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. 2014 May 9;289(19):13186-96.
doi: 10.1074/jbc.M113.523530. Epub 2014 Mar 19.

Regulation of the Histone Deacetylase Hst3 by Cyclin-Dependent Kinases and the Ubiquitin Ligase SCFCdc4

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Free PMC article

Regulation of the Histone Deacetylase Hst3 by Cyclin-Dependent Kinases and the Ubiquitin Ligase SCFCdc4

Neda Delgoshaie et al. J Biol Chem. .
Free PMC article

Abstract

In Saccharomyces cerevisiae, histone H3 lysine 56 acetylation (H3K56ac) is a modification of new H3 molecules deposited throughout the genome during S-phase. H3K56ac is removed by the sirtuins Hst3 and Hst4 at later stages of the cell cycle. Previous studies indicated that regulated degradation of Hst3 plays an important role in the genome-wide waves of H3K56 acetylation and deacetylation that occur during each cell cycle. However, little is known regarding the mechanism of cell cycle-regulated Hst3 degradation. Here, we demonstrate that Hst3 instability in vivo is dependent upon the ubiquitin ligase SCF(Cdc4) and that Hst3 is phosphorylated at two Cdk1 sites, threonine 380 and threonine 384. This creates a diphosphorylated degron that is necessary for Hst3 polyubiquitylation by SCF(Cdc4). Mutation of the Hst3 diphospho-degron does not completely stabilize Hst3 in vivo, but it nonetheless results in a significant fitness defect that is particularly severe in mutant cells treated with the alkylating agent methyl methanesulfonate. Unexpectedly, we show that Hst3 can be degraded between G2 and anaphase, a window of the cell cycle where Hst3 normally mediates genome-wide deacetylation of H3K56. Our results suggest an intricate coordination between Hst3 synthesis, genome-wide H3K56 deacetylation by Hst3, and cell cycle-regulated degradation of Hst3 by cyclin-dependent kinases and SCF(Cdc4).

Keywords: CDK (Cyclin-dependent Kinase); Chromatin Histone Modification; E3 Ubiquitin Ligase; Histone Deacetylase; Histones; Ubiquitination.

Figures

FIGURE 1.
FIGURE 1.
Degradation of Hst3 can occur before anaphase. A, wild-type cells expressing TAP-tagged Hst3 were released from G1 in the presence of nocodazole at 30 °C, and aliquots were collected as a function of time. Cell cycle progression was monitored by FACS. Hst3 levels were assessed by immunoblotting with an antibody against the TAP epitope tag. Ponceau S staining is shown as a loading control. B, cdc23-1 HST3-TAP cells were released from G1 at 23 °C and switched to 37 °C after 45 min to inactivate APC. Samples were analyzed as in A. C, PDS1-HA HST3-TAP cells were released from G1 in the presence of nocodazole at 30 °C. Samples were processed as in A.
FIGURE 2.
FIGURE 2.
Hst3 is phosphorylated at Thr-380 and Thr-384 in vivo. A, Hst3 contains two Cdk1 sites at Thr-380 and Thr-384, highlighted in red. These Cdk1 sites are located outside of the predicted catalytic core of Hst3, residues 53–340, shown in boldface type. B, two threonines of Hst3 and their spacing are conserved in the pathogenic fungi C. albicans, C. tropicalis, and C. glabrata. C, Hst3 is phosphorylated at Thr-380 and Thr-384 in vivo. The phosphorylated Hst3 peptide was identified from a phosphoproteome analysis of cells labeled with [13C6,15N4]arginine without prior purification of Hst3. The doubly phosphorylated peptide had a mass of 1423.602 Da that was within 2 ppm of its theoretical mass. The MS/MS spectrum contains abundant y- and b-fragment ions that confirm the peptide sequence DSIGpTPPpTPLR, where the residues preceded by “p” are phosphorylated. The fragment ion at m/z 954.47 (y8*) is caused by a loss of H3PO4 (98 Da) from the corresponding y8 fragment ion, which provides additional evidence for phosphorylation of Thr-380.
FIGURE 3.
FIGURE 3.
Mutation of either Thr-380 or Thr-384 increases the abundance of Hst3 at late stages of the cell cycle. A–D, cells expressing TAP-tagged wild-type or mutant forms of Hst3 were synchronized in G1 and released into the cell cycle in the presence of nocodazole at 30 °C. Aliquots of cells were processed to monitor DNA contents by FACS and Hst3-TAP proteins by immunoblotting. Ponceau S is shown as a loading control.
FIGURE 4.
FIGURE 4.
Mutation of either Thr-380 or Thr-384 reduces the rate of Hst3 degradation. A–D, asynchronous populations of cells expressing wild-type or mutant forms of Hst3-TAP were exposed to 35 μg/ml cycloheximide, and aliquots of cells were harvested at 5-min intervals to monitor the abundance of Hst3-TAP proteins by immunoblotting. Phosphoglycerate kinase (Pgk1) is shown as loading control.
FIGURE 5.
FIGURE 5.
Cdc34 and SCFCdc4 are required for Hst3 degradation in vivo. A, inactivation of the SCF subunit Cdc53 stabilizes Hst3 in G2/M. cdc53-1 HST3-TAP cells were released from G1 in the presence of nocodazole at 23 °C and were switched to 37 °C after 120 min to inactivate SCF. B, Hst3 cannot be degraded in the absence of the E2 enzyme Cdc34. cdc34-2 HST3-TAP cells were released from G1 at 23 °C in the presence of nocodazole and were switched to 37 °C after 120 min to inactivate Cdc34. C–E, Hst3 degradation requires the F-box protein Cdc4. cdc4-1, cdc4-10, and cdc4-12 mutants expressing Hst3-TAP were released from G1 in the presence of nocodazole at 23 °C and were switched to 37 °C. A–E, aliquots of cells were analyzed by FACS to follow cell cycle progression, and Hst3 levels were monitored by immunoblotting with an antibody that detects the TAP epitope tag. Ponceau S staining is shown as loading control.
FIGURE 6.
FIGURE 6.
Clb5-Cdk1 phosphorylates Hst3 on Thr-380 and Thr-384 in vitro. A, kinase assays were performed with [γ-32P]ATP and either wild-type GST-Hst3 or the GST-Hst3-Thr-380A, Thr-384A (Hst3 2A) mutant as substrates. The reaction products were resolved through an SDS-12% polyacrylamide gel, and incorporation of radiolabeled phosphate was detected by autoradiography. Time 0 corresponds to samples collected before the addition of CDKs. B, GST-Hst3 was phosphorylated with cold ATP and Clb5-Cdk1, and the reaction products were analyzed by mass spectrometry. The doubly phosphorylated peptide that eluted at 34.8 min had a mass of 1413.592 Da that was within 2 ppm of its theoretical mass. The MS/MS spectrum shows abundant y- and b-fragment ions confirming the peptide sequence DSIGT(ph)PPT(ph)PLR, where the residues followed by (ph) are phosphorylated. Fragment ions at m/z 944.4 (y8*) and m/z 932.4 (b9*) are caused by losses of H3PO4 (98 Da) from the corresponding y8 and b9 fragment ions and provide additional evidence for phosphorylation of Thr-380 and Thr-384.
FIGURE 7.
FIGURE 7.
CDK-phosphorylated Hst3 is polyubiquitylated by SCFCdc4in vitro. Recombinant GST-Hst3 wild-type or GST-Hst3 2A was phosphorylated with either Clb5-Cdk1 or Clb2-Cdk1 (lower panel) and subsequently ubiquitylated with Cdc34 and SCFCdc4 (upper panel). The reaction products were detected after SDS-PAGE and immunoblotting (IB) with an antibody against GST. Polyubiquitylation is observed as smears of high molecular weight forms of GST-Hst3. The bands labeled non-specific are present in reactions lacking GST-Hst3 (lane 1) and caused by cross-reaction of the GST antibody with some of the proteins present in the ubiquitylation reactions.
FIGURE 8.
FIGURE 8.
Stabilization of Hst3 reduces cell fitness. A, colony formation assays. The 5-fold serial dilutions of each strain were plated on SC plates with or without MMS, but containing 5-fluoroorotic acid (5-FOA) to select against the URA3 plasmid encoding wild-type HST3, thereby leaving LEU2 plasmids as the only source of Hst3. The SC-leucine plate containing MMS was incubated at 25 °C for 5 days, whereas the SC-uracil plates were incubated at 25 °C for 3 days. B, continuous monitoring of cell density. Cultures were grown to saturation at 25 °C in SC medium lacking leucine. Each strain was inoculated at A595 = 0.1 in 96-well plates containing YPD medium with or without MMS. Plates were incubated in a Sunrise (Tecan) plate reader, and cell density was monitored at 10-min intervals for up to 48 h. The average A595 of three replicates was plotted as a function of time. The growth curves corresponding to the three Hst3 mutants are superimposable. C, lag times are defined as the amount of time necessary for saturated cell populations to start proliferating following dilution to A595 = 0.1. The corresponding numerical data are shown in Table 2. D, doubling times of cells in exponential phase. The corresponding numerical data are shown in Table 3.

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