Multimodal Analysis of the Genome Architecture Using Expansion Microscopy

SUMMARY The three-dimensional organization of the genome regulates gene expression, impacting fundamental biological processes in development, homeostasis and disease. Deep understanding of the relationship between genome structure and function requires insight into 3D chromatin architecture at individual gene loci, at high resolution and at genome-wide scale. Current approaches to visualize chromatin architecture are limited by trade-offs between depth and breadth and cannot link 3D position with sequence information with simultaneous profiling of transcription and epigenetic marks in single cells. Genomic methods such as Hi-C typically analyze a population average of genomic contact information and imaging approaches such as ChromEMT4 provide nucleosome level visualization with 1 nanometer resolution, but lack locus identification and genome scale. Thus, there is a critical need for a broadly applicable method that incorporates both sequence and nuclear position to reveal the spatial structure of the genome at nanometer scale. Ideally such a method would have nucleosome resolution (~10 nm) at the single-cell level and simultaneously provide functional readouts of epigenetic marks, transcription factors (TFs), and transcriptional activity using multimodal labeling in the same cell. The goal of this proposal is to develop, validate and benchmark such a method to provide genome-wide chromatin architecture analysis with sequence information by combining iterative expansion microscopy (PanExM) with genome-wide barcoding. This method will provide nucleosome resolution imaging of the genome with kilobase sequence resolution while allowing multimodal protein labeling of the chromatin thanks to the protein retention during PanExM. To achieve this: in Aim 1, we will adapt PanExM to visualize the chromatin (ChromExM), develop multimodal labeling of DNA, RNA, TFs and epigenetic marks, develop benchmarking tools to test isotropic expansion, structural perturbations and labeling efficiency and develop analytical and quantitative methods to characterize how local chromatin structure regulates function. In Aim 2 we will adapt fluorescence in situ hybridization (FISH)-based chromatin tracing to ChromExM using genome-wide barcodes as well as specific probes to label sites of interest such as promoters and enhancers, we will develop approaches to systematically examine enhancer-promoter interactions during transcription activation, and we will benchmark ChromExM to established methods of super resolution microscopy, chromatin tracing and HiC. The methods developed here will provide unprecedented resolution of the chromatin structure at the single cell level and genome-wide, and will be broadly applicable by the research community. ChromExM will provide much-needed tools to allow a large community of researchers to address the structural basis of how epigenetic modifications, DNA-sequence, and chromatin proteins regulate gene expression.