FIGURE 4.

Both histone H4 Lys⁴⁴ and H2A Leu¹¹⁶ and Leu¹¹⁷ are needed for trans-histone H3 Lys³⁶ di- and trimethylation in vivo and in vitro. A, yeast whole cell extracts generated from cells expressing WT or the indicated histone mutants, including histone H4 single mutations, H2A double mutations, or combination of H4 and H2A triple mutations, are immunoblotted against H3 Lys⁴, H3 Lys³⁶, and H3 Lys⁷⁹ methyl-specific antibodies. The asterisks denote nonspecific bands. Immunoblots for histone H3 and H2A are used as loading controls. For detecting H3 Lys³⁶ trimethylation, both short and long exposures to film are shown. The Set2 protein levels are detected by probing with an α-Set2 antibody. B, in vitro HMT assays are performed using the indicated yeast chromatin substrates incubated with or without recombinant Set2. The amounts of chromatin substrates used in the assays are normalized by the Branford method. HMT reactions are analyzed by scintillation counting.