SEARCH

SEARCH BY CITATION

Keywords:

  • histone acetylation;
  • histone deacetylation;
  • mitosis;
  • histone H3 serine 10 phosphorylation;
  • trichostatin A;
  • tobacco

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Protoplast isolation and cell cycle progression
  8. Antibodies
  9. Immunofluorescence
  10. Acknowledgements
  11. References

Post-translational modifications of core histone proteins play a key role in chromatin structure and function. Here, we study histone post-translational modifications during reentry of protoplasts derived from tobacco mesophyll cells into the cell cycle and evaluate their significance for progression through mitosis. Methylation of histone H3 at lysine residues 4 and 9 persisted in chromosomes during all phases of the cell cycle. However, acetylation of H4 and H3 was dramatically reduced during mitosis in a stage-specific manner; while deacetylation of histone H4 commenced at prophase and persisted up to telophase, histone H3 remained acetylated up to metaphase but was deacetylated at anaphase and telophase. Phosphorylation of histone H3 at serine 10 was initiated at prophase, concomitantly with deacetylation of histone H4, and persisted up to telophase. Preventing histone deacetylation by the histone deacetylase inhibitor trichostatin A (TSA) led to accumulation of protoplasts at metaphase–anaphase, and reduced S10 phosphorylation during anaphase and telophase; in cultured tobacco cells, TSA significantly reduced the frequency of mitotic figures. Our results indicate that deacetylation of histone H4 and H3 in tobacco protoplasts occurs during mitosis in a phase-specific manner, and is important for progression through mitosis.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Protoplast isolation and cell cycle progression
  8. Antibodies
  9. Immunofluorescence
  10. Acknowledgements
  11. References

The nucleosome consists of DNA wrapped around a histone octamer containing two copies of each of the four core histone proteins, H2A, H2B, H3 and H4 (reviewed in Kornberg and Lorch, 1999). This basic structure is further folded into a higher chromatin structure by the aid of multiple proteins or protein complexes (reviewed in Grewal and Moazed, 2003). Chromatin constitutes a highly dynamic structure, which is directly influenced by modifications of DNA (e.g. methylation) as well as by post-translational modifications of core histone proteins. The latter include modifications of specific amino acid residues, particularly at the amino-terminal tail, by acetylation, methylation, phosphorylation, (ADP)ribosylation as well as ubiquitination (van Holde, 1989; Wolffe, 1992). These modifications generate ‘codes’ for the recruitment of proteins or protein complexes that affect chromatin structure and function (reviewed by Grewal and Moazed, 2003; Jenuwein and Allis, 2001; Zhang and Reinberg, 2001). For example, histone H3 can be methylated at lysine 9 by a specific histone methyltransferase, SUV39H1 in humans (Rea et al., 2000) or Kryptonite in Arabidopsis (Jackson et al., 2002), thus generating a ‘code’ for the recruitment of heterochromatin protein 1 (HP1) (Bannister et al., 2001; Lachner et al., 2001), a chromo-domain protein that is involved in heterochromatin formation and gene silencing (Cavalli and Paro, 1998). On the other hand, acetylation of histones is often associated with ‘open’ chromatin configuration and gene transcription (reviewed by Eberharter and Becker, 2002).

The transition from interphase to mitosis is accompanied by dramatic changes in chromosome structure and function. Mitosis involves condensation of chromosomes and the formation of repressive chromatin associated with inhibition of transcriptional activity operated by all three RNA polymerases (Gottesfeld and Forbes, 1997). Chromosome condensation is associated with substantial reduction in histone acetylation; indirect immunofluorescence microscopy of various mammalian cell lines showed a reduction in K9H3 and K5H4 acetylation in mitotic cells compared with interphase cells (Kruhlak et al., 2001). Using trichostatin A (TSA), an inhibitor of histone deacetylases (HDACs), Kruhlak et al. (2001) also found that the resulting hyperacetylated histones did not affect chromosome condensation nor normal progression through mitosis. In a recent study, however, Cimini et al. (2003) demonstrated that histone hyperacetylation impaired chromosome condensation and sister chromatid separation during mitosis. In barley and in the field bean Vicia faba, reduction in histone H4 acetylation has been reported during mitosis (Belyaev et al., 1997; Jasencakova et al., 2001; Wako et al., 2003), although certain chromosomal regions may remain highly acetylated during metaphase (Jasencakova et al., 2000).

In the present report, we study histone post-translational modifications during the cell cycle and their significance for progression through mitosis. Using tobacco protoplasts induced to reenter the cell cycle, we followed the dynamic changes in tetraacetylated histone H4, dimethylated K4H3, dimethylated K9H3, acetylated K9/14H3 as well as S10H3 phosphorylation. The results showed that methylation at K4H3 and K9H3 remained associated with chromosomes during interphase and mitosis, whereas acetylation of H4 and H3 was dramatically reduced during mitosis, each being deacetylated at a specific phase. Prevention of histone deacetylation by TSA impaired progression through mitosis.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Protoplast isolation and cell cycle progression
  8. Antibodies
  9. Immunofluorescence
  10. Acknowledgements
  11. References

To investigate dynamic changes in histone modifications and their significance for cell cycle progression, we used tobacco protoplasts induced by phytohormones to reenter the cell cycle (Zhao et al., 2001). Immunolabeling experiments were performed on cells collected at various times after phytohormone induction using commercially available antibodies to tetraacetylated histone H4 (4KAcH4), dimethylated K4H3 (K4m2H3), dimethylated K9H3 (K9m2H3), acetylated K9/14 H3 (K9/14AcH3), and phosphorylated S10H3 (S10PH3). Control immunolabeling using only secondary antibody showed no specific labeling of nuclei (data not shown).

Immunolabeling experiments show (Figure 1) that in interphase cells both dimethylated K4H3 and K9H3 are dispersed within the nucleus but omitted from the nucleolus. Both methylated K4H3 and K9H3 were associated with chromosomes during mitosis. Thus, methylated K4H3 and K9H3 are permanently associated with chromatin during interphase and mitosis, a phenomenon which might be attributed to the stable, irreversible nature of this modification. We next analyzed the dynamic changes in histone acetylation during the cell cycle. Unlike methylation, histone acetylation is a reversible modification that is regulated by two opposing groups of enzymes: histone acetyltransferases (HATs) and HDACs.

image

Figure 1. Histone H3 remains methylated at lysine 4 and 9 during interphase and mitosis. Tobacco protoplasts were induced to reenter the cell cycle and subjected to immunofluorescence 96 h after induction. DAPI was used as a counterstain. Bar = 10 μm.

Download figure to PowerPoint

Figure 2 shows that acetylated histone H4 as well as acetylated K9/14H3 were associated with chromatin during interphase, being dispersed within the nucleus and absent from the nucleolus. During mitosis, deacetylation of histones H4 and H3 was temporally regulated in a phase-specific manner: while deacetylation of histone H4 was initiated at prophase and persisted up to telophase, deacetylation of histone H3 commenced at the metaphase/anaphase transition and persisted up to telophase.

image

Figure 2. Temporal deacetylation of histones H3 and H4 during mitosis. Protoplasts were subjected to immunofluorescence 96 h after induction using anti-tetraacetylated histone H4 (4KAcH4) and anti-acetylated lysine 9/14 histone H3 (K9/14AcH3). Note that deacetylation of histone H4 initiates at prophase and persists to the end of mitosis, whereas histone H3 undergoes deacetylation at the metaphase/anaphase transition. DAPI was used as a counterstain. Bar = 10 μm.

Download figure to PowerPoint

Consistent with previous reports (reviewed by Prigent and Dimitrov, 2003), histone H3 was not phosphorylated at serine 10 (S10) during interphase; it became phosphorylated at prophase and persisted up to telophase (Figure 3). Phosphorylation at S10 appears to be restricted to chromocenters during prophase up to early anaphase, but at late stages of mitosis, concomitantly with deacetylation of histone H3, S10 phosphorylation becomes widespread along chromosomes.

image

Figure 3. Histone H3 is phosphorylated at serine 10 during mitosis. Protoplasts were subjected to immunofluorescence 96 h after hormonal induction using anti-phosphorylated serine 10 histone H3 (S10PH3). Note that phosphorylation of H3S10 is evident at prophase at (peri)centromeric regions and it persists up to telophase; no phosphorylation is detected during interphase. Bar = 10 μm.

Download figure to PowerPoint

The importance of histone deacetylation for progression through mitosis in mammalian cells is not clear. Histone hyperacetylation either had no effect (Kruhlak et al., 2001) or a dramatic effect on chromosome condensation and segregation (Cimini et al., 2003). We used TSA, a histone deacetylase inhibitor, to test the significance of histone deacetylation for progression of plant cells through mitosis. To reduce possible effects on gene expression and progression through S phase, we applied TSA 72 h after induction, a time point where a high proportion of cells are at the G2 phase of the cell cycle (Zhao et al., 2001). Cells were collected at 96 h for immunolabeling and microscopic inspection. We first analyzed the efficiency of TSA treatment on histone acetylation by immunolabeling with anti-acetylated H4 (AcH4). The results showed (Figure 4) that TSA treatment led to histone H4 acetylation during all phases of mitosis from prophase to telophase. Similar results were obtained for histone H3 (data not shown). We could not detect any effect of TSA treatment on the initiation of phosphorylation of H3 at S10 (Figure 5). However, at late stages of mitosis, the period at which histone H3 becomes deacetylated, S10 phosphorylation was significantly reduced (Figure 5, late anaphase and telophase). Progression through mitosis with hyperacetylated histones resulted in mitotic arrest: compared with untreated cells, a higher proportion of mitotic cells (81% versus 43%) were found at the metaphase/anaphase transition (Figure 6a), suggesting that histone deacetylation is essential for progression through mitosis. We also analyzed the effect of TSA on cell cycle progression in tobacco suspension culture derived from Nicotiana sylvestris (Figure 6b,c). In TSA-treated cells the frequency of mitotic figures was dramatically reduced (0.4%) compared with untreated cells (4%) (Figure 6b). TSA-treated cultured cells also showed higher proportion of metaphases (Figure 6b) and the occurrence of abnormal anaphases (37% of all anaphases) displaying chromosomal bridges (arrows in Figure 6c).

image

Figure 4. Histone H4 hyperacetylation during mitosis following treatment with trichostatin A (TSA). Protoplasts were subjected to TSA 72 h after induction and subjected to immunofluorescence with anti-acetylated H4 (AcH4) 24 h after TSA application. Bar = 10 μm.

Download figure to PowerPoint

image

Figure 5. Prevention of histone deacetylation by trichostatin A (TSA) reduced H3S10 phosphorylation at late stages of mitosis. Protoplasts were subjected to TSA 72 h after induction and subjected to immunofluorescence with anti-phosphorylated serine 10 histone H3 (S10PH3) 24 h after TSA application. Bar = 10 μm.

Download figure to PowerPoint

image

Figure 6. Trichostatin A (TSA)-treated cells are impaired in mitosis. (a) Protoplasts were subjected to TSA 72 h after induction and inspected under a fluorescence microscope 24 h after TSA application. The combined numbers of mitotic figures counted at metaphase and early anaphase or at late anaphase and telophase are indicated with their percentages in brackets. (b) Low frequency of mitoses in cultured tobacco cells (Nicotiana sylvestris) treated with TSA. Percentages of the mitotic phases were calculated out of 2000 control (−TSA) and 20 000 TSA-treated (+TSA) cells counted. Note the increased frequency of metaphases in TSA-treated cells. (c) Abnormal anaphases in cultured tobacco cells (N. sylvestris) treated with TSA. Note that among the anaphases counted in TSA-treated cells, 37% were abnormal displaying chromosomal bridges (arrows).

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Protoplast isolation and cell cycle progression
  8. Antibodies
  9. Immunofluorescence
  10. Acknowledgements
  11. References

The results presented here demonstrate the dynamic nature of histone post-translational modifications during cell cycle progression in tobacco. While histone methylation showed no detectable changes during the cell cycle, histone acetylation was dramatically affected being under rigorous regulation during mitosis. Histone H3 and H4 were differentially deacetylated during mitosis: H4 was deacetylated as early as prophase and up to telophase, while histone H3 was deacetylated at the metaphase/anaphase transition persisting up to telophase. Such temporal control over deacetylation of H3 and H4 suggests that different HDACs may operate during different phases of mitosis to control H4 and H3 acetylation level. This proposition is in line with the finding that the different classes of HDACs in maize embryos displayed specificity for substrate and for distinct acetylation pattern on nucleosomes (Kolle et al., 1999). In a variety of eukaryotic cycling cells, HDAC expression level is constant (Bartl et al., 1997; Brandtner et al., 2002), whereas HDAC activity fluctuates during the cell cycle (Brandtner et al., 2002). HDAC activity is controlled in multiple ways including protein–protein interaction, post-translational modifications (e.g. phosphorylation), as well as by subcellular localization (reviewed in Sengupta and Seto, 2004). In maize, the specificity of HDACs was regulated by phosphorylation; specificity of HDAC was increased for histone H2A and decreased for H3 upon phosphorylation (Brosch et al., 1992). Yet, the identity of the HDAC members (16 HDAC genes were identified in Arabidopsis thaliana, Pandey et al., 2002) acting during mitosis in plants and the way by which their activities are controlled need to be elucidated.

It has been well documented that highly acetylated histones are associated with transcriptionally active chromatin, while hypoacetylated histones usually accumulate in transcriptionally silent heterochromatin (reviewed by Eberharter and Becker, 2002). Acetylated histones induced chromatin relaxation and gene transcription (Tse et al., 1998). Accordingly, deacetylation of histone H4 in tobacco cells at early stages of mitosis (prophase) correlates well with the condensation of chromosomes and may facilitate their compaction. Obviously, deacetylation of histone H3 at the metaphase/anaphase transition does not account for chromosome compaction but rather for sister chromatid separation. Indeed, application of TSA at 72 h after inducing protoplasts to reenter the cell cycle led to accumulation of cells at the metaphase/anaphase transition (Figure 6), suggesting that histone deacetylation may be required for chromatid separation and progression through anaphase. When TSA, however, was applied to non-synchronized tobacco cultured cells the major effect was a dramatic drop in the frequency of mitotic figures. These differences between protoplasts and cultured tobacco cells may be attributed to the timing and duration of TSA application during the cell cycle. Similar to the effects of TSA in tobacco cultured cells, in pea TSA also halted mitosis (Murphy et al., 2000). Moreover, in various animal cell lines, the major effect of treatment with histone deacetylase inhibitors is arrest at G2/M transition and/or G1 phase (Noh and Lee, 2003; Qiu et al., 2000). In human primary fibroblasts, however, TSA treatment resulted in impaired chromosome compaction and sister chromatid separation (Cimini et al., 2003). This is similar to our observation of abnormal anaphases in cultured tobacco cells treated with TSA (Figure 6c).

Cells possess at least two populations of histone H3 during interphase: K9-methylated and K9-acetylated. During mitosis (from prophase to metaphase), S10 phosphorylation in plants is confined mainly to pericentromeric regions (Houben et al., 1999; see also Figure 3), which are heterochromatic and enriched in K9-methylated histone H3 (Fransz et al., 2003; Soppe et al., 2002). This mode of phosphorylation is puzzling as S10 phosphorylation is very efficient, at least in vitro when K9 is acetylated, and inefficient when K9 is methylated (Rea et al., 2000). Notably, K9H3 methylation is targeted by HP1 (Bannister et al., 2001; Lachner et al., 2001), a chromo-domain protein that induces chromatin compaction and gene silencing (Cavalli and Paro, 1998). It appears, however, that K9-methylated H3 is the in vivo preferred target for S10 phosphorylation. Indeed, both modifications were reported to coexist on the same molecule (Fass et al., 2002; Fischle et al., 2003), leading to increased affinity of HP1 to histone H3 (Mateescu et al., 2004), which may lead to further chromosome compaction. It is only when K14 of histone H3 is acetylated that HP1 is no longer able to bind histone H3 (Mateescu et al., 2004). It is therefore possible that the reduction in S10H3 phosphorylation during late stages of mitosis observed in TSA-treated cells (Figure 5) may be attributed to dephosphorylation of S10H3 induced by K14 acetylation.

Protoplast isolation and cell cycle progression

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Protoplast isolation and cell cycle progression
  8. Antibodies
  9. Immunofluorescence
  10. Acknowledgements
  11. References

Cultured tobacco cells of N. sylvestris were grown in MS-based medium supplemented with 0.6 μg ml−1α-naphthalene acetic acid (NAA) and 0.2 μg ml−1 6′-benzylaminopurine (BAP). Five days after subculturing, cells were treated for 40 h with 200 nm TSA, an inhibitor of HDACs, and inspected under a fluorescence microscope after staining with propidium iodide (1 μg ml−1). The percentage of mitotic cells was determined by counting the number of prophases, metaphases, anaphases and telophases in a population of 2000 control cells or 20 000 TSA-treated cells.

Protoplasts were isolated from sterile leaves of Nicotiana tabacum (Samsun NN) essentially as described (Zelcer and Galun, 1976). Freshly prepared protoplasts were washed and induced to reenter the cell cycle by incubation at 22°C in VKM medium containing 0.5 μg ml−1 BAP and 2 μg ml−1 NAA as described (Zhao et al., 2001). In certain experiments, TSA was added (100 nm) at 72 h after hormonal induction, and cells were harvested 24 h later and fixed with ethanol:acetic acid (3:1). Progression of protoplasts through mitosis was inspected after staining of fixed cells with diamidino-phenyl-indole (DAPI) using a fluorescence microscope (Olympus, Hamburg, Germany) equipped with a CCD camera (Imago; Photonics, Hamburg, Germany). Images were pseudo-colored and merged using TILL Vision version 3.3 software.

Antibodies

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Protoplast isolation and cell cycle progression
  8. Antibodies
  9. Immunofluorescence
  10. Acknowledgements
  11. References

All antibodies to modified histones, anti-phospho (S10) histone H3, anti-acetyl (K9/K14) histone H3, anti-tetraacetylated H4, anti-dimethyl (K9) histone H3, and anti-dimethyl (K4) histone H3 were purchased from Upstate Biotechnology (Lake Placid, NY, USA). Secondary antibody goat anti-rabbit IgG conjugated with FITC was from Sigma (St Louis, MO, USA).

Immunofluorescence

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Protoplast isolation and cell cycle progression
  8. Antibodies
  9. Immunofluorescence
  10. Acknowledgements
  11. References

Cells collected at various time points after hormonal induction were fixed in 4% paraformaldehyde dissolved in PBS for 15 min at room temperature, followed by washing twice with PBS. Cells were permeabilized in cold acetone (100%) for 7 min at −20°C, washed twice with PBS, blocked in 2% BSA in PBS for 2 h at room temperature, followed by overnight incubation at 4°C with 100 μl primary antibody (1–2 μg in 2% BSA). Cells were washed three times, 5 min each, in PBS, followed by 2 h incubation at room temperature with secondary antibody (goat anti-rabbit IgG tagged with FITC, Sigma, diluted 1:100). In control experiments cells were incubated with secondary antibody alone. Cells were washed as above, dehydrated with methanol (100%) for 20 min, then fixed in ethanol:acetic acid (3:1) and spread on a slide. Cells were stained with DAPI, mounted with Vectashield and inspected by a fluorescence microscope (Olympus) equipped with a CCD camera (Imago, Photonics). Images were pseudo-colored and merged using TILL Vision version 3.3 software. All images were processed using Adobe Photoshop software.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Protoplast isolation and cell cycle progression
  8. Antibodies
  9. Immunofluorescence
  10. Acknowledgements
  11. References

We thank Y. Avivi for critical reading of this manuscript and R. Vunsh for providing N. sylvestris cultured cells. We also thank anonymous reviewers for helpful comments. This work was supported by the Israel Science Foundation (ISF) to G. Grafi (grant no. 385/02-1).

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Protoplast isolation and cell cycle progression
  8. Antibodies
  9. Immunofluorescence
  10. Acknowledgements
  11. References