Description

Nano-Fish Enables Robust Visualization and Precise Spatial Annotation of Regulatory DNA in Single Cells

Tobias Ragoczy¹, Vivek Nandakumar¹, Konstantin Queitsch¹, Hartmann Harz², David Hörl², Heinrich Leonhardt², and John Stamatoyannopoulos^1,3,4, Shreeram Akilesh⁵

¹Altius Institute for Biomedical Sciences, Seattle, WA, 98121, USA;

²Department of Human Biology and Imaging, Ludwig Maximilian University of Munich, Germany;

³Department of Medicine, University of Washington, Seattle, WA, 98195, USA;

⁴Department of Genome Sciences, University of Washington, Seattle, WA, 98195, USA;

⁵Department of Pathology, University of Washington, Seattle, WA, 98195, USA

Our current understanding of transcriptional regulation mechanisms is largely inferred from bulk cell sequencing-based assays such as chromatin conformation capture, DNaseI-seq and/or ChIP-seq. By their nature, these assays destroy information regarding allele-specific patterns and cell-to-cell variability which are key to understanding the spatiotemporal mechanics of gene regulation. Current methods to label and detect gene loci in single cells in situ use a combination of fluorescence in situ hybridization (FISH) and microscopy imaging with BAC/fosmid-based probes that typically range in size from 10s – 100s of kilobases (kb). These large probes lack the sensitivity and precision required to accurately localize regulatory DNA elements (e.g. DNaseI hypersensitive sites) that typically measure ~1kb in size. Furthermore, routine application of DNA-FISH has been hampered by wide variability among protocols used by various laboratories, finicky and specialized microscope configurations and low throughput. These disadvantages have hampered the widespread adoption of these methods and rigorous statistical analyses of the results.

To address these deficits, we have developed a robust and standardized DNA-FISH methodology (Nano-FISH) coupled with an automated image informatics pipeline to enable high-throughput detection and spatial localization of small genomic DNA elements (~1 kb) in situ in hundreds to thousands of individual cells per experiment. Nano-FISH utilizes defined pools of synthetic fluorescent dye-labeled oligonucleotides in an optimized 3D-FISH protocol to reliably detect small (~1 kb) genomic regions in large numbers of adherent or suspension cells in situ. We have successfully applied Nano-FISH in conventional wide-field microscopic imaging as well as in challenging super-resolution imaging applications. To facilitate rigorous statistical analyses of the resulting large image data sets, we have developed a scalable image analysis software suite to reliably identify and quantitatively annotate labeled loci on a single-cell basis. We demonstrate the utility of Nano-FISH to precisely localize specific regulatory genomic elements in 3D nuclear space, to identify small-scale structural genomic variations (gains/losses), to quantitate the spatial interactions between regulatory elements and their putative target gene(s), and to detect genomic conformational changes that induce stimulus-dependent gene expression. Nano-FISH thus provides a new and powerful tool to allow direct visualization of the fine-scale physical organization and dynamics of genome regulation at single cell resolution.

Funding: This study was funded in part by NHGRI RM1-HG007743-02 (CEGS-Center for Photogenomics)