Mechanisms controlling human microglia gene expression

 描述（由申请人提供）：小胶质细胞是存在于中枢神经系统（CNS）中的组织巨噬细胞，具有对中枢神经系统发育、体内平衡、免疫和修复重要的独特且关键的辅助功能。尽管它们很重要，但对控制小胶质细胞发育和功能的机制知之甚少，特别是在人类中。在这里，我们建议利用最近的技术和计算的突破，研究谱系决定和信号依赖性转录因子之间的相互作用如何调节人类和小鼠小胶质细胞的基因表达。具体目标1将测试的假设，人类大脑微环境是一个重要的决定因素的小胶质细胞基因表达。小胶质细胞转录组将在分离后立即获得，并在补充有小胶质细胞表型的推定调节剂的组织培养环境中一周后获得。重要的是，将在全基因组序列的背景下分析所有样品。这些研究将为小胶质细胞的核心转录特征、大脑微环境的影响以及基因型与基因表达模式的关系提供新的定性信息。具体目标2将检验以下假设：PU.1和一组有限的替代谱系决定因子驱动了决定小胶质细胞身份和调控潜力的大多数增强子的选择。我们将使用ChIP-Seq和ATAC-Seq方法来定义新鲜分离的人小胶质细胞中的增强子图谱。这些信息将提示关键的小胶质细胞谱系决定因子，并确定小胶质细胞超级增强子。我们将利用ChIP测序和自然遗传变异的组合来确定选择和激活小胶质细胞特异性增强子所需的核心转录因子组合。这些发现将为人类小胶质细胞发育和功能的分子机制提供定性的新见解，并将为更忠实地将人类iPS细胞重编程为小胶质细胞表型的努力提供信息。具体目标3将测试以下假设：环境因素和转录回路是 小胶质细胞特异性基因表达在小鼠和人之间是保守的。小鼠小胶质细胞转录组和增强子图谱将在特定目的1和2中针对人小胶质细胞所述的相同条件下获得。不同品系的小鼠将被用作天然遗传变异的来源，以定义增强子选择和激活所需的核心转录因子组合。预测具有保守作用的特定转录因子的作用 在调节人和小鼠小胶质细胞发育和功能方面的作用将通过功能丧失实验来评估。这些实验将提供重要的信息，在何种程度上小鼠小胶质细胞可用于模拟人类小胶质细胞和建立保守的转录因子的功能的重要性。

 DESCRIPTION (provided by applicant): Microglia are tissue macrophages that reside in the central nervous system (CNS) and perform unique and critical auxiliary functions important to CNS development, homeostasis, immunity and repair. Despite their importance, remarkably little is known about the mechanisms that control microglia development and function, particularly in humans. Here we propose to leverage recent technical and computational breakthroughs to investigate how interactions between lineage-determining and signal-dependent transcription factors regulate gene expression in human and mouse microglia. Specific Aim 1 will test the hypothesis that the human brain microenvironment is an important determinant of microglia gene expression. Microglia transcriptomes will be obtained immediately upon isolation and after one week in a tissue culture environment supplemented with putative regulators of microglia phenotypes. Importantly, all samples will be analyzed in the context of full genomic sequence. These studies will provide qualitatively new information on core transcriptional signatures of microglia, the influence of the brain microenvironment, and the relationships of genotype to gene expression patterns. Specific Aim 2 will test the hypothesis that PU.1 and a limited set of alternative lineage determining factors drive the selection of the majority of enhancers that determine microglia identity and regulatory potential. We will use ChIP-Seq and ATAC-Seq methods to define enhancer atlases in freshly isolated human microglia. This information will suggest key microglia lineage determining factors and identify microglia super enhancers. We will exploit the combination of ChIP sequencing and natural genetic variation to identify core transcription factor combinations needed for the selection and activation of microglia-specific enhancers. These findings will provide qualitatively new insights into molecular mechanisms that underlie human microglia development and function and will inform efforts to more faithfully reprogram human iPS cells to microglia phenotypes. Specific Aim 3 will test the hypothesis that environmental factors and transcriptional circuits required for microglia-specific gene expression are conserved between mouse and human. Mouse microglia transcriptomes and enhancer atlases will be obtained under the same conditions as described for human microglia in Specific Aims 1 and 2. Different strains of mice will be used as a source of natural genetic variation to define core transcription factor combinations needed for enhancer selection and activation. Roles of specific transcription factors predicted to have conserved roles in regulating human and mouse microglia development and function will be assessed by loss of function experiments. These experiments will provide important information on the extent to which mouse microglia can be used to model human microglia and establish the functional importance of conserved transcription factors.