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 repeating 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 each 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 numerous chromatin regulatory factors, and have their functions regulated by various 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 initial published high-resolution MAS NMR studies revealed that N-terminal domains of histones H3 and H4 are conformationally flexible 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 modulation of the dynamic histone tail conformational landscapes functions to regulate their interactions within chromatin while remaining accessible to chromatin regulatory complexes. To investigate this hypothesis we will pursue the following three aims: (1) determine the influence of higher order chromatin structure regulatory factors on histone tail conformation and dynamics, (2) determine how acetylation of histone H4 lysine 16 regulates chromatin compaction, and (3) determine the regulation of H3 tail conformation and dynamics by trimethyl lysine 36 and PHF1. The proposed studies will provide the first high-resolution insights into involvement of H3 and H4 tails in 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 native chromatin environment.
Regulation of gene expression and genomic stability by epigenetic factors such as histone tail post- translational modifications (PTMs), chromatin architectural proteins and chromatin dynamics has become a dominant field in cancer research because of the reversible nature of epigenetic alterations. In this proposal large chromatin molecules will be investigated by using magnetic resonance spectroscopy and computational techniques to determine with atomic resolution the conformation and dynamics of histone protein tails, which serve as essential epigenetic regulators of chromatin, as well as the influence of histone PTMs and PTM- binding protein partners on histone tail function. These studies will provide molecular level insights into how histone tails control chromatin structure, dynamics and function, which are key for understanding tumorigenesis and drug resistance, and designing new cancer drugs and therapies.
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