Differential regulation of mouse hippocampal gene expression sex differences by chromosomal content and gonadal sex: RNA-Seq Data

Sex differences in the brain as they relate to health and disease are often overlooked in experimental models. Many neurological disorders, like Alzheimer's disease (AD), multiple sclerosis (MS), and autism, differ in prevalence between males and females. Sex differences originate either from differential gene expression on sex chromosomes or from hormonal differences, either directly or indirectly. To disentangle the relative contributions of genetic sex (XX v. XY) and gonadal sex (ovaries v. testes) to the regulation of hippocampal sex effects, we use the "sex-reversal" Four Core Genotype (FCG) mouse model which uncouples sex chromosome complement from gonadal sex. Transcriptomic and epigenomic analyses of hippocampal RNA and DNA from ~12 month old FCG mice, reveals differential regulatory effects of sex chromosome content and gonadal sex on X- versus autosome-encoded gene expression and DNA modification patterns. Gene expression and DNA methylation patterns on the X chromosome were driven primarily by sex chromosome content, not gonadal sex. The majority of DNA methylation changes involved hypermethylation in the XX genotypes (as compared to XY) in the CpG context, with the largest differences in CpG islands, promoters, and CTCF binding sites. Autosomal gene expression and DNA modifications demonstrated regulation by sex chromosome complement and gonadal sex. These data demonstrate the importance of sex chromosomes themselves, independent of hormonal status, in regulating hippocampal sex effects. Future studies will need to further interrogate specific CNS cell types, identify the mechanisms by which sex chromosome regulate autosomes, and differentiate organizational from activational hormonal effects. RNA isolated from snap-frozen Four Core Genotype mouse hippocampus (n=5-6/group) using Qiagen AllPrep Mini Kit. Illumina's TruSeq Stranded mRNA Library Prep Kit (#20020594, Illumina) was used on 500 ng of total RNA for the preparation of strand-specific sequencing libraries according to manufacturer's guidelines. As previously described44, rRNA depletion was performed prior to library construction. Libraries were then normalized to 4 nM, pooled, denatured, and diluted for sequencing on Illumina Hiseq2500 in a 2x100 bp fashion.大脑性别差异与健康和疾病的关联在实验模型中常被忽视。诸多神经系统疾病，如阿尔茨海默病(Alzheimer's disease, AD)、多发性硬化症(multiple sclerosis, MS)与自闭症，其患病率在男女群体中存在显著差异。性别差异的来源可分为两类：一是性染色体上的差异基因表达，二是直接或间接由激素差异所介导。为厘清遗传性别（XX vs. XY）与性腺性别（卵巢 vs. 睾丸）对海马体性别相关效应的相对调控贡献，我们采用了“性别反转”四核心基因型(Four Core Genotype, FCG)小鼠模型，该模型可将性染色体组成与性腺性别解耦。我们对约12月龄FCG小鼠的海马体RNA与DNA开展转录组学与表观基因组学分析，结果显示，性染色体组成与性腺性别对X染色体及常染色体编码基因的表达、DNA修饰模式的调控效应存在差异。X染色体上的基因表达与DNA甲基化模式主要由性染色体组成驱动，而非性腺性别。绝大多数DNA甲基化变化表现为XX基因型（相较于XY基因型）在CpG位点的高甲基化，其中以CpG岛、启动子区域及CTCF结合位点的差异最为显著。常染色体基因表达与DNA修饰则同时受性染色体组成与性腺性别调控。本研究数据证实，性染色体本身在不依赖激素状态的情况下，对海马体的性别相关效应具有重要调控作用。未来研究需进一步探究中枢神经系统(central nervous system, CNS)的特定细胞类型，明确性染色体调控常染色体的具体机制，并区分激素的组织效应与激活效应。本研究使用Qiagen AllPrep Mini试剂盒从快速冷冻的四核心基因型小鼠海马体中提取RNA（每组样本量n=5-6）。取500 ng总RNA，按照制造商说明书，使用Illumina TruSeq Stranded mRNA文库制备试剂盒（货号#20020594，Illumina）构建链特异性测序文库。如前文所述44，文库构建前需进行核糖体RNA(ribosomal RNA, rRNA)去除步骤。随后将文库归一化至4 nM，混合、变性并稀释，随后在Illumina HiSeq2500平台上以2×100 bp双端测序模式进行测序。