Portal de Periódicos da CAPES

The N-terminus of histone H2B, but not that of histone H3 or its phosphorylation, is essential for chromosome condensation

2001; Springer Nature; Volume: 20; Issue: 22 Linguagem: Inglês

10.1093/emboj/20.22.6383

1460-2075

Anne-Elisabeth de la Barre, Dimitar Angelov, Annie Molla, Stéfan Dimitrov,

RNA Interference and Gene Delivery

Article15 November 2001free access The N-terminus of histone H2B, but not that of histone H3 or its phosphorylation, is essential for chromosome condensation Anne-Elisabeth de la Barre Anne-Elisabeth de la Barre Laboratoire de Biologie Moléculaire et Cellulaire de la Différenciation, INSERM U 309, Institut Albert Bonniot, Domaine de la Merci, 38706 La Tronche, Cedex, France Search for more papers by this author Dimitri Angelov Dimitri Angelov Institute of Solid State Physics, Bulgarian Academy of Sciences, 1784 Sofia, Bulgaria Search for more papers by this author Annie Molla Annie Molla Laboratoire de Biologie Moléculaire et Cellulaire de la Différenciation, INSERM U 309, Institut Albert Bonniot, Domaine de la Merci, 38706 La Tronche, Cedex, France Search for more papers by this author Stefan Dimitrov Corresponding Author Stefan Dimitrov Laboratoire de Biologie Moléculaire et Cellulaire de la Différenciation, INSERM U 309, Institut Albert Bonniot, Domaine de la Merci, 38706 La Tronche, Cedex, France Search for more papers by this author Anne-Elisabeth de la Barre Anne-Elisabeth de la Barre Laboratoire de Biologie Moléculaire et Cellulaire de la Différenciation, INSERM U 309, Institut Albert Bonniot, Domaine de la Merci, 38706 La Tronche, Cedex, France Search for more papers by this author Dimitri Angelov Dimitri Angelov Institute of Solid State Physics, Bulgarian Academy of Sciences, 1784 Sofia, Bulgaria Search for more papers by this author Annie Molla Annie Molla Laboratoire de Biologie Moléculaire et Cellulaire de la Différenciation, INSERM U 309, Institut Albert Bonniot, Domaine de la Merci, 38706 La Tronche, Cedex, France Search for more papers by this author Stefan Dimitrov Corresponding Author Stefan Dimitrov Laboratoire de Biologie Moléculaire et Cellulaire de la Différenciation, INSERM U 309, Institut Albert Bonniot, Domaine de la Merci, 38706 La Tronche, Cedex, France Search for more papers by this author Author Information Anne-Elisabeth de la Barre1, Dimitri Angelov2, Annie Molla1 and Stefan Dimitrov 1 1Laboratoire de Biologie Moléculaire et Cellulaire de la Différenciation, INSERM U 309, Institut Albert Bonniot, Domaine de la Merci, 38706 La Tronche, Cedex, France 2Institute of Solid State Physics, Bulgarian Academy of Sciences, 1784 Sofia, Bulgaria ‡A.-E.de la Barre and D.Angelov contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:6383-6393https://doi.org/10.1093/emboj/20.22.6383 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info We have studied the role of individual histone N-termini and the phosphorylation of histone H3 in chromosome condensation. Nucleosomes, reconstituted with histone octamers containing different combinations of recombinant full-length and tailless histones, were used as competitors for chromosome assembly in Xenopus egg extracts. Nucleosomes reconstituted with intact octamers inhibited chromosome condensation as efficiently as the native ones, while tailless nucleosomes were unable to affect this process. Importantly, the addition to the extract of particles containing only intact histone H2B strongly interfered with chromosome formation while such an effect was not observed with particles lacking the N-terminal tail of H2B. This demonstrates that the inhibition effect observed in the presence of competitor nucleosomes is mainly due to the N-terminus of this histone, which, therefore, is essential for chromosome condensation. Nucleosomes in which all histones but H3 were tailless did not impede chromosome formation. In addition, when competitor nucleosome particles were reconstituted with full-length H2A, H2B and H4 and histone H3 mutated at the phosphorylable serine 10 or serine 28, their inhibiting efficiency was identical to that of the native particles. Hence, the tail of H3, whether intact or phosphorylated, is not important for chromosome condensation. A novel hypothesis, termed 'the ready production label' was suggested to explain the role of histone H3 phosphorylation during cell division. Introduction Although mitotic chromosomes were described more than a century ago, their structure and assembly are still poorly understood. However, during the last few years real progress towards the understanding of the complex process of chromosome formation has been made thanks to two complementary approaches: genetics in yeast and biochemical manipulations in extracts isolated from Xenopus eggs (for a recent review see Hirano, 2000). This last approach has been extremely useful since the in vitro condensed chromosomes can be manipulated in their natural conditions allowing a causal relationship between the different events involved in their assembly to be studied. In addition, the structure and properties of these chromosomes are identical to those formed in cells in culture (Houchmandzadeh et al., 1997; Houchmandzadeh and Dimitrov, 1999; Poirier et al., 2000). Therefore, the dissection of the mechanism of chromosome condensation in the Xenopus egg extract is relevant to the study of physiological mechanisms operating in vivo. Indeed, the use of the extracts has allowed the identification and isolation of the condensin complex, which is required for chromosome condensation (Hirano and Mitchison, 1994; Hirano et al., 1997; Hirano, 2000). This complex exists in vertebrate somatic cells and in yeast and is associated with chromatin exclusively during mitosis, as observed in the case of Xenopus egg extracts (Hirano and Mitchison, 1994; Saitoh et al., 1994; Sutani et al., 1999). Moreover, the study of chromosome assembly in these extracts showed that topoisomerase II had an enzymatic but not a structural role in the maintenance of mitotic chromosome organization (Hirano and Mitchison, 1993). The basic unit of chromatin, the nucleosome, contains an octamer of core histones (two each of H2A, H2B, H3 and H4) around which two superhelical turns of DNA are wrapped. The crystallographic structure of both the histone octamer and the whole nucleosome has been solved. Each histone within the octamer consists of a structured domain (the histone fold) and non-structured N-terminal tails (Arents et al., 1991; Luger et al., 1997). The linker histone interacts mainly with the linker DNA between the nucleosomes. Since the early seventies it has been assumed that linker histones were main players in chromosome condensation (Bradbury et al., 1973; Bradbury, 1992). However, more recent experiments have shown that their absence affected neither chromosome condensation nor nucleus assembly both in Xenopus egg extract (Ohsumi et al., 1993; Dasso et al., 1994) and in vivo (Shen et al., 1995; Shen and Gorovsky, 1996; Barra et al., 2000). These data argued strongly against the above hypothesis. In contrast, it has recently been reported that the flexible N-termini of core histones play an essential role in chromosome assembly (de la Barre et al., 2000). The N-terminal tails of histones are subjected to a large number of post-translational modifications, which are believed to have important functions in numerous cellular processes (reviewed in Wolffe and Hayes, 1999; Cheung et al., 2000; Strahl and Allis, 2000; Turner, 2000). During the last few years particular attention has been paid to the phosphorylation of the histone H3 tail at serine 10. In fact, this modification was observed in two different processes: the first is the activation of transcription (Mahadevan et al., 1991; Thomson et al., 1999a, b), which requires substantial chromatin decompaction, and the second is chromosome assembly during mitosis and meiosis (Hendzel et al., 1997; Van Hooser et al., 1998; Wei et al., 1998, 1999; de la Barre et al., 2000; Hsu et al., 2000; Kaszas and Cande, 2000; Adams et al., 2001; Giet and Glover, 2001). The function of histone H3 phosphorylation is not clear. Indeed, during cell division histone H3 phosphorylation was related either to chromosome condensation (Hendzel et al., 1997; Wei et al., 1998, 1999; Hsu et al., 2000) or to chromosome cohesion (Kaszas and Cande, 2000). However, in both cases the evidence was mostly correlative. For example, during mitosis in cultured vertebrate cells, the phosphorylation of histone H3 occurred in a precise spatio-temporal order (Hendzel et al., 1997; Van Hooser et al., 1998; Sauve et al., 1999). Indeed, initially detected in late G2 phase on pericentromeric heterochromatin, it spreads along the chromosome arms as mitosis proceeds. A correlation was observed between the initial chromatin condensation and the phosphorylation of histone H3 (Hendzel et al., 1997; Van Hooser et al., 1998). In plants, however, H3 phosphorylation in both mitosis and meiosis was initiated on already compacted chromosomes, a finding arguing against a role of this H3 modification in chromosome condensation (Kaszas and Cande, 2000). Instead, experiments with wild-type and mutant maize, where chromatid cohesion at metaphase II was absent, strongly suggested an association of histone H3 phosphorylation with chromosome cohesion (Kaszas and Cande, 2000). The Xenopus sperm nucleus DNA is tightly packaged through interactions with the histones H3 and H4 (present in equimolar amounts in sperm nuclei, for details see Dimitrov and Wolffe, 1995) and protamine-like proteins (Philpott et al., 1991; Dimitrov et al., 1994). Upon incubation in the egg extract, the demembranated sperm nuclei undergo a complete remodeling (Dimitrov and Wolffe, 1995, 1996). During the first 5–10 min, a dramatic decondensation takes place, followed by a series of well defined condensation steps culminating in the formation of compact chromosomes (Lohka and Masui, 1983; Hirano and Mitchison, 1993; de la Barre et al., 1999). The chromosome assembly is accompanied by phosphorylation of histone H3 at serine 10 (de la Barre et al., 2000). In this work, the role of the N-terminal tails of the different histone species was studied in chromosome condensation by using nucleosome-mediated competitive inhibition of chromosome assembly. Chromosomes were formed in Xenopus egg extracts in the presence of reconstituted chimeric nucleosomes containing different combinations of recombinant full-length and tailless histones. Evidence is presented here that the N-terminus of histone H2B is a main player in the process of chromosome formation. On the contrary, the tail of histone H3, whether intact or phosphorylated, did not interfere with chromosomal condensation. Results Inhibition of chromosome assembly by trypsin- and clostripain-digested nucleosomes Incubation of demembranated sperm nuclei in extracts isolated from Xenopus eggs allowed the assembly of mitotic chromosomes (Lohka and Masui, 1983; Hirano and Mitchison, 1993). Recently, we have demonstrated that adding exogenous native nucleosomes to the assembly reaction resulted in the inhibition of chromosome formation (de la Barre et al., 2000). Importantly, tailless nucleosomes, prepared by trypsin digestion, failed to inhibit the formation of mitotic chromosomes. This suggests that the flexible histone tails are involved in the recruitment of the putative chromosome assembly factor(s) and consequently play an essential role in chromosome assembly (de la Barre et al., 2000). The main objective of this study was to determine which of the tails of the individual histones within the nucleosome is/are required for this inhibition process and, therefore, is/are important for the assembly itself. First, we produced 'partially' tailless nucleosomes by digestion of native particles with the protease clostripain (Figure 1). Indeed, it was reported that clostripain was able to cleave the N-termini of histones H3 and H4, leaving H2A and H2B essentially intact (Encontre and Parello, 1988). However, we have not been able to completely reproduce these data (Figure 1B, 1/200 and other results not shown). The full elimination of the tails of H3 and H4 was always accompanied by significant digestion of histone H2B (>60%) and some cleavage of H2A with probably the first three amino acids being removed from the N-terminus (Encontre and Parello, 1988; Banéres et al., 1997). Upon digestion with higher amounts of clostripain (enzyme/nucleosomes ratio of 1/40; Figure 1B), the histone tails were completely cleaved (Encontre and Parello, 1988; Banéres et al., 1997). Chromosome formation was inhibited in the presence of 16 pmol of 'partially' tailless nucleosomes (compare Figure 1C with Figure 1D, ratio 1/200) while 26 pmol of clostripain-prepared tailless nucleosomes were necessary to observe the same effect (Figure 1D, ratio 1/40). Inhibition of chromosome condensation by tailless nucleosomes prepared by trypsin digestion took place at 33 pmol (Figure 1D and de la Barre et al., 2000). We attributed the slight difference (1.25×) in the inhibitory efficiency of the two types of tailless nucleosomes, respectively obtained by cleavage with each of the two proteases to the fact that trypsin digestion of the histone octamer was less controlled (for details see Encontre and Parello, 1988). Figure 1.Effect of native as well as trypsin- and clostripain-digested nucleosomes on chromosome condensation. (A) An 18% SDS–PAGE of histones isolated from native (a) and trypsin-digested (b) nucleosomes. P1−5 designate the trypsin resistant peptides (mainly the histone fold domains, see van Holde, 1988). (B) As (A), but for histones isolated from clostripain-digested nucleosomes at histone: clostripain molar ratio 1:200 (b) and 1:40 (c). On (a) are shown the histones prepared from the control non-digested nucleosomes. (C) Controls. Demembranated sperm nuclei and chromosomes assembled after 180 min of incubation of sperm nuclei in Xenopus egg extract. (D) A high concentration of proteolized nucleosomes is required for inhibition of chromosome condensation. Demembranated sperm nuclei were incubated in the extract in the absence (control) or in the presence of native or trypsin- (upper part of the panel) or clostripain-digested nucleosomes (lower part of the panel) at the indicated amounts. Chromosome assembly was carried out for 180 min, the samples were fixed, stained with Hoechst 33258 and observed by fluorescence microscopy. Bars, 5 μm. Download figure Download PowerPoint Two conclusions can be drawn from these results: (i) the inhibitory amounts of the two types of tailless nucleosomes are very close and are much higher than that of native nucleosomes (2 pmol, Figure 1D and de la Barre et al., 2000). Thus, regardless of how the tails were removed, the tailless nucleosomes have lost their capacity to inhibit chromosome condensation. These findings confirmed our previous data showing the important function of the histone tails in chromosome condensation (de la Barre et al., 2000). (ii) A removal of the histone N-termini, total for H3 and H4 and in high proportions of H2B, has led to the loss of the inhibiting effect, suggesting that the tail(s) of some of these histones (or the tails of all three) within the nucleosome is/are essential for chromosome assembly. The N-termini of histones H2A and H2B interfere with chromosome condensation The above results, however, do not discriminate between these different possibilities. In addition, the degree of proteolysis of the histones is experimentally difficult to control, i.e. particles containing histones digested to different extents may be present in the nucleosome preparations. In order to overcome these difficulties, we have then reconstituted 'chimeric' nucleosomes by using bulk nucleosomal DNA and different combinations of well defined recombinant full-length and mutant histones (Figure 2). The recombinant proteins were expressed in bacteria and purified to homogeneity (Figure 2B). A trace amount of 32P-end-labeled 152 bp EcoRI–RsaI fragment comprising a Xenopus borealis somatic 5S RNA gene was added to the reconstitution reactions. In this way the efficiency of reconstitution and the structure of the reconstituted particles could be checked (Figure 2A and C). Under the conditions used, all added DNA had formed a complex with the histones: no free DNA was detected by electrophoretic mobility shift analysis (EMSA) (Figure 2A). The DNase I footprinting demonstrated a clear 10 bp nucleosomal repeat (Figure 2C), an evidence for well-structured particles. Therefore, the reconstituted nucleosomes showed an organization which was highly similar to the native ones, a result which was in agreement with the available data (Luger et al., 1999). Figure 2.Characterization of the reconstituted 'chimeric' nucleosomes. Nucleosomes were reconstituted by using bulk DNA isolated from mononucleosomes and different combinations of recombinant histones and their mutants. A tracer amount of 32P-end-labeled 152 bp EcoRI–RsaI fragment containing a Xenopus borealis 5S RNA gene was present in the reconstitution mixture; (ΣH): nucleosomes, reconstituted with intact histones; (ΣGH): particles containing only the globular domains of the four histones; (H2B), (H3): chimeric nucleosomes, containing either intact H2B, or H3 and the globular parts of the three remaining histones; (H3/H4) and (H2A/H2B): particles reconstituted either with intact H3 and H4 or H2A and H2B and the two other histones tailless; (GH3), (GH2B): reconstituted nucleosomes either with the histone fold domain of H3 or that of H2B and the three remaining histones intact. (A) EMSA of the reconstituted nucleosomes in 2% agarose gel. After completion of the electrophoresis the gel was stained with ethidium bromide. (B) SDS–PAGE (18%) of the recombinant histones, isolated from the reconstituted chimeric nucleosomes. (C) DNase I footprinting of the reconstituted samples. The arrow shows the dyad axis of the nucleosomes. For simplicity the biochemical and structural characterization of only some of the used chimeric nucleosomes are shown. Download figure Download PowerPoint Further on, the reconstituted structures were used in chromosome assembly inhibition experiments. The nucleosomes reconstituted with tailless histones (ΣGH) did not affect chromosome condensation, whereas those assembled with full-length proteins (ΣH) completely inhibited the process (Figure 3). Importantly, the inhibition was observed at 2 pmol, an inhibitory amount equal to that of native nucleosomes. Thus, the reconstituted particles not only showed very close structure to the native ones, but they also behaved functionally like them. This validated the use of nucleosome particles reconstituted with several combinations of full-length and mutant histones to study their inhibition properties. Surprisingly, the particles (H2A/H2B) containing intact histones H2A and H2B and the globular domains GH3 and GH4 of H3 and H4 respectively, demonstrated a very strong inhibitory effect (Figure 3): 3.5 pmol only were sufficient to arrest the chromosome assembly process. Thus, the tails of H2A and H2B are likely to be major players in chromosome assembly. Figure 3.(A) The tails of histones H2A and H2B, but not those of H3 and H4 interfere with chromosome condensation. Chromosome assembly was performed as described in Figure 1 in the presence of the different chimeric nucleosomes. After fixation and staining with Hoechst 33258, the structures formed were observed by fluorescence microscopy. The amount of the competitor particles is shown on the left upper corner of each picture. The reconstituted particles are schematically drawn on the left side of the respective panels. The tails of the histones are presented in grey. For simplicity the tail of only one of the two homologous histones is shown. Bar, 5 μm. (B) Quantification of the data presented in (A). Download figure Download PowerPoint The N-terminus of histone H2B is a main player in chromosome condensation To understand whether the tails of both histones H2A and H2B, or the terminus of only one of these histones are essential for chromosome condensation, nucleosomes were prepared which contained either intact H2A (H2A particles) or intact H2B (H2B particles), the other three histones being tailless. Under these conditions, only the H2B particles were able to inhibit chromosome assembly (Figure 4A), demonstrating that the tail of histone H2B is essential for this process. This conclusion was further confirmed by the experiments with particles reconstituted with tailless H2B (GH2B) and the three other full-length histones (Figure 3B, GH2B particles). Indeed, these reconstitutes were unable to inhibit chromosome assembly. Figure 4.The tail of histone H2B is responsible for the nucleosome-induced inhibition of chromosome assembly. (A) The experiments were carried out as described in Figure 1, but in the presence of nucleosomes containing either full-length histone H2A, or H2B, or H3 or H4 and the globular domains of the three remaining histones. The assembled structures were visualized by fluorescence microscopy. (B) Same as (A), but in the presence of reconstituted nucleosomes comprising either the globular domain of histone H2B (GH2B) or that of histone H3 (GH3) and the three other full-length histones. (C) Quantification of the data presented in (A) and (B). For comparison the inhibition effect of (ΣH) and (ΣGH) reconstituted nucleosomes are also shown. Bar, 5 μm. Download figure Download PowerPoint Interestingly, the H2B nucleosome inhibition was observed at 6 pmol, an amount about two times higher than that of the H2A/H2B particles (see Figure 3) suggesting that the presence of intact H2A terminus, although dispensable, increased the inhibitory properties of the chimeric particles. The tail of histone H3, whether intact or phosphorylated, is not essential for chromosome condensation A widely held hypothesis claimed that the phosphorylation of histone H3 at serine 10 and hence the tail of histone H3 are required for chromosome condensation during mitosis (for recent reviews see Cheung et al., 2000; Hans and Dimitrov, 2001). However, this hypothesis was essentially based on correlative evidence. Our experimental procedure is a unique approach allowing the direct study of a causal relationship between these two events. We have found that chromosome formation was not inhibited by chimeric nucleosomes containing intact H3 and/or intact H4 and the other tailless histones (Figures 3 and 4), which strongly suggested that the tails of these proteins were not important for chromosome assembly. This was further confirmed by the properties of the GH3 and GH4 particles (comprising the globular domain of H3 or of H4 and the three other full-length histones, Figure 4B and C): the lack of the N-terminus of H3 or of H4 had no effect on the inhibitory ability of these reconstituted nucleosomes. Besides, the degree of phosphorylation of histone H3 of the exogenous nucleosomes added to the extract was essentially the same as that of the endogenous nucleosomes of the remodeled sperm nuclei (Figure 5A). Thus, the presence of nucleosomes with a phosphorylated H3 tail exhibited the same inhibition capacity as nucleosome samples devoid of a H3 tail, demonstrating that the tail of histone H3, whether intact or phosphorylated, does not play an important role in chromosome condensation. This was further confirmed by experiments with particles containing the four histones full length but with H3 mutated either at serine 10 or at serine 28 (the two sites of phosphorylation of H3 during cell division): the inhibition with both ΣH(S10A) and ΣH(S28A) reconstitutes was observed with the same amount (2 pmol) as that of phosphorylated native nucleosomes (Figure 5). Figure 5.The mutation of the phosphorylable serines of histone H3 tail does not interfere with the nucleosome inhibition effect on chromosome assembly. (A) The degree of phosphorylation of exogenous nucleosomes is the same as that of the nucleosomes of the remodeled sperm nuclei. Identical amounts of bulk nucleosomes and sperm nuclei were added to equal volumes of extract and, after incubation for 30 min at 22°C, the samples were run on an 18% SDS–PAGE gel. The phosphorylation of histone H3 was visualized by western blotting. The results of two independent experiments are shown. (B) Effect on the mutation of the phosphorylable serines of histone H3 on the nucleosome inhibition efficiency. Chromosome assembly was carried out in the presence of nucleosomes reconstituted either with histone H3 mutated at serine 10, ΣH(S10A), or with H3 mutated at serine 28, ΣH(S28A), and the three remaining non-mutated full-length histones. The experiments as well as the visualization of the formed structures were as in Figure 3. Download figure Download PowerPoint Effect of competitor nucleosomes on the kinetics of chromosome assembly Above, we have demonstrated that some of the reconstituted nucleosomes did not affect the final compact state of mitotic chromosomes. Nonetheless, a possibility exists that these particles could be able to interfere with the time course of chromosome assembly. To check this hypothesis, the kinetics of chromosome condensation in the presence of such different reconstitutes was followed. As seen in Figure 6, all reconstitutes were able to slightly delay the time course of chromosome formation. It should be pointed out that even the tailless nucleosomes were able to induce the same delay in the kinetics of chromosome condensation. Figure 6.Chromosome assembly kinetics is delayed in the presence of competitor nucleosomes. Chomosome assembly was carried out under standard conditions (Figure 3) in the absence (A) or the presence (B) of different types of reconstituted nucleosomes. At the times indicated an aliquot of the reaction was removed, fixed and stained with Hoechst 33258. Bar, 5 μm. Download figure Download PowerPoint Phosphorylation of histone H3 correlates with the initial stages of sperm nucleus decondensation in Xenopus egg extracts We have recently shown that the assembly of chromosomes in the Xenopus extract is accompanied by phosphorylation of histone H3 at serine 10 (de la Barre et al., 2000). However, it is not known whether this histone H3 modification correlates with chromosome condensation in the extract. To address this question the kinetics of chromosome assembly was followed and histone H3 phosphorylation was visualized by both immunofluorescence and western blotting (Figure 7). The decondensation of sperm nuclei was accompanied by histone H3 phosphorylation. Importantly, upon completion of the decondensation (at ∼10 min after the initial incubation), a saturation of the histone H3 phosphorylation was recorded (Figure 7B). Therefore, in contrast to cells in culture, a straightforward correlation between sperm decondensation and phosphorylation of histone H3 at serine 10 is observed. Figure 7.Decondensation of sperm nuclei in Xenopus egg extract is accompanied by phosphorylation of histone H3 at serine 10. (A) Chromosomes were assembled under standard conditions in mitotic egg extract and aliquots were removed and fixed at the times indicated. Chromosomal DNA was stained with Hoechst 33258. Phosphorylation of histone H3 was visualized by indirect immunofluorescence using an antibody against phosphorylated histone H3 at serine 10. Bar, 5 μm. (B) Immunoblotting analysis of the kinetics of histone H3 phosphorylation during chromosome assembly. Sperm nuclei were incubated in the extract for the times indicated and the chromosome intermediates were pelleted by centrifugation on a bench-top centrifuge. The proteins from the pellet were separated on a 15% SDS–PAGE gel and after transfer the phosphorylated histone H3 was detected using anti-phosphorylated histone H3 antibody. Download figure Download PowerPoint Discussion Chromosome assembly in Xenopus egg extracts was used to assess the role of each individual N-terminal histone tail in chromosome condensation. Initially, tailless nucleosome particles were prepared by digestion of native nucleosomes either with trypsin or with clostripain. Both types of tailless nucleosomes lost their ability to inhibit chromosome assembly, confirming that the N-histone termini play an essential role in chromosome condensation (de la Barre et al., 2000). Effect of the individual histone tails on chromosome assembly This question was addressed by using chimeric nucleosomes reconstituted with full-length and tailless histones in different combinations. The (H2B) particle, containing intact H2B and the three other tailless histones, was the only one able to inhibit chromosome condensation, identifying the N-terminus of this histone as a main player in this process. This was further confirmed by an experiment with the G2B nucleosome (reconstituted with the globular domain of H2B and the remaining histones intact), which was unable to interfere with chromosome assembly. Interestingly, the inhibition with H2B reconstitute was observed at 6 pmol, an amount three times higher than the inhibitory amount of native or reconstituted with intact histone ΣH particles. Furthermore, (H2A/H2B) chimeric nucleosomes containing full-length H2A and H2B were found to inhibit chromosome formation at 3.5 pmol. Since the H2A N-terminus alone did not have the ability to interfere with chromosome assembly, these data showed that its presence in the nucleosome containing intact H2B increased the inhibitory capacity of the particle. The nucleosomes GH3 (intact H2A, H2B and H4, and tailless H3) impeded chromosome condensation at 2.5 pmol, i.e. almost as efficiently as the ΣH structures, further demo