Measuring genome organization during stem cell differentiation with super-resolution microscopy

在发育和分化过程中，细胞特性的建立、维持和变化由复杂的信号通路控制，其中包括细胞因子的相互作用和表观遗传修饰。越来越多的证据表明，除了研究良好的DNA和组蛋白修饰外，空间基因组结构也可能有助于定义单个细胞的身份和潜力的整体表观遗传信息内容。我们现在希望系统地研究早期发育过程中基因组组织的变化，其中全基因组转录的变化伴随着特定的表观遗传变化。对于已定义的干细胞培养系统，我们想要重述从原始的多能性到启动的多能性以及随后的细胞分化的定义步骤。我们将专注于Nanog多能性基因簇的基因组组织，并使用改进的荧光杂交方案测量干细胞分化过程中凝聚水平的变化和调节元件(增强子和启动子)之间的远程相互作用，并使用超分辨率显微镜测量活细胞。这种微观方法不能达到构象捕获方法(如HIC)的分子分辨率，但提供了物理距离和单细胞分辨率。此外，自动化高通量显微镜能够识别稀有细胞群体以及与形态特征和生理状态的功能联系。我们将引入特定的突变来剖析顺式作用的DNA序列元件在调节Nanog多能性基因簇的活性和空间组织中的作用。同时，我们将研究反式作用的表观遗传因子(Dnmts、Tets和HMT)及其在局部基因组浓缩、折叠和活性中的作用。我们的研究应该补充这一优先项目的其他方法学方法，并有助于阐明空间基因组结构在早期发育和细胞分化中的作用和调节。

The establishment, maintenance and change of cellular identities during development and differentiation is controlled by complex signaling pathways that include interactions of cellular factors and epigenetic modifications. There is growing evidence that in addition to the well-studied DNA and histone modifications also spatial genome architecture might contribute to the overall epigenetic information content that defines the identity and potential of individual cells.We now want to systematically investigate changes in genome organization during early development where changes in genome-wide transcription are accompanied by specific epigenetic changes. With defined stem cell culture systems, we want to recapitulate the defined steps from naïve pluripotency to primed pluripotency and subsequent cellular differentiation. We will focus on the genome organization of the Nanog pluripotency gene cluster and measure changes in condensation levels and long-range interactions between regulatory elements (enhancers and promoters) during stem cell differentiation using refined fluorescent hybridization protocols and live cell measurements with super-resolution microscopy. This microscopic approach does not reach the molecular resolution of conformation capture methods (like e.g. HiC) but provides physical distances and single cell resolution. Moreover, automated high-throughput microscopy enables the identification of rare cell populations and functional links with morphological features and physiological states. We will introduce specific mutations to dissect the role of cis-acting DNA sequence elements in regulating the activity and spatial organization of the Nanog pluripotency gene cluster. In parallel we will study trans-acting epigenetic factors (DNMTs, TETs and HMT) and their role in local genome condensation, folding and activity. Our study should complement other methodological approaches of this priority program and should help to elucidate the role and regulation of spatial genome architecture during early development and cellular differentiation.