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SummaryTriple-negative breast cancer is associated with the worst prognosis and the highest risk of recurrence among all breast cancer subtypes1. Residual disease, formed by cancer cells persistent to chemotherapy, remains one of the major clinical challenges towards full cure2,3. There is now consensus that non-genetic processes contribute to chemoresistance in various tumor types, notably through the initial emergence of a reversible chemotolerant state4–6. Understanding non-genetic tumor evolution stands now as a prerequisite for the design of relevant combinatorial approaches to delay recurrence. Here we show that the repressive histone mark H3K27me3 is a determinant of cell fate under chemotherapy exposure, monitoring epigenomes, transcriptomes and lineage with single-cell resolution. We identify a reservoir of persister basal cells with EMT markers and activated TGF-β pathway leading to multiple chemoresistance phenotypes. We demonstrate that, in unchallenged cells, H3K27 methylation is a lock to the expression program of persister cells. Promoters are primed with both H3K4me3 and H3K27me3, and removing H3K27me3 is sufficient for their transcriptional activation. Leveraging lineage barcoding, we show that depleting H3K27me3 alters tumor cell fate under chemotherapy insult – a wider variety of tumor cells tolerate chemotherapy. Our results highlight how chromatin landscapes shape the potential of unchallenged cancer cells to respond to therapeutic stress.

Modulation of chromatin structure via histone modification is a major epigenetic mechanism and regulator of gene expression. However, the contribution of chromatin features to tumor heterogeneity and evolution remains unknown. Here we describe a high-throughput droplet microfluidics platform to profile chromatin landscapes of thousands of cells at single-cell resolution. Using patient-derived xenograft models of acquired resistance to chemotherapy and targeted therapy in breast cancer, we found that a subset of cells within untreated drug-sensitive tumors share a common chromatin signature with resistant cells, undetectable using bulk approaches. These cells, and cells from the resistant tumors, have lost chromatin marks-H3K27me3, which is associated with stable transcriptional repression-for genes known to promote resistance to treatment. This single-cell chromatin immunoprecipitation followed by sequencing approach paves the way to study the role of chromatin heterogeneity, not just in cancer but in other diseases and healthy systems, notably during cellular differentiation and development.