In the first aim, we developed systems and procedures for high-throughput decoding of combinatorial chromatin and DNA modifications at the single-molecule level. This single-molecule imaging technology allow for direct imaging and decoding of several epigenetic modifications on histones and DNA within the same complex. Thus, for the first time, we were able to explore directly and systematically the cross-talk, interplay and interactions of higher-order combinations of epigenetic marks. We applied this technology to study the combinatorial epigenetic states of mutant nucleosomes in pediatric gliomas (Furth et al., Cell Reports, 2022): A lysine-to-methionine substitution of residue 27 on histone H3 (K27M) is a driver mutation in high-grade pediatric gliomas, aggressive and fatal brain tumors affecting children. Our single-molecule system allowed us to image individual nucleosomes and delineate the combinatorial epigenetic patterns associated with H3-K27M expression. We found that mutant nucleosomes directly interact with MLL1, leading to a genome-wide redistribution of the active epigenetic mark H3K4me3. H3-K27M-mediated deregulation of repressive and active chromatin marks led to an unbalanced chromatin state, supporting the poorly differentiated phenotype of these tumors. Our study provided evidence for a direct effect of H3-K27M oncohistone on the MLL1-H3K4me3 pathway and highlighted the capability of single-molecule tools to reveal mechanisms of chromatin deregulation in cancer. In ongoing research, which is based on our findings, we are testing combinations of epigenetic drugs as a potential therapeutic strategy for these gliomas, with promising data in primary cell lines and mouse model (Algranati et al., iScience, in revisions).
In the second aim, we developed single-molecule strategies to explore mechanisms of transcription factors and chromatin regulators binding and regulation. This work is still under progress, yet we were able to identify conditions to analyze the binding of MBD and CXXC domains, and apply this knowledge to the liquid biopsy technology developed in Aim 3.
In the third aim, our goal was to apply the principles learned above and implement the single-molecule systems to explore the epigenetic code of circulating cell-free nucleosomes and identify their tissue-of-origin. Indeed, we were highly successful in that project, and developed the first technology for multiplexed single-molecule epigenetic analysis of plasma-isolated nucleosomes for cancer diagnostics (Fedyuk et al., Nature Biotechnology, 2022): We established a revolutionary proof-of-concept of a liquid-biopsy approach to cancer diagnostics. ‘EPINUC’ is a single-molecule multi-parametric assay that comprehensively profiles the Epigenetics of Plasma Isolated Nucleosomes, DNA methylation and cancer-specific protein biomarkers. Our system allows high-resolution detection of six active and repressive histone modifications, their ratios, and combinatorial patterns, on millions of individual nucleosomes by single-molecule imaging. We applied this analysis to a cohort of plasma samples and showed that it detects colorectal cancer at high accuracy and sensitivity, even at early stages. Finally, combining EPINUC with direct single-molecule DNA sequencing revealed the tissue-of-origin of colorectal, pancreatic, lung and breast tumors.
