Xq22 deletions and correlation with distinct neurological disease traits in females: Further evidence for a contiguous gene syndrome.

Xq22 deletions that encompass PLP1 (Xq22-PLP1-DEL) are notable for variable expressivity of neurological disease traits in females ranging from a mild late-onset form of spastic paraplegia type 2 (MIM# 312920), sometimes associated with skewed X-inactivation, to an early-onset neurological disease trait (EONDT) of severe developmental delay, intellectual disability, and behavioral abnormalities. Size and gene content of Xq22-PLP1-DEL vary and were proposed as potential molecular etiologies underlying variable expressivity in carrier females where two smallest regions of overlap (SROs) were suggested to influence disease. We ascertained a cohort of eight unrelated patients harboring Xq22-PLP1-DEL and performed high-density array comparative genomic hybridization and breakpoint-junction sequencing. Molecular characterization of Xq22-PLP1-DEL from 17 cases (eight herein and nine published) revealed an overrepresentation of breakpoints that reside within repeats (11/17, ~65%) and the clustering of ~47% of proximal breakpoints in a genomic instability hotspot with characteristic non-B DNA density. These findings implicate a potential role for genomic architecture in stimulating the formation of Xq22-PLP1-DEL. The correlation of Xq22-PLP1-DEL gene content with neurological disease trait in female cases enabled refinement of the associated SROs to a single genomic interval containing six genes. Our data support the hypothesis that genes contiguous to PLP1 contribute to EONDT.

The wmN1 Enhancer Region of the Mouse Myelin Proteolipid Protein Gene (mPlp1) is Indispensable for Expression of an mPlp1-lacZ Transgene in Both the CNS and PNS.

The myelin proteolipid protein gene (PLP1) encodes the most abundant protein in CNS myelin. Expression of the gene must be strictly regulated, as evidenced by human X-linked leukodystrophies resulting from variations in PLP1 copy number, including elevated dosages as well as deletions. Recently, we showed that the wmN1 region in human PLP1 (hPLP1) intron 1 is required to promote high levels of an hPLP1-lacZ transgene in mice, using a Cre-lox approach. The current study tests whether loss of the wmN1 region from a related transgene containing mouse Plp1 (mPlp1) DNA produces similar results. In addition, we investigated the effects of loss of another region (ASE) in mPlp1 intron 1. Previous studies have shown that the ASE is required to promote high levels of mPlp1-lacZ expression by transfection analysis, but had no effect when removed from the native gene in mouse. Whether this is due to compensation by another regulatory element in mPlp1 that was not included in the mPlp1-lacZ constructs, or to differences in methodology, is unclear. Two transgenic mouse lines were generated that harbor mPLP(+)Z/FL. The parental transgene utilizes mPlp1 sequences (proximal 2.3 kb of 5'-flanking DNA to the first 37 bp of exon 2) to drive expression of a lacZ reporter cassette. Here we demonstrate that mPLP(+)Z/FL is expressed in oligodendrocytes, oligodendrocyte precursor cells, olfactory ensheathing cells and neurons in brain, and Schwann cells in sciatic nerve. Loss of the wmN1 region from the parental transgene abolished expression, whereas removal of the ASE had no effect.

The wmN1 enhancer region in intron 1 is required for expression of human PLP1.

The myelin proteolipid protein gene (PLP1) encodes the most abundant protein present in myelin from the central nervous system (CNS). Its expression must be tightly controlled as evidenced by mutations that alter PLP1 dosage; both overexpression (elevated PLP1 copy number) and lack thereof (PLP1 deletion) result in X-linked genetic disorders in man. However, not much is known about the mechanisms that govern expression of the human gene. To address this, transgenic mice were generated which utilize human PLP1 (hPLP1) sequences (proximal 6.2 kb of 5'-flanking DNA to the first 38 bp of exon 2) to drive expression of a lacZ reporter cassette. LoxP sites were incorporated around a 1.5-kb section of hPLP1 intron 1 since it contains sequence orthologous to the wmN1 region from mouse which, previously, was shown to augment expression of a minimally-promoted transgene coincident with the active myelination period of CNS development. Eight transgenic lines were generated with the parental, 6.2hPLP(+)Z/FL, transgene. All lines expressed the transgene appropriately in brain as evidenced by staining with X-gal in white matter regions and olfactory bulb. Removal of the "wmN1" region from 6.2hPLP(+)Z/FL with a ubiquitously expressed Cre-driver caused a dramatic reduction in transgene activity. These results demonstrate for the first time that the wmN1 enhancer region: (1) is functional in hPLP1; (2) works in collaboration with its native promoter-not just a basal heterologous promoter; (3) is required for high levels of hPLP1 gene activity; (4) has a broader effect, both spatially and temporally, than originally projected with mPlp1.

Targeted deletion of the antisilencer/enhancer (ASE) element from intron 1 of the myelin proteolipid protein gene (Plp1) in mouse reveals that the element is dispensable for Plp1 expression in brain during development and remyelination.

Myelin proteolipid protein gene (Plp1) expression is temporally regulated in brain, which peaks during the active myelination period of CNS development. Previous studies with Plp1-lacZ transgenic mice demonstrated that (mouse) Plp1 intron 1 DNA is required for high levels of expression in oligodendrocytes. Deletion-transfection analysis revealed the intron contains a single positive regulatory element operative in the N20.1 oligodendroglial cell line, which was named ASE (antisilencer/enhancer) based on its functional properties in these cells. To investigate the role of the ASE in vivo, the element was deleted from the native gene in mouse using a Cre/lox strategy. Although removal of the ASE from Plp1-lacZ constructs profoundly decreased expression in transfected oligodendroglial cell lines (N20.1 and Oli-neu), the element was dispensable to achieve normal levels of Plp1 gene expression in mouse during development (except perhaps at postnatal day 15) and throughout the remyelination period following cuprizone-induced (acute) demyelination. Thus, it is possible that the ASE is non-functional in vivo, or that loss of the ASE from the native gene in mouse can be compensated for by the presence of other regulatory elements within the Plp1 gene.

Plp1 in the enteric nervous system is preferentially expressed during early postnatal development in mouse as DM20, whose expression appears reliant on an intronic enhancer.

Recently, the myelin proteolipid protein gene (Plp1) was shown to be expressed in the glia of the enteric nervous system (ENS) in mouse. However, beyond this, not much is known about its expression in the intestine. To address this matter, we investigated Plp1 expression at the mRNA and protein levels in the intestine of mice at different ages (postnatal days 2, 9, 21, and 88). In this study, we show that Plp1 expression preferentially occurs during early postnatal development, primarily as the DM20 isoform. Western blot analysis indicated that DM20 migrated according to its formula weight when isolated from the intestine. However, mobilities of both PLP and DM20 were faster than expected when procured from the brain. The 6.2hPLP(+)Z/FL transgene, which uses the first half of the human PLP1 gene to drive expression of a lacZ reporter gene, recapitulated the developmental pattern observed with the native gene in the intestine, indicating that it can be used as a proxy for Plp1 gene expression. As such, the relative levels of ß-galactosidase (ß-gal) activity emanating from the 6.2hPLP(+)Z/FL transgene suggest that Plp1 expression is highest in the duodenum, and decreases successively along the segments, toward the colon. Moreover, removal of the wmN1 enhancer region from the transgene (located within Plp1 intron 1) resulted in a dramatic reduction in both transgene mRNA levels and ß-gal activity in the intestine, throughout development, suggesting that this region contains a regulatory element crucial for Plp1 expression. This is consistent with earlier studies in both the central and peripheral nervous systems, indicating that it may be a common (if not universal) means by which Plp1 gene expression is governed.

PLP1-lacZ transgenic mice reveal that splice variants containing "human-specific" exons are relatively minor in comparison to the archetypal transcript and that an upstream regulatory element bolsters expression during early postnatal brain development.

Much of what is known about the mechanisms that control the developmental expression of the myelin proteolipid protein gene (PLP1) has been attained through use of transgenic animal models. In this study, we analyzed expression of related transgenes which utilize PLP1 genomic DNA from either human or mouse to drive expression of a lacZ reporter. Human PLP1 (hPLP1) sequence span either the proximal 6.2 or 2.7 kb of 5'-flanking DNA to an internal site in Exon 2, while those from mouse comprise the proximal 2.3 kb of 5'-flanking DNA to an analogous site in Exon 2. Transgenes with hPLP1 sequence were named, in part, to the amount of upstream sequence they have [6.2hPLP(+)Z/FL and 2.7hPLP(+)Z]. The transgene containing mouse sequence is referred to here as mPLP(+)Z, to denote the species origin of PLP1 DNA. Mice which harbor the 6.2hPLP(+)Z/FL transgene were used as a model system to investigate the developmental expression of splice variants that incorporate supplementary exons from what is classically defined as PLP1 intron 1. While expression of the splice variants were detected in brain through RT-PCR analysis, they are present at much lower levels relative to the archetypal (classic) transcript. Additionally, we show that mice which harbor the 6.2hPLP(+)Z/FL transgene demonstrate wide-ranging expression throughout brain at P2, whereas expression of mPLP(+)Z is quite limited at this age. Therefore, we generated new transgenic mouse lines with the 2.7hPLP(+)Z transgene, which contains hPLP1 sequence orthologous to just that in mPLP(+)Z. Of the seven lines analyzed, six showed higher levels of 2.7hPLP(+)Z expression in brain at P21 compared to P2; the other line expressed the transgene, only weakly, at either age. This trend, coupled with the robust expression observed for 6.2hPLP(+)Z/FL at P2, suggests that the distal 3.5 kb of 5'-flanking PLP1 DNA specific to 6.2hPLP(+)Z/FL contains regulatory element(s) important for promoting early postnatal expression in brain.

Expression of myelin genes: comparative analysis of Oli-neu and N20.1 oligodendroglial cell lines.

The use of immortalized cells has been instrumental as a tool with which to study gene regulation. However, it is crucial to understand the status of a given cell line, especially when investigating the regulation of genes whose expression is developmentally regulated. Several immortalized cell lines have been derived from primary cultures of mouse oligodendrocytes. Two such cell lines, N20.1 and Oli-neu, were characterized here in terms of their relative expression of myelin genes at both the mRNA level and the protein level. Analysis of the splice isoforms expressed by the myelin proteolipid protein (Plp1), myelin basic protein (Mbp), and 2',3'-cyclic nucleotide 3'-phosphodiesterase (Cnp) genes, along with the relative amount of protein expressed by these genes, suggests that the cell lines are representative of immature oligodendrocytes, although Oli-neu cells appear to be farther along the differentiation pathway compared with N20.1 cells. Previous studies have shown that the developmental increase in Plp1 gene expression that occurs during the active myelination period is governed by transcription regulatory elements present within the first intron. The responsiveness of one of these elements, the so-called antisilencer/enhancer (ASE), was investigated in both cell lines. Results presented here suggest that the ASE has a much more potent effect in Oli-neu cells. Thus, the two cell lines appear to be at different stages and will be useful as a means to study transcription regulatory elements whose influence changes during development.

Ethanol modulation of hippocampal neuroinflammation, myelination, and neurodevelopment in a postnatal mouse model of fetal alcohol spectrum disorders.

Fetal alcohol spectrum disorders (FASD) are alarmingly common and result in significant personal and societal loss. Neuropathology of the hippocampus is common in FASD leading to aberrant cognitive function. In the current study, we evaluated the effects of ethanol on the expression of a targeted set of molecules involved in neuroinflammation, myelination, neurotransmission, and neuron function in the developing hippocampus in a postnatal model of FASD. Mice were treated with ethanol from P4-P9, hippocampi were isolated 24 h after the final treatment at P10, and mRNA levels were quantitated by qRT-PCR. We evaluated the effects of ethanol on both pro-inflammatory and anti-inflammatory molecules in the hippocampus and identified novel mechanisms by which ethanol induces neuroinflammation. We further demonstrated that ethanol decreased expression of molecules associated with mature oligodendrocytes and greatly diminished expression of a lacZ reporter driven by the first half of the myelin proteolipid protein (PLP) gene (PLP1). In addition, ethanol caused a decrease in genes expressed in oligodendrocyte progenitor cells (OPCs). Together, these studies suggest ethanol may modulate pathogenesis in the developing hippocampus through effects on cells of the oligodendrocyte lineage, resulting in altered oligodendrogenesis and myelination. We also observed differential expression of molecules important in synaptic plasticity, neurogenesis, and neurotransmission. Collectively, the molecules evaluated in these studies may play a role in ethanol-induced pathology in the developing hippocampus and contribute to cognitive impairment associated with FASD. A better understanding of these molecules and their effects on the developing hippocampus may lead to novel treatment strategies for FASD.

A Transgenic Mouse Model to Selectively Identify α3 Na,K-ATPase Expressing Cells in the Nervous System.

The α3 Na+,K+-ATPase (α3NKA) is one of four known α isoforms of the mammalian transporter. A deficiency in α3NKA is linked to severe movement control disorders. Understanding the pathogenesis of these disorders is limited by an incomplete knowledge of α3NKA expression in the brain as well as the challenges associated with identifying living cells that express the isoform for subsequent electrophysiological studies. To address this problem, transgenic mice were generated on the C57BL/6 genetic background, which utilize the mouse α3 subunit gene (Atp1a3) promoter to drive the expression of ZsGreen1 fluorescent protein. Consistent with published results on α3NKA distribution, a ZsGreen1 signal was detected in the brain, but not in the liver, with Atp1a3-ZsGreen1 transgenic mice. The intensity of ZsGreen1 fluorescence in neuronal cell bodies varied considerably in the brain, being highest in the brainstem, deep cerebellar and select thalamic nuclei, and relatively weak in cortical regions. Fluorescence was not detected in astrocytes or white matter areas. ZsGreen1-positive neurons were readily observed in fresh (unfixed) brain sections, which were amenable to patch-clamp recordings. Thus, the α3NKA-ZsGreen1 mouse model provides a powerful tool for studying the distribution and functional properties of α3NKA-expressing neurons in the brain.

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 Puralpha and Purbeta rank highest among the candidate factors as constituents of the multi-protein complex formed on the ASE.

Effects of Intron 1 Sequences on Human PLP1 Expression: Implications for PLP1-Related Disorders.

Alterations in the myelin proteolipid protein gene ( PLP1) may result in rare X-linked disorders in humans such as Pelizaeus-Merzbacher disease and spastic paraplegia type 2. PLP1 expression must be tightly regulated since null mutations, as well as elevated PLP1 copy number, both lead to disease. Previous studies with Plp1-lacZ transgenic mice have demonstrated that mouse Plp1 ( mPlp1) intron 1 DNA (which accounts for slightly more than half of the gene) is required for the mPlp1 promoter to drive significant levels of reporter gene expression in brain. However not much is known about the mechanisms that control expression of the human PLP1 gene ( hPLP1). Therefore this review will focus on sequences in hPLP1 intron 1 DNA deemed important for hPLP1 gene activity as well as a couple of "human-specific" supplementary exons within the first intron which are utilized to generate novel splice variants, and the potential role that these sequences may play in PLP1-linked disorders.

Control of human PLP1 expression through transcriptional regulatory elements and alternatively spliced exons in intron 1.

Although the myelin proteolipid protein gene (PLP1) encodes the most abundant protein in central nervous system (CNS) myelin, not much is known about the mechanisms that govern expression of the human gene (hPLP1). Much more is known about the processes that regulate Plp1 gene expression in rodents. From studies with Plp1-lacZ transgenic mice, it was determined that the first intron of mouse Plp1 (mPlp1) is required to attain high levels of expression in brain, concurrent with the active myelination period. Other studies have suggested that within mPlp1 intron 1 (>8 kb) lie several regions with enhancer-like activity. To test whether these sequences (and possibly others) in hPLP1 intron 1 are functional, deletion-transfection analysis was performed with hPLP1-lacZ constructs that contain various portions of the intron, or lack it altogether. Results presented here demonstrate the importance of hPLP1 intron 1 in achieving maximal levels of expression in the immortalized oligodendroglial cell line, Oli-neu. Deletion analysis indicates that the intron contains multiple positive regulatory elements which are active in Oli-neu cells. Some of these elements appear to be functionally conserved between human and mouse, while others are not. Furthermore, our studies demonstrate that multiple splice variants can be formed due to inclusion of extra (supplementary) exons from what is classically thought of as hPLP1 intron 1. Thus, splicing of these novel exons (which are not recognized as such in mPlp1 due to lack of conserved splice sites) must utilize factors common to both human and mouse since Oli-neu cells are of mouse origin.

Enhanced gastrointestinal expression of cytosolic malic enzyme (ME1) induces intestinal and liver lipogenic gene expression and intestinal cell proliferation in mice.

The small intestine participates in lipid digestion, metabolism and transport. Cytosolic malic enzyme 1 (ME1) is an enzyme that generates NADPH used in fatty acid and cholesterol biosynthesis. Previous work has correlated liver and adipose ME1 expression with susceptibility to obesity and diabetes; however, the contributions of intestine-expressed ME1 to these conditions are unknown. We generated transgenic (Tg) mice expressing rat ME1 in the gastrointestinal epithelium under the control of the murine villin1 promoter/enhancer. Levels of intestinal ME1 protein (endogenous plus transgene) were greater in Tg than wildtype (WT) littermates. Effects of elevated intestinal ME1 on body weight, circulating insulin, select adipocytokines, blood glucose, and metabolism-related genes were examined. Male Tg mice fed a high-fat (HF) diet gained significantly more body weight than WT male littermates and had heavier livers. ME1-Tg mice had deeper intestinal and colon crypts, a greater intestinal 5-bromodeoxyuridine labeling index, and increased expression of intestinal lipogenic (Fasn, Srebf1) and cholesterol biosynthetic (Hmgcsr, Hmgcs1), genes. The livers from HF diet-fed Tg mice also exhibited an induction of cholesterol and lipogenic pathway genes and altered measures (Irs1, Irs2, Prkce) of insulin sensitivity. Results indicate that gastrointestinal ME1 via its influence on intestinal epithelial proliferation, and lipogenic and cholesterologenic genes may concomitantly impact signaling in liver to modify this tissue's metabolic state. Our work highlights a new mouse model to address the role of intestine-expressed ME1 in whole body metabolism, hepatomegaly, and crypt cell proliferation. Intestinal ME1 may thus constitute a therapeutic target to reduce obesity-associated pathologies.

YY1 negatively regulates mouse myelin proteolipid protein (Plp1) gene expression in oligodendroglial cells.

YY1 (Yin and Yang 1) is a multifunctional, ubiquitously expressed, zinc finger protein that can act as a transcriptional activator, repressor, or initiator element binding protein. Previous studies have shown that YY1 modulates the activity of reporter genes driven by the myelin PLP (proteolipid protein) (PLP1/Plp1) promoter. However, it is known that Plp1 intron 1 DNA contains regulatory elements that are required for the dramatic increase in gene activity, coincident with the active myelination period of CNS (central nervous system) development. The intron in mouse contains multiple prospective YY1 target sites including one within a positive regulatory module called the ASE (anti-silencer/enhancer) element. Results presented here demonstrate that YY1 has a negative effect on the activity of a Plp1-lacZ fusion gene [PLP(+)Z] in an immature oligodendroglial cell line (Oli-neu) that is mediated through sequences present in Plp1 intron 1 DNA. Yet YY1 does not bind to its alleged site in the ASE (even though the protein is capable of recognizing a target site in the promoter), indicating that the down-regulation of PLP(+)Z activity by YY1 in Oli-neu cells does not occur through a direct interaction of YY1 with the ASE sequence. Previous studies with Yy1 conditional knockout mice have demonstrated that YY1 is essential for the differentiation of oligodendrocyte progenitors. Nevertheless, the current study suggests that YY1 functions as a repressor (not an activator) of Plp1 gene expression in immature oligodendrocytes. Perhaps YY1 functions to keep the levels of PLP in check in immature cells before vast quantities of the protein are needed in mature myelinating oligodendrocytes.

Leydig cells express the myelin proteolipid protein gene and incorporate a new alternatively spliced exon.

Although the myelin proteolipid protein gene (Plp1) is highly expressed in the central nervous system encoding the most abundant myelin protein in oligodendrocytes, it is also expressed in other tissues, including testis. Transgenic studies with mice that harbor Plp1-lacZ fusion genes suggest that Leydig cells are the source of Plp1 gene expression in testis. However, virtually nothing is known about Plp1 gene regulation in Leydig cells, which is the focus of this study. The first intron contains both positive and negative regulatory elements that are important in regulating Plp1 gene expression in oligodendrocytes. To test whether these elements are functional in Leydig cells, a battery of Plp1-lacZ fusion genes with partial deletion of Plp1 intron 1 sequence was transfected into the mouse Leydig cell line, TM3. Results presented here suggest that an enhancer, which is very potent in oligodendrocytes, is only nominally active in TM3 cells. The intron also contains several negative regulatory elements that are operative in TM3 cells. Moreover a new exon (exon 1.2) was identified within the first 'intron' resulting in novel splice variants in TM3 cells. Western blot analysis suggests that these splice variants, along with those containing another alternatively spliced exon (exon 1.1) derived from intron 1 sequence, give rise to multiple Plp1 gene products in the mouse testis.

Expression of a myelin proteolipid protein (Plp)-lacZ transgene is reduced in both the CNS and PNS of Plp(jp) mice.

Jimpy (Plp(jp)) is an X-linked recessive mutation in mice that causes CNS dysmyelination and early death in affected males. It results from a point mutation in the acceptor splice site of myelin proteolipid protein (Plp) exon 5, producing transcripts that are missing exon 5, with a concomitant shift in the downstream reading frame. Expression of the mutant PLP product in Plp(jp) males leads to hypomyelination and oligodendrocyte death. Expression of our Plp-lacZ fusion gene, PLP(+)Z, in transgenic mice is an excellent readout for endogenous Plp transcriptional activity. The current studies assess expression of the PLP(+)Z transgene in the Plp(jp) background. These studies demonstrate that expression of the transgene is decreased in both the central and peripheral nervous systems of affected Plp(jp) males. Thus, expression of mutated PLP protein downregulates Plp gene activity both in oligodendrocytes, which eventually die, and in Schwann cells, which are apparently unaffected in Plp(jp) mice.

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 (approximately 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.

Potentiation of myelin proteolipid protein (Plp) gene expression is mediated through AP-1-like binding sites.

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 DNA-protein 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.

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.

Repression of myelin proteolipid protein gene expression is mediated through both general and cell type-specific negative regulatory elements in nonexpressing cells.

The myelin proteolipid protein gene (Plp ) is expressed primarily in oligodendrocytes. Yet how the gene remains repressed in nonexpressing cells has not been defined, and potentially could cause adverse effects in an organism if the mechanism for repression was impaired. Previous studies suggest that the first intron contains element(s), which suppress expression in nonexpressing cells, although the identity of these elements within the 8 kb intron was not characterized. Here we report the localization of multiple negative regulatory elements that repress Plp gene expression in nonexpressing cells (+/+ Li). Two of these elements (regions) correspond to those used by Plp expressing cells (N20.1), whilst another acts in a cell type-specific manner (i.e. operational in +/+ Li liver cells, but not N20.1 cells). By gel-shift and DNase I footprinting analyses, the factor(s) that bind to the cell type-specific negative regulatory region appear to be far more abundant in +/+ Li cells than in N20.1 cells. Thus, Plp gene repression is mediated through the combinatorial action of both "general" and cell type-specific negative regulatory elements. Additionally, repression in +/+ Li cells cannot be overcome via an antisilencer/enhancer element, which previously has been shown to function in N20.1 cells.