Fig. 4.

(A) Assembly of histone tetramers from synthetic histone H3 (cH3, prepared by NCL) and recombinant histone H4 (rH4) as monitored by size exclusion chromatography. Lane 1, molecular mass marker proteins; lane 2, standard of HeLa cell core histone octamers. Numbers above the gel indicate elution fraction number and the approximate protein mass range for each fraction. (B) Micrococcal nuclease digestion analysis of nucleosome formation catalyzed by RSF. Lanes 1 and 2 show the formation of nucleosomes, an indicator of successful chromatin assembly with reaction mixtures containing RSF, a DNA plasmid template, the cH3/rH4 tetramer, and two different concentrations of HeLa histones H2A and H2B. In contrast, control lanes 3 and 4 do not form chromatin as histones H2A and H2B were omitted from these reaction mixtures. (C) Synthetic histone H3 sequence indicating original acetylation modification sites, the NCL ligation junction, and the H3 K9 preferred site of methylation by the G9a methyltransferase. (D) Enzymatic methylation and deacetylation of a cH3/rH4 tetramer. Autoradiographic analysis of cH3/rH4 methylation by G9a with and without prior treatment with human histone deacetylase Sirt1. Lane 1, control experiment depicting methylation of unacetylated recombinant H3/H4 tetramer with G9a; lane 2, failure of G9a to methylate hyperacetylated cH3/rH4 tetramer because K9 is acetylated; lane 3, brief treatment of acetylated cH3/rH4 tetramer with Sirt1 followed by incubation with G9a results in the removal of AcK9 and the subsequent G9a-catalyzed methyl transfer to the newly liberated K9 side chain amine.