Regulation of chromosome stability by the histone H2A variant Htz1, the Swr1 chromatin remodeling complex, and the histone acetyltransferase NuA4

Abstract

NuA4, the only essential histone acetyltransferase complex in Saccharomyces cerevisiae, acetylates the N-terminal tails of histones H4 and H2A. Affinity purification of NuA4 revealed the presence of three previously undescribed subunits, Vid21/Eaf1/Ydr359c, Swc4/Eaf2/Ygr002c, and Eaf7/Ynl136w. Experimental analyses revealed at least two functionally distinct sets of polypeptides in NuA4: (i) Vid21 and Yng2, and (ii) Eaf5 and Eaf7. Vid21 and Yng2 are required for bulk histone H4 acetylation and are functionally linked to the histone H2A variant Htz1 and the Swr1 ATPase complex (SWR-C) that assembles Htz1 into chromatin, whereas Eaf5 and Eaf7 have a different, as yet undefined, role. Mutations in Htz1, the SWR-C, and NuA4 cause defects in chromosome segregation that are consistent with genetic interactions we have observed between the genes encoding these proteins and genes encoding kinetochore components. Because SWR-C-dependent recruitment of Htz1 occurs in both transcribed and centromeric regions, a NuA4/SWR-C/Htz1 pathway may regulate both transcription and centromere function in S. cerevisiae.

Fig. 1.

Isolation of the NuA4 HAT complex. (A) TAP of the NuA4 complex was carried out on strains containing either TAP-tagged Eaf7 or no tagged protein. The purified protein was analyzed by SDS/PAGE and silver staining. The indicated proteins were identified by trypsin digestion of each stained band followed by MALDI-TOF mass spectrometry or tandem mass spectrometry after subjecting an aliquot of the eluate from the final column directly to trypsin. Contaminating bands are indicated: ***, Sse1; **, Ssa1; *, Ssb1; †, TEV protease. The Srp1/Kap95 importin complex, which transports NLS-containing proteins into the nucleus (56), also copurified with NuA4 and is often found associated with nuclear protein complexes (N.J.K., A.E. and J.F.G., unpublished data). (B) Summary of purified proteins identified by mass spectrometry. Proteins that were present in at least two of the six purifications are represented. Protein size (kDa) was as predicted by amino acid composition (Saccharomyces Genome Database; www.yeastgenome.org) and not determined experimentally. (†, N.J.K., A.E., and J.F.G., unpublished data.)

Fig. 2.

Genetic similarity of the SWR-C and a subset of NuA4. (A) Synthetic genetic interactions for the SWR-C and NuA4. SGA technology (19) was used to cross Nat^r strains harboring individual deletions of genes encoding Htz1 or subunits of the SWR-C or NuA4 with a transcription-targeted array of 384 Kan^R deletion strains to create sets of Nat^r Kan^r haploid double mutants. Growth rates were assessed by automated image analysis of colony size. Lines connect genes with synthetic genetic interactions. The lengths of lines and proximity of boxes in this diagram and in Fig. 3A are unrelated to the strengths of the indicated synthetic genetic interactions. (B) Microarray analysis of gene expression was performed for the indicated deletion strains. Pearson correlation coefficients were then calculated for each pair of deletions, and the deletions were organized by 2D hierarchical clustering according to the similarities of their effects. (C) Immunoblots of whole-cell protein extracts from wild-type (WT, NJK28), esa1-L245P (LPY3500), vid21Δ (NJK1042), eaf7Δ (NJK1254), yng2Δ (NJK1482), eaf5Δ (NJK1259), htz1Δ (NJK1527), and swr1Δ (NJK1665) cells (see Table 3) grown to log phase in yeast extract/peptone/dextrose at 30°C and transferred to 37°C for 4 h. The membranes were probed with antihyperacetylated histone H4 (Penta) antibodies (α Ac-H4) and then reprobed with antibodies to Cdc28 to verify approximately equal protein loading.

Fig. 3.

Htz1, SWR-C, and the NuA4 complex function to regulate chromosome stability/transmission. (A) SGA analysis (19) using either a transcription-targeted 384-deletion-strain array or genome-wide kinetochore screens (K.B. and V. Measday, unpublished data) identified numerous synthetic genetic interactions between deletions of genes encoding Htz1, SWR-C, or the NuA4 subunits Vid21 and Yng2, and known chromosome stability/transmission factors (see text for details). Genome-wide screens were carried out with four essential kinetochore genes [skp1–3 (42), cep3–1 (43), ctf13–30 (44), and okp1–5 (45)], whereas the nonessential components (mcm21Δ, mcm22Δ, ctf19Δ, bub1Δ, bub3Δ, mad1Δ, and mad2Δ) were present on the targeted miniarray. (B) Effects of the microtubule destabilizing agent benomyl on the growth of wild-type (NJK28), vid21Δ (NJK1042), eaf5Δ (NJK1259), eaf7Δ (NJK1254), yng2Δ (NJK1482), yaf9Δ (NJK1240), htz1Δ (NJK1527), and swr1Δ (NJK1665) strains. Five-fold serial dilutions of strains starting from an OD₆₀₀ of 0.1 were plated onto yeast extract/peptone/dextrose plates containing 5 or 15 μg/ml benomyl and incubated for 2 days at 30°C.

Fig. 4.

Htz1 recruitment is Swr1-dependent and NuA4-independent at all tested regions. (A) ChIP analysis at the ADH1 gene. A schematic of the ADH1 locus is shown with the location of the ChIP primer pairs 1–3 (Upper) and the major polyadenylation/cleavage site at +1137 (Lower) (57). The asterisks indicate a subtelomeric region of chromosome V (9716–9823) that we frequently use as a nontranscribed control when analyzing transcriptionally active regions (5). Chromatin samples from each strain were analyzed after the indicated precipitation. The bottom row is input, used to normalize the PCR amplification efficiency of each primer pair. Equivalent crosslinking of Rpb3 in each case was used as a control for sample integrity (not shown). Whereas swr1Δ leads to a loss of Htz1 occupancy at all tested regions, deletion of members of the NuA4 complex (vid21Δ, yng2Δ, and eaf5Δ) has no detectable effect. (B) A schematic of the chromosome III centromere (CEN-III), with the location of coding sequences, structural elements and ChIP primer pairs depicted. Asterisk indicates a subtelomeric region of chromosome 5 as above. Transcriptional frequency of Ycl001w-b (mRNA/hr) is from ref. (http://web.wi.mit.edu/young/expression/halflife.html). The histone H3 variant Cse4 and the kinetochore component Cbf2 crosslink at the centromere, as reported (–61). (C) ChIP analysis at CEN-III. Chromatin samples from the indicated strains were analyzed after precipitation via the TAP tag. Htz1 and H2A occupancy at CEN-III (and CEN-V, not shown) is equivalent to that at the subtelomeric (asterisk) region. Htz1 recruitment at this region depends on the SWR-C (indicated by swr1Δ), but independent of NuA4 (indicated by vid21Δ and yng2Δ). H2A recruitment is independent of both complexes. The reduced occupancy at primer pair 3 directly over CEN-III is not unique to Htz1 and is also exhibited by H2A, H2B, H3, and H4 (latter three not shown). This may be a combined function of relative primer efficiency, reduced nucleosome density at this region, and steric occlusion by the kinetochore complex.