High-resolution spatial multi-omics reveals cell-type specific nuclear compartments

The mammalian nucleus is compartmentalized by diverse subnuclear structures. These subnuclear structures, marked by nuclear bodies and histone modifications, are often cell-type specific and affect gene regulation and 3D genome organization. Understanding nuclear organization and its role in gene expression requires identifying the molecular constituents of subnuclear structures and mapping their associations with specific genomic loci and their transcriptional levels in individual cells, within complex tissues. Here, we introduce two-layer DNA seqFISH+, which allows simultaneous mapping of 100,049 genomic loci, together with nascent transcriptome for 17,856 genes and a diverse set of immunofluorescently labeled subnuclear structures all in single cells in both cell lines and complex tissues. These data enable imaging-based chromatin profiling of diverse subnuclear markers and capture changes in chromatin organization at genomic scales from 100–200 kb to approximately 1 Mb, depending on the subnuclear marker and DNA locus. Using multi-omics datasets in the adult mouse cerebellum, we showed that repressive chromatin regions are more variable by cell type than active regions across the genome. We also discovered RNA polymerase II (RNAPII)-enriched foci were locally associated with long, cell-type specific genes (> 200kb), in a manner distinct from nuclear speckles. Further, our analysis revealed that cell-type specific facultative and constitutive heterochromatin regions marked by H3K27me3 and H4K20me3 are enriched at specific genes and gene clusters, respectively, and shape radial chromosomal positioning and inter-chromosomal interactions in neurons and glial cells. Together, our results provide a single-cell high-resolution multi-omics view of subnuclear structures and chromatin marks, associated genomic loci, and their impacts on gene regulation, directly within complex tissues. CUT&Tag for RNA Polymerase II phospho Ser5 (RNAPIISer5-P) and histone modfication H3K27me3 and CUT&RUN for Lamin B1 and histone modfications H3K4me3, H3K9me9 in mouse embryonic stem cells and mammary gland cells.