Periodic Reporting for period 4 - SM-Epigen (Revealing the Epigenetic Regulatory Network with Single-Molecule Precision)
Periodo di rendicontazione: 2023-05-01 al 2023-10-31
Within this proposal, we developed and applied single-molecule methodologies to elucidate basic principles and mechanisms in the combinatorial action of histone modifications, and the crosstalk with DNA methylation and chromatin regulators. We did so by encompassing experimentation on multiple levels, ranging from development of biochemical methods for preparation of samples to adaptation of surface chemistry and single-molecule DNA sequencing technologies, followed by computational pipelines for data analysis. This integrative approach is critical for addressing long-standing questions in epigenetics and transcription regulation, and establishing new toolsets for functional genomics. Furthermore, our work may pave the way for novel clinical applications.
Our overall objectives were:
In the first aim, we developed systems and procedures for high-throughput decoding of combinatorial chromatin and DNA modifications. We established a new standard for functional genomics, by developing and applying single-molecule systems that would 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.
In the second aim, we aimed to develop and apply single-molecule strategies to explore mechanisms of transcription factor binding and regulation. We will also examine the interplay between TFs binding to the underlying chromatin structure.
In the third aim, we aimed 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. Analysis of the tissue-of-origin of cell-free DNA (cfDNA) has been shown to be extremely useful for clinical applications, mainly the screening for fetal genetic abnormalities in pregnant women. However, all current applications require genetic differences in order to distinguish between the contributing tissues. It has recently been shown that cfDNA is actually in the form of nucleosomes; thus, it can be classified not only based on changes in its sequence but also by distinctive epigenetic marks. Here, our goal was to establish single-molecule systems for identifying these epigenetic marks on circulating nucleosomes and determining their tissue-of origin. This will pave the way for the development of non-invasive diagnostic tests for several clinical conditions, as well as the elucidation of the mechanisms underlying the release of these nucleosomes to the blood.
Our overall objectives were:
In the first aim, we developed systems and procedures for high-throughput decoding of combinatorial chromatin and DNA modifications. We established a new standard for functional genomics, by developing and applying single-molecule systems that would 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.
In the second aim, we aimed to develop and apply single-molecule strategies to explore mechanisms of transcription factor binding and regulation. We will also examine the interplay between TFs binding to the underlying chromatin structure.
In the third aim, we aimed 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. Analysis of the tissue-of-origin of cell-free DNA (cfDNA) has been shown to be extremely useful for clinical applications, mainly the screening for fetal genetic abnormalities in pregnant women. However, all current applications require genetic differences in order to distinguish between the contributing tissues. It has recently been shown that cfDNA is actually in the form of nucleosomes; thus, it can be classified not only based on changes in its sequence but also by distinctive epigenetic marks. Here, our goal was to establish single-molecule systems for identifying these epigenetic marks on circulating nucleosomes and determining their tissue-of origin. This will pave the way for the development of non-invasive diagnostic tests for several clinical conditions, as well as the elucidation of the mechanisms underlying the release of these nucleosomes to the blood.
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.
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.
The single-molecule technology developed within this grant is highly powerful in detecting a limited number of epigenetic modifications (2-6) at very high resolution, as well as analyzing their combinations. During the development of the project, we realized that in addition to this single-molecule technology, a complementary methodology is needed for low-resolution analysis of many histone modifications per single cells. This is complementary to the single-molecule work as it allows us to systematically screen many epigenetic modifications at the biological systems we study, and then focus on the specific epigenetic marks that show interesting behavior by applying our single-molecule analysis. The methodology we developed is an adaption of Cytometry by Time of Flight (CyTOF), with a custom-designed panel of antibodies targeting histone modifications and chromatin regulators. We also established new modes for the analysis of this unique CyTOF data. We expect this methodology to be of great use to epigenetics studies in single cells in diverse biological systems.
Moreover, we now established our liquid biopsy technology for the diagnosis of several different cancers, and are working on a project to apply it also to identify minimally residual disease in the context of rectal cancer. We expect these results to have high value for the community.
Moreover, we now established our liquid biopsy technology for the diagnosis of several different cancers, and are working on a project to apply it also to identify minimally residual disease in the context of rectal cancer. We expect these results to have high value for the community.