1. Cell Cycle and Checkpoint Regulation of Histone H3 K56 Acetylation by Hst3 and Hst4 
Nancy L. Maas, Kyle M. Miller, Lisa G. DeFazio, David P. Toczyski 
Molecular Cell 
Volume 23, Issue 1, Pages (July 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1 Regulation of Hst3 in Response to Damage
(A) Cultures were grown to log phase in rich medium and either harvested or treated with 10 μg/ml nocodazole, 0.05% MMS, or 200 mM HU for 3 hr. Hst3-TAP levels were detected by Western blot using anti-CBP antibody, which recognizes the TAP tag.
(B) Strains were arrested in G1 with α factor (α) and released into rich medium in the absence or presence of 0.05% MMS. Samples were collected at indicated time points and analyzed by Western blotting for Hst3-TAP with IgG. The same blots were probed for Cdc28 as a loading control.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 MEC1-Dependent Transcriptional Repression of HST3 upon Damage
(A) HST3-TAP was placed under control of the GAL1 promoter. Asynchronous cells (asy) were synchronized in G1 with α factor, and Hst3 expression was simultaneously induced in 2% galactose. After 2 hr, transcription of HST3 was then blocked by releasing into glucose in the absence or presence of 0.05% MMS. Whole-cell extracts were analyzed for Hst3-TAP by Western blot at the indicated time points.
(B) Log phase wild-type and mec1Δsml1Δ cultures were synchronized in G1 with α factor and released into rich medium in the absence or presence of 0.05% MMS. Forty minutes after release, α factor was added in order to rearrest cells in the following G1. Cell cycle position was monitored by FACS, and HST3 transcript levels were analyzed by Northern blotting. RNA was loaded based on quantification, and equal loading was verified by examining actin levels (data not shown).
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 Hst3 Regulates Histone H3 K56 Acetylation
(A) Strains with wild-type histone H3, or a K56R or K56Q H3 mutation, were examined for damage sensitivity with or without GAL1-driven overexpression of HST3. Each strain was analyzed by 7-fold serial dilutions on plates containing either HU, MMS, or CPT. Growth was analyzed on plates containing either 2% dextrose or 2% galactose to assess the effects of repressing or inducing, respectively, the expression of Hst3. The full dilution series is shown in Figure S1.
(B) Cells were synchronized in G1 with α factor and released into rich medium containing 2% glucose or 2% galactose. Hst3-TAP levels were detected by Western blotting with anti-CBP antibody, and K56 acetylation was detected with anti-acetyl-H3Lys56 (Upstate). Western blotting, probing, and exposing were done in parallel for each antibody employed. Passage through S phase was monitored for the first 60 min by FACS.
(C) Two-fold dilutions of samples from the H3 K56-Ac peak (30 and 40 min time points pooled) from the experiment shown in Figure 3B were Western blotted and probed for total H3 and H3 K56-Ac.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Hst4 Is Downregulated in a MEC1-Dependent Manner upon Damage
(A) An HST3-HA HST4-GFP strain was arrested in G1 with α factor and released. Samples were taken at the indicated times and processed for FACS or Western blotted and probed as shown.
(B) HST4-GFP and HST4-GFP mec1Δsml1Δ strains were analyzed as in Figure 1B.
(C) The indicated TAP-tagged strains were grown to log phase and treated as in Figure 1A. Samples were analyzed by Western blotting using IgG, with Cdc28 as a loading control.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Regulation of K56 Acetylation by Hst3 and Hst4 in Response to DNA Damage
(A) Cultures were synchronized in G1 with α factor and released into rich medium in the absence or presence of 0.05% MMS. Samples were taken at 10 min intervals. Forty minutes after release, α factor was added to rearrest cells in the following G1. K56 acetylation was detected by Western blotting with anti-acetyl-H3Lys56 (Upstate). Cell cycle progression was monitored by FACS.
(B) The 50–90 min time points from the experiment shown in Figure 5A were examined on a single Western blot probed for total H3 and H3 K56-Ac.
(C) Wild-type and hst3Δhst4Δ strains were arrested in G2/M with nocodazole for 3 hr and harvested. Two-fold dilutions were Western blotted and probed for total H3 and H3 K56-Ac.
(D) HST3/hst3Δ HST4/hst4Δ diploids either wild-type for HHT1 or homozygous for hht1-K56R were dissected, grown at 23°C, replica plated, and transferred to 37°C. hst3Δhst4Δ double mutants are indicated with circles on the 23°C plates.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Alk1, Ynl058c, and Hst3 Are Similarly Regulated upon DNA Damage
(A) Cultures were synchronized in G1 with α factor and released into rich medium in the absence or presence of 0.05% MMS. Samples were collected at indicated time points and analyzed by Western blotting for the TAP-tagged proteins with anti-CBP. The same blots were probed for Cdc28 as a loading control.
(B) Experiments were performed as described in (A), except cells were released from G1 into 10 μg/ml nocodazole or 0.05% MMS.
(C) Log phase wild-type and mec1Δsml1Δ cultures were synchronized in G1 with α factor and released into rich medium in the absence or presence of 0.05% MMS. Forty minutes after release, α factor was added in order to rearrest cells in the following G1. ALK1 and YNL058C transcript levels were analyzed by Northern blot, and cell cycle position was monitored by FACS (data not shown).
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
A Model for the Cell Cycle Control of Histone H3 K56 Acetylation by Hst3 and Hst4
In an unperturbed cell cycle, H3 K56 is acetylated during S phase. This acetylation is removed by Hst3, which is expressed at the end of S phase, and is subsequently kept at low levels until the following S phase by Hst4. In response to DNA-damaging agents, Mec1/hATR and Rad53/hChk2 function to repress transcription of HST3 and HST4, allowing H3 K56 acetylation to persist.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions