Single-molecule, single-cell profiling of linked chromatin states [Single_cell_CUT&Tag]

Genomic methods for profiling multiple chromatin features face tradeoffs between specificity, sensitivity, and the ability to measure chromatin co-occupancy. As a result, context-dependent interactions between chromatin proteins remain difficult to resolve in rare cell types and primary tissues. Here, we present CoCUT&Tag, which uses synthetic antigen-peptide repeats paired with nanobodies to amplify Tn5 transposase targeting and directly measure co-occupancy of two chromatin features in single cells. We applied CoCUT&Tag to test the model that bivalent chromatin, marked by the coexistence of H3K27me3 and H3K4 methylation, preserves stem-cell multipotency by keeping developmental genes poised for future activation. Instead, we find that during human hematopoiesis, stem cells are defined by a more active chromatin landscape, whereas lineage-restricted populations contain more bivalent genes. Bivalent and unmarked genes are activated with similar timing, but bivalent genes achieve higher expression, are associated with more numerous and stronger enhancers, and are preferentially derepressed following BMI1 loss. Our study shows that bivalent chromatin increases enhancer demand to enable high-amplitude gene activation, demonstrating that CoCUT&Tag can resolve longstanding questions about combinatorial chromatin regulation.