It is a long established fact that sex is an important factor that influences the transcriptional regulatory processes of an organism. However, understanding sex-based differences in gene expression has been limited because existing studies typically sequence and analyze bulk tissue from female or male individuals. Such analyses average cell-specific gene expression levels where cell-to-cell variation can easily be concealed. We therefore sought to utilize data generated by the rapidly developing single cell RNA sequencing (scRNA-seq) technology to explore sex dimorphism and its functional consequences at the single cell level. Our study included scRNA-seq data of ten well-defined cell types from the brain and heart of female and male young adult mice in the publicly available tissue atlas dataset, Tabula Muris. We combined standard differential expression analysis with the identification of differential distributions in single cell transcriptomes to test for sex-based gene expression differences in each cell type. The marker genes that had sex-specific inter-cellular changes in gene expression formed the basis for further characterization of the cellular functions that were differentially regulated between the female and male cells. We also inferred activities of transcription factor-driven gene regulatory networks by leveraging knowledge of multidimensional protein-to-genome and protein-to-protein interactions and analyzed pathways that were potential modulators of sex differentiation and dimorphism. For each cell type in this study, we identified marker genes with significantly different mean expression levels or inter-cellular distribution characteristics between female and male cells. These marker genes were enriched in pathways that were closely related to the biological functions of each cell type. We also identified sub-cell types that possibly carry out distinct biological functions that displayed discrepancies between female and male cells. Additionally, we found that while genes under differential transcriptional regulation exhibited strong cell type specificity, six core transcription factor families responsible for most sex-dimorphic transcriptional regulation activities were conserved across the cell types, including ASCL2, EGR, GABPA, KLF/SP, RXRα, and ZF. We explored novel gene expression-based biomarkers, functional cell group compositions, and transcriptional regulatory networks associated with sex dimorphism with a novel computational pipeline. Our findings indicated that sex dimorphism might be widespread across the transcriptomes of cell types, cell type-specific, and impactful for regulating cellular activities.

中文翻译：

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通过对十种小鼠细胞类型的单细胞 RNA 测序数据进行多级分析，研究转录组范围内的性别二态性

性别是影响生物体转录调节过程的重要因素，这是一个长期确立的事实。然而，对基于性别的基因表达差异的理解有限，因为现有研究通常对来自女性或男性个体的大块组织进行测序和分析。这种分析平均细胞特异性基因表达水平，其中细胞间变异很容易被隐藏。因此，我们试图利用快速发展的单细胞 RNA 测序 (scRNA-seq) 技术产生的数据来探索性别二态性及其在单细胞水平上的功能后果。我们的研究包括来自公开可用的组织图谱数据集 Tabula Muris 中雌性和雄性年轻成年小鼠的大脑和心脏的十种明确定义的细胞类型的 scRNA-seq 数据。我们将标准差异表达分析与单细胞转录组差异分布的鉴定相结合，以测试每种细胞类型中基于性别的基因表达差异。在基因表达中具有性别特异性细胞间变化的标记基因构成了进一步表征在雌性和雄性细胞之间差异调节的细胞功能的基础。我们还通过利用多维蛋白质-基因组和蛋白质-蛋白质相互作用的知识推断转录因子驱动的基因调控网络的活动，并分析了作为性别分化和二态性潜在调节剂的途径。对于本研究中的每种细胞类型，我们鉴定了在雌性和雄性细胞之间具有显着不同平均表达水平或细胞间分布特征的标记基因。这些标记基因富含与每种细胞类型的生物学功能密切相关的通路。我们还确定了可能执行不同生物学功能的亚细胞类型，这些功能显示出雌性和雄性细胞之间的差异。此外，我们发现虽然差异转录调控下的基因表现出很强的细胞类型特异性，但负责大多数性别二态性转录调控活动的六个核心转录因子家族在细胞类型中是保守的，包括 ASCL2、EGR、GABPA、KLF/SP、RXRα , 和 ZF。我们探索了新的基于基因表达的生物标志物、功能细胞群组成、以及与具有新计算管道的性别二态性相关的转录调控网络。我们的研究结果表明，性别二态性可能广泛存在于细胞类型、细胞类型特异性的转录组中，并且对调节细胞活动有影响。