Structural and dynamic studies of histone tails in chromatin by magnetic resonance spectroscopy



 DESCRIPTION (provided by applicant): PROJECT SUMMARY Chromatin is the eukaryotic complex of DNA with proteins that regulates transcription, replication and repair through dynamic changes in its structure. The DNA in chromatin is packaged into repeat nucleosome building blocks, with each nucleosome consisting of ~147 bp of DNA wrapped nearly twice around a histone protein octamer containing two copies each of histones H2A, H2B, H3 and H4. All histones contain disordered N- terminal tail domains, corresponding to ~15-30% of their amino acid sequences that protrude out from the nucleosome. The N-terminal tails of histones H3 and H4 are essential regulators of chromatin function. These domains interact with DNA and other histones to mediate chromatin compaction, recruit a variety of chromatin regulatory factors, and have their functions regulated by numerous post-translational modifications (PTMs). While the atomic structure of the nucleosome and arrangements of nucleosomes within evenly spaced arrays representative of chromatin fibers have been resolved by X-ray crystallography and cryo-electron microscopy, the histone N-terminal tails have escaped high-resolution characterization in densely packed nucleosome arrays. The latter is due to their intrinsic disorder coupled with the fact that they are an integral part of large multi-megadalton protein-DNA assemblies. To address these challenges and directly investigate histone tail domains in chromatin at physiological concentrations, we have applied magic-angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) to recombinant nucleosome arrays reconstituted with 13C,15N-enriched histones. Our recently published initial high-resolution MAS NMR studies revealed that N-terminal domains of histones H3 and H4 are conformationally dynamic even in highly condensed chromatin. These findings strongly suggest that histone tails do not act as static tethers to compact chromatin and recruit PTM-binding proteins and have caused us to reevaluate their function in chromatin. The central hypothesis of this proposal is that histone tails in chromatin function through the modulation of their conformational dynamics by different factors, which allows these domains to mediate interactions within chromatin while remaining accessible to chromatin regulatory complexes. To investigate this hypothesis we will pursue the following three aims: (1) determine how the conformational flexibility of histone tails functions with nucleosome positioning and linker histones to regulate higher order chromatin structure and dynamics, (2) determine how acetylation of histone H4 lysine 16 regulates chromatin compaction, and (3) determine the regulation of H3 tail dynamics by trimethylated lysine 36 and PHF1. The proposed studies will provide the first high-resolution insights into how H3 and H4 tails control critical events that regulate transcription including chromatin compaction and recruitment of an essential PTM-binding protein, and are highly significant for understanding the function of histone tails in chromatin. Finally, these studies will provide an important foundation for future work on key histone PTM-binding complexes in the chromatin environment.