DNA is organized into chromatin by the formation of nucleosomes: 147 base pairs of DNA wrapped around an octamer of histone proteins. Post-translational histone modifications in the DNA- histone interface are thought to perturb interactions between the histone protein and the DNA and play a vital role in processes such as DNA repair and transcription regulation. However, it is not known how these modifications function within the nucleosome core.
To investigate this model, we examined two modifications that are buried under the DNA in the dyad symmetry region of the nucleosome core: the acetylation of Lys115 and Lys122 on histone H3. Three differentially-modified peptides were synthesized, corresponding to residues 110-135 of histone H3 using solid phase peptide synthesis. Expressed protein ligation was used to link each of the synthetic peptides to a recombinant histone thioester using the native Cys110 as a ligation junction to yield three semi-synthetic modified histone H3 proteins, H3-K115Ac, H3-K122Ac and H3-K115Ac/K122Ac.
All three semi-synthetic proteins as well as corresponding glutamine mutations were incorporated into single nucleosomes. Biochemical and biophysical studies including competitive reconstitutions, thermal repositioning, mapping and restriction enzyme digestion were conducted with the semi-synthetic modified nucleosomes. These studies demonstrate that our glutamine mimics do not reproduce the full effects of the genuine post-translational modification. They also reveal that modifications in the DNA-histone interface of the nucleosome reduce the free energy of DNA-octamer binding, and increase the rate of thermal repositioning which may play a role in nucleosome assembly, disassembly and repositioning.