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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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要众所周知，女性和男性患神经精神疾病的倾向不同，但神经精神疾病的根源这些差异不被理解。特别是，决定功能二态性的分子事件女性和男性大脑之间的界限需要明确。我们将研究女性特有的长非编码 RNA Xist 及其新发现的与前 mRNA 剪接调节因子 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、PTB 蛋白的神经元剪接调节以及使用计算方法进行基因表达和选择性剪接。来自指定区域的 RNA-seq 数据将使用新的 rMATS 分析工具对人类和小鼠的大脑进行分析，以创建大型统计数据男性和女性之间差异基因表达和选择性前体 mRNA 剪接的强大数据库女性。表达和剪接变化将与 PTBP1/2 mRNA 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节的基因转录水平。由 XX 基因型引起的女性特异性表达和剪接模式将是与使用四种核心基因型小鼠的雌性激素驱动的事件区分开来。 Xist 目标将是使用条件 Xist 等位基因证实，该等位基因允许在大脑发育过程中去除或激活 Xist 并测量由此产生的拼接变化。 Xist 相对于 PTBP2 的表达为对培养物中的神经元分化进行量化。 PTBP靶向Xist依赖性剪接变化将在 PTBP2 敲除和 PTBP1 转基因小鼠中以及通过全转录组结合分析得到证实由 iCLIP 提供。选择性剪接是一种广泛存在的基因调控机制，但目前只得到了最低限度的研究。检查与雌性细胞的XX基因型的关系。使用复杂的新全基因组方法和分子工具，这些研究有望确定性别二态性的新遗传决定因素哺乳动物大脑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检查提供入口这些二态性的后果。