Side-by-side comparison of histone purities.

<p>Histones were purified according to the RHP protocol (RHP) and according to a published protocol that started with the preparation of inclusion bodies (IB; ref. 5). Both purification procedures started from the same amount of bacteria that were grown on the same day. (A) SDS-PAGE analysis. H2A showed the weakest overexpression (Fig. S1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104029#pone.0104029.s001" target="_blank">File S1</a>) and is consequently the least pure. M: protein marker. (B) Purities.</p

Histone extraction.

<p>Whole cell extracts were prepared under denaturing conditions from bacteria expressing <i>Drosophila</i> H2B by French Press and sonication. Cell debris and residual insoluble material were pelleted by centrifugation. Efficiency of the histone extraction was analyzed on Coomassie-stained SDS gels by loading equivalent amounts of the supernatant containing the solubilized histones (SN) and the corresponding pellet fraction (P). Most H2B was present in the supernatant.</p

Histone purification by cation exchange chromatography.

<p>The whole cell extract from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104029#pone-0104029-g002" target="_blank">Figure 2</a> containing solubilized <i>Drosophila</i> H2B (SN) was filtered and applied to cation exchange chromatography under denaturing conditions. (A) Equivalent amounts of the filtered whole cell extract (Input) and the flow-through fraction of the cation exchange column were analyzed by SDS-PAGE. Most H2B bound to the chromatography resin. (B) H2B was eluted by a NaCl gradient as indicated. Fractions 4–8 were pooled and processed further as described in the main text.</p

Quality control of the histone octamers.

<p>Stoichiometry and purity of the octamers assembled from the histones purified according to the RHP method outlined in the main text were analyzed by SDS-PAGE (RHP). An octamer preparation assembled from histones purified from inclusion bodies according to published protocols was loaded in parallel (IB; ref. 5). The asterisk marks a contamination that is present to a lesser extent in RHP octamers.</p

Histone purification strategies.

<p>Schematic depiction of the workflow of (A) the conventional histone purification method according to Luger and coworkers <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104029#pone.0104029-Luger2" target="_blank">[2]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104029#pone.0104029-Luger3" target="_blank">[3]</a> and (B) our RHP protocol. For further details see the main text. Footnotes indicate variations and simplifications of the initial protocol. * The gel filtration step was successfully omitted in simplified purification schemes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104029#pone.0104029-Clapier1" target="_blank">[5]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104029#pone.0104029-Vary1" target="_blank">[7]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104029#pone.0104029-Gelbart1" target="_blank">[9]</a>. # These steps can be replaced by dilution into or dialysis against SAU 200 buffer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104029#pone.0104029-Clapier1" target="_blank">[5]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104029#pone.0104029-Gelbart1" target="_blank">[9]</a>. ‡ To remove possible DNA contaminations, it was suggested to filter the sample through an anion exchange resin prior to applying it to the cation exchange chromatography <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104029#pone.0104029-Vary1" target="_blank">[7]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104029#pone.0104029-Gelbart1" target="_blank">[9]</a>. § Anion exchange filtering and cation exchange chromatography can be combined. See note in step 3.2 and Figure S2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104029#pone.0104029.s001" target="_blank">File S1</a>.</p

Histone octamer assembly.

<p>Histones purified according to the RHP protocol were assembled into octamers. The elution profile of the size exclusion chromatography column is depicted (upper panel). The protein content of selected elution fractions was analysed by SDS-PAGE (lower panel and Fig. S3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104029#pone.0104029.s001" target="_blank">File S1</a>). Octamers eluted with a tailing shoulder, which contained a contaminating protein (asterisk).</p

Extinction coefficients of <i>Drosophila</i> histones at 280 nm.

<p>*Molecular weights do not include the initial methionine.</p>#<p>The extinction coefficients were calculated using the ProtParam tool with water as solvent (Swiss Institute of Bioinformatics; <a href="http://web.expasy.org/protparam/" target="_blank">http://web.expasy.org/protparam/</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104029#pone.0104029-Artimo1" target="_blank">[17]</a>.</p

Nucleosome array reconstitution.

<p>(A) Schematic depiction of the nucleosome arrays (DNA: grey line; nucleosome positions: ovals). The array DNA comprised 25 repeats of a 197 bp fragment (magnification) harbouring the Widom-601 nucleosome positioning sequence (dashed line). Numbers indicate positions of restriction enzyme sites and nucleosome boundaries with respect to the nucleosomal dyad axis (0). (B) AvaI digests of purified nucleosome arrays reconstituted with increasing amounts of unmodified (H4) or acetylated (H4K16ac) octamers. The reactions were loaded onto a native agarose gel and DNA visualized by ethidium bromide stain. (bp: base pairs).</p

Reconstitution of chromatosome arrays.

<p>(A) AvaI digests of chromatosome arrays carrying H4K16ac. Chromatosome arrays were reconstituted with increasing amounts of H1, purified by MgCl₂ precipitation and digested to monomers with AvaI. Mononucleosomes were separated from chromatosomes on native agarose gels and visualized by ethidium bromide stain. Faint additional bands may contain coprecipitating competitor DNA or multimers arising from incomplete AvaI digest. (kb: kilobases). (B) Analysis of the relative ratio of H1 to core histones in chromatosome arrays. Top: Chromatosome arrays from the H1 titration shown in A were loaded onto SDS gels and the protein content visualized by Coomassie stain. The gel of the acetylated chromatosome arrays is depicted. Bottom: Quantification of the relative ratio of H1 to core histones. The theoretically expected ratio of 0.24 for a 1:1 stoichiometry of H1 to octamers was not reached, presumably because the Coomassie staining did not linearly correlate with the molecular weight of the proteins <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088411#pone.0088411-Tal1" target="_blank">[79]</a>. (kDa: kilodaltons; a.u.: arbitrary units).</p

ISWI ATPase activity is not influenced by H4K16ac.

<p>(A) Steady-state ATPase assay. ATPase activity of ISWI (100 nM) was stimulated with saturating concentrations of nucleosome arrays (600 nM) carrying unmodified (H4) or acetylated H4 (H4K16ac). ATP hydrolysis rates in presence of saturating concentrations of ATP (1 mM) were determined. Control reactions did not contain nucleosome arrays. Error bars represent standard deviations (No arrays: n = 4; H4 and H4K16ac: n = 5). (B) ISWI (350 nM) was stimulated with DNA (1.2 mg/ml salmon sperm DNA) and increasing concentrations of an unmodified or H4K16ac-carrying histone H4 N-terminal peptide (H4 tail peptide). ATP hydrolysis rates were determined as above at 1 mM ATP. A peptide with scrambled amino acid sequence of the H4 tail harbouring an acetylated lysine residue served as control. Data were fit to lines to extract slopes (dashed lines; H4: 4.1*10³ s⁻¹ M⁻¹; H4K16ac: 3.5*10³ s⁻¹ M⁻¹). Error bars display standard deviations (n = 3–4).</p