Elucidation of Mechanisms Controlling Human and Mouse Myelin PLP1 Gene Expression

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PROJECT SUMMARY/ABSTRACT Accurate expression of myelin proteolipid protein (PLP) is essential as illustrated by the fact that mutations in PLP1 result in either Pelizaeus-Merzbacher disease (PMD) or spastic paraplegia type 2 (SPG2), which are X- linked leukodystrophies. Mutations include duplications and deletions of PLP1, indicating the need for stringent regulatory control of PLP1 gene expression; however, relatively little is known about the mechanisms of this regulation. We have generated PLP1-lacZ transgenic mice, in which the first half of the PLP1 gene from either human (hPLP1) or mouse (mPlp1) is used to drive expression of the lacZ reporter gene to investigate PLP1 gene regulation. Our data indicate that a portion of PLP1 intron 1, called the wmN1 region, is required for substantial expression of the human- and mouse-based PLP1-lacZ transgenes. Hence, mutations that disrupt activity of the wmN1 enhancer region may be the root cause of PMD/SPG2 in unexplained cases; ~20% of males with PMD/SPG2 do not have alterations in PLP1 gene dosage or in the coding sequence, suggesting that mutations may occur in gene regions that are not routinely analyzed (e.g., introns, promoter). Intriguingly, expression of the mouse-based PLP1-lacZ transgene occurred later in development than the one with hPLP1 sequences. Whether this is due to lack of an important regulatory element in the mouse-based transgene as it contains considerably less 5′-flanking PLP1 DNA, or to a true species difference needs to be resolved. Recently, two exons were identified in hPLP1 intron 1, which when incorporated, result in “human-specific” splice variants. Little is known about these splice variants as the supplementary exons were only recently discovered. Notably, these exons are utilized by our hPLP1-lacZ transgenic mice; thus our transgenic mice constitute the first (and only) animal model available to investigate the “human-specific” splice variants. Based on our preliminary data, we hypothesize that the wmN1 region is essential for PLP1 expression and that expression of the “human-specific” splice isoforms occurs predominantly during early development. Aim 1 will use PLP1-lacZ mice to determine the cell types that express the transgene, whether the wmN1 region is required for PLP1 expression during times of remyelination, and if the removal of most of PLP1 intron 1 DNA from the transgene has any additional effects beyond the loss of the wmN1 region alone. Deletion of the wmN1 region from the native gene in human iPSCs, and in mouse, will determine whether the enhancer is essential for PLP1 expression. Aim 2 will determine the origin of enhancer activity in the wmN1 region and its important target sites by deletion-transfection and mutational analyses. The enhancer’s cognate factors will then be identified with protein-DNA interaction studies. We will sequence the DNA of hPLP1 intron 1 and the promoter from deidentified patients with unexplained PMD/SPG2 to identify possible mutations in noncoding regions (e.g., wmN1). Aim 3 will determine whether the early expression noted with human-based transgene is due to the extra 3.5 kb of 5′-flanking PLP1 DNA or expression of “human-specific” splice variants.