Unbiased proteomic screen for binding proteins to modified lysines on histone H3

Abstract

We report a sensitive peptide pull-down approach in combination with protein identification by LC-MS/MS and qualitative abundance measurements by spectrum counting to identify proteins binding to histone H3 tail containing dimethyl lysine 4 (H3K4me2), dimethyl lysine 9 (H3K9me2), or acetyl lysine 9 (H3K9ac). Our study identified 86 nuclear proteins that associate with the histone H3 tail peptides examined, including seven known direct binders and 16 putative direct binders with conserved PHD finger, bromodomain, and WD40 domains. The reliability of our proteomic screen is supported by the fact that more than one-third of the proteins identified were previously described to associate with histone H3 tail directly or indirectly. To our knowledge, the results presented here are the most comprehensive analysis of H3K4me2, H3K9me2, and H3K9ac associated proteins and will provide a useful resource for researchers studying the mechanisms of histone code effector proteins.

Figure 1

Strategy to isolate binders to modified K4 and K9 of histone H3. Unmodified H3 or modified H3 peptides containing dimethylated K4, dimethylated K9 or acetylated K9 were incubated with HeLa nuclear extracts and washed with low stringency buffers to enhance the isolation of low affinity binding proteins. The resulting proteins were resolved by SDS-PAGE for protein identification and quantification.

Figure 2

SDS-PAGE of binders isolated in the H3 pull-downs. (A) Biotinylated H3 peptides containing amino acids 1–21 and the indicated modifications (me2 = dimethylation; ac = acetylation) were used to isolate binders from HeLa nuclear extracts. The isolated proteins were analyzed by SDS-PAGE separation and stained with CBB. (B) The unmodified H3 peptide pull-down is highlighted to show the identity of the major proteins bound to this peptide.

Figure 3

Cluster analysis of protein abundance among modified H3 pull-downs. For each protein, spectral counts from Table 1 are scaled to Z-scores, calculated across the various H3 modifications and control beads listed below. As shown in the legend, red indicates abundance that is above average among the eight pull-downs; green indicates below-average abundance, with various shades indicating intermediary abundance levels. To highlight proteins present at similar levels in the same samples, Z-scores for each protein were clustered along the horizontal axis.

Figure 4

Confirmatory binding of PHF2 to H3K4me2. Western blot for PHF2 in H3 peptide pull-downs from HeLa nuclear extracts with the various methylations states in K4 and K9.