Different combinations of histone modifications have been proposed to signal distinct gene regulatory functions, but this area is poorly addressed by existing technologies. We applied high-throughput single-molecule imaging to decode combinatorial modifications on millions of individual nucleosomes from pluripotent stem cells and lineage-committed cells.We identified definitively bivalent nucleosomes with concomitant repressive and activating marks, as well as other combinatorial modification states whose prevalence varies with developmental potency. We showed that genetic and chemical perturbations of chromatin enzymes preferentially affect nucleosomes harboring specific modification states. Last, we combined this proteomic platform with single-molecule DNA sequencing technology to simultaneously determine the modification states and genomic positions of individual nucleosomes. This single-molecule technology has the potential to address fundamental questions in chromatin biology and epigenetic regulation.
Bibliographical noteFunding Information:
We thank I. Tirosh, P. Blainey, K. Xiong, and S. Elledge for constructive discussions; J. Reifenberger for help with imaging technology; M. Bray for help with image analysis; I.-S. Kim, K. Bouazoune, C. Epstein, and J. Kaur for their experimental contributions; and S. Deindl, B. Liau, and R. Ryan for valuable experimental advice. E.S. is supported by the Jane Coffin Childs Memorial Fund for Medical Research. D.J. is partially supported by National Human Genome Research Institute Small Business Innovation Research grant R44HG005279. This research was supported by funds from the National Human Genome Research Institute, the Klarman Cell Observatory, and the Howard Hughes Medical Institute. Sequencing data are available in the supplementary materials (database S1). D.J. is a founder and the president of SeqLL, which contributed to the technologies described in this study. E.S., D.J., and B.E.B. have filed a provisional patent covering single-molecule methodologies described in the paper.