Individual lysine acetylations on the N terminus of Saccharomyces cerevisiae H2A.Z are highly but not differentially regulated

Abstract

The multi-functional histone variant Htz1 (Saccharomyces cerevisiae H2A.Z) is acetylated on up to four N-terminal lysines at positions 3, 8, 10, and 14. It has thus been posited that specific acetylated forms of the histone could regulate distinct roles. Antibodies against Htz1-K8(Ac), -K10(Ac), and -K14(Ac) show that all three modifications are added by Esa1 acetyltransferase and removed by Hda1 deacetylase. Completely unacetylatable htz1 alleles exhibit widespread interactions in genome scale genetic screening. However, singly mutated (e.g. htz1-K8R) or singly acetylable (e.g. the triple mutant htz1-K3R/K10R/K14R) alleles show no significant defects in these analyses. This suggests that the N-terminal acetylations on Htz1 are internally redundant. Further supporting this proposal, each acetylation decays with similar kinetics when Htz1 transcription is repressed, and proteomic screening did not find a single condition in which one Htz1(Ac) was differentially regulated. However, whereas the individual acetylations on Htz1 may be redundant, they are not dispensable. Completely unacetylatable htz1 alleles display genetic interactions and phenotypes in common with and distinct from htz1Δ. In addition, each Htz1 N-terminal lysine is deacetylated by Hda1 in response to benomyl and reacetylated when this agent is removed. Such active regulation suggests that acetylation plays a significant role in Htz1 function.

FIGURE 1.

Htz1 lysines 8, 10, and 14 are acetylated by NuA4 as a component of chromatin. A, peptides used to raise αHtz1^Ac antibodies (see “Experimental Procedures”), with the four acetylatable lysines (at positions 3, 8, 10, and 14) on the Htz1 N terminus underlined. The terminal cysteine (–C) on each peptide was used to couple to KLH for immunization or a sulfolink resin for affinity purification. B, αK8^Ac, αK10^Ac, and αK14^Ac are highly specific. C-terminally HA₃-tagged forms of the indicated htz1 alleles were individually expressed as the sole source of the histone and WCEs analyzed by immunoblotting. Specific mutation of the appropriate lysine to unacetylatable arginine (e.g. K8R) abolishes recognition by the relevant affinity-purified antibody. Δ is an htz1Δ strain. αHA demonstrates equivalent expression of each htz1.HA₃. αRpn8 (19 S proteasome subunit) serves as a loading control. C, each acetylated form of Htz1 is chromatin-associated. WT and swr1Δ cells were spheroblasted (total, T); fractionated into cytoplasm (Cy), nucleus (N), and chromatin (Ch); and immunoblotted as indicated. αHA monitors the localization of Htz1.HA₃. Appropriate segregation of histone H2B and Rpn8 indicates efficient fractionation; the former is primarily localized in insoluble chromatin, and the latter is primarily in soluble cytoplasm. D, all of the tested forms of Htz1^Ac are Esa1-dependent and Gcn5-independent. Left panel, ESA1 or esa1-L254P strains were incubated at a nonpermissive temperature (37 °C, 2 h), WCEs were harvested and immunoblotted as indicated. Esa1-dependent H4-K12^Ac is a positive control. Rpn8 is a loading control. Right panel, BY4741. The indicated WCEs were harvested at 30 °C. E, all of the tested forms of Htz1^Ac are dependent on indicated members of the NuA4 acetyltransferase complex. These deletions also reduce H4^Ac levels but have no impact on the abundance of Htz1 (16). F, each Htz1^Ac is increased on deletion of the Hda1 deacetylase.

FIGURE 2.

The primary function of the Htz1 N terminus is to harbor lysine acetylations. A, Pearson CC plot compares the genetic interaction profiles of htz1Δ or htz1-NΔ (X- or Y-axes) with 2,255 profiles performed to date. Blue and black spots, as labeled; green spots, SWR-C components (swr1Δ, yaf9Δ, vps71Δ, vps72Δ, swc3Δ, swc5Δ, and arp6Δ); white spot, WT control; red spots, all other mutants on array. B, distribution plot of the CCs of NΔ against 2,245 mutant profiles. The most highly correlated profiles include those derived from 4KR and 4KQ. C, a correlation plot of CCs for 2,245 individual genetic screens relative to 4KR or 4KQ. D, as expected from their strong but imperfect pairwise CCs htz1Δ, NΔ, 4KR, and 4KQ show both common and unique genetic interactions. Blue and yellow correspond to negative or positive genetic interactions.

FIGURE 3.

Htz1 acetylation does not regulate turnover of the histone variant. A, cycloheximide was used to repress translation in a HTZ1.HA₃ strain, with samples taken for WCE isolation and immunoblotting at times indicated. Quantitation measures each immunoblotted species relative to their abundance at T₀ (precycloheximide). B, glucose (final concentration, 2%) was added to repress GAL1 promoter-driven transcription in a GAL1_p.HTZ1.HA₃ strain, with samples taken for WCE isolation and immunoblotting at times indicated. Quantitation measures each immunoblotted species relative to Rpn8 at the same time point. C, glucose was added to repress transcription from GAL1_p.HTZ1.HA₃ (WT or hda1Δ backgrounds) or GAL1_p.htz1–4KR.HA₃, with samples taken for WCE isolation and immunoblotting at times indicated. Quantitation measures each htz1.HA₃ relative to Rpn8 at the same time point in the same background.

FIGURE 4.

Htz1^Ac is regulated in asf1Δ cells by an indirect mechanism. A, each Htz1^Ac (but not total Htz1.HA₃) is decreased in asf1Δ cells. Rpn8 serves as a loading control. B, asf1Δ reduces each Htz1^Ac but has no effect on the amount of total Htz1 associated with chromatin. WT and asf1Δ cells were spheroblasted (total, T); fractionated into cytoplasm (Cy), nucleus (N), and chromatin (Ch); and immunoblotted as indicated. Appropriate localization of H2B and Rpn8 indicates efficient fractionation (as in Fig. 1C). C, schematic depicts the various roles of Asf1, a histone H3-H4 donor to various acetyltransferase complexes (substrates indicated), CAF/proliferating cell nuclear antigen (PCNA) at replication forks (for replication-dependent deposition (RDD)), and HIR in actively transcribed regions (for replication-independent deposition (RID)). D, glucose (final concentration, 2%) was added to repress GAL1_p-driven transcription in GAL1_p.ASF1.HA₃ or GAL1_p.ASF1.HA₃.PEST strains, with samples taken for WCE isolation and immunoblotting at times indicated. Rpn8 serves as a loading control. E, GAL1_p.ASF1.HA₃ or GAL1_p.ASF1.HA₃.PEST strains were maintained in glucose (G, 2%) or galactose/raffinose (GR, 2%/1%) containing medium for >24 h before the samples were collected for WCE isolation and immunoblotting. Rpn8 serves as a loading control.

FIGURE 5.

Htz1^Ac is reduced in response to benomyl. A, htz1Δ (and to a lesser degree NΔ) cells are sensitive to benomyl (BEN, 15 μg/ml). In contrast htz1Δ but not NΔ cells are sensitive to camptothecin (CPT, 20 μm). Spots are 10-fold serial dilutions on YPD plates ± each agent after 2 days at 30 °C. B, unacetylatable htz1 strains (NΔ, 4KR, or 4KQ) are weakly sensitive to benomyl. Growth curves were analyzed on a Bioscreen (see “Experimental Procedures”) during culture in YPD + benomyl (0–60 μg/ml as indicated). The table indicates the time taken (in minutes) for each strain to traverse A₆₀₀ 0.45 to 0.9 (i.e. exponential part of growth curve) in YPD ± benomyl (60 μg/ml). ND, not determined. C, each Htz1^Ac is reduced on benomyl treatment. WT cells were grown in YPD to A₆₀₀ ∼0.5 and benomyl, TBZ, or camptothecin was added (concentrations indicated) before growth for another 2 h. WCEs were harvested and immunoblotted as indicated. Rpn8 is a loading control. U, untreated. D, the decrease of each Htz1^Ac in response to benomyl is reversible. WT cells were grown in YPD to A₆₀₀ ∼0.5, and benomyl was added (40 μg/ml) for 2 h (T2). The cells were then washed, resuspended in YPD, and allowed to recover for 2 (R2) or 5 (R5) h. WCEs were harvested and immunoblotted as indicated. Rpn8 is a loading control. U, untreated; B, benomyl.

FIGURE 6.

The benomyl-induced decrease of each Htz1^Ac is Hda1-dependent. A, cultures were grown in YPD to A₆₀₀ ∼0.5, benomyl was added (40 μg/ml) and incubated as indicated before WCEs were harvested and immunoblotted. B, Htz1^Ac is reduced within 30 min of benomyl treatment. The cultures were grown in YPD to A₆₀₀ ∼0.5 before benomyl addition (40 μg/ml). WCEs were harvested and immunoblotted as indicated. C, acetylations on histones H3 (K9^Ac and K27^Ac) and H4 (K5^Ac and K12^Ac) are reduced by benomyl but independently of Hda1. The cultures were grown in YPD to A₆₀₀ ∼0.5 before benomyl addition (40 μg/ml, 2 h). WCEs were harvested and immunoblotted as indicated. U, untreated; B, benomyl.