Telomeric heterochromatin boundaries require NuA4-dependent acetylation of histone variant H2A.Z in Saccharomyces cerevisiae

Abstract

SWR1-Com, which is responsible for depositing H2A.Z into chromatin, shares four subunits with the NuA4 histone acetyltransferase complex. This overlap in composition led us to test whether H2A.Z was a substrate of NuA4 in vitro and in vivo. The N-terminal tail of H2A.Z was acetylated in vivo at multiple sites by a combination of NuA4 and SAGA. H2A.Z acetylation was also dependent on SWR1-Com, causing H2A.Z to be efficiently acetylated only when incorporated in chromatin. Unacetylatable H2A.Z mutants were, like wild-type H2A.Z, enriched at heterochromatin boundaries, but were unable to block spreading of heterochromatin. A mutant version of H2A.Z that could not be acetylated, in combination with a mutation in a nonessential gene in the NuA4 complex, caused a pronounced decrease in growth rate. This H2A.Z mutation was lethal in combination with a mutant version of histone H4 that could not be acetylated by NuA4. Taken together, these results show a role for H2A.Z acetylation in restricting silent chromatin, and reveal that acetylation of H2A.Z and H4 can contribute to a common function essential to life.

Figure 1.

H2A.Z was acetylated by NuA4 in vitro and in vivo. (A) ³H-Acetyl-CoA was incorporated into H2A and H2A.Z by NuA4. (B) ³H-Acetyl-CoA was incorporated into H2A.Z-containing nucleosomes by NuA4. (C) H2A.Z was acetylated in vivo primarily by NuA4. The image represents an anti-Flag immunoblotting of an AU gel with times referring to minutes past shift to 37°C.

Figure 2.

Other enzymes contributed to H2A.Z acetylation. (A–C) Shown are anti-Flag immunoblots of an AU gel. (A) Several genes encoding the catalytic subunits of HATs and the SWR1-Com were deleted and analyzed for defects in H2A.Z acetylation. (B) Epistasis analysis of esa1-L357H and gcn5Δ showed that NuA4 acetylated H2A.Z prior to SAGA acetylation. (C) gcn5Δ and eaf1Δ were synthetic lethal as shown by four tetra-type tetrads. The white circles represent where double mutants should be observed.

Figure 3.

Soluble and chromatin-associated H2A.Z was analyzed for acetylation in wild-type and swr1Δ. Shown is an anti-Flag immunoblot of an AU gel.

Figure 4.

H2A.Z was acetylated in the N-terminal tail. (A,B) Shown are anti-Flag immunoblots of an AU gel. (A) H2A.Z is acetylated at the N-terminal Lys 3, Lys 8, Lys 10, and Lys 14. Lanes 1 and 2 are chromosomal HTZ1-3Flag. Lanes 3 and 4 are plasmid-borne HTZ1-3Flag alleles. (B) Lys 3, Lys 8, Lys 10, and Lys 14 can all be acetylated in vivo. All HTZ1-3Flag alleles are plasmid-borne.

Figure 5.

H2A.Z acetylation was important for a boundary to silent chromatin. (A) qRT–PCR of a gene cluster near the telomere of chromosome IX-R and the genes adjacent to the left and right boundaries of HMR. (B) ChIP analysis of Sir3p at chromosome IX-R. (C) ChIP analysis of H2A.Z at chromosome IX-R described in A. Error bars represent standard deviation between four independent RNA purifications (A) or two independent ChIPs (B,C).

Figure 6.

Genetic interactions between HTZ1 alleles and mutations affecting histone H4 acetylation. (A) htz1-K3,8,10,14R-3Flag∷KanMX was crossed to eaf1Δ∷His3MX, and tetrads were analyzed. Tenfold serial dilutions were spotted on minimal media and grown for 3 d at the indicated temperatures. (B) Wild-type HHF2 was carried on a URA3 plasmid in a background with either wild-type HTZ1 or htz1-K3,8,10,14R-3Flag. These strains were transformed with either wild-type HHF2, hhf2-K5,8,12R, or hhf2-K5,8,12,16R on a TRP1 plasmid. Cells were grown in medium containing tryptophan. Tenfold serial dilutions were spotted on either minimal medium lacking tryptophan and lacking uracil or minimal medium lacking tryptophan and containing FOA, to select against the URA3 plasmid. Cells were grown for 3 d at 23°C prior to imaging.

Figure 7.

A model linking SWR1-Com and NuA4 in H2A.Z function. Following deposition into chromatin, H2A.Z is first acetylated by NuA4 and subsequently by NuA4 and SAGA.