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.

项目摘要众所周知，男女对神经精神疾病有不同的政策，但是这些差异尚不理解。特别是确定功能二态性的分子事件在男性和男性之间，需要定义男性大脑。我们将研究女性特定的长时间非编码RNA Xist及其与前MRNA剪接调节器PTBP1和2的新鉴定的相互作用影响雌性大脑中的基因表达和替代剪接。该项目将使用专业知识和工具在三个实验室中开发，用于研究Xist RNA，由PTB蛋白和使用计算方法的基因表达和替代剪接。来自定义区域的RNA-seq数据将使用新的RMAT分析工具分析人和小鼠大脑，以创建大型统计差异基因表达和雄性和替代性mRNA剪接的强大数据库女性。表达和剪接变化将与PTBP1/2 mRNA的变化相关相同的数据集来定义这些分子可能调节的基因转录水平。由XX基因型引起的女性特定表达和剪接模式将是与使用四个核心基因型小鼠驱动的事件区别。 xist定位将是使用条件XIST等位基因确认，允许在大脑发育过程中去除或激活XIST 并测量所得剪接变化。 xist相对于ptbp2的表达将是对培养中的神经元分化进行了量化。剪接中XIST依赖性变化的PTBP靶向将在PTBP2敲除和PTBP1转基因小鼠中确认，并通过转录组结合分析进行确认由ICLIP。替代剪接是基因调节的宽度机制，但仅是最小的根据雌性细胞的XX基因型进行了检查。使用复杂的新基因组方法和分子工具，这些研究有望确定性二态性的新遗传决定者哺乳动物的大脑并阐明其潜在的分子机制。从长远来看，已确定的由XIST和PTBP驱动的分子变化将提供检查功能这些二态性的后果。