Combinatorial single molecule analysis of chromatin

Abstract

The present invention provides for single-molecule profiling of combinatorial protein modifications and single-molecule profiling of combinatorial protein modifications combined with single-molecule sequencing of protein/nucleic acids complexes. High-throughput single-molecule imaging was applied to decode combinatorial modifications on millions of individual nucleosomes from pluripotent stem cells and lineage-committed cells. Applicants identified bivalent nucleosomes with concomitant repressive and activating marks, as well as other combinatorial modification states whose prevalence varies with developmental potency. Applying genetic and chemical perturbations of chromatin enzymes show a preferential affect on nucleosomes harboring specific modification states. The present invention also combines this proteomic platform with single-molecule DNA sequencing technology to simultaneously determine the modification states and genomic positions of individual nucleosomes. This novel single-molecule technology can be used to address fundamental questions in chromatin biology and epigenetic regulation leading to novel therapeutics and diagnostics.

1 . A method for analyzing chromatin comprising:
 (a) covalently linking an oligonucleotide sequence to a plurality of isolated chromatin fragments, wherein the oligonucleotide sequence is configured to bind to a capture molecule;   (b) purifying the isolated chromatin fragments linked to an oligonucleotide sequence by size exclusion;   (c) binding the purified chromatin fragments to a solid support comprising the capture molecule;   (d) incubating the solid support with a first set of at least one labeling ligand with specific binding affinity for a target molecule and wherein the labeling ligand includes a marker; and   (e) imaging the solid support, whereby the isolated chromatin fragments comprising the target molecule are visualized.   
     
     
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