Histone phosphorylation in native chromatin induces local structural changes as probed by electric birefringence

In order to understand how the phosphorylation of histones affects the chromatin structure, we used electron microscopy, sedimentation velocity, circular dichroism and electric birefringence to monitor the salt-induced filament reversible solenoid transition of phosphorylated and native chromatin. Phosphorylation in vitro of chicken erythrocyte chromatin by cyclic-AMP-dependent protein kinase from porcine heart led to the modification of the histones H3 and H5 only, which were modified at a level of one phosphate and about three phosphate groups per molecule, respectively. In contrast to circular dichroism and sedimentation studies, which tend to suggest that phosphorylation of H3 and H5 does not affect chromatin structure, electron microscopy reveals that phosphorylation causes a relaxation of structure at low ionic strength. Electric birefringence and relaxation time measurements clearly prove that local structural changes are induced in chromatin: we observe a decrease of the steady-state birefringence with the appearance of a negative contribution in the signal and a marked increase of the flexibility of fibres. The component with the negative birefringence presents very short relaxation times, like those exhibited by small DNA fragments or individual nucleosomes. Two possibilities are then suggested. First, the conformational change is consistent with what would be expected from the presence of DNA segments loosely associated with the core histone H3. That the length of such segments could correspond to about one to two base-pairs per nucleosome strongly suggests that phosphorylation induces changes affecting some specific H3-DNA interactions only. This result could corroborate previous observations indicating that the N-terminal region of H3, where the site of phosphorylation is located, plays a decisive role in maintaining the superstructure of chromatin. Second, phosphorylation could introduce hinge points between each nucleosome. In this case, the negative birefringence results from partial orientation of the swinging nucleosomes. A possible mode of action of phosphorylation might be to weaken structural restraints imposed by histone H3, thus facilitating further condensation of chromatin.