Linker histones are involved in chromatin higher-order structure and gene regulation. We have successfully achieved partial phosphorylation of linker histones in chicken erythrocyte soluble chromatin with CDK2, as indicated by HPCE, MALDI-TOF and Tandem MS. We have studied the effects of linker histone partial phosphorylation on secondary structure and chromatin condensation. Infrared spectroscopy analysis showed a gradual increase of β-structure in the phosphorylated samples, concomitant to a decrease in α-helix/turns, with increasing linker histone phosphorylation. This conformational change could act as the first step in the phosphorylation-induced effects on chromatin condensation. A decrease of the sedimentation rate through sucrose gradients of the phosphorylated samples was observed, indicating a global relaxation of the 30-nm fiber following linker histone phosphorylation. Analysis of specific genes, combining nuclease digestion and qPCR, showed that phosphorylated samples were more accessible than unphosphorylated samples, suggesting local chromatin relaxation. Chromatin aggregation was induced by MgCl2 and analyzed by dynamic light scattering (DLS). Phosphorylated chromatin had lower percentages in volume of aggregated molecules and the aggregates had smaller hydrodynamic diameter than unphosphorylated chromatin, indicating that linker histone phosphorylation impaired chromatin aggregation. These findings provide new insights into the effects of linker histone phosphorylation in chromatin condensation.
In the presence of 1.7 mM Mg2+, the diameter of the circular structures produced by small chromatin fragments isolated from chicken erythrocytes remains essentially unchanged when the number of nucleosomes in these fragments increases from 10 to 36. In contrast, the results obtained in unidirectional shadowing experiments show that under the same conditions the height of the chromatin fragments increases with the number of nucleosomes. These observations indicate that the electron microscope images studied in this work correspond to a top view of small chromatin fragments. Rotary-shadowed chromatin fragments show three parts: (a) a contour with a heavy deposition of platinum; (b) an annular zone between the central region and the periphery; and (c) a central hole. The heterogeneous ring generated by the deposition of platinum in the periphery suggests that nucleosomes form a one-start helix (5-7 nucleosomes per turn) that apparently can be left- or right-handed. The annular region (thickness of about 11 nm) shows spokes probably due to flat faces and core DNA of radially oriented nucleosomes. The central hole (8-12 nm) is clearly seen in many images but it is not empty because some deformed fragments show coated material (probably linker DNA) that protrudes from this central depression. We have observed that these structural elements directly detected in short chromatin fragments are also present in long chromatin fibers. This allows us to conclude that these elements are basic structural components of the 30 nm chromatin fiber.
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