Eukaryotic DNA exists in the cell nucleus via chromatin, which acts to both compact and regulate the genome. The basic subunit of chromatin is the nucleosome, which is comprised of a histone protein core, wrapped by ~147bp of DNA. The N-terminal regions of each of the histones protrude from the wrapped core and are referred to as the histone tails. Histone tails harbor a plethora of post-translational modifications (PTMs) that direct the function of chromatin regulators as they remodel chromatin during development and in response to external factors. Recognition of histone PTMs by these regulatory complexes is mediated through the action of reader sub-domains. The interaction of reader domains with modified histone tails has been extensively studied using peptide fragments of the tails. However, we have very little knowledge of how these domains associate with the full nucleosome. We are using NMR spectroscopy and complimentary orthogonal techniques to investigate this, and have found that the conformation of the histone tails in the context of the nucleosome has a dramatic effect on reader domain binding. As a model system, we are investigating the interaction of the BPTF PHD finger with its known cognate modification, methylated lysine 4 on histone H3 (H3K4me3). Here, we show that the conformation adopted by the histone H3 tails within the context of the nucleosome is inhibitory to binding of the BPTF PHD finger to H3K4me3, as compared to histone peptides. Using NMR spectroscopy and MD simulations, we find that the H3 tails interact robustly but dynamically with nucleosomal DNA, and demonstrate that this inhibits PHD finger association. Modifications and mutations of the H3 tail outside the binding region increase the accessibility to PHD finger binding, indicating that PTM crosstalk can regulate reader domain binding by altering the nucleosome conformation. Together, our results demonstrate that the nucleosome context has a dramatic impact on signaling events at the histone tails, and highlights the importance of studying histone binding in the context of the nucleosome.
Catherine Musselman (University of Iowa)