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. 1999 Apr;19(4):2515-26.
doi: 10.1128/MCB.19.4.2515.

Esa1p is an essential histone acetyltransferase required for cell cycle progression

A S Clarke  1 , J E Lowell, S J Jacobson, L Pillus
Affiliations

Esa1p is an essential histone acetyltransferase required for cell cycle progression

A S Clarke et al. Mol Cell Biol. 1999 Apr.

Abstract

Histones are dynamically modified during chromatin assembly, as specific transcriptional patterns are established, and during mitosis and development. Modifications include acetylation, phosphorylation, ubiquitination, methylation, and ADP-ribosylation, but the biological significance of each of these is not well understood. For example, distinct acetylation patterns correlate with nucleosome formation and with transcriptionally activated or silenced chromatin, yet mutations in genes encoding several yeast histone acetyltransferase (HAT) activities result in either no cellular phenotype or only modest growth defects. Here we report characterization of ESA1, an essential gene that is a member of the MYST family that includes two yeast silencing genes, human genes associated with leukemia and with the human immunodeficiency virus type 1 Tat protein, and Drosophila mof, a gene essential for male dosage compensation. Esa1p acetylates histones in a pattern distinct from those of other yeast enzymes, and temperature-sensitive mutant alleles abolish enzymatic activity in vitro and result in partial loss of an acetylated isoform of histone H4 in vivo. Strains carrying these mutations are also blocked in the cell cycle such that at restrictive temperatures, esa1 mutants succeed in replicating their DNA but fail to proceed normally through mitosis and cytokinesis. Recent studies show that Esa1p enhances transcription in vitro and thus may modulate expression of genes important for cell cycle control. These observations therefore link an essential HAT activity to cell cycle progression, potentially through discrete transcriptional regulatory events.

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Figures

FIG. 1
FIG. 1
Esa1p has sequence similarity (gray boxes) with human Tip60 and D. melanogaster MOF. A region of strong sequence similarity to acetyltransferases including yeast Hat1p and Gcn5p is indicated (black boxes). Similar and identical residues in this region are highlighted. Residues most conserved among known and predicted acetyltransferases in the A motif as defined by Neuwald and Landsman (42) are noted with asterisks. Positions of temperature-sensitive esa1 alleles (see text) are noted by arrows above the Esa1p cartoon; the position of the esa1-L327S allele is also shown by an arrow in the sequence expansion. The conserved G→E mutation in mof (25) is marked with an arrowhead below the sequence. An N-terminal chromo domain (1, 25, 31, 47) is found in these three SAS family members (diagonal stripes). The number of amino acids in each protein is indicated at the right.
FIG. 2
FIG. 2
Recombinant Esa1p acetylates histones. Products of HAT assays using either yeast or calf histones as substrates were resolved on an SDS–18% polyacrylamide gel that was stained with Coomassie brilliant blue (A) and treated for fluorography (B). The activities of recombinant Esa1p were compared with that of recombinant Gcn5p; whereas Esa1p preferentially acetylates H4, Gcn5p preferentially acetylates H3 (32). (C) Bacterial extracts containing equivalent amounts of recombinant Esa1p, vector control, or recombinant Esa1-L327Sp were incubated with 3H-acetyl coenzyme A and either calf histones or BSA. Esa1p acetylates calf histones but not BSA. No activity was observed with vector control or Esa1-L327Sp lysates or with two other mutant proteins tested (data not shown). The preferred targets of Esa1p are Lys5 of histone H4 (D), Lys14 of histone H3 (E), and Lys4 and Lys7 of histone H2A (F). Note that lysines 8, 12, and 16 in H4 were also acetylated. When repetitive yields for the cycles of Edman degradation were calculated, the extent of acetylation at each of these sites was equivalent and significantly less than that of Lys5.
FIG. 3
FIG. 3
esa1 mutants are temperature sensitive. Temperature sensitivity was observed by plating wild-type ESA1 (LPY3498) and esa1 mutant (esa1-L254P [LPY3500], esa1-L327S [LPY3430], and esa1-414 [LPY3291]) strains in fivefold serial dilutions at 28°C (left) and 37°C (right). All three alleles are recessive mutations.
FIG. 4
FIG. 4
esa1 mutants display conditional decreases in the level of histone H4-Lys5 acetylation in vivo. Whole-cell lysates from wild-type (WT; lanes 2, 6, and 10) or esa1 temperature-sensitive (lanes 3 to 5, 7 to 9, and 11 to 13) strains grown at 28°C (lanes 2 to 5) or 37°C (lanes 6 to 13) were separated on an SDS–18% polyacrylamide gel and either transferred to nylon and probed with an antiserum directed against Ly5-acetylated H4 (A) or probed with a control antiserum directed against histones (B; upper panel shows H4 immunoreactive band) or stained with Coomassie brilliant Blue (B, lower panel). Purified yeast histones (YH; approximately 6 μg; lane 1) were run in parallel. esa1-L254P (LPY3500; lanes 3, 7, and 11), esa1-414 (LPY3291; lanes 4, 8, and 12), and esa1-L327S (LPY3430; lanes 5, 9, and 13) strain displayed decreased Lys5-acetylated immunoreactivity at the nonpermissive temperature of 37°C. Arrowheads denote migration of histone H4.
FIG. 5
FIG. 5
esa1 temperature-sensitive mutants arrest in the G2/M phase of the cell cycle after DNA replication. Wild-type ESA1 (LPY3498) (left) and mutant esa1-L254P (LPY3500) (right) cells were incubated at 28 and 37°C for 4 h and analyzed for DNA content by flow cytometry. The x and y axes represent relative fluorescence intensity and number of cells, respectively. esa1 mutants arrested with G2/M DNA content after DNA replication at the restrictive temperature of 37°C. This mutant phenotype was also observed for esa1-L327S, esa1-414, and four other temperature-sensitive alleles tested (data not shown). The time to reach the arrest varied somewhat depending on the allele of ESA1.
FIG. 6
FIG. 6
esa1 mutants display improper DNA segregation. ESA1 (LPY3498), esa1-L254P (LPY3500), and esa1-414 (LPY3291) cells were incubated at 37°C for 4 h, and esa1-L327S (LPY3430) cells were incubated at 37°C for 8 h, fixed, and stained with DAPI. esa1 mutant cells were predominantly large budded as visualized by Nomarski optics (left and center columns, bottom), with some triple-budded cells (right column, bottom). DAPI staining indicates that esa1 mutant cells either failed to segregate or abnormally segregated their chromatin in comparison to wild-type cells. Bar, 5 μm.
FIG. 7
FIG. 7
esa1 mutants disrupt mitotic cell cycle progression. ESA1 (LPY3498) and esa1-L254P (LPY3500) cells were incubated at 37°C for 4 h and prepared for indirect immunofluorescence microscopy. esa1 mutant cells exhibited aberrant nuclear and short spindle morphologies at the restrictive temperature. As visualized by double-label immunofluorescence microscopy, antisera directed against Kar2p detected the nuclear envelope and ER and α-tubulin detected microtubules. Both the nuclear envelope and the microtubules extended only partially through the bud neck in comparison to the wild type but coincided with the DAPI-stained region. Bars represent 5 μm.
FIG. 8
FIG. 8
esa1 mutants have aberrant ultrastructural morphology. Wild-type ESA1 (LPY3498) (A and B) and esa1-L254P (LPY3500) (C and D) cells were incubated at 37°C for 4 h and prepared by rapid freezing for electron microscopy. In wild-type cells, the nucleolar electron-dense material was at both poles of the dividing nucleus (n) (A) or in a crescent shape in a nondividing cell (B, closed arrowhead). However, in the esa1 mutant (C and D, open arrowheads), the electron-dense material was dispersed throughout the nucleus. The majority of the mutant large-budded cells showed the nucleus on one side of the bud neck, in contrast to wild-type cells, where the nucleus extended equally through the bud neck. The cytoplasm of the esa1 mutant (C, asterisk) was also more highly vesiculated than in wild-type cells. Each panel shows a different cell, and scale bars shown represent 1 μm (A and C) and 0.5 μm (B and D). v, vacuoles.
FIG. 9
FIG. 9
The esa1 mutant G2/M arrest is dependent on the RAD9 checkpoint gene. (A) rad9Δ (LPY3784), (B) esa1-L254P (LPY3785), (C) rad9Δ esa1-L254P (LPY3780), (D) mad3Δ esa1-L254P (LPY4223), and (E) mad3 (LPY4222) mutant cells were incubated at 37°C and then analyzed for DNA content by flow cytometry. The x and y axes represent relative fluorescence intensity and number of cells, respectively. The esa1-L254P and mad3Δ esa1-L254P mutants arrested after DNA replication with approximately 80% of the cells containing 2C DNA content, whereas the rad9Δ and mad3 cells have normal DNA content for dividing cells. The rad9Δ esa1-L254P double mutant had both 1C and 2C peaks, indicating that esa1-L254P cell cycle arrest is dependent on RAD9 but not on MAD3.
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