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Revealing the Epigenetic Regulatory Network with Single-Molecule Precision

Periodic Reporting for period 2 - SM-Epigen (Revealing the Epigenetic Regulatory Network with Single-Molecule Precision)

Reporting period: 2020-05-01 to 2021-10-31

Genes and genomic elements are packaged by chromatin structures that regulate their activity. The fundamental unit of chromatin is the nucleosome, composed of an octamer of histones. The large numbers of histone modifications, chromatin remodelers and transcription factors (TFs) that interact with our genome has fueled speculation that multiple elements act combinatorially to direct specific outcomes. However, the field lacks technologies for detection and analysis of such combinations, thus impeding our ability to test this hypothesis and shed light on human genome regulation.
Our goal is to establish robust single-molecule systems for investigating combinatorial chromatin and TF interactions with single-molecule precision and genome-wide coverage. With these systems, we aim to unravel the complex, nuanced and intriguing chromatin language, which is the basis for transcriptional regulation.

We will develop and apply single-molecule methodologies to elucidate basic principles and mechanisms in the combinatorial action of histone modifications, and the crosstalk with DNA methylation, as well as TF binding and dynamics. We will do 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 are:

In the first aim, we will develop systems and procedures for high-throughput decoding of combinatorial chromatin and DNA modifications. We will establish 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 will be able to explore directly and systematically the cross-talk, interplay and interactions of higher-order combinations of epigenetic marks.

In the second aim, we will 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. This work will enable the charting of combinatorial TF interactions at scale, and provide transformative tools to study gene regulation in diverse biological systems.

In the third aim, we will 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. This work will establish single-molecule systems for identifying these epigenetic marks on circulating nucleosomes and determining their tissue-of origin. Our single-molecule systems are uniquely adequate to tackle this goal, due to the low input requirements and the comprehensive output data. 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.
We have advanced significantly in each of the aims described in the proposal. For Aims 1 and 2, we have successfully scaled up and optimized the single-molecule nucleosome profiling technology. We mostly focused on histone modifications, and recently also initiated work on the analysis of transcription factors and chromatin regulators. We have modified the sample preparation procedures to allow analysis of very low input material, both from plasma and cells in culture. We developed procedures for concurrent mapping of 5-mC by two alternative approaches: (1) enzymatic labeling using synthetic co-factors, and (2) application of protein binding domains that bind specifically to methylated or unmethylated DNA. In addition, we established a fluidics system in the lab as well as automatic scripts to run single-molecule DNA sequencing, and thus significantly increased our capacity in that regard.

For Aim 3, we developed EPINuc, a novel single-molecule multi-parametric-based assay to comprehensively profile the Epigenetics of Plasma Isolated Nucleosomes. Our approach, based on the single-molecule technology, allows high resolution profiling of combinatorial modification states on millions of individual nucleosomes by single-molecule imaging. EPINuc is unique in its capacity to generate multi-layered information from very limited liquid biopsy material. As proof of concept, we applied it to diagnose colorectal cancer (CRC), which is one of the most common cancers worldwide, necessitating expensive and invasive screening tests for all adults older than 50. Strikingly, EPINuc revealed significant differences in the epigenetic states and protein biomarkers’ levels between healthy individuals versus CRC patients. We are in the process of preparing these results for publication. In addition, we secured additional funding for this project (Horizon 2020 - ERC Proof of Concept grant) to commercialize the technology.
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.