Title: Simultaneous measurement of DNA methylation and genome-nuclear lamina interactions in single cells

Abstract

DNA methylation (5-methylcytosine or 5mC) and the 3-dimensional organization of the genome within the cell nucleus are two critical epigenetic features that regulate gene expression and cellular behavior, and aberrant patterns of these epigenetic features are associated with cancer and disease. In cancer, widespread regions of DNA methylation loss, termed hypomethylation, have previously been found positioned at the periphery of the nucleus, the nuclear lamina (NL), suggesting DNA methylation and genome organization could be linked epigenetic states. However, the direct genome-wide relationship between 5mC and genome-NL interactions remains obscured in bulk measurements. To overcome this limitation, we developed a newsingle-cell sequencing technology (sc5mC+DamID-seq) to simultaneously measure DNA methylation and genome-NL interactions from the same cell. sc5mC+DamID-seq uses mark- specific barcoded adapters to identify both epigenetic features and individual cells, enabling us to profile thousands of single cells per day. By applying sc5mC+DamID-seq to chronic myelogenous leukemia cells (KBM7), we observed hypomethylation at regions contacting the NL, known as lamina associated domains (LADs). Interestingly, we also discovered that genomic regions that contact the NL more frequently in single cells display the greatest loss and variability in 5mC. Further, while LADs appear as continuous stretches of contact with the NL in bulk sequencing, LADs at the single-cell level frequently display segments of noncontact with the NL, which we found influences the amount of hypomethylation in that domain. Finally, to test the connection between these two epigenetic features, we globally demethylated the epigenome, which relocated more variable genome-NL contact regions towards the nuclear interior. Thus, simultaneous single-cell measurements with sc5mC+DamID-seq has enabled us to systematically uncover the relationship between DNA methylation and genome organization in cancer cells.