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Previous Article | Next Article 
Molecular and Cellular Biology, July 2006, p. 5325-5335, Vol. 26, No. 14
0270-7306/06/$08.00+0 doi:10.1128/MCB.00584-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Undine Mechold,1,,
Franck Brouillard,3
Cristele Gilbert,1
Philippe Leclerc,2 and
Vasily Ogryzko1*
Oncogenese, Differenciation et Transduction du Signal, CNRS UPR 9079, Institut Andre Lwoff, Batiment B, 1er Etage, 7 rue Guy Moquet, 94800 Villejuif, France,1 ,2 Proteomic Core Facilities, Faculté demédecine René Descartes, 156, rue de Vaugirard, Paris F-75015, France,3 Service de Microscopie Confocale, INSERM IFR93, 80, rue du Général Leclerc, F-94276 Bicêtre CEDEX, France4
Received 4 April 2006/ Accepted 27 April 2006
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ABSTRACT |
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INTRODUCTION |
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An important aspect of the histone code is its postulated epigenetic role. Currently, the term "epigenetic" is used to describe heritable or stable changes of phenotype that do not depend on changes in primary DNA sequence (34). Chromatin is considered a principal carrier of epigenetic information (9). However, unlike DNA replication, which relies on the complementary base-base recognition (44), the postulated epigenetic templating mechanisms depend on preferential recruitment of the enzymes that deposit particular epigenetic marks (DNA methylation, histone acetylation, or methylation) on chromatin to sites containing the same mark (13, 14, 28, 42).
The replacement histone variants add another dimension to the field of histone code studies (16, 33, 35, 43). Unlike their canonical counterparts, the replacement histones are expressed in a replication-independent manner and employ specialized machineries for their deposition into chromatin (21, 24, 40). They can be associated with active or silenced chromatin and are involved in the regulation of gene expression and the organization of chromatin structure (16, 17, 22). Recent studies also suggest a role for replacement histones in epigenetic templating. One example is provided by neocentromeres in higher eukaryotes. Their formation and propagation do not require a particular DNA sequence (1, 5). CenpA, the specialized version of H3 histone, replaces H3 in the centromeric nucleosomes and likely serves as an epigenetic mark determining the maintenance of the centromere-specific nucleoprotein complex independent of DNA sequence (1, 5).
Compared to that for the H3-like replacement histones, an epigenetic role for the H2A-like or H2B-like replacement histones remains relatively unexplored. The H2A/H2B pair differs from the H3/H4 pair of the core histones in a number of important aspects, such as in the deposition mechanisms (37) and in having a more peripheral location in the nucleosome (20) and a less stable association with DNA (15).
In this study, we focus our attention on the mammalian histone H2AZ. This replacement histone variant is essential for survival and is highly conserved through the eukaryotic domain. Recent studies on Saccharomyces cerevisiae demonstrate that H2AZ is nonrandomly distributed throughout the genome of this organism, concentrating mostly in intergenic regions (12, 19, 31, 45). The mapping of the position of H2AZ with nucleosome-level resolution shows that H2AZ-containing nucleosomes are highly localized (12, 31, 45) and in many cases do not spread to more than two nucleosome positions (12, 31).
In this work, we use H2AZ as a model to develop a novel strategy to study the chromatin environment of a particular replacement histone variant in mammalian cells. Consistent with the data obtained with yeast, we show that neither H2AZ itself nor other features of the H2AZ-containing nucleosome spread to the neighboring nucleosomes in vivo, arguing against a role for H2AZ as a self-perpetuating epigenetic mark.
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MATERIALS AND METHODS |
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MS. The peptide mixtures obtained from tryptic digestion of the bands on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel were analyzed by nano-high-pressure liquid chromatography (LC) (LC Packing) directly coupled to an ion-trap mass spectrometer (MS) (ThermoFinnigan LCQ Deca XP) equipped with a nanoelectrospray source. The samples were run in two different modes. For peptide identification, the ion trap acquired successive sets of six scan modes consisting of full-scan MS over a range of 200 to 2,000 m/z, followed by five data-dependent tandem MS (MS-MS) scans on the five most abundant ions in the full scan. The MS-MS spectra were acquired with relative collision energy of 35% and an isolation width of 2.0 Da. Their interpretation was performed with the Bioworks software package. Alternatively, for the confirmation and quantification of the presence of a particular peptide in the sample, the ion trap was set in a selection reaction monitoring mode. The quantities of the corresponding peptides were estimated by use of the peak intensities.
Recombinant expression plasmids. To generate H2A and H2AZ expression plasmids, cDNAs for these histones (purchased as expressed sequence tag clones from Invitrogene; 4702210 for H2AZ and 3353785 for H2A) were PCR amplified and subcloned in frame into a pOZFHHN or pOZFHHC vector (V. Ogryzko and U. Mechold, unpublished data) or as cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP) fusions into the pOZFHHN vector.
Antibodies. Antibodies against trimethyl H4K20, H3K9, and H3K27 were gifts from T. Jenuwein. Anti-trimethyl H3K4 was a gift from B. Turner. Anti-hemagglutinin (anti-HA) was purchased from Roche, anti-H2AZ from Abcam, and anti-methylcytosine from VWR. Secondary goat anti-rabbit antibody was from Daco.
Purification of the epi:H2AZ-containing octamer and its neighborhood. The nuclear pellet was prepared according to the protocol described in reference 6. It was further washed with 600 mM NaCl in 20 mM Tris (pH 8.0), 10% glycerol, 0.1% Tween and then resuspended in an equal volume of nuclease digestion buffer (0.34 M sucrose, 10 mM Tris [pH 7.5], 3 mM MgCl2, 1 mM CaCl2). The nuclei were incubated with micrococcal nuclease for 20 min at 37°C until the reaction was stopped with 4 mM EDTA. To release the undigested chromatin, the pellet was sonicated and the size of chromatin fragments was monitored by use of a 2% agarose gel. The unsolubilized material was removed by centrifugation at 16,000 x g for 30 min, and the supernatant was dialyzed against 300 mM KCl in 20 mM Tris (pH 8.0), 10% glycerol, 0.1% Tween overnight. The precipitated material was removed by centrifugation at 45,000 x g for 30 min. The double-tag purification was performed as described in reference 26, except that at the second step Ni-nitrilotriacetic acid (NTA)-agarose (QIAGEN) was used. Briefly, 5 ml of chromatin solution was incubated overnight with 500 µl (packed volume) of the anti-FLAG agarose beads (Sigma) and washed five times with 300 mM KCl in 20 mM Tris (pH 8.0), 10% glycerol, 0.1% Tween, and the epi:H2AZ-containing chromatin fragments were eluted with 500 µl of FLAG peptide (1 mg/ml FLAG peptide in 300 mM KCl in 20 mM Tris [pH 8.0], 10% glycerol, 0.1% Tween). Fifty microliters of Ni-NTA-agarose was added to the FLAG eluate and incubated for 2 h. The Ni-NTA beads were washed with the same washing buffer. The final elution with 250 mM imidazole (in 20 mM Tris [pH 8.0], 10% glycerol, 0.1% Tween) was done after the elution with 2 M NaCl in 20 mM Tris (pH 8.0), 10% glycerol, 0.1% Tween. Alternatively, for the preparation of mononucleosomes containing epitope-tagged histones, we performed more-intensive micrococcal nuclease digestion of purified chromatin and isolated mononucleosome-sized chromatin fragments after 10 to 35% glycerol gradient fractionation. The double-tag purification was performed as described above, except the 2 M NaCl elution step was omitted and the epi:H2AZ-containing chromatin was eluted with imidazole.
Immunofluorescence and confocal microscopy. C2C12 cells were chosen as a convenient positive control for euchromatin and heterochromatin staining, due to the presence of prominent heterochromatin domains in these cells. Confocal images of C2C12 cells stained with anti-histone antibodies and epi:H2A-/H2AZ-expressing HeLa cells were taken with a Leica TCS SP microscope equipped with argon and UV lasers. The cells were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton, and blocked with 0.3% bovine serum albumin before being treated with primary and secondary antibodies. The colocalization analysis was performed on NIH 3T3 cells grown on chambered coverslips and transfected with Polyfect (QIAGEN) according to the manufacturer's protocol and performed 2 days after transfection by use of a confocal Zeiss LSM510 microscope. Alternatively, 293T cells were transfected by use of the calcium phosphate method and analyzed 1 or 2 days after transfection. The colocalization analysis was performed with LSM Examiner software.
Western and slot blot analyses. Separation of proteins was performed in 18% Tris-glycine SDS-polyacrylamide gels for silver or Coomassie staining and Western analysis. Proteins were transferred to nitrocellulose membranes and probed with antibodies according to the standard procedure. For analysis of DNA methylation, serial dilutions of DNA from epi:H2AZ- and epi:H2A-containing chromatin fragments were transferred to nitrocellulose membrane via a slot blotting device and subsequently cross-linked for 2 h at 80°C in vacuum. The membranes were blocked in 5% fat-free milk and probed with anti-methylcytosine antibody according to the standard procedure.
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RESULTS |
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According to their association with mitotic chromosomes, the triple-tagged H2AZ and H2A (epi:H2AZ and epi:H2A) were incorporated into chromatin (Fig. 1A shows the N-terminal constructs; the C-terminal constructs are not shown). The expression level of epi:H2A was on the order of 1% of that of the endogenous protein (not shown), as judged by Coomassie staining. The expression level of epi:H2AZ, estimated by Western analysis with anti-H2AZ antibody, was less than 30% of that of the endogenous protein (Fig. 1B). Because of the relatively low expression level of epi:H2AZ, it should be significantly diluted in the pool of endogenous H2AZ, ensuring that most of the other H2AZ molecules, if present in the same chromatin fragment, should be untagged (also see Discussion).
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H2AZ-containing nucleosomes are enriched in histone posttranslational modifications marking both active and silenced chromatin. Active and silenced chromatin states are marked by different posttranslational modifications (14, 42). Accordingly, we tested the H2AZ-containing histone octamer with a set of antibodies highly specific for different activation- and silencing-associated trimethylated histones H3 and H4.
As expected, the H4K20, H3K9, and H3K27 antibodies stained mostly heterochromatin in control immunofluorescence experiments, while H3K4 did not (Fig. 2A). Surprisingly, Western analysis revealed that compared to the epi:H2A-containing octamers, which represent an average nucleosome, the epi:H2AZ-containing octamers are enriched in both repression-specific K9 and K27 trimethylation and activation-specific K4 tri- and dimethylation of histone H3 (Fig. 2B). On the other hand, the H4 trimethylated in K20, which also marks repressed chromatin (Fig. 2A), was detected preferentially in the octamers with canonical H2A histone. Impressively, the same detection pattern was seen in both N-terminally and C-terminally tagged H2AZ/H2A pairs (Fig. 2A, compare left and right panels). Thus, in the context of the H2AZ-containing nucleosome, we were able to dissociate different modifications associated with silencing. Equally intriguing is a finding that some repression- and activation-associated modifications (K9/27 and K4) of H3 histone were both enhanced in the H2AZ-containing octamer.
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In accordance with the complex pattern of the H3 histone methylation in the H2AZ-enriched octamer, we could not assign H2AZ to either heterochromatin or euchromatin by microscopy (regular immunofluorescence with epitope-tagged proteins and YFP-H2AZ fusions in NIH 3T3, HeLa, and 293 cells was used; not shown). However, when YFP-H2AZ was cotransfected with CFP-H2A and their localizations were compared in the same cell, the distinct localization of H2AZ could be indirectly inferred by the analysis of the CFP-YFP colocalization coefficients (Fig. 3, compare colocalization coefficients for the CFP-H2A/YFP-H2A and CFP-H2A/YFP-H2AZ cotransfections).
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Importantly, omission of the salt elution step gave practically the same result as the two-step elution scheme (not shown), ruling out salt elution as a cause of a possible artifact. In addition to analysis of oligonucleosomes containing epi:H2AZ, we also performed more-intensive micrococcal nuclease digestion and glycerol gradient fractionation to purify single mononucleosomes containing epi:H2AZ (Fig. 5C). The MS-MS analysis of the untagged H2A(H2AZ) band from this preparation showed practically the same amount of H2AZ-specific peptides as the oligonucleosome analysis (Fig. 5B, Mono panel).
Similar mass spectrometry analysis was also performed on chromatin fragments (from regular HeLa cells) of lengths of 2 or 3 nucleosomes that were purified with antibody against native H2AZ. In this case, the ratio of H2AZ-specific peptide to common peptide reflected that H2AZ comprised 20% of the total H2A (Fig. 5B, IP aH2AZ panel). This corresponds to approximately one H2AZ molecule per two or three nucleosomes and is consistent with the conclusions based on tandem affinity purification.
The data above indicate that the H2AZ deposition in vivo is a highly localized phenomenon limited to only one H2AZ/H2B dimer per several nucleosomes. Consistent with the absence of any enrichment of H2AZ in the neighborhood of the H2AZ-containing nucleosome, the analysis of posttranslational modifications shows differences much less pronounced between the H2A and H2AZ neighborhoods (Fig. 5D) than between the H2A and H2AZ octamers.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A crucial requirement of our approach is a low level of the epitope-tagged histone expression compared to its endogenous H2AZ counterpart (Fig. 1B). This should make deposition of several epitope-tagged histones side by side very unlikely. The expression and eventual deposition of epitope-tagged H2AZ include transcription of the epi:H2AZ gene, mRNA processing, transport to the cytoplasm, mRNA translation to epitope-tagged H2AZ, and incorporation of the H2AZ into the deposition complexes and their transport back to the nucleus. At least one of these steps is expected to include randomization and mixing of the epi:H2AZ mRNA or protein with endogenous H2AZ mRNA or H2AZ histone. Thus, the low relative expression of epi:H2AZ should ensure that most other H2AZ molecules, if present in the same chromatin fragment, are untagged.
As histones are highly conserved proteins, a potential problem with our approach is the introduction of an artifact due to epitope tagging. We addressed this problem by using both N-terminal and C-terminal tagging of H2AZ to make our conclusions less dependent on the potential effects of this procedure. While in other cases the site of tagging could significantly affect the protein properties, we did not detect any dramatic differences for the H2AZ/H2A histones in this respect.
The strategy described here can be applied directly to any histone variant. The list of the replacement histone variants is growing (2, 3, 11), and analysis of the sequence databases reveals the existence of many as-yet-uncharacterized open reading frames encoding new histone variants (not shown). Our procedure should contribute to better understanding of their biological roles and their involvement in epigenetic coding of the chromatin state.
Implications for the histone code hypothesis. Using our purification scheme, we were able to compare different histone modifications in the context of the H2AZ-containing nucleosome and its neighboring nucleosomes. This analysis revealed that the H2AZ-containing octamers are enriched in some posttranslational modifications marking both active and silenced chromatin. Consistent with the finding of the repression-specific histone marks, we also observe enhanced cytosine methylation in the H2AZ-containing nucleosomes. Notably, we also observed dissociation between the repression-specific H3 K9/K27 and H4 K20 in the H2AZ-containing nucleosome. As H2AZ constitutes approximately 3% of the total H2A in mammalian cells, the dissociation between the H3 K9/K27 and H4 K20 methylations and the correlation between the H3K4 and K9/27 methylations, both clearly seen in the context of the H2AZ-containing nucleosome, are hard to observe in an analysis of bulk chromatin. Our finding underscores the importance of analyzing different histone modifications in the context of other histone variations.
Implications for epigenetic maintenance. Compared to the H3/H4 pair of core histones, the H2A/H2B pair of histones has relatively high mobility. Since recent findings are consistent with H2AZ playing a role as an epigenetic mark poising promoters for transcriptional activation (12, 19, 31, 45), the question of how this mark could be maintained on a particular chromatin site despite its relatively high mobility attains crucial importance. This possibility is not without a precedent; for example, the dynamic association of HP1 with chromatin is consistent with its crucial role in the maintenance of stable heterochromatin states (4, 10).
To account for the possibility of H2AZ acting as an epigenetic chromatin mark, the simplest model to consider is the direct recruitment model depicted in Fig. 6. Similar to the other epigenetic templating mechanisms (proposed for DNA methylation, histone acetylation, and methylation [13, 14, 28, 42]), H2AZ could be directly involved in the maintenance of a particular chromatin state by serving as a mark that determines preferential recruitment of the H2AZ deposition machinery to the H2AZ-containing chromatin. This mechanism would ensure recovery of the H2AZ-containing chromatin state following its loss in processes such as transcription, DNA damage/repair, and thermal noise.
This model predicts either the presence of a second H2AZ in the same octamer (homotypic nucleosome) or its spread to the neighboring nucleosome. In fact, the homotypic nucleosome composition in vivo was observed in the case of two H3-like replacement histones, H3.3 and CenpA, consistent with their suggested role in epigenetic maintenance (25, 36, 40). The prediction was also consistent with the crystallographic analysis of an in vitro-reconstituted nucleosome containing H2AZ, which led to the conclusion that H2AZ cannot coexist in the same nucleosome with regular H2A (39). Contrary to the second prediction of the model and contrary to the conclusions from the crystallographic analysis, we found almost no presence of another H2AZ molecule either in the H2AZ-containing nucleosome purified directly from cells or in its nucleosome neighborhood. Furthermore, the fact that we found trace amounts of the second H2AZ in both cases simply reflects the probability of finding H2AZ in an average HeLa nucleosome and therefore strongly argues against preferential concentration of the H2AZ molecules in close vicinity to one another.
Experimental evidence indicates that H2AZ is enriched in some areas in the nucleus directly compared to regular H2A (Fig. 3) (8, 18, and 32). However, the resolution of a confocal microscope cannot discriminate how close to each other the H2AZ molecules are in these areas. Our biochemical data argue against a particular model that suggests the direct involvement of H2AZ in epigenetic templating (Fig. 6). Nevertheless, we cannot exclude the possibility that the domains enriched in H2AZ histone are maintained via an epigenetic mechanism that involves H2AZ in a less direct way.
Given the evidence toward a role for the H3-like variant histones in epigenetic stability (25, 36, 40), the fact that we could not find similar evidence for an H2A class variant histone could reflect yet another essential difference between the H3/H4 and H2A/H2B-like histones. However, we cannot rule out the possibility that the direct recruitment model will be correct for other replacement H2A/H2B variants. We hope that the methodology introduced in this work will be useful in addressing the role of replacement histone variants and chromatin in general in the maintenance and transmission of epigenetic information.
. . .
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ACKNOWLEDGMENTS |
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The work was supported by Association pour la Recherche sur le Cancer and la Ligue Nationale contre le Cancer.
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FOOTNOTES |
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These authors contributed equally to this work.
Present address: Génétique des Génomes Bactériens, Genetics of Bacterial Genomes, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France.
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H2AZ*). (B) Results of a mass spectrometry analysis of the H2A(H2AZ) bands in identification mode. The bands corresponding to the H2A(H2AZ) histone from different samples (shown boxed on the left panel) were excised and analyzed by nano-LC/MS-MS in identification mode. Top left panel: A, e:H2A octamer and neighborhood; Z, e:H2AZ octamer and neighborhood. The positions and intensities of the peaks corresponding to the H2A- and H2AZ-specific peptides are shown on the chromatograms (right panel and bottom). (C) Alignment of the H2A and H2AZ peptide sequences. The locations of all peptides used for identification of the endogenous H2A bands shown in panel B are boxed. Indicated are the A/Z and the Z peptides used for quantification of the H2AZ content in the endogenous H2A/Z bands shown in Fig. 