Preparation of Recombinant Histones and Chaperones

6XHis-Yeast Nap1 histone chaperone (yNap1, or Nap1 in this report) was expressed and purified with Ni-NTA resin (Thermo Fisher Scientific, Waltham, MA) as reported elsewhere.^{37 (link)}Xenopus laevis core histones were separately prepared as detailed elsewhere,^{38 (link)} and H2B T112C^{39 (link)} was labeled with an Atto647N fluorophore functionalized with maleimide (Sigma-Aldrich, St. Louis, MO). As for H3K56ac and H4K16ac preparation, the thiol–ene coupling reaction was performed on H3K56C/C110A and H4K16C, respectively, as reported earlier,^{30 (link),40 (link)} which was modified from earlier publications.^{41 ,42} This modification strategy has been utilized to reproduce results obtained with specific acetylations via native chemical ligation.^{40 (link),42} H3K56ac and H4K16ac in our nucleosomes reported here refer to H3K_s56ac and H4K_s16ac. Briefly, H3K56ac and H4K16ac were prepared by reacting the cysteine thiols with N-vinyl-acetamide (NVA). Mass-spectrometric analyses of the acetylated histones confirm near complete acetylation (Figure S2). H2A-H2B dimer and (H3–H4)₂ tetramer were refolded and purified as described in ref 38 (link).