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

项目摘要 众所周知，女性和男性对神经精神疾病有不同的倾向，但是， 这些差异不被理解。特别是决定功能二型性的分子事件 女性和男性大脑之间的差异需要被定义。我们将研究女性特有的长 非编码RNA Xist及其与前mRNA剪接调节因子PTBP 1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。该项目将利用专门知识和工具 由三个实验室开发，用于研究Xist RNA，PTB蛋白对神经元剪接的调节， 基因表达和选择性剪接。RNA-seq数据来自定义的区域 将使用新的rMATS分析工具对人类和小鼠的大脑进行分析， 男性和女性之间差异基因表达和选择性前mRNA剪接的强大数据库 女性表达和剪接变化将与PTBP 1/2 mRNA和Xist的变化相关。 相同的数据集，以确定基因可能受到这些分子在转录和后 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被 区别于使用四个核心基因型小鼠的由雌性激素驱动的事件。Xist目标将是 使用条件性Xist等位基因证实，该等位基因允许在大脑发育期间去除或激活Xist 并测量拼接中产生的变化。Xist相对于PTBP 2的表达将是 在培养的神经元分化中定量。PTBP靶向Xist依赖的剪接变化 将在PTBP 2敲除和PTBP 1转基因小鼠中以及通过全转录组结合分析来证实 通过iCLIP。选择性剪接是一种广泛的基因调控机制，但仅在最低程度上受到关注。 检查与雌性细胞的XX基因型的关系。使用复杂的新的全基因组方法， 分子工具，这些研究有望确定新的遗传决定因素的性别二型性， 哺乳动物脑，并阐明其潜在的分子机制。从长远来看， 由Xist和PTBP驱动的分子变化将为功能性 这些二态性的后果。

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.