Characterization of the histone H2A.Z-1 and H2A.Z-2 isoforms in vertebrates
© Dryhurst et al; licensee BioMed Central Ltd. 2009
Received: 22 October 2009
Accepted: 14 December 2009
Published: 14 December 2009
Within chromatin, the histone variant H2A.Z plays a role in many diverse nuclear processes including transcription, preventing the spread of heterochromatin and epigenetic transcriptional memory. The molecular mechanisms of how H2A.Z mediates its effects are not entirely understood. However, it is now known that H2A.Z has two protein isoforms in vertebrates, H2A.Z-1 and H2A.Z-2, which are encoded by separate genes and differ by 3 amino acid residues.
We report that H2A.Z-1 and H2A.Z-2 are expressed across a wide range of human tissues, they are both acetylated at lysine residues within the N-terminal region and they exhibit similar, but nonidentical, distributions within chromatin. Our results suggest that H2A.Z-2 preferentially associates with H3 trimethylated at lysine 4 compared to H2A.Z-1. The phylogenetic analysis of the promoter regions of H2A.Z-1 and H2A.Z-2 indicate that they have evolved separately during vertebrate evolution.
Our biochemical, gene expression, and phylogenetic data suggest that the H2A.Z-1 and H2A.Z-2 variants function similarly yet they may have acquired a degree of functional independence.
In the eukaryotic cell, the template for all DNA metabolic activities including DNA repair, replication, recombination and transcription, is chromatin. Chromatin is a nucleoprotein complex in which approximately 147 base pairs of DNA are wrapped around a histone core consisting of two each of the core histones H2A, H2B, H3 and H4, resulting in a repetitive structure called the nucleosome. Linker histones of the H1 family bind to the linker DNA regions connecting adjacent nucleosomes in the chromatin fiber. Histones represent the major protein component of chromatin and most of the synthesis of the canonical forms takes place during S phase of the cell cycle to allow for efficient packaging of the newly replicated DNA. In contrast, a subset of histone variants is synthesized throughout the cell cycle and can replace canonical histones to specify chromatin domains for specific functions [1, 2]. Histone H2A.Z is one such replacement histone variant and while it is very widely studied, its structural and functional roles have not only proven to be many and diverse but also controversial [2, 3].
From a functional perspective, histone H2A.Z has been found to be present in heterochromatin, where it participates in the formation of pericentric and centric chromatin, [4, 5] and in euchromatin [6, 7], where it is usually found associated with promoters of active genes . Recent work has revealed that promoters are marked by complexes that contain H2A.Z in conjunction with the H3.3 variant indicating that replication-independent histone variant replacement plays a role in generating an appropriate chromatin landscape at these loci . The concept that H2A.Z serves to poise promoters for transcriptional activation but is displaced from chromatin once transcription is initiated has been widely documented [10–13].
Recently, another very interesting function has been ascribed to H2A.Z, which is that it aids in localizing genes to the nuclear periphery and in doing so marks them for more rapid reactivation even after several cell divisions . Many of these functions of H2A.Z, although seemingly very different, may not be entirely unrelated in a broad sense if H2A.Z is considered a mark that is required to specify the nuclear location of a given region of the genome perhaps with respect to the nuclear periphery, or the nuclear matrix in general.
At the structural level, H2A.Z has been shown to enhance the stability of the nucleosome [15, 16] despite the fact that the H2A.Z-H2B dimer exhibits a reduced stability compared to H2A-H2B dimers [16, 17]. H2A.Z has also been shown to alter nucleosome mobility . When present in nucleosome arrays, H2A.Z increases the intramolecular interactions and promotes HP1α-mediated folding of the fiber [19, 20]. Furthermore, several well-positioned H2A.Z-containing nucleosomes flank sites occupied by the insulator binding protein CTCF which prevents the spread of heterochromatin .
How H2A.Z is able to participate in these many cellular events could be based on whether it is placed within isolated nucleosomes or within contiguous stretches of chromatin, as is the case at many polycomb group genes in embryonic stem cells . Alternatively, post translational modifications shown to exist on H2A.Z, including acetylation, ubiquitination and SUMOylation, may direct certain populations of H2A.Z for a specific function.
Recently, we identified the presence of two H2A.Z protein isoforms in chicken that differ by 3 amino acids . Our group has also provided evidence of distinct phylogenetic patterns for the H2A.Z-1 and H2A.Z-2 variants during vertebrate evolution . Importantly, H2A.Z is the only histone variant that has been shown to be indispensable for survival in Drosophila  and mice . However, in this latter study, only H2A.Z-1 was knocked out. This indicates that H2A.Z-2 is incapable of compensating for the loss of H2A.Z-1 in mice. Whether this is because of lower amounts of total H2A.Z or because of differences in the nuclear localization, post translational modification, biochemical interactions or temporal expression of the H2A.Z-1 and H2A.Z-2 genes is unknown. In the present work, we show that both isoforms are expressed across a wide range of human tissues and that they display a similar nuclear distribution and levels of N-terminal acetylation. Furthermore, we show that the distribution of H2A.Z-1 and H2A.Z-2 within chromatin differs, as does their association with histone H3 trimethylated at lysine 4. Despite the high degree of amino acid sequence similarity between these H2A.Z isoforms, they display very divergent promoter sequences that could result in temporal and tissue-specific differences in gene expression.
The N-terminal tails of H2A.Z-1 and H2A.Z-2 are acetylated in vivoin chicken cells
H2A.Z-1 and H2A.Z-2 are mainly distributed in euchromatin in mouse fibroblasts and in HEK 293 cells
H2A.Z-1 and H2A.Z-2 associate with different forms of post-translationally modified H3 and H4 within the nucleosome
H2A.Z-1 and H2A.Z-2 are differentially expressed among tissues
The promoter sequences of the H2A.Z-1 and H2A.Z-2 genes are substantially different
The characterization of the H2A.Z-2 promoter emphasizes the existence of a clear differentiation between H2A.Z-1 and H2A.Z-2. The H2A.Z-2 promoter contains no TATA box and the positions of the CAAT elements and GC-boxes do not coincide with those identified in H2A.Z-1 (Figure 6A). Furthermore, it seems that H2A.Z-2 promoters show a lesser degree of conservation across the mammalian species studied, as indicated by the lack of the positional consensus in the elements identified (see Additional files 1 and 2). Searches for potential regulatory elements in the proximal promoter region resulted in the identification of three CAAT elements as well as several binding sites for transcription factors including c-Myc and N-Myc. However, the positioning of these latter binding sites at the proximal region suggests that they may function in constitutive gene expression rather than in inducible regulation in response to agents such as hormones [36, 37].
The differentiation between the H2A.Z-1 and H2A.Z-2 promoters has also been analysed from a phylogenetic perspective (Figure 6B). Our results reveal the differentiation of two major evolutionary lineages based on H2A.Z promoter sequences, one encompassing the H2A.Z-1 promoter sequences and the other including H2A.Z-2 promoter sequences (Figure 6B). Such topology is in agreement with the phylogenetic inferences reconstructed on the basis of H2A.Z protein sequences and nucleotide coding regions, which also show an evolutionary differentiation between H2A.Z-1 and H2A.Z-2 variants. The presence of such differentiation at promoter regions strongly supports the presence of evolutionary constraints which act with different direction and intensity on the H2A.Z-1 and H2A.Z-2 proteins.
H2A.Z is the most extensively studied histone variant and it has been shown to be involved in several seemingly unrelated and divergent processes. Understanding how this protein participates in different cellular events has undoubtedly been further complicated by the use of different biological systems, since, while certain general functions of H2A.Z may be universal in all organisms, the specific details and fine-tuning in higher eukaryotes may not be present, or may be the work of other proteins, in yeast or flies. Part of this fine-tuning of H2A.Z in vertebrates could be due to the co-existence in the cell of two H2A.Z protein isoforms (H2A.Z-1/H2A.Z-2) that was first determined by mass spectrometry in total H2A.Z isolated from chicken erythrocytes . An evolutionary and phylogenetic analysis of these isoforms revealed a substantial conservation at the protein level where they differ by only three amino acids (see Figure 6C), but a marked divergence at the nucleotide sequence level . It also revealed that the two H2A.Z isoforms had a common origin early in deuterostome evolution that was followed by a subsequent process of differentiation . Indeed, it appears that the strength of the evolutionary constraints operating at the nucleotide level are not equal between the isoforms, with H2A.Z-2 being significantly more constrained than H2A.Z-1 . Cumulatively, the evolutionary evidence points toward a possible functional diversification of these two proteins.
Recently, our group has characterized the sites of acetylation within the N-terminus of H2A.Z-2 from chicken erythrocytes and from chicken erythroleukemic (MSB) cells treated with sodium butyrate to inhibit histone deacetylases . Notably in the chicken, H2A.Z-2 is the variant that has traditionally been called H2A.Z or H2A.F/Z. In mouse and human H2A.Z-1 has customarily been considered H2A.Z, while H2A.Z-2 was previously known as H2A.V/F. In the current work, we determined that H2A.Z-1 is acetylated at at least three lysine residues within the N-terminus, as is H2A.Z-2. The patterns of acetylation are similar for both isoforms, where a triply-acetylated (K4, K7 and K11) form as well as doubly-acetylated (K4 + K7, K7 + K11, and K4 + K11) forms of the N-terminal peptides were identified within the sample. Singly-acetylated N-terminal peptides were also detected. However, the sites of acetylation were not able to be determined owing to the low abundances of these species. In a previous study, we identified multiple singly-acetylated H2A.Z-2 peptides, with the abundance of acetyl K4-containing species being relatively equal to the acetyl K7 species , and this is likely to be the case with the singly-acetylated forms of H2A.Z-1. Previously, H2A.Z-1 has been shown to be monoubiquitinated at one of several lysine residues within the C-terminus, as well as SUMOylated. However, neither modification was observed on either H2A.Z variant in chicken cells, although this could be a result of the purification method used here, or the cell type [30, 38].
Our results regarding the nuclear localization of the H2A.Z isoforms indicate that they are identically distributed mainly in the euchromatin of mouse embryonic fibroblasts, as seen by other groups [6, 30]. However, both H2A.Z-1 and H2A.Z-2 staining are also present within DNA-dense regions that represent the centres of chromosomes. This is most probably due to the known presence of H2A.Z in pericentric and centric chromatin [4, 5]. This distribution of H2A.Z is corroborated by our biochemical analysis of chromatin fractions indicating that endogenous H2A.Z is present in greater amounts within the S1 (euchromatin) and SE (heterochromatin) fractions compared to the Pellet (insoluble chromatin). In accordance with the distribution of endogenous H2A.Z within these fractions, we see that both stably transfected H2A.Z-1-Flag and H2A.Z-2-Flag are also present in all fractions. However, interestingly, H2A.Z-2-Flag protein is present in greater amounts within the S1 fraction compared to H2A.Z-1-Flag. This suggests that H2A.Z-2 may play a greater role in the function of H2A.Z within the nuclease accessible euchromatin than does H2A.Z-1. Several systems have shown that H2A.Z functions in part to poise promoter chromatin for transcriptional activation, since it is remodelled from promoters of actively transcribing genes [10–13, 39, 40]. H2A.Z functioning in this manner would probably fractionate within the S1 fraction, since this chromatin would be in a more open conformation. Furthermore, the low levels of H2A.Z present within the P fraction also agree with its not playing a significant role after the initiation stage of transcription. Therefore, in HEK 293 cells H2A.Z-2 may be playing a greater role than H2A.Z-1 in poising chromatin for transcription, which agrees with our results indicating that H2A.Z-2 also shows a greater association with H3 Tri-Me K4 than H2A.Z-1 (see below).
Genome-wide distribution patterns indicate that H3 Tri-Me K4 and H2A.Z are both present at the promoters and 5' ends of genes in yeast and humans . Therefore, it is not surprising that H2A.Z and H3 Tri-Me K4 are present within the same nucleosome [30, 31]. The fact that H2A.Z-2 associates with H3 Tri-Me K4 to a greater extent than H2A.Z-1 is interesting because it indicates that there is a greater overlap in the regions of chromatin where H2A.Z-2 is incorporated in conjunction with the H3 Tri-Me K4 mark than H2A.Z-1. This reinforces the idea that H2A.Z-1 and H2A.Z-2 could be incorporated at different locations within the genome. The remodelling of H2A.Z into chromatin in mammals is known to occur via the action of several distinct complexes that contain the SRCAP protein, p400 or TIP 48/49 as the adenosine triphosphate-dependent remodelling subunits and evidence suggests that the deposition patterns of these complexes may differ [39, 41–43]. It is tempting to speculate that one complex may prefer a specific isoform over the other, or that one complex may be responsible for incorporating H2A.Z at low levels across the genome and another for the increased levels of H2A.Z that are necessary to promote appropriate chromatin architecture at promoters . Furthermore, H2A.Z nucleosomes flank nucleosome free regions whose formation is DNA-sequence dependent in several organisms including humans . Whether one of the human complexes that integrates H2A.Z into chromatin contains a subunit that recognizes a specific DNA sequence is unclear but points to the potential for more than one mechanism to specify the location of H2A.Z nucleosomes . Indeed, a recent in silico analysis suggests that both genetic and epigenetic factors can predict whether a nucleosome will contain H2A.Z .
The functional output of the increased association of H2A.Z-2 with H3 Tri-Me K4 is unclear, but it is highly unlikely that it results in a structural alteration of the nucleosome that would otherwise not be observed in those containing H2A.Z-1 with H3 Tri-Me K4 given the high sequence similarity of the isoforms. At least one of the effects of having H2A.Z and H3 Tri-Me K4 within the same nucleosome could be related to the recruitment or function of the RNA Pol II complex, since both the C-terminal region of H2A.Z and H3 Tri-Me K4 have been shown to interact with components of this complex [47, 48]. Another possibility is that nucleosomes containing both H2A.Z and the H3 Tri-Me K4 mark protect less DNA than other nucleosomes, making the DNA more accessible for transcription [21, 49]. Nevertheless, it is possible that any differences in the genomic localization and association with post translationally modified forms of other histones of the endogenous H2A.Z-1 and H2A.Z-2 could reflect spatial and temporal differences in their nuclear availability as determined by their levels of expression, or by their import into the nucleus.
Our results also reveal that H2A.Z-1 and H2A.Z-2 nucleosomes are enriched in H3 phosphorylated at S10 compared to H2A nucleosomes in asynchronous and mitotic HeLa. This association is interesting because like H2A.Z, H3 PhosS10 has been shown to be involved in processes requiring open and condensed chromatin, namely in transcription and chromosome condensation during mitosis . The structural and functional consequences of H3S10 phosphorylation are not entirely clear; however, this modification does correlate with increased expression of immediate early genes after induction of MAP Kinase cascades [51, 52]. Furthermore, it is not surprising that H4AcK16 is found to associate with H2A.Z-1 and H2A.Z-2 nucleosomes because it has been shown that this modification of H4 is required for incorporation of H2A.Z into subtelomeric chromatin in budding yeast .
Our analysis of the transcript levels of H2A.Z-1 and H2A.Z-2 indicate that there is no one dominant form, rather they are both expressed across a wide range of human tissues and that the expression levels vary depending on developmental stage. This pattern of expression probably reflects the significant sequence variation within the promoter regions of these genes and the differences in abundance of specific transcription factors within the tissues. However, it could also reflect subtle changes in the requirements of the tissues for one isoform over the other, based on any functional differences between H2A.Z-1 and H2A.Z-2. Indeed, it has recently been shown that H2A.Z-2 is specifically upregulated during macrophage differentiation and activation  and our analysis of the promoter of H2A.Z-2 indicates the presence of putative binding sites for the Ikaros transcription factor which is known to play a role in hematopoietic cell development . Also as previously mentioned, genetic knockout studies in mice show that H2A.Z-1 is essential for development, indicating that H2A.Z-2 cannot compensate for the loss of H2A.Z-1 at least at early stages of mouse embryo differentiation .
The evolutionary evidence presented here suggests that it is highly possible that H2A.Z-1 and H2A.Z-2 appeared in vertebrates as a result of the entire genome duplication that took place between the divergence of the prochordates from the ancestral chordate lineage and the evolution of vertebrates, as stated by the 2R hypothesis . While most of the genes arising from this duplication event were silenced, some of them were retained and their protein products either acquired a neofunctionalization (a completely new function) or a subfunctionalization in which the two new proteins acquired specialized functions . This latter situation appears to be the case in going from H2A.Z-e to H2A.Z-1 and H2A.Z-2 in vertebrates.
We present evidence that the two forms of H2A.Z present in mammals display similar genomic localization patterns mainly within euchromatin but that subtle differences in their association with post translationally modified forms of other histones exist. Thus, it is possible that throughout the course of vertebrate evolution, the two H2A.Z isoforms have acquired a degree of independent function that may contribute to the increased complexity and large diversity of roles for this histone variant in higher organisms. It is also possible that differences in chromatin localization patterns of the isoforms could be due to the temporal regulation of their genes, especially considering the dissimilarity in the promoter regions.
Mouse embryonic fibroblasts were grown in Dulbecco's modified eagle medium (DMEM) with 10% fetal calf serum, plated on glass coverslips and allowed to attach and grow until between 40% and 80% confluent. The coding region of H2A.Z-1 was fused in frame to the 3' end of cyan fluorescent protein (CFP) into the pAmCyan1-N1 vector (Clontech) and the coding region of H2A.Z-2 was fused in frame to the 3' end of yellow fluorescent protein (YFP) into the pZsYellow1-N1 vector (Clontech, CA, USA). The cells were then co-transfected with H2A.Z-1-CFP and H2A.Z-2-YFP using effectene transfection reagent (Qiagen, CA, USA) according to the manufacturer's instructions. The cells were grown for an additional 16-20 h followed by replacement of the medium and the addition of 1 μg/ml Hoechst 33342. After 10 min, the medium was replaced with fresh medium containing no Hoechst dye. The coverslips were then mounted onto glass slides after using vacuum grease to create a small reservoir for media and imaged immediately using a Zeiss Axiovert 200 M inverted fluorescence microscope. Images were collected with a 100 × 1.4 N.A. PlanApo objective and a Photometrics CoolSnap fx CCD.
Native H2A.Z purification
Native H2A.Z was purified from chicken MSB cells (chicken erythroleukemic cells transformed by Marek's virus) treated with 5 mM sodium butyrate for 16 h as in . Isolated nuclei were extracted with HCl to solubilize the histones as described in . Approximately 30-40 mg of total histones was loaded onto a 1.5 cm × 120 cm BioGel P-60 (Bio-Rad) column and eluted in 50 mM NaCl and 20 mM HCl . H2A.Z eluted within a peak that also contained H2A and these fractions were selected for further purification by several rounds of reverse phase HPLC on a C18 column (Vydac, GA, USA).
Upon purification of H2A.Z, as described above, the H2A.Z fraction purified from sodium butyrate treated MSB cells was lyophilized and reconstituted in 100 mM ammonium bicarbonate, pH 8. An aliquot (10%) of each fraction was treated with propionic anhydride to derivatize unmodified ε-amino groups of lysine residues. Chemical derivatization with propionic anhydride converts the amino groups to their corresponding propionyl amides, and these methods have been detailed previously . In brief, equal volumes of propionylation reagent (15 μL) and H2A.Z (15 μL) were reacted and derivatization was performed twice to ensure full conversion. The sample was vacuum-dried after each derivatization. The derivatized H2A.Z samples were then digested with 4 ng trypsin (Promega, WI, USA) for 8 h at 37°C. Derivatization blocks lysine residues from cleavage and, thus, trypsin cleaves C-terminal to arginine residues only. Following digestion, the samples were again reacted with propionic anhydride to derivatize the amino-termini of the trypsin-generated H2A.Z peptides. Both samples were dried a final time in a speed-vac concentrator, and were subsequently reconstituted in 0.1% acetic acid.
The resulting H2A.Z peptide mixtures were analyzed via tandem MS/MS. Each sample was pressure-loaded onto a capillary pre-column (360 μm outside diameter [(OD] × 75 μm inside diameter [ID] fused silica) packed with 4 cm of C18 reverse phase resin (5-20 μm diameter). After washing for 5 min with 0.1% acetic acid, the pre-column was connected to an analytical column (360 μm OD × 50 μm ID fused silica) packed with 8 cm of C18 (5-μm diameter) resin and equipped with an electrospray emitter tip as previously described . H2A.Z peptides were eluted using nanoflow HPLC with an 1100 series high-performance liquid chromography (HPLC) pump (Agilent Technologies, CA, USA) and a 60 nL/min flow rate was achieved by splitting the flow from the HPLC. The gradient consisted of 0-60%B in 50 min and 60-100%B in 10 min (solvent A: 0.1 M acetic acid, solvent B: 70% acetonitrile, 0.1 M acetic acid). Gradient-eluted peptides were ionized using an electrospray ionization source, modified for nanospray, and were analysed using a hybrid quadrupole linear ion trap Fourier transform (LTQ-FT) mass spectrometer (Thermo Scientific, MA, USA). The LTQ-FT instrument was operated with a data-dependent method, which consisted of acquisition of a full mass spectrum using the FT as analyser followed by 10 MS/MS acquisitions of the 10 most abundant ions in the initial full spectrum. Full mass spectra were acquired for m/z 300-2000 using a resolution of 100,000. Upon collision-activated dissociation (CAD) of precursor ions, MS/MS spectra were acquired using the ion trap as the analyser. Dynamic exclusion was enabled, by which precursor ions were selected for dissociation twice within 20 s before they were added to the exclusion list for 30 s. All CAD MS/MS spectra were interpreted via manual validation for identification of the N-terminal peptides of both H2A.Z isoforms. Accurate mass measurements acquired in the FT were utilized to generate selected ion chromatograms (SICs). A window of +/- 0.005 Da around the theoretical monoisotopic m/z values of the [M+2H]+2 and [M+3H]+3 ions was used to generate the SICs for the N-terminal peptides of H2A.Z-1 and H2A.Z-2.
Stable transfection and chromatin fractionation
H2A, H2A.Z-1 and H2A.Z-2 sequences were amplified from HeLa cell cDNA and Flag tags were added in frame to the 3' ends by PCR. The sequences were then cloned into pcDNA3.0 vector (Invitrogen, CA, USA) and the plasmid was linearized by digesting with Sca1. HEK 293 cells were transfected using PolyFect reagent (Qiagen) and cells stably expressing the Flag-tagged protein were selected for by the addition of G418 (Gibco, CA, USA) to cell culture medium (DMEM containing 10% fetal bovine serum). After several weeks, stable colonies expressing similar levels of the proteins were selected. Nuclei were isolated and digested with Micrococal Nuclease (Worthington, NJ, USA) at 30 U/mg of DNA for 5 min at 37°C. The reaction was stopped by the addition of EDTA to a final concentration of 10 mM on ice, and the sample was centrifuged at 10,000 g for 10 min at 4°C to yield an S1 supernatant and a pellet. The pellet was resuspended and lysed in 0.25 mM EDTA pH 8 and stirred for 1 h at 4°C. Upon centrifugation as before, a supernatant SE and a final pellet P were thus obtained. Under the experimental conditions used here, S1, SE and P correspond to approximately 5%-10%, 25%-30% and 60%-65%, respectively, of the total nuclear DNA. Western Blotting was performed on the normalized protein component of these fractions using SDS-PAGE and standard procedures. Flag antibody (Sigma, NY, USA) dilution was 1:5000, H2A.Z antibody (Abcam) dilution was 1:1000, H3 Tri-Me K4 antibody (Millipore, MA, USA) dilution was 1:5000, and H4 antibody was made in-house and used at a 1:10,000 dilution.
Preparation of mononucleosomes and immunoprecipitation
Mononucleosomes were prepared from HeLa cells transiently transfected with H2A-Flag, H2A.Z-1-Flag or H2A.Z-2-Flag within the pcDNA3.0 vector following the procedure of Sarcinella and colleagues . When required, cells were treated with 100 mg/ml nocodazole for 16 h after 24 h of transfection and an aliquot was monitored by fluorescence acitvated cell sorting after treatment with propidium iodide to ensure mitotic arrest. Briefly, isolated nuclei were digested with Micrococal Nuclease (Worthington) at a concentration of 100 U/mg of DNA at 37°C for 30 min. The reaction was stopped by the addition of EGTA to a final concentration of 1 mM and the suspension was centrifuged at 600 g for 10 min. The resulting pellet was resuspended in 20 mM Tris pH 7.5, 0.2 mM EGTA, 420 mM NaCl and 1.5 mM MgCl2 and incubated on ice for 1 h then spun at 1000 g for 10 min. The supernatant was then brought to 150 mM NaCl by addition of 20 mM Tris pH 7.5, 1.5 mM MgCl2, 0.2 mM EGTA and 25% glycerol dropwise on the vortex. This suspension was centrifuged at 1000 g for 10 min and monitored on 4% native acrylamide gels to ensure complete digestion of the chromatin to mononucleosomes. For the immunoprecipitation, 10 ul of anti-Flag agarose beads (Sigma) was used to immunoprecipitate Flag-containing mononucleosomes from H2A-Flag, H2A.Z-1-Flag, H2A.Z-2-Flag or mock transfected control HeLa cells. The beads were washed eight times in 1 ml 20 mM Tris pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 0.2 mM EGTA, 0.2% Triton X-100, resuspended in SDS sample buffer without β-mercaptoethanol, boiled, followed by the addition of β-mercaptoethanol to the supernatant. Immunoprecipitated nucleosomes were run on 15% SDS-PAGE and transferred to polyvinylidene fluoride membrane (BioRad, CA, USA). Anti H3 AcK9K14, anti H3 Tri-MeK27, anti H3 PhosS10 and anti H4 AcK16 were all from Millipore and used at a 1:1000 dilution. Anti H2A antibody was from Abcam and used at 1:1000 dilution.
Total histones from HeLa cell nuclei were extracted in 0.6N HCl as described in . These histones were electrophoresed on a 10% polyacrylamide AUT gel  in several lanes, one of which was cut out and soaked in 125 mM Tris-HCl pH 6.8, 4% SDS, 20% glycerol and 1.43 M β-mercaptoethanol for 10 min at room temperature while the other was stained with Coomassie blue solution. The unstained gel strip was laid horizontally and electrophoresed in a 6% polyacrylamide stacking, 15% polyacrylamide separating SDS gel prepared according to .
Samples from three human cDNA panels derived from various human fetal and adult tissues were obtained from Clontech Laboratories Inc (CTL; CA, USA). These included normal tissue from adult (CTL MTC panel I, CTL MTC panel II) and fetal (CTL MTC fetal MTC panel) sources. Each cDNA sample represented a pool of individuals of either gender. For quantitative PCR (QPCR) analysis, cDNA was diluted 20-fold in RNase/DNAse free water. H2A.Z-1- and H2A.Z-2-specific primers were designed based on the 5' and 3' untranslated regions and amplicon sequences were confirmed by DNA sequencing. H2A.Z-1 forward primer: TTGCTTGAGCT TCAGCGGAATT, reverse primer: TTCCTTGTTATCTCAGGACTCT H2A.Z-2 forward primer: GCGGCCGAGCGGAGGCGGAG, reverse primer: TGCTTAGAGGGATGCTTTAAC. The levels of H2A.Z transcripts were analysed by SYBR Green incorporation using a Stratagene MX3005P QPCR system and MXPro software. Each 15 μl DNA amplification reaction consisted of 2 μl of diluted cDNA and 13 μl of Platinum SYBR Green qPCR SuperMix-UDG with Rox (Invitrogen) containing 2.5 pmol of each primer. Thermocycling conditions for both primer sets were: 9 min 95°C followed by 40 cycles of 15 s 95°C, 30 s 60°C and 45 s 72°C. Reactions were performed in quadruplicate and averaged cycle threshold was converted to transcript copy number by interpolation from a standard curve. The standard curve was constructed using a dilution series of purified H2A.Z-1 or H2A.Z-2 amplicon.
Nucleotide sequences corresponding to promoter regions of H2A.Z-1 and H2A.Z-2 histones were retrieved from the GenBank database through recurrent BLAST searches performed on general nucleotide collections as well on completed genomes . A total of 27 nonredundant sequences belonging to mammals were compiled and properly classified as either H2A.Z-1 or H2A.Z-2 (see Additional File 3). Multiple alignments of the promoter sequences were conducted using the CLUSTAL W and the BIOEDIT programs [66, 67]. Conserved regulatory elements have been simultaneously identified from the alignments of mammalian H2A.Z-1 and H2A.Z-2 sequences by using the program Transcription Regulatory Element Search (TRES, http://bioportal.bic.nus.edu.sg/tres/) to perform searches in the object-oriented transcription factors database (ooTFD) . Phylogenetic relationships among H2A.Z promoter regions were reconstructed using the neighbour-joining method using uncorrected nucleotide p-distances using the complete deletion option. The reliability of the resulting topology was tested by both the bootstrap and the interior-branch test methods, producing the BP and CP values, respectively, for each interior node after 1000 replicates . The tree was rooted with the H2A.Ze sequence from sea urchin, representing an early chordate in which H2A.Z-1 and H2A.Z-2 variants are not yet differentiated .
cyan fluorescent protein
Dulbecco's modified eagle medium
linear ion quadrupole trap Fourier transform
polyacrylamide gel electrophoresis
polymerase chain reaction
sodium dodecyl sulphate
selected ion chromatogram
yellow fluorescent protein.
We thank Andra Li and Lindsay Frehlick for reviewing the manuscript. This work was supported by a Canadian Institute of Health Research (CIHR) grant MOP-97878 (JA), a National Institute of Health Grant, GM 37537 (DFH), a CIHR grant (MH) and by a contract within the Ramon y Cajal Subprogramme (MICINN, Spain) (JME-L). MH is an Alberta Heritage Foundation for Medical Research Senior Scholar.
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