Linker histone binding to superhelical DNA: Electrophoretic and filter binding studies

It has been known for years that linker histones bind preferentially to supercoiled DNA. This preference has been demonstrated by a number of different techniques including deoxyfibonucleoprotein electrophoresis, sedimentation, and filter binding under non-competitive conditions. In an attempt to further study this issue, we used one- and two-dimensional electrophoretic gels and filter binding under competitive conditions, with all DNA forms of interest being simultaneously present in the incubation mixture. Comparison between results obtained by the two methods showed that whereas the preference for superhelical molecules was clearly seen in the electrophoretic gels, the filter binding assay failed to reveal this preference. These results reveal limitations to the filter binding technique, which must be borne in mind in studies involving superhelical DNA molecules.     

Linker histone function in chromatin: dual mechanisms of action.

Aspects pertaining to linker histone structure and function are discussed, including the extent to which these proteins are essential, their ability to regulate specific gene expression, and recent structural data that provides a potential molecular basis for understanding how linker histones can have both repressive and stimulatory effects on genomic functions in vivo.     

Linker histone tails and N-tails of histone H3 are redundant: scanning force microscopy studies of reconstituted fibers.

The mechanisms responsible for organizing linear arrays of nucleosomes into the three-dimensional structure of chromatin are still largely unknown. In a companion paper (Leuba, S. H., et al. 1998. Biophys. J. 74:2823-2829), we study the contributions of linker histone domains and the N-terminal tail of core histone H3 to extended chromatin fiber structure by scanning force microscopy imaging of mildly trypsinized fibers. Here we complement and extend these studies by scanning force microscopy imaging of selectively reconstituted chromatin fibers, which differ in subtle but distinctive ways in their histone composition. We demonstrate an absolute requirement for the globular domain of the linker histones and a structural redundancy of the tails of linker histones and of histone H3 in determining conformational stability.     

Linker histone partial phosphorylation: effects on secondary structure and chromatin condensation

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.