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Elucidation of Mechanisms Controlling Human and Mouse Myelin PLP1 Gene Expression

阐明控制人和小鼠髓磷脂 PLP1 基因表达的机制

基本信息

批准号：
10599083
负责人：
Patricia A. Wight
金额：
$ 32.2万
依托单位：
UNIV OF ARKANSAS FOR MED SCIS
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-04-01 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10599083
关键词：
Animal Model Binding Biological Assay Brain CRISPR/Cas technology Code Compensation DNA DNA Sequence DNA sequencing DNA-Protein Interaction Data Development Disease Distal EMSA Elements Enhancers Enzymes Excision Exons Funding Gene Dosage Gene Expression Gene Expression Regulation Genes Histocytochemistry Human In Situ Hybridization Introns LacZ Genes Link Mediating Messenger RNA Mus Mutation Mutation Analysis Myelin Myelin Proteolipid Protein Patients Pelizaeus-Merzbacher Disease Perinatal Positioning Attribute Protein Isoforms Protein Overexpression Proteolipids Quantitative Reverse Transcriptase PCR RNA Splicing Regulation Regulatory Element Reporter Reporter Genes Site Spastic Paraplegia Stains Testing Time Transfection Transgenes Transgenic Mice United States National Institutes of Health Untranslated RNA Variant cell type dosage gene product genome editing induced pluripotent stem cell leukodystrophy link protein male overexpression perinatal period postnatal promoter remyelination spatiotemporal species difference

项目摘要

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.

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。 PLP1导致Pelizaeus-Merzbacher病（PMD）或痉挛性截瘫2型（SPG2），它们是X- 连锁脑白质营养不良突变包括PLP1的重复和缺失，这表明需要对PLP1基因进行严格的测序。 PLP1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PLP1-lacZ转基因小鼠，其中PLP1基因的前半部分来自人（hPLP1）或小鼠（mPlp1）用于驱动lacZ报告基因的表达以研究PLP1 基因调控我们的数据表明，PLP1内含子1的一部分，称为wmN1区，是必需的，基于人和小鼠的PLP1-lacZ转基因的大量表达。因此，在不明原因的病例中，wmN1增强子区域的活性可能是PMD/SPG2的根本原因; 患有PMD/SPG2的男性在PLP1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PLP1-lacZ转基因的表达在发育中比hPLP1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PLP1 DNA，或真正的物种差异需要解决。最近，在hPLP1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。由于补充外显子最近才出现，因此人们对这些剪接变体知之甚少发现了值得注意的是，这些外显子被我们的hPLP1-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wmN1区域对于PLP1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PLP1-lacZ小鼠来确定表达转基因的细胞类型，wmN1区域是否在髓鞘再生期间PLP1表达所需的，如果去除大部分PLP1内含子1 DNA，除了wmN1区域的单独损失之外，转基因的任何其他影响。删除wmN1 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， PLP1的表达。目的2将确定wmN1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hPLP1内含子1和启动子的DNA，从不明原因的PMD/SPG2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PLP1 DNA或"人类特异性"剪接变体的表达。