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

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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.