Elsevier

Experimental Cell Research

Volume 316, Issue 13, 1 August 2010, Pages 2123-2135
Experimental Cell Research

Research Article
Acetylation of core histones in response to HDAC inhibitors is diminished in mitotic HeLa cells

https://doi.org/10.1016/j.yexcr.2010.05.003Get rights and content

Abstract

Histone acetylation is a key modification that regulates chromatin accessibility. Here we show that treatment with butyrate or other histone deacetylase (HDAC) inhibitors does not induce histone hyperacetylation in metaphase-arrested HeLa cells. When compared to similarly treated interphase cells, acetylation levels are significantly decreased in all four core histones and at all individual sites examined. However, the extent of the decrease varies, ranging from only slight reduction at H3K23 and H4K12 to no acetylation at H3K27 and barely detectable acetylation at H4K16. Our results show that the bulk effect is not due to increased or butyrate-insensitive HDAC activity, though these factors may play a role with some individual sites. We conclude that the lack of histone acetylation during mitosis is primarily due to changes in histone acetyltransferases (HATs) or changes in chromatin. The effects of protein phosphatase inhibitors on histone acetylation in cell lysates suggest that the reduced ability of histones to become acetylated in mitotic cells depends on protein phosphorylation.

Introduction

Histones are the most abundant proteins associated with DNA in eukaryotic chromosomes. The core histones H2A, H2B, H3 and H4 wind the DNA into bead-like nucleosome cores [1], [2], while histone H1 organizes the “string of beads” into a 30 nm chromatin fiber [3], [4]. In addition to packaging the DNA and neutralizing some of its negative charge, the histones also play roles in DNA replication, repair and transcription.

Histone function is modulated by several different post-translational modifications, one of which is the reversible acetylation of lysine residues in the N-terminal tails of the four core histones. Histone acetylation is a dynamic process involving a balance between histone acetyltransferase (HAT) and histone deacetylase (HDAC) activities, and it is important because it correlates with gene transcription or readiness for transcription [5], [6], [7], [8], [9], [10], [11].

Normally only a small percentage of core histone molecules are more than mono-acetylated. However, histone acetyl groups turn over rapidly [12]. In cultured cell lines, 70% of the acetate incorporated into histones in a short pulse turns over with a half-life of about 3 min, and the rest with a half-life of 30–40 min [12]. As a result, treatment of interphase cells for a few hours with HDAC inhibitors such as sodium butyrate, valproic acid, trichostatin A (TSA), apicidin or oxamflatin leads to significant histone hyperacetylation [13], [14], [15], [16], [17], [18], [19], [20]. Evidence indicates that butyrate and TSA are competitive inhibitors of HDACs [21], [22], and based on structural similarity to butyrate or acetyllysine, apicidin [23] and valproate probably are also competitive inhibitors of HDACs. The mode of inhibition by oxamflatin has not been reported.

Histone acetylation levels are lower during mitosis than during interphase in mammalian cells [24], [25], [26], [27], [28], [29], [30], [31], [32], [33] and in Physarum polycephalum [34], [35]. Histone acetylation is also decreased during the later stages of meiosis in Lilium microsporocytes [36] and in mouse oocytes [37]. The reason for the decrease in histone acetylation at mitosis is not known. However, it could be related to the cessation of transcription during mitosis in higher eukaryotes (e.g., [38], [39], [40], [41]), either as a cause or a consequence.

In the work reported here, we have explored the possible reasons for underacetylation of histones at mitosis by treating metaphase-arrested HeLa cells with HDAC inhibitors. We find that this treatment results in little or no increase in core histone acetylation. Since the effect is seen in bulk chromatin, it is not due merely to the cessation of transcription. Our results suggest that the causes may be complex, but that the phenomenon reflects reduced turnover of histone acetates in mitotic cells and decreased ability of HATs to act on histones in mitotic chromatin. In vitro experiments suggest that decreased histone acetylation at mitosis is dependent on mitosis-specific protein phosphorylation of an as-yet unknown target.

Section snippets

Chemicals, media and antibodies

Microcystin LR was dissolved at 1 mM in 50 mM Tris–Cl pH 7.0 and stored in aliquots at − 20 °C. Calyculin A was prepared as a 100 μM solution in methanol and stored at 2 °C. Cantharidin was prepared as a 200 mM solution in N,N-dimethylformamide (DMF) and stored at 2 °C. Sodium butyrate was made as a 5 M stock solution in 0.9% NaCl and 20 mM sodium phosphate and adjusted to pH 7.4. Trichostatin A (TSA), oxamflatin and apicidin were prepared as 1 mg/mL solutions in dimethylsulfoxide (DMSO) and stored at − 20 

HDAC inhibitors do not induce hyperacetylation of histone H4 in metaphase-arrested HeLa cells

Treatment of interphase mammalian cells with histone deacetylase (HDAC) inhibitors is known to induce hyperacetylation of core histones (e.g., [13], [14], [15], [16], [17], [18], [19]). In order to determine whether the same is true of mitotic cells, HeLa cultures were synchronized in S-phase by treatment with 2.5 mM thymidine for 20–24 h, released from the thymidine block, and treated 4 h later with 0.25 μg/mL nocodazole to arrest in metaphase [45], [46]. At 16 h after release from thymidine, the

Decreased acetylation of histones at mitosis in response to HDAC inhibitors

We have shown that the ability of histones to become acetylated following treatment of HeLa cells with HDAC inhibitors is significantly diminished during mitosis. Multiacetylated histone H4 isoforms are induced in interphase cells, but not in metaphase-arrested cells, by exposure to sodium butyrate [13], trichostatin A [17], apicidin [18] or oxamflatin [19]. The effect is seen with all four core histones and to varying degrees at all individual acetylation sites that have been examined. Our

Acknowledgments

We are grateful to Dr. Maria Vogelauer and Dr. Hiroshi Kimura for providing acetylhistone-specific antibodies. This work was supported by the University of Wisconsin-Oshkosh Faculty Development Board and by grants R15 GM39915 and R15 GM46040 (to J. R. Paulson) and grant R01 CA69008 (to W. C. Earnshaw and S. H. Kaufmann) from the National Institutes of Health, U.S. Public Health Service. The work was also supported by grants to B.M. Turner from Cancer Research UK (programme grant C1015/A9077)

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    Present address: R&D Systems Inc, 614 McKinley Place N.E., Minneapolis, MN 55413, USA.

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