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

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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.