Elucidation of Mechanisms Controlling Human and Mouse Myelin PLP1 Gene Expression

项目总结/摘要 髓磷脂蛋白脂质蛋白（PLP）的准确表达是必不可少的，正如髓磷脂蛋白脂质蛋白（PLP）突变的事实所说明的那样。 PLP 1导致Pelizaeus-Merzbacher病（PMD）或痉挛性截瘫2型（SPG 2），它们是X- 连锁脑白质营养不良突变包括PLP 1的重复和缺失，这表明需要对PLP 1基因进行严格的测序。 PLP 1基因表达的调节控制;然而，关于这一点的机制知之甚少。 调控我们已经产生了PLP 1-lacZ转基因小鼠，其中PLP 1基因的前半部分来自 人（hPLP 1）或小鼠（mPlp 1）用于驱动lacZ报告基因的表达以研究PLP 1 基因调控我们的数据表明，PLP 1内含子1的一部分，称为wmN 1区，是必需的， 基于人和小鼠的PLP 1-lacZ转基因的大量表达。因此， 在不明原因的病例中，wmN 1增强子区域的活性可能是PMD/SPG 2的根本原因; 患有PMD/SPG 2的男性在PLP 1基因剂量或编码序列上没有改变，这表明 突变可能发生在不被常规分析的基因区域（例如，内含子、启动子）。有趣的是， 基于小鼠的PLP 1-lacZ转基因的表达在发育中比hPLP 1转基因的表达晚 序列的这是否是由于缺乏一个重要的调控元件，在小鼠为基础的转基因，因为它 包含相当少的5′-侧翼PLP 1 DNA，或真正的物种差异需要解决。 最近，在hPLP 1内含子1中鉴定了两个外显子，当它们被整合时，导致“人类特异性”的表达。 剪接变体。关于这些剪接变异体知之甚少，因为补充外显子只是最近才被发现。 发现了值得注意的是，这些外显子被我们的hPLP 1-lacZ转基因小鼠利用;因此我们的转基因小鼠 构成了第一个（也是唯一的）可用于研究“人类特异性”剪接变体的动物模型。基于 根据我们的初步数据，我们假设wmN 1区域对于PLP 1的表达是必需的， “人特异性”剪接异构体的表达主要发生在早期发育期间。目标1将 使用PLP 1-lacZ小鼠来确定表达转基因的细胞类型，wmN 1区域是否 在髓鞘再生期间PLP 1表达所需的，如果去除大部分PLP 1内含子1 DNA， 除了wmN 1区域的单独损失之外，转基因的任何其他影响。删除wmN 1 在人类iPSC和小鼠中，来自天然基因的区域将决定增强子是否是必需的， PLP 1的表达。目的2将确定wmN 1区域增强子活性的来源及其重要性。 通过缺失-转染和突变分析靶向位点。增强子的同源因子将被 通过蛋白质-DNA相互作用研究鉴定。我们将测序hPLP 1内含子1和启动子的DNA， 从不明原因的PMD/SPG 2患者中识别非编码区可能的突变 (e.g., wmN1）。目的3将确定基于人的转基因的早期表达是否是由于 额外的3.5 kb的5′侧翼PLP 1 DNA或“人类特异性”剪接变体的表达。

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.