The overall objective of this proposal is to provide direct evidence for the effects of H1 histone phosphorylation on chromatin structure and function, and to establish the molecular events responsible for these effects. Currently proposed projects are concentrated on the study of the effects of highly phosphorylated H1 histone subcomponents on the structure of reconstituted chromatin. New procedures for the reconstitution of chromatin to high fidelity will be studied, including reconstitution of H1 into native chromatin by exchange reactions. The effects of H1 molecules phosphorylated to different levels and containing phosphate in specific sites will be determined by nuclease digestion, thermal denaturation, sedimentation, and in collaboration with others, nuclear magnetic resonance and neutron scattering. In addition, the effect of phosphorylation on the rate of exchange of H1 between sites in chromatin will be investigated, in order to determine whether phosphorylation affects dynamic interactions between H1 histone and chromatin. The enzyme responsible for the phosphorylation of H1 histone which occurs during interphase and at mitosis in growing cells will be purified to homogeneity from both interphase and mitotic cells, in attempts to identify two interconvertible forms of this enzyme and to establish the mechanism of interconversion. Inhibitory antibodies to this enzyme will be raised and microinjected into cells in order to provide direct evidence for the function of H1 histone phosphorylation in different cell cycle events. The studies should provide information on the biochemistry and molecular biology of chromatin structure and function related to processes involved in the regulation of cell growth and replication.
NIH ExPorter
NIH ExPorter