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.