The Saccharomyces cerevisiae Piccolo NuA4 histone acetyltransferase complex requires the Enhancer of Polycomb A domain and chromodomain to acetylate nucleosomes

Abstract

Chromatin modification complexes are key gene regulatory factors which posttranslationally modify the histone component of chromatin with epigenetic marks. To address what features of chromatin modification complexes are responsible for the specific recognition of nucleosomes compared to naked histones, we have performed a functional dissection of the Esa1-containing Saccharomyces cerevisiae Piccolo NuA4 histone acetyltransferase complex. Our studies define the Piccolo determinants sufficient to assemble its three subunits into a complex as well as Piccolo determinants sufficient to specifically acetylate a chromatin template. We find that the conserved Enhancer of Polycomb A (EPcA) homology region of the Epl1 component and the N-terminal 165 amino acids of the Yng2 component of Piccolo are sufficient with Esa1 to specifically act on nucleosomes. We also find that the Esa1 chromodomain plays a critical role in Piccolo's ability to distinguish between histones and nucleosomes. In particular, specific point mutations in the chromodomain putative hydrophobic cage which strongly hinder growth in yeast greatly reduce histone acetyltransferase activity on nucleosome substrates, independent of histone methylation or other modifications. However, the chromodomain is not required for Piccolo to bind to nucleosomes, suggesting a role for the chromodomain in a catalysis step after nucleosome binding.

FIG. 1.

Representative selection of Piccolo deletion complexes used for HAT assays. The Coomassie-stained SDS-PAGE gel shows recombinant Piccolo complexes coexpressed in E. coli and purified by Talon chromatography. Constituent polypeptides in each deletion are described in Fig. 2. Piccolo v3 in lane 1 was additionally purified by Source Q and S chromatography. Epl1, Yng2, and Esa1 polypeptides for each deletion are indicated with a black square, black triangle, and open circle, respectively, to the right of the appropriate band. An E. coli contaminant with apparent size of 43 kDa that copurifies over Talon chromatography is labeled with a double-headed arrow between lanes 2 and 3. Molecular size standards are shown in lane 18 with corresponding sizes to the right (in kilodaltons).

FIG. 2.

Deletion analysis of Piccolo subunits. The chart shows the identity of subunits present in each Piccolo deletion construct, with evolutionarily conserved regions (EPcA, PHD, and chromodomain), structurally determined regions (Esa1 HAT domain), and hexahistidine tags shown as black and shaded regions. Naked histone and nucleosome HAT activities were normalized by ELISA-determined Esa1 content using anti-Esa1 antibodies. The preference for nucleosome ratio shown on the right side is the ratio of naked histone HAT activity to nucleosome HAT activity, with Piccolo v7 set to 1.00.

FIG. 3.

Piccolo variants containing Esa1 chromodomain mutations. (a) Space-filling representation of H3 peptide containing trimethylated K9 bound to Drosophila HP1 chromodomain. The HP1 chromodomain is shown in light blue, while the H3 peptide backbone and the trimethylated K9 are shown in pink and maroon, respectively. The residues that create or line the hydrophobic cage are displayed as follows: Y24 in light blue, Y48 in dark blue, W45 in yellow, and T54 in green. The corresponding Esa1 residues are also provided. Figure prepared using MidasPlus molecular graphics software (10) and PDB coordinates 1KNE (16). (b) Effect of Esa1 chromodomain point mutations on Piccolo HAT activity. Piccolo v55 and v68 to v74 each contain an N-terminal hexahistidine-tobacco etch virus nuclear inclusion a (NIa) protease site tag (HISN) on the Epl1 component, and all complexes were expressed and purified to similar levels. The preference for nucleosome ratio shown on the right side is the ratio of nucleosomal HAT activity to naked histone HAT activity, with Piccolo v55 set to 1.00 for all eight Piccolo variants.

FIG. 4.

Specific mutations in Esa1 chromodomain severely affect growth of yeast cells. Yeast strains deleted for Esa1 and containing a wild-type Esa1 gene on a low-copy-number URA3 plasmid were transformed with a LEU2 plasmid expressing wild-type Esa1 or Esa1 containing the indicated point mutations in the chromodomain. The left panels show growth of 10-fold serial dilutions on YPD rich medium, while the right panels show similar growth on 0.1% fluoroorotic acid plates. Rows 6 and 8 document the severe growth defects of yeast cells expressing the Y56A and E65L Esa1 chromodomain mutations.

FIG. 5.

Specific effects of chromodomain mutations do not require histone modifications. Piccolo v55, v68, v69, and v74 were assayed using native chicken (black bars), recombinant Xenopus (grey bars), and recombinant yeast (white bars) nucleosomes. Equivalent amounts of nucleosome substrates determined by Coomassie-stained SDS-PAGE gels of the histone proteins were used in the assays. The preference for nucleosome ratio shown on the right side is the ratio of nucleosomal HAT activity to naked histone HAT activity, with Piccolo v55 set to 1.00.

FIG. 6.

Esa1 chromodomain is not necessary for Piccolo to bind nucleosomes. (a) Gel filtration chromatograms of recombinant nucleosome core particles alone (cyan) and nucleosome core particles incubated with Piccolo v7 (red), Piccolo v72 (blue), Piccolo v69 (yellow), and Piccolo v74 (green). All Piccolo samples form stable complexes with nucleosomes, as shown by the appearance of a larger peak, distinct from the nucleosome peak and from the Piccolo-only peak at approximately 27 min (data not shown). SDS-PAGE gels of the peaks confirm the assignment of the gel filtration peaks. For example, fractions for the Piccolo/nucleosome peaks show all three Piccolo subunits and all four nucleosome histone subunits (data not shown). (b) Gel filtration chromatograms of recombinant nucleosome core particles (NCP) incubated with Piccolo v7 (red), which contains full-length Esa1, or Piccolo v14 (green), which lacks the Esa1 chromodomain. Both samples form the Piccolo/nucleosome complex, establishing that the chromodomain is not required for stable binding of Piccolo to nucleosomes. (c) Piccolo/nucleosome pulldown experiment confirms that the Esa1 chromodomain is not necessary for binding to nucleosomes. Blank (lanes 1 to 3), Piccolo v78 (lanes 4 to 6), or v82 (lanes 7 to 9) was immobilized on Strep-Tactin beads via their Strep-tagged Yng2 subunits and incubated with nucleosome core particles, and unbound and bound fractions were analyzed by Western blots to detect the histone H3 component of nucleosomes. Input samples are shown in lanes 1, 4, and 7; unbound (supernatant) fractions in lanes 2, 5, and 8; and bound fractions in lanes 3, 6, and 9.