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

Abstract

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.

Separate Proteolipid Protein/DM20 Enhancers Serve Different Lineages and Stages of Development

“…ASE is not prominently conserved but is part of a SINE repeating element. Although it has been shown to confer enhancer activity in the context of reporter constructs transfected into oligodendroglial cell lines (Meng et al, 2005), constructs containing the ASE2538 and ASE300 sequences did not express in the oligodendrocytes of the transgenic mice derived here. Rather, both ASE2538 and ASE300 conferred postnatal expression to a subpopulation of spinal cord cells concentrated in the lumbar enlargement near the central canal in ventrolateral gray matter with mice bearing the longer sequence Figure 4.…”

“…ASE contains several AP-1 like binding sites (Dobretsova et al, 2004), differentially binds nuclear proteins from diverse cell types, and functions in reporter constructs with both the plp/DM20 proximal promoter, and, in a position-dependent manner, with a thymidine kinase (TK ) promoter when transfected into a plp/DM20-expressing oligodendrocyte cell line (Meng et al, 2005). In contrast, neither the ASE2538 nor ASE300 constructs expressed in oligodendrocytes, Schwann cells, satellite cells, or OECs in transgenic mice.…”

“…However, the location and function of plp/DM20 enhancers remain essentially unknown. Here we interrogated the in vivo regulatory potential of multiple plp/ DM20 sequences containing evolutionarily conserved nonprotein-coding modules as well as the anti-silencer enhancer (ASE) sequence shown previously to have in vitro regulatory activity (Wight et al, 1997;Meng et al, 2005).…”

Expression of a Myelin Proteolipid Protein (Plp)-lacZ Transgene is Reduced in both the CNS and PNS of Plp jp Mice

“…The transgene contains Plp genomic DNA extending from the proximal 2.4 kb of 5′-flanking DNA, downstream to the first 37 bp of exon 2, which were used to drive expression of a lacZ reporter gene cassette. Thus the transgene contains the Plp promoter as well as a tissue-specific enhancer located in Plp intron 1 DNA [24,25]. Expression of the PLP(+)Z transgene was evaluated in affected Plp jp males, which express a mutated Plp gene (Plp jp ), and compared to that produced in wild-type male littermates containing the normal Plp gene.…”

A tyrosine hydroxylase–yellow fluorescent protein knock-in reporter system labeling dopaminergic neurons reveals potential regulatory role for the first intron of the rodent tyrosine hydroxylase gene

“…Intron sequences have been shown to contain regulatory elements (Galvagni and Oliviero, 2000;Karadsheh and Delpire, 2001;Hermann and Heckert, 2005;Lee and Friedman, 2005;Meng et al, 2005), and the difference in this sequence could explain the differences in expression pattern between the YFP and TH alleles in our knock-in system. Indeed, we identified TH-expressing cellspecific DHSs in the first intronic sequence of the rat TH gene, which is highly homologous to the mouse sequence.…”