Elucidating an Xist-dependent program of sexually dimorphic alternative splicing in the mammalian brain

阐明哺乳动物大脑中依赖于 Xist 的性二态选择性剪接程序

• 批准号: 9305157
• 负责人: Douglas L Black
• 金额: $ 64.17万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9305157
• 关键词: Affect; Alleles; Alternative Splicing; Automobile Driving; Binding; Binding Sites; Biological Assay; Brain; Cells; Computing Methodologies; Data; Data Set; Databases; Dependence; Development; Disease; Event; Excision; Exhibits; Female; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genes; Genetic; Genetic Determinism; Genetic Transcription; Genotype; Genotype-Tissue Expression Project; Gonadal Steroid Hormones; Hormone use; Human; Immunoprecipitation; Knock-out; Measurement; Measures; Mental disorders; Messenger RNA; Methods; Molecular; Mus; Neuronal Differentiation; Neurons; Pattern; Polypyrimidine Tract-Binding Protein; Predisposition; Proteins; RNA; RNA Splicing; RNA-Binding Proteins; Regulation; Reverse Transcriptase Polymerase Chain Reaction; Role; Source; Structure; Tissue-Specific Gene Expression; Tissues; Transcript; Transgenic Mice; Transgenic Organisms; Untranslated RNA; Woman; X Chromosome; X Inactivation; base; brain tissue; crosslink; dimorphism; gene function; genome-wide; mRNA Precursor; male; men; mutant; nervous system disorder; neuropsychiatry; prediction algorithm; programs; relating to nervous system; sexual dimorphism; tool; transcriptome; transcriptome sequencing

PROJECT SUMMARY Women and men are well known to have different propensities to neuropsychiatric illness, but the source of these differences is not understood. In particular, the molecular events that determine functional dimorphism between the female and male brain need to be defined. We will examine how the female-specific long noncoding RNA Xist and its newly identified interaction with the pre-mRNA splicing regulators PTBP1 and 2 affect gene expression and alternative splicing in the female brain. The project will use expertise and tools developed in three labs for the study of Xist RNA, of neuronal splicing regulation by the PTB proteins, and of gene expression and alternative splicing using computational methods. RNA-seq data from defined regions of both human and mouse brain will be analyzed using the new rMATS analysis tool to create a large statistically robust database of differential gene expression and alternative pre-mRNA splicing between males and females. Expression and splicing changes will be correlated with changes in PTBP1/2 mRNA and Xist across the same datasets to define genes potentially regulated by these molecules at the transcriptional and post- transcriptional levels. Female specific patterns of expression and splicing caused by the XX genotype will be distinguished from events driven by female hormones using four core genotype mice. Xist targeting will be confirmed using conditional Xist alleles that allow either removal or activation of Xist during brain development and measurement of the resulting changes in splicing. The expression of Xist relative to PTBP2 will be quantified over neuronal differentiation in culture. The PTBP targeting of Xist-dependent changes in splicing will be confirmed in PTBP2 knockout and PTBP1 transgenic mice, and by transcriptome-wide binding analyses by iCLIP. Alternative splicing is a widespread mechanism of gene regulation, but has been only minimally examined in relation to the XX genotype of female cells. Using sophisticated new genome-wide methods and molecular tools, these studies promise to identify new genetic determinants of sexual dimorphism in the mammalian brain and to elucidate their underlying molecular mechanisms. In the longer term, the identified molecular changes driven by Xist and PTBP will provide entrée to the examination of the functional consequences of these dimorphisms.

项目总结 众所周知，女性和男性患神经精神疾病的倾向不同，但 这些差异是不被理解的。特别是，决定功能二态的分子事件 女性和男性大脑之间的关系需要界定。我们将考察女性特有的长时间 非编码RNA Xist及其与前mRNA剪接调控因子PTBP1和2的相互作用 影响女性大脑中的基因表达和选择性剪接。该项目将使用专业知识和工具 由三个实验室开发，用于研究Xist RNA，研究PTB蛋白对神经元剪接的调节，以及 使用计算方法进行基因表达和选择性剪接。来自定义区域的RNA-SEQ数据 人类和小鼠的大脑都将使用新的rMATS分析工具进行分析，以创建一个大型统计 雄性和雄性之间差异基因表达和替代前-mRNA剪接的强大数据库 女性。表达和剪接的变化将与PTBP1/2mRNA和Xist的变化相关 相同的数据集来定义在转录和转录后可能受这些分子调控的基因。 转录水平。由XX基因引起的女性特有的表达和剪接模式将是 区别于由雌性荷尔蒙驱动的事件，使用四个核心基因型鼠。XIST目标将是 证实使用了条件Xist等位基因，允许在大脑发育期间移除或激活Xist 以及对由此产生的剪接变化的测量。Xist相对于PTBP2的表达式将为 在培养中对神经元分化进行了量化。PTBP靶向Xist依赖的剪接改变 将在PTBP2基因敲除和PTBP1转基因小鼠中得到证实，并通过转录组范围的结合分析得到证实 由iCLIP提供。选择性剪接是基因调控的一种广泛的机制，但还只是最低限度的。 检查与雌性细胞的XX基因型的关系。使用复杂的全基因组新方法和 分子工具，这些研究有望识别新的性别二型性决定因素 并阐明其潜在的分子机制。从长远来看，已确定的 XIST和PTBP驱动的分子变化将为功能的研究提供入口 这些二态现象的后果。