp genes were swapped for the mouse sequence. Results presented here demonstrate that the orthologous sequence from rat can substitute for the mouse ASE, unlike those from human, cow, or pig.

Regulation of murine myelin proteolipid protein gene expression

To identify putative sequences that direct cell type‐specific expression and/or enhance proteolipid protein (PLP) gene expression, glial or nonglial cells were transfected with various PLP‐luciferase constructs that collectively span the entire mouse PLP‐specific DNA present in a transgene known to direct cell type specificity in transgenic mice. These constructs were transfected into murine oligodendrocyte cell lines that transcribe the PLP gene and, hence, should contain the requisite trans‐acting factors necessary for PLP gene expression. Mouse NIH/3T3 fibroblasts were used as a nonglial model. We have finely mapped the PLP promoter region for transcriptional regulatory elements and demonstrate both positive and negative elements, none of which appear to extinguish expression in nonglial cells. The 5′‐flanking PLP DNA tested did not enhance the basal herpes simplex‐1 virus thymidine kinase (TK) promoter, nor did PLP sequences present in the distal half of intron 1. The 5′ portion of intron 1 did enhance TK promoter activity, suggesting that this region of the gene may contain enhancer elements that modulate PLP gene expression; however, the enhancement did not appear to be cell type‐specific. Intriguingly, a 541 bp region of the intron that significantly enhanced TK promoter activity contains multiple JC virus repeated elements and other elements known to be important in restricting the virus to oligodendrocytes. These results suggest that intron 1 sequences may modulate expression of the PLP gene. J. Neurosci. Res. 50:917–927, 1997.

Myelin proteolipid protein (Plp) intron 1 DNA is required to temporally regulate Plp gene expression in the brain

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.

Functional Characterization of a cis-Acting DNA Antisilencer Region that Modulates Myelin Proteolipid Protein Gene Expression

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 over‐coming 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 β‐galactosidase activity when inserted into Plp‐lacZ constructs, which originally exhibited low levels of β‐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.

Proteomic analysis of nuclear factors binding to an intronic enhancer in the myelin proteolipid protein gene

The myelin proteolipid protein gene (Plp1) encodes the most abundant protein found in CNS myelin, accounting for nearly one‐half of the total protein. Its expression in oligodendrocytes is developmentally regulated – peaking during the active myelination period of CNS development. Previously, we have identified a novel enhancer (designated ASE) in intron 1 DNA that appears to be important in mediating the surge of Plp1 gene activity during the active myelination period. Evidence suggests that the ASE participates in the formation of a specialized multi‐protein/DNA complex called an enhanceosome. The current study describes an optimized, five‐step, DNA affinity chromatography purification procedure to purify nuclear proteins from mouse brain that bind to the 85‐bp ASE sequence, specifically. Electrophoretic mobility shift assay analysis demonstrated that specific DNA‐binding activity was retained throughout the purification procedure, resulting in concomitant enrichment of nucleoprotein complexes. Identification of the purported regulatory factors was achieved through mass spectrometry analysis and included over 20 sequence‐specific DNA‐binding proteins. Supplementary western blot analyses to determine which of these sequence‐specific factors are present in oligodendrocytes, and their developmental and regional expression in whole brain, suggest that Purα and Purβ rank highest among the candidate factors as constituents of the multi‐protein complex formed on the ASE.

Myelin proteolipid protein (Plp) intron 1 DNA is required to temporally regulate Plp gene expression in the brain: Temporal regulation of Plp-lacZ transgenes

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, ascompared to the brain, in all of the transgenic lines generated.