CoCUT&Tag enables single-cell, single-molecule measurement of linked chromatin states, revealing that bivalent chromatin persists and increases during human hematopoietic differentiation rather than being maximal in stem cells, and functions not to accelerate gene activation but to establish an enhancer threshold that enables high-amplitude expression by requiring accumulation of more numerous and stronger lineage-specific enhancers before Polycomb restraint is overcome.

Genomic methods for profiling multiple chromatin features face tradeoffs in specificity, sensitivity, and the ability to measure chromatin co-occupancy in rare cell types and primary tissues. Here, we present CoCUT&Tag, which uses synthetic antigen-peptide repeats paired with nanobodies to enhance Tn5 transposase targeting and enable single-cell measurement of the co-occupancy of two chromatin features on the same DNA molecule. We apply 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. We find that during human hematopoietic differentiation, global bivalency increases, whereas the number of genes in an active chromatin state is highest in stem and multipotent progenitor cells. Bivalent and unmarked genes activate with similar timing, but bivalent genes define a distinct regulatory class that achieves higher expression, is associated with more numerous and stronger enhancers, and is preferentially derepressed following loss of Polycomb-repressor proteins. 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.