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Diverse cell types in tissues have distinct gene expression programs, chromatin states, and nuclear architectures. To correlate such multimodal information across thousands of single cells in mouse brain tissue sections, we use integrated spatial genomics, imaging thousands of genomic loci along with RNAs and epigenetic markers simultaneously in individual cells. We reveal that cell type–specific association and scaffolding of DNA loci around nuclear bodies organize the nuclear architecture and correlate with differential expression levels in different cell types. At the submegabase level, active and inactive X chromosomes access similar domain structures in single cells despite distinct epigenetic and expression states. This work represents a major step forward in linking single-cell three-dimensional nuclear architecture, gene expression, and epigenetic modifications in a native tissue context.

Identifying the relationships between chromosome structures, nuclear bodies, chromatin states and gene expression is an overarching goal of nuclear-organization studies 1-4 . Because individual cells appear to be highly variable at all these levels 5 , it is essential to map different modalities in the same cells. Here we report the imaging of 3,660 chromosomal loci in single mouse embryonic stem (ES) cells using DNA seqFISH+, along with 17 chromatin marks and subnuclear structures by sequential immunofluorescence and the expression profile of 70 RNAs. Many loci were invariably associated with immunofluorescence marks in single mouse ES cells. These loci form 'fixed points' in the nuclear organizations of single cells and often appear on the surfaces of nuclear bodies and zones defined by combinatorial chromatin marks. Furthermore, highly expressed genes appear to be pre-positioned to active nuclear zones, independent of bursting dynamics in single cells. Our analysis also uncovered several distinct mouse ES cell subpopulations with characteristic combinatorial chromatin states. Using clonal analysis, we show that the global levels of some chromatin marks, such as H3 trimethylation at lysine 27 (H3K27me3) and macroH2A1 (mH2A1), are heritable over at least 3-4 generations, whereas other marks fluctuate on a faster time scale. This seqFISH+-based spatial multimodal approach can be used to explore nuclear organization and cell states in diverse biological systems.