Global reorganization of chromatin fibers during stem cell differentiation

Chromatin structure and organization determine genome activity and output in the nucleus. In this study, we developed an approach that allowed us to gain insights into how chromatin fibers are structured and change dynamically during human embryonic stem (ES) cell lineage specification in vivo. Specifically, we simultaneously stenciled the chromatin fiber architecture and the epigenetic information encoded in individual fibers onto the underlying DNA templates using a combination of nonspecific DNA GpC– and N6-adenine–methyltransferases. Applying nanopore ultra-long-read sequencing to the chromatin stencils (hereafter, Ex-nanoHiMe-seq) enabled a readout of the architecture and the epigenetic information carried by ultra-long individual chromatin fibers. By applying Ex-nanoHiMe-seq to ES cells, neural progenitor cells and cortical glutamatergic neurons, we revealed that chromatin fibers were formed by heterogeneous clusters of nucleosomes in vivo and that nucleosome densities inside clusters were cell-type- and chromatin-state-specific. By analyzing the epigenetic information along individual chromatin fibers, we also uncovered the all-or-none actuation of a chromatin domain on single fibers during epigenetic reprogramming, as well as the DNA elements responsible for the inheritance of facultative heterochromatin during DNA replication and cell division in human ES cells. Our approach and results enable a novel understanding of how DNA elements shape chromatin structure via modulating epigenetic information.