The objective of this propose is to elucidate the molecular details of the organization of nucleosomes into higher-order structure of chromatin, the 30 nm filament. The 30 nm filament is the predominant form of chromatin in the eukaryotic cell. With increasing evidence for the importance of chromatin structure in gene regulation becomes essential to understand the molecular details of chromatin higher-order structure, and how this structure is affected by proteins such as H5, which is thought to play a role in chromatin inactivation or HMG 14/17, which are found to be associated with actively transcribed chromatin. The function of the various domains of the linker histone H1/H5 will be elucidated by determining the structure of chromatin reconstituted with fragments of H5 that lack specific domains or parts of domains. Efforts to crystallize the globular domain of H5 will be initiated. The main techniques to be used are neutron scattering scanning transmission electron microscopy (STEP). Neutron scattering will be used to (i) locate the position of H1/H5 in the 30 nm filament by selective deuteration (ii) test current models for chromatin structure by contrast variation (iii) measure and characterize changes in chromatin structure on association with linker histones III/H5 and the proteins HMG 14/17 (iv) characterize the structure of 30 nm filaments reconstituted to totally from histones and DNA and (v) treasure changes in chromatin structure such as fiber diameter and mass per unit length as a function of DNA linker length. STEM will be used in the initial stages to measure the diameter and mass per unit length of totally reconstituted chromatin fibers. Time-resolved X-ray synchrotron scattering will be used to measure the kinetics of formation of the 30 nm filament from depleted chromatin and histone H5. The kinetics will provide an independent test of whether several contiguous neocleosomes must have bound H5 in order to form a higher order structure. The results we help to resolve current controversies about the structure of the 30 nm filament, and provide a starting point for understanding how chromatin structure influences gene expression.
Showing the most recent 10 out of 13 publications
