The Saccharomyces cerevisiae histone H2A variant Htz1 is acetylated by NuA4

Abstract

The histone H2A variant H2A.Z (Saccharomyces cerevisiae Htz1) plays roles in transcription, DNA repair, chromosome stability, and limiting telomeric silencing. The Swr1-Complex (SWR-C) inserts Htz1 into chromatin and shares several subunits with the NuA4 histone acetyltransferase. Furthermore, mutants of these two complexes share several phenotypes, suggesting they may work together. Here we show that NuA4 acetylates Htz1 Lys 14 (K14) after the histone is assembled into chromatin by the SWR-C. K14 mutants exhibit specific defects in chromosome transmission without affecting transcription, telomeric silencing, or DNA repair. Function-specific modifications may help explain how the same component of chromatin can function in diverse pathways.

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The Htz1-K14R mutant is selectively sensitive to benomyl. (A) Cross-species alignment of the H2A.Z N-terminal tail; upper set is multiple Saccharomyces species (Cliften et al. 2003), lower set is metazoans. Residues different from S. cerevisiae Htz1 are boxed. The four modifiable lysines in the N-terminal tail of S. cerevisiae Htz1 (K3, K8, K10, and K14) are indicated with asterisks. The peptide used to raise the Htz1 K14^Ac antibody is boxed and shaded. (B) Htz1 K3, K8, K10, and K14 were individually mutated to arginine (R). Mutants were expressed as the sole source of Htz1 and 10-fold serial dilutions of each strain were spotted onto the indicated plates (HU [100mM]; benomyl [15 μg/mL] in YPD). Plates were incubated for 3 d at 30°C. (C) As in B, except the sensitivity of the indicated strains to 6AU (75 μg/mL) or MPA (15 μg/mL) was examined after 72 h.

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Htz1-K14 is acetylated by NuA4. (A) Specificity of the Htz1-K14^Ac antibody for the acetylated (K14Ac) relative to the unmodified (K14) Htz1 peptide. Indicated amounts (in picomoles) of each peptide were spotted onto nitrocellulose before immunoblotting. (B) Htz1-K14 is acetylated in vivo. HA-tagged forms of the indicated Htz1 proteins were individually expressed as the sole source of the histone and strains were analyzed by immunoblotting. Deletion of Htz1 or mutation of K14 to arginine (K14R) or glutamine (K14Q) abolishes recognition by the anti-K14^Ac antibody. In contrast, mutation of residues K3, K8, or K10 has no effect on K14^Ac levels. Total Htz1 was detected with anti-HA. Anti-Rpt1 and anti-Rpn8 were used as loading controls. (C) Esa1 is required for acetylation of Htz1-K14 in vivo. Strains containing ESA1 or the temperature-sensitive mutants esa1-L327S or esa1-L254P were grown at room temperature (RT°) or for 4 h at the nonpermissive temperature (37°C). (Bottom) Growth was assayed after 2 d at room temperature (RT°) or 37°C. Cell extracts were assayed by immunoblotting with the indicated antibodies. (D) Htz1-K14 acetylation is differentially dependent on subunits of NuA4. Extracts from indicated deletion strains were immunoblotted with the antibodies shown to the right of each panel. (E) NuA4 efficiently acetylates Htz1-K14 in vitro. (Top) Partial TAP purifications of indicated HATs were monitored by reactivity of the protein A tag (αPAP). (Bottom) An untagged strain (NT) was used as a control. IgG-purified HATs were added to reactions containing recombinant Htz1/H2B dimer and acetyl CoA. Htz1-K14^Ac and Htz1 were detected by immunoblotting.

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Htz1-K14 is acetylated after assembly into chromatin (A) Htz1-K14 acetylation is reduced in SWR-C mutants. Extracts from the indicated deletion strains were assayed for total and acetylated Htz1 by immunoblotting. Histone H3 and Rpt1 were used as loading controls. (B) Htz1 localization to chromatin does not require K14 acetylation. Cells containing HA-tagged forms of the indicated Htz1 proteins were separated into total (T), cytoplasmic (C), nuclear (N), or chromatin (Ch) fractions and immunoblotted with the indicated antibodies. Cytoplasmic segregation of the proteasome components Rpt1 and Rpn8 and the translation initiation factor eIF-5A demonstrates efficient fractionation. (C) Htz1 K14 acetylation does not regulate genomic distribution of the histone variant. HA-tagged Htz1 proteins (wild type [WT], K14R, or K14Q) were individually expressed as the sole source of the histone. The relative recruitment of each at the telomere of chromosome V (TEL-V), the highly transcribed ADH1 gene, and the centromere of chromosome III (CEN3) was then determined by ChIP. A schematic of each location is shown (size in kilobases is indicated), along with the relative location of the primer pairs used (see Supplementary Table 3; Krogan et al. 2003, 2004). In each case, occupancy is expressed relative to *, a subtelomeric region of chromosome V (9716–9823; see TEL-V schematic) (Krogan et al. 2003). Results are the mean ± standard deviation (SD) of three independent ChIPs. (D) Htz1 K14 mutants in chromatin have normal stability. The chromatin pellets from B were extracted with increasing salt concentrations as indicated and proteins remaining in the pellet assayed by immunoblotting.

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Chromosome missegregation rates are increased in htz1-K14R mutants. (A) Diploid htz1Δ/htz1Δ cells containing empty vector (pRS313) or Htz1 plasmids (wild type [WT], K14R, or K14Q) were plated to single colonies, with selection maintained for the plasmids. Segregation of a reporter chromosome fragment was scored visually by counting sectored colonies (Koshland and Hieter 1987; Krogan et al. 2004). Frequencies of events are listed, with relative rate (normalized to the pHTZ1 control) shown in parentheses. (B) Htz1 is not required for kinetochore localization at the centromere. Epitope-tagged forms of the centromere-specific histone H3 variant Cse4, or representatives of the inner (Cbf1, Cbf2/Ndc10) or central (Ctf3, Ctf19) kinetochore were analyzed by ChIP at CEN3. Schematic shows a model of kinetochore components; arrows indicate interactions between complexes. Location of the ChIP primers at CEN3 and means of calculating relative occupancy are as in Figure 3C.