Site-specific Binding Affinities within the H2B Tail Domain Indicate Specific Effects of Lysine Acetylation

Jeffrey J. Hayes,Xiaodong Wang

doi:10.1074/jbc.m706035200

Abstract

Acetylation of specific lysines within the core histone tail domains plays a critical role in regulating chromatin-based activities. However, the structures and interactions of the tail domains and the molecular mechanisms by which acetylation directly alters chromatin structures are not well understood. To address these issues we developed a chemical method to quantitatively determine binding affinities of specific regions within the individual tail domains in model chromatin complexes. Examinations of specific sites within the H2B tail domain indicate that this tail contains distinct structural elements and binds within nucleosomes with affinities that would reduce the activity of tail-binding proteins 10-50-fold from that deduced from peptide binding studies. Moreover, we find that mutations mimicking lysine acetylation do not cause a global weakening of tail-DNA interactions but rather the results suggest that acetylation leads to a much more subtle and specific alteration in tail interactions than has been assumed. In addition, we provide evidence that acetylation at specific sites in the tail is not additive with several events resulting in similar, localized changes in tail binding.

Highlights

75% of the mass of each of the core histones is organized into a largely ␣-helical domain that is assembled into the protein spool onto which the DNA is wrapped [1, 9]
Acetylation of lysine 16 within the H4 tail domain abrogates an interaction between the H4 tail and a surface of H2A/H2B, resulting in a reduction in the ability of nucleosome arrays to condense into higher order structures [24]
We reasoned that evaluation of the binding parameters for several sites within a tail domain would reveal fundamental aspects of tail function and of post-translational modifications within these domains

Summary

EXPERIMENTAL PROCEDURES

Preparation of Core Histones—Coding sequences for H2B mutants were obtained from the wild type Xenopus H2B sequence by the Stratagene QuikChange kit and were cloned into the pET3a expression plasmid. Samples of 0.8 ␮M NCPs containing different NaCl concentrations were reacted with 5.6 ␮M FM for different times, and the reaction was stopped by adding DTT to a final concentration of 5 mM. That the reaction of NCPs with FM can be described by overall rate constant kNCP, as shown, kNCP ϭ ϪNϪ1dN/dt (Eq 10). We find that the rate of reaction of the free dimer and the NCP at elevated salts (0.5 M) where the tails are expected to be completely released from binding sites within nucleosomes [17] are equivalent (see text)

RESULTS

Linker DNA and Specific DNA

Methods