Establishment of epigenetic maps of plasma circulating nucleosomes to identify their tissue-of-origin

The analysis of cell-free DNA (cfDNA) in plasma represents a rapidly advancing field in medicine, with developing methods aiming to utilize it for diagnostics of cancer and additional pathologies. As blood cfDNA consists predominantly of nucleosome-protected DNA shed into the bloodstream by dying cells, it remains associated with histones that may retain the covalent modifications present in the cell of origin. In my research, I am utilizing a novel single-molecule technology developed in our lab in order to obtain multi-layered data from cell-free circulating nucleosomes (cfNUC). This technology consists of immobilizing nucleosomes in a spatially distributed manner on microscopic coverslips, following their incubation with fluorescently labeled antibodies to histone modifications and direct single-molecule DNA sequencing-by-synthesis. Total internal reflection (TIRF) microscopy is used to record the modification state and sequence of each nucleosome.

I have developed methodologies to extract nucleosomes from plasma and anchor them to the surface, followed by single-molecule detection of the combinatorial pattern of histones H3K4me3 and H3K27me3. I am further developing methods to examine additional histone modifications as well as DNA methylation. Finally, I plan to apply single-molecule DNA sequencing in order to map these epigenetically decoded cfNUCs to the genome. By performing a correlation analysis of the integrated data with reference datasets corresponding to different tissues and cancer types, I intend to establish highly informative epigenetic maps of plasma cfNUC indicative of their tissue of origin. This approach has immense potential for non-invasive diagnostics along with improved therapy stratification through real-time monitoring of disease progression.