Identification of cell-type-specific multi-way regulatory hubs during vertebrate development
General Genetics and Functional Genome Biology
Bioinformatics and Theoretical Biology
Final Report Abstract
Genetic information is packed into a DNA-protein molecular complex called chromatin which is highly organized in three dimensions (3D) inside the nucleus of every cell. Local and longrange chromatin architecture, often referred to as the epigenome, is largely responsible for regulating gene expression dynamically over time and tissue space during development, disease, and in response to environmental changes. Genomics methods have been developed to measure chromatin architecture and 3D organization, but deeper understanding of regulatory dynamics in tissues made up of multiple cell types has been largely limited by available tools to isolate cell-type resolved material in sufficient quantities. Single-cell-resolved measurements of RNA and chromatin with high-throughput sequencing have allowed in silico reconstruction of cell states within heterogeneous tissues and a better understanding of chromatin and gene expression dynamics. We have used state-of-the-art single-cell genomics tools to deeply characterize gene regulatory network dynamics during zebrafish embryo segmentation and in response to the loss of a key developmental transcription factor, npas4l. In contrast, 3D genome organization has proven difficult to characterize with cell-type resolution when performed on native tissues. To address this need, we have developed epigenome-C (Epi-C), a multimodal long-read sequencing assay that simultaneously captures 3D genome organization, chromatin accessibility, and DNA methylation within single molecules. We show that Epi-C is highly reproducible and gives signals comparable to state-of-the-art individual assays for all three modalities. Epi-C molecules provide substantial coverage of sparse genomic annotations, allowing deconvolution of allele- and cell-type-resolved regulatory dynamics in native, heterogeneous tissues. By taking advantage of a unique aspect of Nanopore sequencing, called adaptive sampling, we show that Epi-C molecules can be enriched to give high coverage of genomic regions of interest, thereby increasing the resolution of Epi-Cs readouts for chromatin dynamics. Furthermore, the current implementation of Epi-C is only a starting point, as the method’s open-ended design will allow immediate expansion to a fourth modality, namely DNA protein occupancy, with potential for even further expansion as parallel technologies are developed for Nanopore sequencing. We expect Epi-C to be widely implemented as it does not require any specialized instrumentation beyond a Nanopore sequencer, and has unique promise to resolve dynamics of chromatin architecture and 3D organization within native tissues of all types. Epi-C will have a high impact in our understanding of gene regulation dynamics, thereby strongly influencing research into all aspects of life science.
