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Originally published In Press as doi:10.1074/jbc.M601975200 on May 17, 2006

J. Biol. Chem., Vol. 281, Issue 29, 20036-20044, July 21, 2006
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H2A.Z Stabilizes Chromatin in a Way That Is Dependent on Core Histone Acetylation*

Anita A. Thambirajah, Deanna Dryhurst, Toyotaka Ishibashi, Andra Li, Allison H. Maffey, and Juan Ausió1

From the Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8W 3P6, Canada

Received for publication, March 1, 2006 , and in revised form, May 17, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The functional and structural chromatin roles of H2A.Z are still controversial. This work represents a further attempt to resolve the current functional and structural dichotomy by characterizing chromatin structures containing native H2A.Z. We have analyzed the role of this variant in mediating the stability of the histone octamer in solution using gel-filtration chromatography at different pH. It was found that decreasing the pH from neutral to acidic conditions destabilized the histone complex. Furthermore, it was shown that the H2A.Z-H2B dimer had a reduced stability. Sedimentation velocity analysis of nucleosome core particles (NCPs) reconstituted from native H2A.Z-containing octamers indicated that these particles exhibit a very similar behavior to that of native NCPs consisting of canonical H2A. Sucrose gradient fractionation of native NCPs under different ionic strengths indicated that H2A.Z had a subtle tendency to fractionate with more stabilized populations. An extensive analysis of the salt-dependent dissociation of histones from hydroxyapatite-adsorbed chromatin revealed that, whereas H2A.Z co-elutes with H3–H4, hyperacetylation of histones (by treatment of chicken MSB cells with sodium butyrate) resulted in a significant fraction of this variant eluting with the canonical H2A. These studies also showed that the late elution of this variant (correlated to enhanced binding stability) was independent of the chromatin size and of the presence or absence of linker histones.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The histone H2A family possesses the largest number of variants of all the core histones. Most of the structural variability resides in the N- and C-terminal unstructured domains (tails) of the H2A molecule, and it confers a defined chromatin function to the members of this highly heterogeneous family (1). One such member that has received a lot of attention in recent years is H2A.Z.

Histone H2A.Z is a replication-independent histone replacement variant that is synthesized from an intron-containing, polyadenylated mRNA transcript (2). In contrast to other histone variants, H2A.Z has been shown to be indispensable for viability in protozoan (3) and metazoan organisms (4, 5). In particular, the C-terminal region has been shown to be critical for survival in Drosophila (6). Although many attempts have been made to connect the structural variability of the molecule to its functional specialization, the one or more mechanisms involved still remain elusive.

Indeed, both the functional and structural roles of H2A.Z have been and still are quite controversial (7). At the functional level, results obtained in yeast show that H2A.Z can be implicated in transcriptional activation (8) and in silencing at the HMR locus (9). It has been shown to mark the 5'-ends of active and inactive genes within euchromatin (10), and it has been involved in preventing the spreading of ectopic heterochromatin (11). In Drosophila, H2A.Z is localized in transcribed regions of euchromatin and in non-transcribed heterochromatin domains (12). In mammals, H2A.Z has been described to be prevalent at pericentric heterochromatin (13) and interact with HP1{alpha}2 (14). To cite an example of the very recent functional (15) debate, using cross-linked chromatin immunoprecipitation and microarray analysis, H2A.Z has been shown to globally localize to the promoters of inactive genes in yeast. However, by using native, non-cross-linked chromatin immunoprecipitation in different chicken cell lines, it has been shown that H2A.Z is present within active genes (16).

These contrasting putative roles for H2A.Z have been mirrored to some extent by earlier structural studies of reconstituted NCP consisting of this histone variant that independently found it to have stabilizing and destabilizing roles. When the crystal structure of an H2A.Z-containing NCP was first published (17), it was pointed out that even though its overall structure was similar to that of the NCP consisting of canonical core histones (18), "distinct localized changes result in the subtle destabilization of the interaction between the (H2A.Z-H2B) dimer and the (H3–H4) (2) tetramer" (17). A subsequent study in solution, using nucleosomes reconstituted with recombinant H2A.Z, provided initial experimental support to this notion (19). However, a more recent biophysical characterization (20), published by the same laboratory that produced the crystallographic data, indicated that H2A.Z produces a subtle but noticeable stabilization of the histone octamer within the NCP.

To shed some additional light onto the current structural controversy, we decided to purify native H2A.Z from chicken erythrocytes to reconstitute several of the integral components (histone octamer and H2A.Z-H2B dimers) of the NCP and the NCPs themselves. Although most of the results reported here support the current data on the stabilizing role of H2A.Z within the NCP, preliminary HAP data using highly acetylated chromatin suggests that this effect can be abolished by histone acetylation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Tissues—Chicken MSB Marek virus-transformed cells (21) were a generous gift from Dr. Vaughn Jackson. Cells were grown and butyrate-treated as described elsewhere (22). Chicken blood and chicken liver were processed as described previously (23, 24).

Native H2A.Z Purification—Native H2A.Z was purified from chicken erythrocyte histones by several rounds of high-performance liquid chromatography, which combined preparative 1 x 25 cm (300 Å and 5 µm) C4 and analytical 0.46 x 25 cm (300 Å and 5 µm) C18 columns from Vydac. Elution was with a gradient from buffer A (0.1% trifluoroacetic acid in water) to buffer B (100% acetonitrile) (25).

Chromatin Preparation—Different chromatin fractions from the different cell sources were obtained by micrococcal nuclease digestion according to protocols previously described (23, 24). In brief, nuclei from different tissues were digested with micrococcal nuclease and centrifuged to yield an SI supernatant (containing 10–20% of chromatin) and a P1 pellet. The nuclear pellet thus obtained was resuspended and lysed in 0.25 mM EDTA (pH 7.5) to produce, after centrifugation, an SE supernatant and a PE pellet (24). Additionally, a chromatin fractionation procedure modified from a method described earlier by Olins and Olins was also used (23, 26). Accordingly, nuclei prepared as above were extensively digested with micrococcal nuclease to produce, after centrifugation, an SI (containing >30% of total chromatin) and P1 pellet. After extensive dialysis of the SI supernatant against 0.1 M KCl in Tris buffer (to precipitate H1/5-containing chromatin complexes), the cloudy suspension was subsequently centrifuged to produce an S2 supernatant and a P2 pellet. Supernatant S2 consisted mainly of linker histone-depleted nucleosome core particles (of ~146-bp DNA) and P2 contained linker-histone-containing chromatosomes (of ~168-bp DNA) in addition to some higher nucleosome oligomers that become negligible as the time of the initial micrococcal nuclease digestion increases (23). Chicken tissues (blood and liver) were used as a source of native chromatin. Chromatin consisting of hyperacetylated histones was obtained from chicken MSB Marek virus-transformed cells as described elsewhere (22).

Nucleosomes and Histone Octamers—NCPs were obtained from chicken erythrocytes and prepared as described elsewhere (24). Histone octamers were usually obtained from NCPs (and occasionally from linker histone-stripped chromatin) adsorbed onto HAP and eluted with 2.5 M NaCl in 20 mM potassium phosphate buffer (pH 6.8) (22). Nucleosomes and histone octamers consisting of native H2A.Z were obtained by reconstitution using high-performance liquid chromatography-purified native chicken H2A.Z and the corresponding complement of chicken core histones (H2B and H3–H4) according to a previous study (25). In some instances, the reconstituted histone octamers were purified by gel filtration on a 120-x 1.5-cm Sephacryl S-300 HR column eluted with 2 M NaCl, 20 mM Tris-HCl (pH 7.5) buffer (see below) prior to their use in NCP reconstitution.

AUT/SDS-Native PAGE and Agarose Electrophoresis—Electrophoretic analyses of histones (AUT- and SDS-PAGE) and of DNA (4.5% native PAGE and 1% agarose gels) were prepared and run as described elsewhere (19, 27).

Western Blot Analysis—Western blot analysis was carried out as described in a previous study (23). An H2A.Z antibody elicited against the acetylated N-terminal region of the molecule was a kind gift of Dr. Colyn Crane-Robinson and was used under the conditions outlined before (16).

Analysis of the pH-dependent Stability of the Histone Octamer—Nucleosome octamers (~2 mg/ml or higher) were dialyzed against 2 M NaCl at different pH at 4 °C. After dialysis, they were fractionated by gel filtration on a 120-x 1.5-cm Sephacryl S-300 HR column and diluted with the corresponding 2 M NaCl buffer at 4–5 ml/h at 4 °C. The buffers for the different pHs (5.5, 6.0, and 7.5) used were prepared using sodium phosphate and or Tris-HCl (28).

Circular Dichroism—Jasco J-720 and Jasco J-810 spectropolarimeters were used for CD. The latter was equipped with a circulating water bath and a temperature controller. For the melting profiles, spectra were recorded at 222 nm with a spectral bandwidth of 1 nm and response time of 1 s. The temperature was increased at a rate of 1 °C per minute. The buffer used for the melting studies was 0.1 mM EDTA, 10 mM HEPES (pH 7.5) in different NaCl concentrations (29). Otherwise, histone spectra were recorded at 20 °C in 100 mM NaCl, 10 mM Tris-HCl (pH 7.5) buffer as described previously (30). An extinction coefficient of A230 = 3.1 cm2 mg–1 was used for histone H2A (31) and H2A.Z.

Sedimentation Analysis—Sedimentation velocity analysis of NCPs at different ionic strengths was carried out in a Beckman XL-I analytical ultracentrifuge as described previously (30). Sedimentation analysis of NCPs at different salt concentrations using sucrose gradients was carried out as described previously (23).

Scanning Densitometry Analysis Methodology—Scanning densitometry was used to quantify the relative distribution of H2A.Z throughout each of the sucrose gradient-fractionated mononucleosome analyses. The ChemiImager 4000 scanner was used with the AlphaEaseTM Version 3.3d software (Alpha Innotech Corp.). Density measurements were made in triplicate of H2A.Z, H2A, and the background within each lane. The density scale was adjusted such that black was assigned a value of 255 and white, 0. The background readings were subtracted from the values for H2A.Z and H2A, and H2A.Z values were normalized to H2A within each respective lane. Normalized H2A.Z/H2A values were plotted using GraphPad Prism (GraphPad Software, Inc.) with error bars indicating the standard mean ± S.E.

Hydroxyapatite Chromatography of Chromatin and Histones—Hydroxyapatite chromatography of chromatin was carried out as described elsewhere (32). Hydroxyapatite fractionation of H2A and H2A.Z, and H2A-H2B and H2A.Z-H2B dimers was carried out under the same conditions as the chromatin fractions, except as indicated in the figure legends. The H2A and H2B histones used in these experiments were obtained from chicken erythrocytes, and H2A.Z was obtained from bacterial expression. Histone H2A-H2B and H2A.Z-H2B dimers were reconstituted and purified by heparin fast-protein liquid chromatography as described previously (33) prior to their HAP fractionation.


Figure 1
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  		FIGURE 1.
Histone octamer dissociation under decreasing pH as assessed by gel filtration chromatography. Chicken erythrocyte histone octamers in 2 M NaCl at pH 5.5, 6.0, and 7.5 were loaded onto a Sephacryl S-300 HR gel-filtration column equilibrated and eluted with the corresponding pH buffers. The absorbance of the eluted fractions at 230 nm (A230) is plotted as a function of the fraction number. Shown underneath are the AUT-PAGE analyses of representative fractions collected from the different elutions. The gels are aligned so that the starting and last electrophoretic lanes shown underneath their corresponding elution profiles coincide with the very first and last fraction of each profile. For the elution profiles at pH 6.0 and 5.5 only the area of the gels corresponding to the H2A-H2A.Z region is shown. The boxes highlight the location of H2A.Z. In the gels shown in this figure, all loadings were normalized as to have approximately equal amounts of H2A in each lane. The gels were stained with Coomassie Blue.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The Histone H2A.Z-containing Octamer Is Less Stable at Low pH—The heterotypic histone core of the NCP consists of an octamer with a tri-partite organization in which a histone H3–H4 tetramer is flanked by two histone H2A-H2B dimers. In solution and at physiological ionic strength and above, the octamer exists in a multiple equilibrium that involves the H2A-H2B dimers, the (H3–H4)2 tetramers, the (H3–H4)2-(H2A-H2B) hexamer, and the octamers themselves (28). In the absence of DNA it is possible to shift the equilibrium toward a stable octamer by increasing the ionic strength of the medium to 2 M NaCl or higher (28). The stability of the histone core octamer in 2 M NaCl at different pH can provide useful information on the interactions between the H2A-H2B dimer and (H3–H4)2 tetramer components (28).

Following a slightly modified version of the method described in a previous study (28) we used gel-filtration chromatography to characterize the stability of the histone octamer at different pH (Fig. 1). Our results with the histone octamer consisting of canonical histone variants (major component of the native octamers used in this experiment) exhibited a very similar behavior to that described before (see Fig. 7a in Ref. 28). In contrast, under physiological pH (pH 7.5), H2A.Z was present in the fractions corresponding to stable octamers, whereas, upon a decrease in pH, its distribution was observed within dissociated fractions. At pH 5.5 most of the H2A.Z partitioned with the dissociated H2A-H2B dimers.

Histidine residues have been involved in the abrupt loss of stability when the pH drops from 6.5 to 5.5 due to the ionization of histidine (as the pK of the histidine imidazole ring is 6.0), which results in the loss of hydrogen bonding (28). In this regard, the crystallographic structure of the H2A.Z NCP (17) has shown that the loop L1 between helix {alpha}1 and {alpha}2 of the histone H2A.Z fold exhibits some significant changes between residues 38 and 44 (38SRTTSHG44) when compared with the equivalent region (36KGNYAE41) in H2A. These regions are at the interface between the two H2A molecules present in the histone octamer (34), and hence the structural differences between these two equivalent domains would presumably prevent the existence of core histone octamer H2A.Z/H2A hybrids (17). Very interestingly, a highly conserved histidine residue (His43) is present at this interface in H2A.Z and another nearby (His35), which is also not present in canonical H2A. In contrast, no differences in histidine amino acids exist at the interface between either H3/H2A or H4/H2A (34).

Histone H2A.Z Destabilizes the H2B-H2A.Z Dimer—The results from the previous section show that, under neutral pH conditions, the native histone H2A.Z-containing octamer is very stable. However, a recent report using recombinant H2A.Z and H2B histones has shown that the reconstituted histone heterodimers are unstable (35). Therefore, before proceeding with the reconstitution of H2A.Z-containing NCPs, we decided next to analyze the stability of the native H2A.Z-H2B dimer. To that end the thermal stability of the dimer at different ionic strengths was analyzed using CD spectroscopy as in previous studies (29, 36).

Fig. 2A shows the CD spectra of H2A.Z in comparison to H2A. The two spectra are quite similar with an estimated {alpha}-helical content of 21% for H2A and 24% for H2A.Z as determined by the ellipticity at 222 nm (37). These values are lower than those expected from the crystallographic data (49%) due to the lack of interaction with H2B, which contributes in part to the stabilization of the secondary structure of the histone fold (32, 36). Nevertheless, the CD spectra shown in this figure provide a strong indication that the purification process did not alter the ability of H2A.Z to fold into a native (monomeric) conformation.

As a preamble to the salt-dependent thermal stability characterization of the H2A.Z-H2B complex interaction, we performed first a similar analysis for the native H2A-H2B histone heterodimer and compared our results with those previously published (36, 38). As shown in Fig. 2B, our results are in good agreement with this earlier data. However, attempts to produce the same data for H2A.Z-H2B completely failed due to the unfolded state adopted by the H2A.Z-H2B dimers under any of the buffer conditions (see Fig. 2C) required to perform this type of analysis. This experimental work was being carried out when a report was just published describing a similar finding (35). Our results hence come in support of this publication, corroborating the unfolded state of the H2A.Z-H2B dimer under physiological ionic and temperature conditions. It is not clear whether this is the result of compositional amino acid differences at the extensive interface between H2A.Z and H2B (34, 35). Alternatively a specialized metazoan H2B variant that specifically interacts with H2A.Z, such as that recently described in Trypanosome (39), could help to maintain the stability of the heterodimer. Whatever the situation, the molecular mechanisms involved in the stability of the H2A.Z-H2B complex remain yet to be elucidated.

H2A.Z-containing NCPs Exhibit a Very Similar Salt-dependent Conformation and Slightly Stabilize the Particle—Our ability to purify native H2A.Z allowed us to reconstitute native H2A.Z-containing NCPs as shown in Fig. 3A. It was thus possible to characterize the ionic strength dependence of the sedimentation coefficient of these particles and compare it to that of the canonical NCP (Fig. 3B). This approach has proven to be very useful in assessing the stability and dynamics of the NCP in solution (40). The results shown in Fig. 3B indicate that native H2A.Z NCPs behave very similarly to canonical NCPs with the sedimentation coefficients of the former being slightly higher than those of the latter at any given salt concentration. This suggests a slightly more compact conformation of the H2A.Z NCP. A subtle increase in compaction is also supported by the slight increase in electrophoretic mobility of these particles in native PAGE (compare lanes 1 and 2 in Fig. 3A, panel 3).


Figure 2
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  		FIGURE 2.
CD analysis of H2A, H2A.Z, and the salt-dependent thermal stability of their dimers with H2B. A, CD spectra of H2A and H2A.Z. The residual molar ellipticity [{theta}] is plotted as a function of the wavelength. The spectra were recorded at 20 °C in 0.1 M NaCl, 10 mM Tris-HCl (pH 7.5). B, salt concentration dependence of the melting temperature (Tm) of the H2A-H2B dimer in solution. The gray squares correspond to the dependence determined previously (36), and the black diamonds are the values experimentally determined in the present report. C, change of the residual molar ellipticity of the H2A-H2B and H2A.Z-H2B dimers as a function of temperature. The experiments in B and C were carried out in 0.1 M NaCl, 0.1 mM EDTA, 10 mM HEPES (pH 7.5) as in previous studies (29, 36). Please notice that the starting ellipticity at 222 nm in C is lower than that of H2A.Z alone in A. The difference is due to the different buffers used: Tris-HCl (panel A) versus HEPES in B and C.

 
To better characterize the salt-dependent stability of H2A.Z NCPs in a native setting, an electrophoretic analysis of native NCPs fractionated by sucrose gradients under different NaCl concentrations was performed as previously described by our laboratory (Fig. 3C) (23). When the PAGE loadings were normalized for approximately equal amounts of H2A, the fractions from the upper part of the gradient (fractions 19–21) in 0.6 M and 0.9 M NaCl exhibited a small but noticeable depletion in H2A.Z. The range between 0.6 M and 0.9 M NaCl corresponds to the range of ionic strength that causes dissociation of the histone H2A-H2B dimers from the NCP to begin. At 0.9 M and 1.2 M NaCl, there was a clear decrease in H2A.Z at lower sucrose concentrations with a concomitant increase in the relative amount of H2A.Z in earlier, more stable mononucleosome populations. Hence, the slight depletion of H2A.Z in the fractions from the upper part of the 0.6–1.2 M sucrose gradients indicated that native H2A.Z-containing NCPs were slightly stabilized by the presence of this histone variant in agreement with earlier results (20) and in disagreement with our own previously published data using NCPs reconstituted with recombinant H2A.Z (19).


Figure 3
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  		FIGURE 3.
Sedimentation analysis of the H2A.Z-containing NCPs. A, electrophoretic analysis of NCP reconstituted with native H2A.Z. Panel 1, SDS-PAGE (lane 1); panel 2, AUT-PAGE (lane 1); panel 3, native (4.5%) PAGE of native H2A-containing NCP (lane 1) and reconstituted H2A.Z-containing NCP (lane 2). CM, chicken erythrocyte histone marker. M, CfoI-cut pBR322 marker. B, NaCl dependence of the sedimentation coefficient (s20,w) of native (open circles) and reconstituted H2A.Z-containing NCP (black circles). C, native chicken erythrocyte NCPs at different NaCl concentrations in 10 mM Tris-HCl (pH 7.5), 0.1 mM EDTA buffer were loaded and fractionated on a 5–20% sucrose gradient made in the same corresponding buffers. The absorbance at 260 nm was measured of fractions collected from the gradients and plotted as a function of the fraction number. AUT-PAGE analyses of the indicated fractions is shown below. Only the area of the gels corresponding to the H2A-H2A.Z region is shown. As in Fig. 1, the gels shown in this figure were normalized so as to contain approximately equal amounts of H2A in each lane. The bar plots show the normalized H2A.Z/H2A amounts measured by scanning densitometry of mononucleosomes fractionated by sucrose gradients under increasing [NaCl]. Error bars indicate the mean ± S.E. At 0.9 M and 1.2 M NaCl, where H2A-H2B dimer interactions within the mononucleosome are disrupted, H2A.Z distribution strongly favors more stable populations.

 
Histone H2A.Z Chromatin Distribution Is Not Affected by the Presence or Absence of Linker Histones—Previous research on the genomic distribution of H2A.Z has been controversial, and this histone variant has been ascribed to either transcriptionally active domains (16, 41) or repressed domains (14, 15) in both yeast and higher eukaryotes. In the former, its location has also been assigned to the boundary between heterochromatin and euchromatin domains and acts to synergistically prevent the spread of the former into the latter (11).

Digestion of chromatin within the intact nuclei with micrococcal nuclease proceeds first through the linker histone-deficient euchromatin domains. A time-course analysis of an extensive digestion with this enzyme can be used to fractionate linker histone-containing nucleosomes from NCPs (26). This can be used to infer cohabitation of histone variants with histone H1 or H5 (23).


Figure 4
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  		FIGURE 4.
Salt (0.1 M KCl) fractionation of chromatin particles upon digestion of chicken erythrocyte nuclei with micrococcal nuclease. A, outline of the fractionation procedure, which is based upon the method described by Olins (26). Chicken erythrocyte nuclei were digested with micrococcal nuclease and, after stopping the reaction by the addition of EDTA, were centrifuged to yield a nuclease-resistant heterochromatin pellet P1 and an SI supernatant. Upon dialysis of SI against a 0.1 M KCl-containing buffer, further centrifugation produced an NCP-containing supernatant (S2), which was depleted of linker histones and a pellet (P2), which consisted of chromatosomes and nucleosome oligomers containing a full complement of linker histones. B, AUT-PAGE analysis of P1, P2, and S2 fractions taken over a time course of micrococcal nuclease digestion. H2A.Z is present in all fractions indicating a widespread chromatin distribution, which is not dependent upon the presence of linker histones.

 
When a similar approach to that described above was used (Fig. 4), H2A.Z was found to be equally distributed in linker histone-containing (Fig. 4, fraction P2) and linker histone-depleted (Fig. 4, fraction S2) nucleosome fractions as well as in the highly nuclease-resistant P1 fraction consisting primarily of tightly folded heterochromatin. This result suggests that histone H2A.Z does not exhibit any selective partitioning between chromatin regions with different linker histone content. This is in agreement with the data obtained in Drosophila where it was shown that H2A.Z is found widely (albeit non-randomly) distributed across the euchromatin and heterochromatin regions of the polytene chromosomes (12).

Non-acetylated H2A.Z Binds More Tightly to Chromatin—One of the most striking structural differences of H2A.Z described to date was the early observation by the Davie's laboratory that showed that this variant eluted from HAP-adsorbed chromatin at much higher NaCl concentrations than its canonical H2A counterpart (42). This suggested that either this variant binds more tightly to the nucleosomal DNA or that it interacts more strongly with H3–H4 in the histone core within the same NCP.

We therefore decided to explore this observation further and in more detail. The results of this approach are shown in Fig. 5. Fig. 5A shows a typical histone elution profile from an SE chromatin fraction (see "Experimental Procedures") from chicken liver. The electrophoretic analysis of some of the fractions along the profile is shown in Fig. 5B (panel 1). The results are in agreement with the data of a previous study (42) and show that most of H2A.Z elutes very closely to histones H3–H4. To determine whether this delayed elution was a result of tissue specificity, the presence/absence of linker histones, and/or the nature of the starting chromatin fraction (size distribution of the micrococcal nuclease fragments), several similar columns were carried out as shown in Fig. 5 (B and C). A delayed elution of H2A.Z was observed in every single instance.

We checked next whether the late elution of this variant could be the result of a differential interaction of H2A.Z with the HAP resin as histones are well known to interact differently with HAP under certain ionic conditions (43). To this end, equimolar mixtures of H2A/H2A.Z as well as H2A-H2B and H2A.Z-H2B dimers were loaded onto two HAP columns of identical characteristics to those used in Fig. 5 (A and B). When a salt gradient from 0–2 M NaCl was used with the H2A/H2A.Z mixture, H2A eluted first and H2A.Z trailed behind (see Fig. 5D). However, when the H2A/H2A.Z mixture and H2A-H2B and H2A.Z-H2B dimers were eluted with a 1–2 M NaCl gradient (because H2A-H2B did not start eluting from HAP-adsorbed chromatin until ~1.2 M NaCl, see Fig. 5A), then only a single peak (see peak 4 in Fig. 5D and peaks 1 and 2 in Fig. 5E) was observed in every instance. The result shows that, in 20 mM phosphate (pH 6.7) buffer and above 1 M NaCl, the differential binding affinities of H2A and H2A.Z for the resin (alone or in complex with H2B) were abolished and hence this had no bearing on the delayed effect observed in Fig. 5B.

We finally decided to analyze the role, if any, that histone acetylation might play in H2A.Z elution (Fig. 5F) using chicken erythroleukemic cells that had been grown in suspension in the presence of 5 mM sodium butyrate (a well known histone deacetylase inhibitor). The elution of H2A.Z in this instance was monitored by AUT-PAGE (Fig. 5F, panel 1) and by using a recently developed antibody against acetylated H2A.Z (16). Much to our surprise, acetylation resulted in H2A.Z eluting in a very similar fashion to the canonical H2A counterpart (Fig. 5F).

Hyperacetylated histones have been reported in some instances, such as H4, to elute from HAP-adsorbed chromatin at lower NaCl concentrations than their non-acetylated counterparts; a fact that has been taken as an indication that hyperacetylation destabilizes the nucleosome (44). Indeed, Fig. 5F (panel 1) shows that the tri- and tetra-acetylated forms of H4 eluted earlier than the less acetylated counterparts. However, the shift in the elution experienced by acetylated H2A.Z was by far much more pronounced (Fig. 5F, panel 3). These results show that acetylation of core histones and/or acetylation of H2A.Z itself can revert the chromatin-enhanced stability imparted by this variant.


Figure 5
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  		FIGURE 5.
Salt-dependent dissociation of histones from HAP-adsorbed chromatin complexes. A, representative elution profile of histones from EDTA-solubilized chicken liver chromatin (fraction SE) eluted from a 1.5-x 15-cm HAP column. The column had been equilibrated in 20 mM potassium phosphate buffer (pH 6.8), and the elution was carried out with a 0–2.5 M NaCl gradient in the same buffer. The black bar indicates the fractions corresponding to H2A.Z elution, and the shaded bar corresponds to the same fractions when chromatin from butyrate-treated chicken MSB cells was eluted under similar conditions. B, AUT-PAGE analyses of the fractions corresponding to the H1/5, H2A/B, and H3/4 eluting regions shown in A for HAP columns loaded with different starting samples: 1, chicken liver SE fraction; 2, chicken erythrocyte SE fraction; 3, linker histone-depleted chicken erythrocyte SE fraction; 4, chicken erythrocyte SI fraction. Only the upper region of the gel is shown in sections 2–4. C, AUT-PAGE analysis of the histones (left) and agarose gel electrophoresis analysis of the DNA (right) from different chicken erythrocyte fractions generated by micrococcal nuclease digestion. SI and SE are the samples used for the analysis shown in A and B. CM, chicken erythrocyte marker; P, insoluble pellet; M1, {lambda} DNA BstEII-digested marker. M2, 123-bp ladder marker. D, HAP chromatography of an equimolar mixture of H2A/H2A.Z eluted from a column with identical characteristics to those used in A. Elution with a 0–2 M NaCl gradient resulted in three regions: 1, 2, and 3. Elution with a 1–2 M NaCl gradient resulted in a single peak (peak 4). The inset shows an SDS-PAGE of regions 1–3 and peak 4. CM, chicken erythrocyte marker. The vertical arrows indicate the loading points of the starting sample for the two different salt gradients. E, same as in D, but using reconstituted H2A-H2B and H2A.Z-H2B dimers (see "Experimental Procedures"). In both instances, elution of either one of these dimers with a 1–2 M NaCl gradient resulted in a single peak consisting of the starting sample loaded onto the column (see lanes 1 and 2 in the inset). F, electrophoretic analysis of the HAP elution profile of butyrate-treated chicken MSB cells. 1, AUT-PAGE analysis (the arrows and numbers indicate the number of acetyl groups); 2, SDS-PAGE analysis; 3, Western blot using an anti-acetylated H2A.Z antibody. The boxes in B and E highlight the regions containing H2A.Z.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The results of the preceding sections show that H2A-Z-containing chromatin complexes exhibit well defined contrasting structural differences. On one hand, some subtle but noticeable differences were observed involving a pH-dependent destabilization of the histone octamer (Fig. 1). Alternatively, the H2A.Z-H2B dimer was found to exhibit an extremely low stability (Fig. 2). The less stable histone-histone interaction results are in contrast to the slightly increased salt-dependent stability of the NCP (Fig. 3) that is enhanced at the level of the chromatin fiber as demonstrated by the HAP elution results (Fig. 5).

The loss of stability of the H2A.Z-containing octamers (at pH below 7) and dimers probably reflect changes in interactions at the different interfaces between this histone variant and the complementary core histones, which may affect the highly dynamic nature of the nucleosome. Such histone instability, particularly as it concerns the H2A.Z-H2B dimer, has been proposed to be responsible for the shift of the equilibrium involving the physiologically relevant sequential dissociation of H2A-H2B dimers from the NCP (45) in a way that contributes to enhance the overall stability of the nucleoprotein complex (35). It is proposed in the same report that the highly charged nature of the H2A-H2B tails can also contribute to the stability of this histone dimer (33, 46) so that partial neutralization of the charge within these regions (as per acetylation) could increase the dimer stability and hence favor its dissociation from the NCP (35). However, even if this was indeed the case, the molecular details of the mechanism involved remain to be established and additional components are likely involved.

Regarding the chromatin stabilization imparted by H2A.Z, two important additional structural considerations need to be made here. Firstly, the N-terminal tail of histone H2A has been shown to bind to two defined positions centered at 25–35 nucleotides from the ends of the DNA in the 146-bp NCP (47, 48). Secondly, these nucleotide stretches are within the region of DNA whose interaction with the octamer in the NCP is loosened as a result of histone acetylation (49, 50).

That the flanking ends of DNA at the entry and exit site of the NCP are potentially involved in chromatin stabilization is supported by the lower salt-dependent stability of the NCP (Fig. 3) when compared with that of full chromatin (Fig. 5). Also, H2A.Z dissociated significantly at lower salts from the SI fraction (Fig. 5B, panel 4), which consisted predominantly of mononucleosomes (Fig. 5C, SI). Hence an acetylation-mediated destabilized interaction in these regions may also contribute to the weakening of the H2A.Z interaction with DNA in the chromatin complex. An implication of this is that the enhanced interaction of H2A.Z with DNA is probably mediated by the rather unique N-terminal region of the H2A.Z molecule, and hence it can also be affected by its own acetylation. Interestingly, in the amino acid swapping mutants between H2A.Z and H2A.1 carried out in Drosophila (6), exchange of the first 12 N-terminal amino acids resulted in the highest lethality after swapping of the indispensable region corresponding to the C-terminal docking domain (amino acids 81–119) (17) of these two molecules (6). Acetylation of H2A.Z mainly affects Lys4, Lys7, and Lys11 (16). The need for acetylation of at least one lysine within the N-terminal tail of H2A.Z has been shown to be essential for survival in Tetrahymena (51, 52). The molecular details involved, although not completely understood, have been proposed to operate through the modification of a charge patch that reduces the charge of the tail domain (51, 52). Our current results do not allow us to determine the extent to which the intrinsic acetylation of H2A.Z itself and/or that of the other core histones contribute to the loss of chromatin binding affinity of the molecule. Current work in our laboratory is being carried out to elucidate the molecular mechanisms involved.

The results presented in this report may have important functional implications. The observation that the binding affinity of histone H2A.Z to chromatin reverts to that of canonical H2A upon histone acetylation is quite interesting and could help explain the functional duality ascribed to this histone variant (7). Indeed, histone H2A.Z has been ascribed to active (8, 10, 16, 41, 53–55), inactive (9, 10, 13, 14, 56), and boundary (11) transcriptional domains. It has been shown that a chromatin-remodeling complex consisting of the Swi2/Snf2-related ATPase Swr1p has been shown to deposit H2A.Z into yeast euchromatin (57). This SWR1-Com complex was shown to contain NuA4, which is able to acetylate H2A/H4 (58). It is thus possible to imagine that acetylation is important for the initial deposition (replacement of canonical H2A) of H2A.Z into chromatin and for its further exchange by H2A/H2B during transcriptional elongation (55), when histones are well known to be highly acetylated (59, 60). This would suggest that acetylation of H2A.Z itself and/or of its proximal nucleosome histone environment would be a hallmark for its dynamic involvement in active regions versus the unacetylated form, which could be present in the passive domains of active/poised genes or in the heterochromatin regions where its chromatin-stabilizing effect may be further enhanced by the interaction with other non-histone proteins such as heterochromatin protein 1 (HP1) (14).


   FOOTNOTES
 
* This work was supported by Canadian Institutes of Health Research Grant MOP-57718 (to J. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Biochemistry and Microbiology, University of Victoria, P. O. Box 3055, Petch Bldg. 220, Victoria, British Columbia V8W 3P6, Canada. Tel.: 250-721-8863; Fax: 250-721-8855; E-mail: jausio{at}uvic.ca.

2 The abbreviations used are: HP1{alpha}, heterochromatin protein 1{alpha}; NCP, nucleosome core particle; AUT, acetic acid-urea-Triton X-100; HAP, hydroxyapatite. Back


   ACKNOWLEDGMENTS
 
The antibody against acetylated H2A.Z was a kind gift from the laboratory of Colyn Crane-Robinson, University of Portsmouth, United Kingdom. We are very thankful to Dr. D. Wade Abbott for kindly providing us with the chromatin fractions that were used to produce the results shown in Fig. 4B. We are also very indebted to Dr. José Maria Eirín López and to Ron Finn for their skillful assistance in the preparation of the figures and for their helpful suggestions.



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