Ion-responsive compaction and decompaction of long reconstituted chromatin

Eukaryotic genomic DNA is hierarchically folded into chromatin, whose higher-order structure is dynamically reorganized in response to the cellular physicochemical environment. Here, we examined structural transitions of long chromatin reconstituted on an 81 297-bp DNA template by single-molecule fluorescence microscopy. Increasing the histone/DNA mass ratio generated heterogeneous conformational populations, including coil, intrachain-segregated, and globule states, and increased the basal globule fraction. Spermidine, a trivalent polyamine, further promoted globule formation in reconstituted chromatin, whereas NaCl induced decompaction in the presence of spermidine. However, unlike naked DNA, reconstituted chromatin retained a residual globule population even at high NaCl concentrations. To quantify the NaCl-dependent reduction of the additional globule population formed upon spermidine addition, we introduced the normalized induced globule fraction, G_norm, relative to the basal globule level. This analysis showed that NaCl preferentially decompacted the globules formed upon spermidine addition, whereas the histone-dependent basal globule population remained largely preserved. These results suggest that spermidine-associated additional compaction and histone-dependent basal compaction exhibit distinct NaCl responses in long reconstituted chromatin. These findings provide a single-molecule basis for understanding how chromatin compaction is regulated by multiple physicochemical mechanisms in complex environments where diverse biomolecules and ions coexist.