G34R cancer mutation alters the conformational ensemble and dynamics of the histone H3.3 tails

Mutations in the histone variant H3.3 have been discovered in pediatric and adult gliomas and osteosarcomas. One of these is G34R in the H3.3 N-terminal tail. While this mutation is known to disrupt epigenomic pathways, the effects on nucleosome structure itself have not been explored. In light of recent studies, which demonstrate that the interaction of the H3 tail with nucleosomal and linker DNA is driven in large part by arginine residues, we sought to determine if the G34R cancer mutation and adjacent G33R mutation, not observed in cancer, directly alter nucleosome structural dynamics. Using nuclear magnetic resonance spectroscopy and molecular dynamics simulations, we investigate the effects of these mutations on the H3 tail in the context of the nucleosome. We show that both of these mutations enhance association of the H3 tails with DNA and decrease conformational dynamics around the site of mutation. Our results also reveal changes in the conformational ensemble of the entire tail, re-positioning it on the nucleosomal DNA and promoting intra-tail interactions. We demonstrate that these changes in the nucleosome, produced by mutations, alter the association of a tandem of plant homeodomain-fingers from CHD4 with the unmodified H3 tails.