Gilbert, N.; Boyle, S.; Allan, J.

Embryonic stem cells (ESCs) are thought to maintain pluripotency through global hyper-transcription, linked to an "open" chromatin structure. To investigate this idea, we analyzed higher-order chromatin fibres from NIH3T3 cells, mouse ESCs, and their differentiated progenitors. Bulk chromatin composition including protein:DNA ratio and nucleosome repeat length varied little between the cell types, but surprisingly biophysical analyses such as linker histone FRAP, hydrodynamic sedimentation and nuclease sensitivity also showed no significant differences in the conformation of purified higher-order chromatin fibres. To better evaluate the structure of higher-order chromatin fibres observed in cells, we developed a novel technique called SPOCC (Sedimentation Properties of Cross-Linked Chromatin). This approach revealed that ESCs and differentiated cells share similar bulk higher-order chromatin fibre structures, whilst ESCs have a slightly more disrupted structure than NIH3T3 cell chromatin. These results indicate that ESC transcriptional activity and plasticity are not driven by a fundamentally "open" higher-order chromatin conformation.