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 (a ntis ilencer/e nhancer) 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.     

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.

     

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.