Biochem. J. (2013) 449, 567–579 (Printed in Great Britain)
567
doi:10.1042/BJ20121646
REVIEW ARTICLE
Organizing the genome with H2A histone variants
Catherine B. MILLAR1
Faculty of Life Sciences, Michael Smith Building, University of Manchester, Manchester M13 9PT, U.K.
Chromatin acts as an organizer and indexer of genomic DNA and
is a highly dynamic and regulated structure with properties directly related to its constituent parts. Histone variants are abundant
components of chromatin that replace canonical histones in a subset of nucleosomes, thereby altering nucleosomal characteristics.
The present review focuses on the H2A variant histones, summarizing current knowledge of how H2A variants can introduce chem-
ical and functional heterogeneity into chromatin, the positions
that nucleosomes containing H2A variants occupy in eukaryotic
genomes, and the regulation of these localization patterns.
INTRODUCTION
between three and nine residues that differ among the canonical
H2As. It is not known whether these small sequence differences
functionally distinguish classes of canonical H2As, but they are
not regarded as histone variants because their cognate genes are so
closely related.
Canonical and variant histones: what’s the difference?
Packaging the eukaryotic genome relies on the organization of
DNA into nucleosomes, an octameric core of histone proteins
around which the genetic material is wound. Nucleosomes are
the simplest subunits of chromosomes, and the properties of
chromatin fibres are influenced by the chemical identity of the
proteins within each nucleosome particle, which usually includes
two molecules of each of the canonical core histones: H2A, H2B,
H3 and H4. These highly conserved proteins are essential in all
eukaryotes and are encoded by multiple genes, often physically
located in clusters. Their expression is timed to coincide with
the major task of packaging the newly synthesized genome
during S-phase (see [1] for a review of canonical histone gene
organization and regulation). In addition to these canonical
histones, separately encoded variant histones are present in
eukaryotic cells. Their cognate genes are evolutionarily distinct
from the canonical histone genes and they are physically located
outside the replication-dependent histone clusters. Histone variant
expression is not restricted to S-phase and protein levels are
much lower than the canonical histones (∼ 5–10 % [2]). Like
the canonical histones, variants are generally highly conserved
between species, although some variants have a restricted species
distribution (see the section below). Importantly, canonical and
variant histone proteins differ in their amino acid sequences
(Figure 1A). This sequence divergence can be significant, with
over half of all residues differing between H2A and its variant
H2A.B (previously called H2A.Bbd), and is particularly notable
in the light of the overall high conservation of histone proteins.
Variant histones are generally denoted by a prefix or suffix to the
histone name, e.g. mH2A (macroH2A) and H2A.B (see [3] for
recommendations on variant histone nomenclature).
Some sequence differences also exist between proteins encoded
by different copies of the canonical histone genes. However, these
are generally conservative changes restricted to a small number
of residues. For example, approximately half of the canonical
H2A proteins in humans have a threonine residue at position 16,
whereas the other half have a serine residue, and this is one of
Key words: chromatin, H2A.B, H2A.X, H2A.Z, histone variant,
mH2A.
Variants of H2A: types and species distribution
Of the four core histones, variants of H3 and H2A are the most
common, with the H2A family containing the highest number of
variant forms. All eukaryotic genomes contain genes encoding
both canonical and variant H2As, with the number of variants
generally increasing with evolutionary complexity (Figure 1B).
Budding and fission yeasts each have one separately encoded H2A
variant, H2A.Z, and H2A.X function is provided by the canonical
H2A in these species. Mammalian genomes have a separate gene
encoding H2A.X, as well as multiple genes encoding H2A.Z,
mH2A and H2A.B. mH2A and H2A.B, although not ubiquitous in
eukaryotes, are found in vertebrates and may have been present
in the ancestor of all animals [4]. Some exceptions to this general
distribution are known, with H2A.X absent from the nematode
lineage and present as a fusion with the canonical H2A in yeasts
or with H2A.Z in Drosophila.
In addition to these four near-ubiquitous H2A variants, a
handful of lineage-specific H2A proteins have been identified.
Examples include Tetrahymena thermophila H2A.Y, which has
a long non-histone N-terminus that regulates H3 S10ph during
mitosis [5], and plant H2A.W proteins containing C-terminal
SPKK motifs [3]. Although these variants are likely to represent
interesting evolutionary solutions to particular environmental
niches, they have not been studied in detail and only the more
generally distributed and conserved H2A.X, H2A.Z, mH2A and
H2A.B will be discussed in the present review.
Variant-specific sequences affect nucleosome properties
The overall histone fold architecture is preserved in all variants
(Figure 1), allowing them to form nucleosomes that differ
surprisingly little in structure from canonical nucleosomes,
Abbreviations used: ChIP, chromatin immunoprecipitation; ES, embryonic stem; HOX, homeobox; HP1, heterochromatin protein 1; IL, interleukin; mH2A,
macroH2A; NDR, nucleosome-depleted region; pol II, polymerase II; SAHF, senescence-associated heterochromatic foci; TSS, transcription start site;
U-PAR, urokinase-type plasminogen activator receptor; Xi, inactive X.
1
email [email protected]
c The Authors Journal compilation c 2013 Biochemical Society
568
Figure 1
C. B. Millar
Features and species distributions of H2A variants
(A) Alignment of mH2A (mH2A.1), H2A.Z (H2A.Z.1), H2A.X, canonical H2A (H2A.C) and H2A.B (H2A.B.1) protein sequences produced using ClustalX and displayed with JalView. The sequences
are ordered by similarity. α-Helical regions are boxed in blue, and residues contributing to the acid patch are boxed in pink. The M6 region of H2A.Z is indicated with a thick pink line. For clarity,
the non-histone portion of mH2A is not shown in the alignment. (B) Schematic diagram of H2A variant protein sequences drawn to scale. The α-helical regions of each H2A variant and the mH2A
macro domain are represented as boxes. The number of genes encoding each H2A type in the yeast (S. cerevisiae ) and human genomes are shown at the right-hand side. In yeast, both canonical
H2A genes encode proteins with the H2A.X SQELF motif at the C-terminus, but there is no separate H2A.X gene, hence 0 (2).
despite significant sequence diversity [6,7]. Notwithstanding this
overall structural conservation, variant-containing nucleosomes
display differences in their stabilities and biochemical properties,
and arrays composed of variant nucleosomes have different
tendencies to fold into compact structures (reviewed in [8,9]).
Certain regions within H2A proteins appear to be particularly
important for nucleosome properties.
One key region that lies on the surface of the nucleosome
core particle is an acidic patch made up of a cluster of
negatively charged residues, the majority of which are contributed
by H2A [10]. This acidic patch is important for folding of
nucleosome arrays and some H2A variants alter the size
of the negatively charged region because of sequence differences
(Figure 1A). H2A.Z-containing nucleosomes have an extended
acidic patch that causes nucleosome arrays to be more compact,
whereas H2A.B has a truncated acidic patch and a consequent
decrease in nucleosome array folding [11,12]. Sequence changes
between variants can therefore have a direct effect on chromatin
conformation. The acidic patch has also emerged as an
important docking site for a number of proteins, including HP1
(heterochromatin protein 1) and IL (interleukin)-33 (reviewed in
[13]). The binding of some of these proteins is dependent on the
identity of the H2A protein; for example IL-33 binds nucleosomes
containing H2A or H2A.Z, but not H2A.B, because of differences
in the acidic patch [14]. Varying the H2A protein is therefore a
way to regulate the binding of proteins to the nucleosome surface
and presumably also to other divergent regions.
Outside of the histone fold, sequence differences between H2A
and its variants are prevalent in the N- and C-terminal tails. The Cterminal tail in particular appears to be an important determinant
of H2A variant identity, and both the length and the sequence
are highly divergent [15]. mH2A is the most extreme example
due to the presence of a basic region and a macro domain that
c The Authors Journal compilation c 2013 Biochemical Society
together are approximately twice the size of the histone portion of
the protein (Figure 1B). This C-terminal extension reduces access
to linker DNA [16] and can influence the binding of remodelling
enzymes [17]. H2A.B conversely has a shorter C-terminus than
canonical H2A (Figure 1), which means that H2A.B-containing
nucleosomes wrap only 118 bp of DNA instead of 146 bp and are
less stable [18]. Both H2A.X and H2A.Z have similar length
C-terminal tails to canonical H2A, but these are divergent at
the sequence level (Figure 1A). The C-terminal tail of H2A.X
includes a number of phospho-acceptor sites that are integral to
the function of H2A.X in the DNA-damage response (reviewed
in [19]), whereas the C-terminus of H2A.Z is important for its
function in budding yeast, flies and human cells [20–22]. Genetic
experiments in fission yeast have also shown that the H2A.Z Nterminal tail is important as its removal affects H2A.Z function,
most probably due to the removal of four acetylation sites [23].
In budding yeast, part of the N-terminus is also required for an
interesting extra-chromatin role of H2A.Z in correctly targeting
the Mps3 (monopolar spindle 3) protein to the inner nuclear
membrane [24]. The roles of the N-termini of other H2A variants
have not yet been examined, but their sequence divergence
indicates that they may be important for variant-specific functions.
Varying the H2A variants
Sequence diversification between H2A variants means that
changing the H2A protein in the nucleosome alters the chemical
environment, thereby affecting nucleosome characteristics and
influencing the binding of other chromosomal proteins. Other
mechanisms that can produce altered H2A variant proteins
therefore have the potential to further diversify nucleosome
function.
Organizing the genome with H2A histone variants
Table 1
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The human complement of H2A variants
The human genome contains 1–3 genes encoding each H2A variant, as well as numerous pseudogenes that are not described here. Some of these genes produce multiple splice isoforms and
consequently multiple proteins. The expression of some genes or splice isoforms is tissue-specific, although not all tissues have been tested. Phenotypes indicate the phenotypes of mouse knockouts
(N.D., not determined). Although multiple proteins are produced for some variants in mammalian cells, the location data does not distinguish between these subtypes. Locations are a compilation of
all reported enriched regions (details and references are in the text), some of which have yet to be verified in more than one species. For H2A.X the locations include both H2A.X and H2A.Xph.
Variant Genes
Protein isoforms
Expression
Phenotypes
Genomic locations
H2A.Z
H2A.Z.1
H2A.Z.1
Widespread [160–162]
Lethality [25]
Promoters of pol II genes and rRNA genes, flanking tRNA genes,
enhancers, transposons, pericentric heterochromatin, other
heterochromatic regions.
H2A.Z.2
H2A.Z.2.1
H2A.Z.2.2
Widespread [160–162]
Brain, skeletal muscle [21,22]
N.D.
H2A.X
H2A.X (two
transcripts) [163]
H2A.X
Widespread [164,165]
Male sterility, genomic
instability [106]
Telomeric heterochromatin, silent mating type heterochromatin, XY body,
sites of DNA damage, tRNA genes, replication origins, rDNA,
transposons; pol II genes.
mH2A
mH2A.1
mH2A.1.1
Differentiated cells [28,29]
Female-specific hepatic
steatosis [39]
Xi, gene-coding sequences and distal upstream regions, large domains,
TSS (differentiated cells).
mH2A.2
mH2A.1.2
mH2A.2
Widespread [28,29]
Widespread [96]
H2A.B1
H2A.B2
H2A.B3
H2A.B1
H2A.B2
H2A.B3
Testis and other tissues [108,166,167]
H2A.B
N.D.
N.D.
N.D.
N.D.
In most vertebrates, H2A variants are encoded by multiple
genes, which often encode different proteins, e.g. mH2A.1 and
mH2A.2 (Table 1). The differences between these proteins are
generally fewer than the differences between them and H2A.
However, genetic evidence indicates that some of these may
play unique roles, as a knockout of the H2A.Z.1 gene (h2afz)
is lethal in mice even though the H2A.Z.2 gene (h2afv) is
present [25]. Protein sub-functionalization may have occurred
after gene duplication, giving the closely related proteins distinct
roles. Alternatively, different expression patterns may underlie the
unique functions for variant gene isoforms and there is evidence
for tissue-specific expression for multiple variant histone isoforms
(Table 1). Either way, the presence of multiple genes is one
mechanism that facilitates further diversification of H2A variants.
In addition to the presence of multiple genes, downstream
processing steps can result in different mature variant proteins.
Unlike the canonical histone genes, some variant genes contain
introns in higher organisms and alternative splicing has been
shown to produce two forms each of mH2A.1 and H2A.Z.2
(Table 1). The H2A.Z.2 gene can produce a minor splice isoform,
H2A.Z.2.2, that encodes a protein with an alternative shorter
C-terminus. This splice isoform is present predominantly in the
brain, and the protein forms nucleosomes that are less stable than
those containing the full-length H2A.Z.2.1 [21,22]. mH2A.1.1
and mH2A.1.2 proteins differ in a short stretch of amino acids
between the histone and macro domains due to alternative splicing. Only mH2A.1.1 can associate with metabolites of NAD, and
this activity is responsible for the recruitment of mH2A.1.1 to sites
of DNA damage and can regulate the conformation of the macro
domain [26,27]. mH2A.1.1 expression is more restricted than that
of mH2A.1.2, being present predominantly in adult differentiated
cells [28,29]. However, alterations to this expression pattern have
been observed in human cancers, where changes to alternative
splicing of the gene encoding mH2A.1 lead to a reduced level of
mH2A.1.1 [30]. The ADP-ribose-binding function of mH2A.1.1
is required for suppression of lung cancer cell proliferation,
indicating that it is loss of this function that contributes
to carcinogenesis when mH2A.1.1 levels are reduced due to
alterations in splicing patterns [30]. Alternative splicing therefore
represents an important way to alter H2A variant functions.
− 1 nucleosome (testis, H2A.Lap1), coding regions (HeLa, H2A.B).
In organisms where there is only a single gene encoding a
H2A variant, a number of chemically distinct proteins can still
be produced. Again, alternative splicing may be used, as has
been found for the H2A.Z transcript in the tunicate Oikopleura
dioica, resulting in proteins containing variable numbers of
N-terminal lysine residues, thereby potentially regulating the
extent of acetylation in this organism [31]. The H2A.Z N-terminus
is also subject to post-synthetic processing, which produces two
forms of the protein in Schizosaccharomyces pombe, one of
which lacks the first two methionine residues [32]. Most other
forms of post-translational regulation involve the addition of
modifications, such as acetylation, methylation, phosphorylation
and ubiquitination. In some cases, such as phosphorylation of
residues within the C-terminus of H2A.X, these modifications are
key to the function of the protein and act through recruitment of
other proteins [19]. Other modifications may alter the biophysical
properties of the nucleosome, with H2A.Z acetylation affecting
nucleosome stability [33]. A more detailed discussion of H2A
variant post-transcriptional modifications is given in [34].
Additional H2A variant isoforms, produced through altered
RNA or protein metabolism, are likely to exist, which may play
ever more specialized roles in chromatin regulation. The general
theme seems to be that evolution continues to act on H2A variants
to produce further variant forms, and the examples so far implicate
the non-histone portion of mH2A and the N- and C-terminal
tails of H2A.Z as functionally important. The fact that the H2A
family has diversified to include highly conserved variant proteins
indicates that these variants play important roles in chromatin
organization. Indeed, deletions of genes encoding H2A variants
generally have severe consequences, with H2A.Z essential for
viability in organisms from nematodes to mice [25,35–37]
and single mH2A gene deletions causing severe developmental
defects in zebrafish and metabolic defects in mice [38,39]. As
the importance of H2A variants derives from the effects they
have on nucleosomal properties, a key to understanding their
roles is to map the nucleosomes that contain H2A variants across
whole genomes. Genome-wide studies have been instrumental in
mapping the distributions of histone modifications as a first step to
understanding their functions [40,41] and recent work has begun
to describe the distribution of H2A variants in a similar manner.
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Figure 2
C. B. Millar
Summary of H2A variant locations in eukaryotic genomes
(A) Illustration of H2A variant distributions around pol II-transcribed genes in mammalian genomes. The start and end of an average gene are depicted, with the transcription start (TSS) and
termination (TTS) sites indicated. Representation of enrichment patterns for mH2A, H2A.B and H2A.Z are shown at active and inactive genes in differentiated cells. Based on data from [50,90,92].
(B) mH2A distribution at genes in stem cells [38,91]. H2A.Lap1 and H2A.Z distributions in mouse round spermatids [57]. (C) H2A.Z and H2A.Xph distributions in the yeast genome are non-overlapping.
A schematic diagram of S. cerevisiae chromosome 3 is shown, with the heterochromatic regions shaded. Enrichment of H2A.Z and H2A.Xph are depicted, with H2A.Z being restricted to euchromatin,
whereas H2A.Xph is enriched in heterochromatin and a few euchromatic peaks that correspond to replication origins. The peaky distribution of H2A.Z corresponds to enrichment around TSSs and
other features when viewed at higher magnification. Based on data from [55,104].
THE H2A LANDSCAPE OF THE GENOME
H2A.Z localization has general and cell-type-specific features
Theoretically, H2A variants could occupy nucleosomes randomly,
with prevalence proportional to the amount of variant protein, or
they could be enriched in certain genomic locations where they
are functionally important. In fact, H2A variant nucleosomes
occupy distinct locations in genomes relative to genes and
other features. For most H2A variants, localization to specific
genomic compartments was initially determined at low resolution
by microscopy and has more recently been examined at high
resolution by ChIP (chromatin immunoprecipitation). ChIP
followed by identification of immunoprecipitated DNA fragments
by microarray hybridization or high-throughput sequencing has
revealed the genomic localizations of many H2A proteins.
Although these studies are limited by the fact that they present
a snap-shot of nucleosome occupancy in a cell or tissue at a
particular time point, and nucleosomes can be highly dynamic,
general localization patterns emerge, particularly when variants
are examined in multiple species.
H2A.Z localization has been examined in representative
organisms from multiple eukaryotic kingdoms (fungi, animalia
and plantae), revealing a conserved localization pattern at the 5
ends of genes across vast distances of evolutionary time [42–
53]. In higher resolution studies, this 5 -end-enrichment resolves
to occupancy in individual nucleosomes surrounding the TSS
(transcription start site) (Figure 2A) [49,50,54,55]. There are
some species-specific differences in the relative abundance of
H2A.Z in the + 1 and − 1 nucleosomes, with the − 1 nucleosome
enriched in Saccharomyces cerevisiae and Homo sapiens, but
not in Drosophila melanogaster or S. pombe [47,56]. Different
tissues within the same species may also turn out to have slightly
different patterns, as for example H2A.Z is absent from the
+ 1 nucleosome in mouse testis but not in other mouse cell
types that have been examined [51,57,58] (Figures 1A and 1B).
Some of these differences may reflect cell-cycle-related changes
in abundance of H2A.Z at TSS-flanking nucleosomes, which have
c The Authors Journal compilation c 2013 Biochemical Society
Organizing the genome with H2A histone variants
recently been observed in mouse trophoblast cells [59]. Away
from the TSS, H2A.Z levels in genes are lower in transcribed
regions (Figure 2A). H2A.Z is also enriched at gene enhancers in
human and mouse cells [50,52,58,60], and the high enrichment of
H2A.Z at genes and gene regulatory elements is the most generally
conserved feature of H2A.Z localization.
The relationship between H2A.Z occupancy and gene activity
levels is complex. H2A.Z levels are generally highest at the most
active genes in higher organisms, and a positive effect on gene
expression has been inferred because depletion of H2A.Z reduces
pol II (polymerase II) recruitment to chromatin [52]. Exceptions
to this general genome-wide trend exist for some genes and in
some cell types. H2A.Z appears to repress transcription at p21
[61], U-PAR (urokinase-type plasminogen activator receptor)
[62] and at Np63α target genes [63] in human cell lines. H2A.Z
is absent from stably repressed promoters in ES (embryonic stem)
cells [58], but it is enriched at many inducible promoters while
they are inactive and is required for activation of these genes
during differentiation [51]. This is similar to the effect of H2A.Z
removal on inducible genes in budding yeast, e.g. GAL1, where
H2A.Z is required for normal induction kinetics [64–66]. At the
genome scale in yeast, H2A.Z-containing nucleosomes are not
positively correlated with gene expression [32,42,43,45,48,54]
and H2A.Z deletion results in both up- and down-regulation of
genes, although some of these effects may be indirect [67]. The
different relationships between H2A.Z and gene activity may
represent alternative modes of gene regulation in different genes,
species or cell types, or may reflect subpopulations of H2A.Z
that are differentially marked by post-translational modifications
or associated with nucleosomes whose other components are
different. For example, acetylated isoforms of H2A.Z are more
enriched at active than inactive genes in yeast and vertebrate cells
[46,68,69] and a doubly modified form of H2A.Z that is acetylated
and ubiquitinated is found specifically at bivalent genes in mouse
ES cells [58]. These modified forms of H2A.Z may facilitate
distinct functions of H2A.Z that result in different transcriptional
outcomes.
Outside of pol II-transcribed genes, budding yeast H2A.Z is
found flanking numerous other genomic features [55], although
levels here tend to be lower than at genes (see Table 1 for a
summary of H2A.Z-enriched genomic regions). Occupancy in
some transposon classes has been observed in yeast, flies and
plants, although in Arabidopsis thaliana only transposons with
low levels of DNA methylation have H2A.Z enrichment [49]. This
anti-correlation between H2A.Z occupancy and DNA methylation
has also been observed in Fugu [70] and mammals [69], although
it is not a strong anticorrelation in mammalian cells [71]. Overall,
many different types of euchromatic features contain some level
of H2A.Z enrichment, but the significance of these H2A.Zcontaining nucleosomes has not yet been tested. H2A.Z has been
localized to heterochromatic regions in some organisms and cell
types. In both budding and fission yeasts H2A.Z is enriched near
telomeres [32,42], but is absent from telomeric heterochromatin
and in fact acts to prevent the spread of heterochromatic
factors into neighbouring euchromatin in S. cerevisiae [67].
Centromeric heterochromatin in S. pombe and A. thaliana also
lacks H2A.Z [32,44,49], but pericentric heterochromatin in mouse
cells from the early embryo and in mouse and human sperm
contains H2A.Z [72–74]. Indeed, high enrichment of H2A.Z is
restricted to pericentric heterochromatin in human sperm and the
protein is unusually absent from euchromatin in this cell type
[74]. Centromeric and pericentromeric heterochromatin differ
in their other chromatin components, e.g. CenH3 or H3K9me3
(histone H3 trimethylated on Lys9 ), and H2A.Z may preferentially
associate with only some classes of heterochromatin. Other
571
examples of H2A.Z occupancy in heterochromatin regions have
been documented in flies [75] and in human osteoscarcoma cells
[52], but overall it is not completely clear which subtypes of
heterochromatin contain H2A.Z. One confounding factor is that
many ChIP studies do not report a complete picture of heterochromatin localization, particularly in higher organisms where the
highly repetitive nature of some heterochromatic regions poses
problems because recovered DNA fragments cannot be attributed
to a unique locus. Lack of H2A.Z causes defects in chromosome
segregation in many organisms [76–78], which in mammalian
cells is due to a defect in HP1α recruitment to pericentric heterochromatin [78], indicating that H2A.Z is a functionally important
component of heterochromatin. Interestingly, levels of H2A.Z at
centromeric heterochromatin increase specifically during mitosis,
the time when the conformation of this region becomes critically
important [59].
ChIP experiments incorporating multiple time points have
revealed the dynamic nature of H2A.Z-containing nucleosomes.
Thus far these have generally been carried out at the single gene
level, e.g. FIG1, PHO5, p21 and U-PAR [40,54,61,62]. These
studies show that H2A.Z is removed from nucleosomes during
gene activation and re-incorporated during repression. H2A.Z
occupancy is also regulated in response to DNA damage and the
cell cycle [59,79,80]. The association of H2A.Z with nucleosomes
is therefore dynamic, and at the whole-genome level H2A.Z
occupancy correlates well with nucleosomes undergoing rapid
turnover in yeast [81]. Whether this is a consequence of H2A.Z
acting as a replacement histone at any nucleosomes undergoing
turnover, due to its cell-cycle-independent presence, or whether
H2A.Z is required for nucleosome dynamics is still unresolved.
However, it is clear that understanding the many roles of H2A.Z
will require the examination of H2A.Z enrichment over multiple
time points and in different cell types.
mH2A is abundant in heterochromatin and large euchromatic
domains
mH2A has historically been linked to heterochromatin and
was originally identified as an abundant component of the
Xi (inactive X) chromosome in female mammalian cells by
immunofluorescence and GFP (green fluorescent protein) tagging
[82,83]. mH2A also associates with the XY bodies that form
during male gametogenesis [84] and with SAHF (senescenceassociated heterochromatic foci), facultative heterochromatic
regions that form in senescent cells [85]. ChIP studies using
human and mouse cells and tissues have validated the presence
of mH2A on the Xi, with fairly uniform chromosome-wide
enrichment [86,87]. Despite this enrichment, mH2A appears to
be dispensable for X inactivation, as female mouse ES cells that
are depleted for both mH2A isoforms do not have apparent defects
in X inactivation [88]. Single gene deletion of mH2A.1 in mice
similarly allows normal X inactivation [89]. It may be that a
small amount of mH2A persists after RNAi (RNA interference)
depletion or deletion of one of the two genes, and that this
is sufficient for X inactivation. Alternatively, mH2A may act
redundantly with other chromatin marks to maintain the Xi in
a silent state.
In addition to the well-known enrichment on the silent X
chromosome, mH2A is also present in euchromatin where it
is found in large (>500 kb) domains that are also enriched for
other repressive chromatin marks such as H3K27me3 (histone H3
trimethylated on Lys27 ) [90]. It is not known whether these large
domains are a feature of higher-order chromosome organization,
but their boundaries often coincide with promoter-proximal
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C. B. Millar
regions. Where mH2A is enriched on genes, it is present both
upstream and downstream of the TSS, including within coding
sequences (Figures 2A and 2B). In ES cells, mH2A is absent
from the region immediately adjacent to the TSS [38,91], whereas
in transformed cell lines mH2A is present at the TSS as well
as upstream and downstream [90,92]. mH2A levels are much
higher in differentiated cells than in pluripotent cells during
mouse embryogenesis, and mH2A acts as a barrier to induced
pluripotent cell reprogramming [93]. It has been proposed that
the different levels and distributions of mH2A in stem cells and
differentiated cells are an important reflection of differences in
their developmental plasticity [94].
mH2A is generally less enriched on active genes, but is present
at low levels on some active genes, including genes involved
in development and cell–cell signalling in human cells, and
lipid metabolism genes in mouse liver [38,87,90]. Genes that
have increased expression in mh2a.1 − / − mice are enriched for
mH2A.1 in mouse liver, indicating that mH2A.1 can act as a direct
repressor [87]. However, not all genes that have mH2A enrichment
are derepressed when it is absent. Indeed, in MCF7 cells, a
small number of genes are down-regulated upon knockdown of
mH2A1 [90,95]. mH2A also contributes to the fine-tuning
of temporal activation of HOXA (homeobox A) cluster genes
during neuronal differentiation [38]. Although the effect of mH2A
depletion on HOX gene expression is subtle, temporal regulation
during development is critical and, consequently, mH2A-depleted
zebrafish embryos display severe defects [38].
Most of the mH2A localization studies to date have been
performed using cells or tissues where mH2A.1 is the predominant
isoform, and some have used reagents specific for mH2A.1 so
that the results may apply only to mH2A.1 [86,91]. mH2A.1
and mH2A.2 are only ∼ 80 % similar at the protein level and
may therefore be targeted to distinct regions to facilitate different
functions. One study has compared mH2A.1 and mH2A.2 at
human gene promoters and found that they have distinct and
overlapping gene targets but that most genes have both isoforms
[38]. However, by immunofluorescence, mH2A.1 and mH2A.2
patterns are distinguishable [96], so it will be interesting to
test whether mH2A.1 and mH2A.2 are differentially targeted
to other genomic sequences, such as different heterochromatic
regions. It will also be of interest to discover whether the splice
isoforms of mH2A.1 that differ in their ability to bind NAD
metabolites occupy distinct genomic locations. It is known that
differently post-translationally modified forms of mH2A are
found at different locations, with mH2A.1 phosphorylated at
Ser137 specifically absent from the Xi [97]. Other modified
forms of mH2A.1 have been identified, including methylated and
ubiquitinated forms [98,99], which may also be restricted to
particular genomic regions.
H2A.X localizes to heterochromatin and collapsed replication forks
The analysis of H2A.X localization at the genome scale has lagged
behind that of the other H2A variants, possibly because most
studies have focused on the localized phosphorylation of H2A.X
that occurs at DNA lesions [100,101]. Another complicating
factor is that H2A.X function is provided by another H2A protein
in some key model organisms, including yeasts and Drosophila,
making analysis less straightforward.
In yeasts, the SQE/DØ consensus motif that is considered characteristic of H2A.X proteins is carried on the canonical H2A proteins. The serine residue within this motif can be phosphorylated,
and recent studies have mapped the phosphorylated form of H2A,
which can be equated to H2A.X (H2A.XS129/28ph), across the
budding and fission yeast genomes. These studies reveal that
c The Authors Journal compilation c 2013 Biochemical Society
H2A.Xph is found in heterochromatin [102–104], although in
fission yeast it is excluded from the centromeric core [102].
The heterochromatin proteins Sir3 and Swi6 are required for
H2A.Xph localization in heterochromatin in S. cerevisiae and
S. pombe respectively, and cells lacking H2A.Xph do not have
detectable defects in heterochromatin formation [102–104]. These
data suggest that H2A.Xph is downstream of heterochromatin
formation or redundant with other factors.
H2A.Xph is also present outside of heterochromatin in yeast,
with enrichment at tRNA genes, replication origins, rDNA, LTRs
(long terminal repeats) and some transposons and protein coding
genes. Many of these are sites that are prone to replication
fork stalling and approximately 30 % of H2A.Xph peaks can be
attributed to collapsed replication forks [103]. At genes, H2A.Xph
is negatively correlated with expression and its localization
at repressed genes is dependent on their lack of expression
[103]. However, it is not known whether H2A.X phosphorylation
regulates gene activity either positively or negatively.
In higher organisms, immunofluorescent staining has shown
that H2A.XS139ph is enriched in the heterochromatic XY
body in mouse sperm, where it is essential for chromosome
condensation and silencing [105]. Lack of H2A.X consequently
leads to male sterility in the mouse [106]. Only one genome-wide
ChIP study examining H2A.X and H2A.XS139ph localizations
in higher organisms has been carried out to date, where a
cell-type difference was detected. In resting T-cells, no peaks
of H2A.X were detected unless the cells were treated with
ionizing irradiation, whereas H2A.X enrichment was present in
sub-telomeric regions and at active TSSs in untreated Jurkat
cells [107]. Sub-telomeric H2A.X is phosphorylated in Jurkat
cells, similar to the pattern in yeasts. The levels of H2A.Xph
increased in T-cells upon treatment with radiation and H2A.Xph
became enriched in transcribed regions [107]. Although further
studies will add to our understanding of the profiles of bulk
and H2A.Xph enrichment before and after DNA damage, it
currently appears that H2A.X has a distinct localization pattern
from the other H2A variants (Figure 2C). The general pattern
of H2A.Xph is conserved between budding and fission yeasts,
and the sub-telomeric localization has also been reported in one
human cell line. Patterns of H2A.X are likely to be dynamic in
response to DNA damage and possibly other signals, and the
relationship between H2A.X, its phosphorylated isoforms and
heterochromatin deserves further attention.
H2A.B is enriched on active genes
In contrast with other histones, H2A.B is rapidly evolving and
appears to be restricted to mammals [108]. Human and mouse
genomes encode three and four H2A.B-like proteins respectively.
Genome-wide localization studies have been carried out for a
mouse H2A.B-like protein (H2A.Lap1) as well as human H2A.B.
H2A.Lap1 has one extra acidic residue compared with the other
H2A.B proteins, which allows nucleosome arrays containing
H2A.Lap1 to partially fold in vitro, while H2A.B arrays remain
uncompacted [12,57].
H2A.Lap1 is highly expressed in the testis, where it is present
during meiosis and in post-meiotic round spermatids [57]. ChIPseq analysis shows that H2A.Lap1 is enriched at the TSSs of
active genes in mouse round spermatids (Figure 2B). H2A.Lap1
occupancy in gene-coding regions is lower than at the TSS or distal
promoter regions, and no relationship to other genomic features
has been described. Although immunofluorescence shows that
H2A.Lap1 is found in the heterochromatic X and Y chromosomes
late in spermatid development, it is not present in constitutive
Organizing the genome with H2A histone variants
heterochromatin and X chromosome enrichment is higher on
active genes, indicating that H2A.Lap1 is an active mark.
Additionally, the levels of H2A.Lap1 at select genes increases
with levels of the cognate transcripts during a time course of
testis development [57]. The enrichment of H2A.Lap1 over the
TSS is distinct from all other variants, and indeed other histone
marks, as this region is usually depleted of nucleosomes.
ChIP-seq analysis of human H2A.B stably expressed in HeLa
cells shows a different pattern to H2A.Lap1, with depletion from
TSSs and enrichment in gene-coding regions (Figure 2A) [92].
Highly expressed genes have more H2A.B, which is also enriched
at intron–exon boundaries. H2A.B co-purifies with a number of
splicing factors, and depletion of the protein leads to an increase
in retention of intronic sequences [92]. These data point to a role
for H2A.B in gene splicing through localization at gene-coding
sequences. Although H2A.B and H2A.Lap1 are highly related
proteins, the mapping data for these two isoforms show that they
have dramatically different localizations. This may be attributable
to tissue-specific functions for H2A.Lap1, or alternatively the
presence of an extra acidic residue in H2A.Lap1 compared with
H2A.B may change the properties and distribution of the protein.
These two studies indicate that the rapid evolution of H2A.Blike proteins or their cellular context can greatly influence their
binding profiles.
Nucleosome occupancy by H2A variants can be homotypic or
heterotypic
A further nuance of H2A variant localization is that both
H2A proteins in the nucleosome can be of the same type
(homotypic) or there may be two different types of H2A
protein in a single nucleosome (heterotypic). It is known that
heterotypic H2A.Z–H2A nucleosomes exist in vivo [109–111]
and homotypic H2A.Z nucleosomes are relatively more enriched
at TSSs in S. cerevisiae and D. melanogaster [110,111].
In mouse cells, H2A.Z-containing nucleosomes at promoters
switch between homo- and hetero-typic states at different cellcycle phases [59]. Homotypic nucleosomes have biochemical
properties that are similar to active chromatin [111], raising the
possibility that homo- and hetero-typic H2A.Z nucleosomes could
have different properties in vivo. Little is known about whether
the other H2A variants form heterotypic nucleosomes in vivo,
although mH2A preferentially forms heterotypic nucleosomes
with H2A [112] and H2A.B can also form heterotypic
nucleosomes in vitro. Further studies of the H2A landscape of the
genome, where homo- and hetero-typic nucleosome combinations
are examined, are likely to reveal further interesting patterns of
H2A variant occupancy.
Collectively, studies of H2A variant occupancies reveal that
different variants have distinct patterns of genome-wide
localizations (Figure 2 and Table 1). H2A.Z and mH2A are
each found in both heterochromatin and euchromatin, but their
localization patterns are different both around genes and in
heterochromatic regions. Despite a generally mutually exclusive
pattern, both H2A.Z and mH2A have been localized to HOX
genes in mouse ES cells [38,51], indicating that they may cooccupy heterotypic nucleosomes at the HOX locus in vivo to
establish a unique chromatin structure. However, further work
is required to establish co-localization of H2A.Z and mH2A, as
similar enrichment patterns could also represent enrichment in
neighbouring nucleosomes or at the same positions in different
cells in the population. mH2A and H2A.X generally associate
with less-transcribed genomic regions, including heterochromatin
and repressed genes, whereas H2A.Z and H2A.B are more
573
enriched at active genes in mammalian cells, indicating that half
of the H2A variants are predominantly associated with gene
repression or silencing while the other two are active marks.
Interestingly, H2A.B localization at genes is drastically different
in two different cell types (Figures 2A and 2B), although the
protein is still associated with active genes and the differences
may also be due to subtle differences in protein sequence between
the two H2A.B isoforms studied to date. H2A.X is predominantly
localized to heterochromatin in yeast and human cells, and both
H2A.Z and mH2A also localize to heterochromatic regions in
human cells. In yeast, where the mapping of heterochromatic
regions is more straightforward, it can be seen that the two H2A
variants present, H2A.X and H2A.Z, predominantly lack any
overlap in their distribution (Figure 2C). It will be of great interest
to map multiple variants in single cell types in higher organisms to
reveal the full extent of their unique and overlapping distributions.
REGULATION OF H2A VARIANT PATTERNS
How are the patterns of H2A variant occupancy in different
genomic loci established and maintained? For this to happen,
H2A variants must be delivered to the nucleus and assembled into
or removed from the appropriate nucleosomes at the correct time.
To generate specific patterns, cells must be able to distinguish
between different H2A variants and indeed regions of the proteins
that are important for targeting have been identified in some cases.
Two classes of proteins, histone chaperones and ATP-dependent
remodelling complexes, have been closely linked to H2A–H2B
assembly and disassembly. Histone chaperones (reviewed in
[113–115]) are involved in several aspects of histone metabolism
and act to partner histones in various subcellular compartments.
ATP-dependent remodelling complexes (reviewed in [116,117])
use energy from ATP hydrolysis to move, remove or assemble
nucleosomes and a subset of these enzymes are involved in
regulating H2A variant nucleosomal occupancies.
Histone chaperones: essential partners but conferring little
specificity
Histone chaperones are proteins that partner histones and
they act, in general, by blocking the interaction sites of the
histones in the nucleosome core particle (reviewed in [118]).
Chaperones are involved in pre-deposition functions, such as
nuclear import, and have post-deposition roles in eviction and
recycling of histones. Both pre- and post-deposition roles could,
hypothetically, be important in regulating the H2A variant
composition of chromatin, as variants could be delivered to
the nucleus in a regulated way by chaperones to enhance
assembly, and chaperones could also determine whether variants
are removed at a certain time point. However, it is difficult
to imagine how chaperones could specify genomic localization
unless their actions are restricted to one or a few variants.
Biochemical and structural studies show that H2A–H2B
chaperones tend to make extensive contacts with both the H2A
and H2B proteins in the dimer [119,120] and any specificity
determinants that might be contributed by H2A variants have
not been identified. The chaperone with the best-characterized
specificity for a single H2A variant is the yeast protein Chz1,
which preferentially binds H2A.Z over H2A and chaperones
H2A.Z in the nucleus [121]. Another chaperone with preferential
activity is nucleolin, which can stimulate the remodelling of
mH2A or H2A-containing nucleosomes, but not those containing
H2A.B [122]. Some chaperones may partner with a number
of H2A variants but not all of them; for example Nap1 can
c The Authors Journal compilation c 2013 Biochemical Society
574
Table 2
C. B. Millar
Known assembly and disassembly factors for each H2A variant
SWR-C assembly activity has been demonstrated in multiple species, including mammals, where
there are two forms of this complex. Apart from ATRX, which is a proposed mH2A disassembly
factor in human cells, all other disassembly factors have been demonstrated only in budding
and/or fission yeasts.
H2A variant
Assembly factor
Disassembly factor
H2A.Z
SWR-C
INO80-C
Fun30*
H2A.X
?
SWR-C
INO80-C
mH2A
?
ATRX*
H2A.Bbd
?
?
*In vitro confirmation of disassembly activity has yet to be shown for Fun30 (H2A.Z) and
ATRX (mH2A).
facilitate removal of dimers containing H2A and H2A.Z, but
not mH2A from nucleosomes [123]. Exhaustive challenges of
chaperone specificities for different H2A proteins have not yet
been performed and it is possible that other chaperones with
preferences for single variants or specific subsets exist.
Even when variant-specific chaperones such as Chz1 are
known to operate, it appears that they do not determine variant
nucleosome patterns, as the enrichment of H2A.Z at several target
genes is not altered in the absence of Chz1 and Nap1 [121]. This
may be due to redundancy in the chaperoning system, with FACT
substituting as a H2A.Z chaperone when Nap1 and Chz1 are
absent [121]. It therefore seems that although histone chaperones
are essential for the normal metabolism of H2A variants, they are
not the prime determinants of the localization of variant proteins.
However, there is still a lot of work to be done to determine how
the delivery of H2A variant dimers to chromatin is regulated by
chaperone proteins.
ATP-dependent remodelling enzymes regulate H2A variant
occupancy in nucleosomes
ATP-dependent chromatin remodellers can be divided into several
categories on the basis of sequence homology and the structural
organization of their catalytic subunit [124]. A few different
subtypes have activity on H2A–H2B dimers, and in particular
members of the ‘split ATPase’ group of remodellers, Swr1
[125] and Ino80 [126], can remove H2A–H2B dimers from
nucleosomes in vitro. Other classes of remodellers, such as
SWI/SNF and RSC also have some H2A–H2B exchange activity
in vitro [127], although this involves moving nucleosomes off
the end of a short piece of DNA, a mechanism that is unlikely
to be used in vivo. The relationship between ATP-dependent
remodellers and the regulation of each H2A variant’s occupancy
in nucleosomes is discussed below and summarized in Table 2.
H2A.Z
The first example of an ATP-dependent remodelling enzyme
showing variant dimer exchange activity was the Swr1 enzyme in
budding yeast, which can swap nucleosomal H2A–H2B dimers
for H2A.Z–H2B dimers in vitro [125]. Swr1 is the catalytic
subunit of a multiprotein complex (SWR-C) that is evolutionarily
conserved and has been biochemically verified in S. pombe,
A. thaliana, D. melanogaster and H. sapiens (reviewed in
c The Authors Journal compilation c 2013 Biochemical Society
[128]). Importantly, the in vivo nucleosomal occupancy pattern of
H2A.Z is dependent on the SWR-C, as ChIP experiments show
reduced H2A.Z occupancy in the absence of SWR-C members
[43,61,125,129]. In H. sapiens, there are two complexes that
contain different Swr1-like subunits, SRCAP or p400 [130,131].
Intriguingly, a smaller SRCAP complex lacking the catalytic
subunits p400 and SRCAP has been isolated from HeLa cells and
found to have H2A.Z deposition activity in vitro [132], indicating
that other ATPase proteins within the SWR-C may also have
dimer-exchange capabilities.
The specific recognition of H2A.Z by the SWR-C in yeast
requires a region near the C-terminus of H2A.Z [133] that is
well conserved in H2A.Z orthologues but different in other
H2A proteins. This M6 region [20] (Figure 1A) is required for
the association of H2A.Z with the SWR-C and most probably
underlies the specificity of the SWR-C for H2A.Z over other
variant types [133,134]. Interestingly, the M6 region includes
residues contributing to the extended acidic patch of H2A.Z that
is important for protein interactions and chromatin compaction,
which may mean that deposition and altered function have been
co-selected within this portion of the H2A.Z sequence.
For the SWR-C to generate a specific pattern of H2A.Zcontaining nucleosomes, the complex must be recruited to those
nucleosomes, which in turn need to be good substrates for
the deposition reaction. Genome-wide ChIP experiments show
that subunits of SWR-C are enriched at the + 1 and − 1
nucleosomes in yeast [135], and in human cells SRCAP is found
at promoters [136], meaning that the SWR-C stably binds at least
a subset of sites where H2A.Z is highly enriched. Factors that
affect the targeting of SWR-C have generally not been directly
tested by examining SWR-C binding, but rather by measuring
H2A.Z enrichment. Although this is a good readout of SWR-C
recruitment, it is also a product of SWR-C activity as well as any
disassembly that may occur at that nucleosome. Such experiments
indicate that acetylation sites in H3 and H4, various HATs, and
the DNA sequence of the adjacent NDR (nucleosome-depleted
region) can all influence H2A.Z occupancy in yeast [48,54,137].
DNA sequence determinants include binding sites for specific
transcription factors, such as Reb1, but also sequences that aid
in the formation of NDRs. The NDR has been proposed to
regulate the activity of the SWR-C and NDR formation depends
on the activity of the RSC remodelling complex, thus RSC
may indirectly regulate H2A.Z occupancy [137]. In mammalian
cells, H2A.Z deposition is dependent on the activation of some
transcription factors including p53, c-Myc [61], ER (oestrogen
receptor) α [138] and Np63α [63], although only Np63α has
been shown to interact with members of the mammalian SWR-C
[63].
While targeting the recruitment of the SWR-C is likely to
be the major regulatory step in determining which nucleosomes
are destined to contain H2A.Z, the components of the substrate
nucleosome can also affect the reaction. The ATPase activity of
Swr1 is stimulated by nucleosomes containing H2A [110] and
acetylation of nucleosomal histones H4 and H2A stimulates the
incorporation of H2A.Z by the SWR-C in vitro [139]. This implies
that the effect of acetyltransferase mutants on H2A.Z occupancy
may be through the regulation of SWR-C activity rather than
recruitment of the complex, and that both enzyme recruitment
and regulation are key to establishing a pattern of H2A.Z in the
genome.
Although assembly of H2A.Z is a critical first step in
establishing nucleosome-specific occupancies, removal of H2A.Z
from nucleosomes will also contribute to the steady-state pattern.
This is analogous to the regulation of histone modifications
(e.g. acetylation) by enzymes that add (acetyltransferases)
Organizing the genome with H2A histone variants
and remove (deacetylases) the modifications. Some H2A.Z
removal may be passive, due to the general disruption of
nucleosomes that occurs during gene transcription and other DNA
operations [140]. However, another remodelling complex with
a split-ATPase family catalytic subunit, the INO80 complex
(INO80-C), has been shown to specifically remove H2A.Z–H2B
dimers from nucleosomes, replacing them with H2A–H2B dimers
to reconstitute canonical nucleosomes [126]. In vivo, G1 -arrested
yeast cells have an altered pattern of H2A.Z when Ino80 is
absent, with less enrichment at promoter nucleosomes and a
corresponding increase in H2A.Z occupancy in gene-coding regions. ChIP-seq experiments confirm that Ino80 binds throughout
gene-coding regions [141], where it could act to remove H2A.Z
from nucleosomes. However, Ino80 is also localized to promoter
nucleosomes [135], indicating that a competition with SWR-C
may occur to establish steady-state H2A.Z occupancy levels.
INO80 complexes are conserved in mammalian cells [142] and
it will be interesting to learn whether mammalian INO80-C also
‘prunes’ H2A.Z from gene-coding regions.
A third ATP-dependent remodelling enzyme, Fun30, which is
related to Swr1 and Ino80 [124], has been linked to the regulation
of H2A.Z nucleosomal occupancies. Budding yeast cells lacking
Fun30 have a disrupted H2A.Z pattern similar to that seen in the
absence of Ino80, with reduced occupancy in nucleosomes near
TSSs and increased localization in coding sequences [143]. In
fission yeast, deletion of the Fun30 orthologue Fft3 results in an
increased occupancy of H2A.Z at centromeres and sub-telomeric
regions [144]. Fun30 may therefore also act to remove H2A.Z,
although in vitro experiments are needed to demonstrate activity
on H2A.Z-containing nucleosomes.
mH2A
Several factors have been shown to be important for mH2A
localization to heterochromatic regions. However, unlike the case
for SWR-C and H2A.Z, it is not clear whether these requirements
are direct, through an assembly activity, or if the factors act further
upstream of mH2A incorporation. For example, the localization
of mH2A to SAHF depends on the chaperones ASF1a and HIR1
[85], but ASF1 and HIR1 are primarily H3–H4 chaperones and
have not been shown to bind mH2A so this is likely to be an
indirect effect. Similarly, mH2A enrichment on the Xi requires
the Xist RNA [145], but this is likely to be an upstream requirement
rather than an assembly mechanism. Recently, a number of
proteins co-purifying with soluble mH2A.1, and which could
therefore be good candidates as assembly factors, were identified
[146]. These included the ATPase protein ATRX, which is in
the Rad54-like family of remodellers and not closely related
to Swr1/Ino80/Fun30. Although no direct biochemical assembly
or disassembly activity was demonstrated, depletion of ATRX
from cells resulted in an increased association of mH2A with
chromatin, indicating that ATRX is either required for the removal
of mH2A from chromatin or acts to inhibit its deposition [146].
Features of the mH2A protein that are important for its
normal localization in chromatin have been studied by examining
enrichment on the Xi. Both the non-histone C-terminal tail and
the core histone domain are required for mH2A.1 targeting to the
Xi [147]. Within the histone domain, several sequences including
a region corresponding to the H2A.Z M6 region are required
for mH2A.1 enrichment on the Xi [148]. Monoubiquitination of
mH2A may also be important for its association with, or retention
on, the Xi, as depletion of the CULLIN3/SPOP ubiquitin E3 ligase
that monoubiquitinates mH2A leads to mH2A dissociation from
the Xi [149]. It will be interesting to see whether the interaction of
575
ATRX and mH2A can be localized to an mH2A-specific sequence
and whether more activities that regulate mH2A localization will
be identified.
H2A.X
The mechanism of H2A.X assembly into nucleosomes has not
been studied in detail. In yeast, as H2A.X is also the canonical
H2A, deposition is replication-dependent and it is not known
what regulates H2A.X assembly in higher organisms. Although
the mechanism of H2A.X incorporation is not known, the last 23
residues of the protein are important for chromatin incorporation
during early embryogenesis [150]. In fact, these residues can
direct H2A.Z to be deposited during this stage when it is normally
absent, which indicates that the C-terminus of H2A.X may contain
a signal that directs its assembly into nucleosomes. A recent
proteomics screen carried out in mammalian cells identified
a number of proteins, including members of the Mi-2/NurD
remodelling complex, as H2A.X-specific interactors making them
candidates for proteins that directly regulate H2A.X nucleosome
occupancy [151].
Removal of H2A.Xph from nucleosomes after DNA damage in
yeast has been linked to the same enzymes that regulate H2A.Z
occupancy, Ino80 and Swr1 [79]. In Drosophila, phosphorylated
H2A.X (H2Av) is removed by a SWR-related complex, dTip60
[152]. As H2Av contains features of both H2A.Z and H2A.X,
the possibility that removal of H2Av is H2A.Z-specific rather
than H2A.X-specific cannot be excluded. However, the fact that
removal depends on a phospho-mimetic mutation at the H2A.Xspecific phospho-acceptor residue Ser137 indicates a dependence
on H2A.X function. Therefore the SWR and INO80 complexes
can remove H2A.X from chromatin, but pathways leading to
H2A.X deposition are as yet undiscovered.
H2A.B
Remodelling enzymes have not yet been linked to the assembly or
disassembly of H2A.B, which occurs via unknown mechanisms,
although NAP-1 can promote the assembly and disassembly of
H2A.B in vitro [153]. As this variant is highly expressed in
the testis, there may be tissue-specific enzymes that direct its
deposition and removal, and further experiments will be required
to identify these.
Regulation of H2A variant deposition and removal is the
key to establishing and maintaining distinct patterns of H2A
variant localization in the genome. This is likely to be
particularly important during development, for example during
early embryogenesis, where H2A variant dynamics seem to be
particularly pronounced ([150,154]; for a review see [155]).
Alterations in H2A variant deposition and removal may also
underlie the aberrant H2A variant protein levels that have been
documented in human cancers [156–159]. It is therefore critical
to understand how H2A proteins are interchanged at different loci
and at particular time points. Although a clear link between ATPdependent remodelling enzymes and H2A variant exchange has
been established, there are many outstanding questions about how
H2A variant patterns are established and maintained.
CONCLUSIONS
The organization and regulation of eukaryotic genomes by
chromatin relies on molecular heterogeneity at the nucleosomal
level and the inclusion of H2A variants is one major mechanism
c The Authors Journal compilation c 2013 Biochemical Society
576
C. B. Millar
to functionally differentiate nucleosomes. H2A and its variants
occupy nucleosomes in different parts of the genome where their
sequence differences result in altered nucleosome properties,
novel post-translational modifications and different protein
interactors. Much progress has been made in understanding the
genomic distributions of H2A variants and future work will shed
light on cell-type-specific differences in variant distributions, the
positions of different heterotypic nucleosomes and the regulation
of these occupancies.
ACKNOWLEDGEMENTS
I thank Yanin Naiyachit and Tom Wood for comments on the paper before submission.
FUNDING
Work in the author’s laboratory is supported by the Wellcome Trust.
REFERENCES
1 Marzluff, W. F., Wagner, E. J. and Duronio, R. J. (2008) Metabolism and regulation of
canonical histone mRNAs: life without a poly(A) tail. Nat. Rev. Genet. 9, 843–854
2 West, M. H. and Bonner, W. M. (1980) Histone 2A, a heteromorphous family of eight
protein species. Biochemistry. 19, 3238–3245
3 Talbert, P. B., Ahmad, K., Almouzni, G., Ausio, J., Berger, F., Bhalla, P. L., Bonner, W. M.,
Cande, W. Z., Chadwick, B. P., Chan, S. W. et al. (2012) A unified phylogeny-based
nomenclature for histone variants. Epigenet. Chromatin 5, 7
4 Talbert, P. B. and Henikoff, S. (2010) Histone variants: ancient wrap artists of the
epigenome. Nat. Rev. Mol. Cell Biol. 11, 264–275
5 Song, X., Bowen, J., Miao, W., Liu, Y. and Gorovsky, M. A. (2012) The nonhistone,
N-terminal tail of an essential, chimeric H2A variant regulates mitotic H3-S10
dephosphorylation. Genes Dev. 26, 615–629
6 Suto, R. K., Clarkson, M. J., Tremethick, D. J. and Luger, K. (2000) Crystal structure of a
nucleosome core particle containing the variant histone H2A.Z. Nat. Struct. Biol. 7,
1121–1124
7 Chakravarthy, S., Gundimella, S. K., Caron, C., Perche, P. Y., Pehrson, J. R., Khochbin,
S. and Luger, K. (2005) Structural characterization of the histone variant macroH2A. Mol.
Cell. Biol. 25, 7616–7624
8 Luger, K., Dechassa, M. L. and Tremethick, D. J. (2012) New insights into nucleosome
and chromatin structure: an ordered state or a disordered affair? Nat. Rev. Mol. Cell Biol.
13, 436–447
9 Bönisch, C. and Hake, S. B. (2012) Histone H2A variants in nucleosomes and
chromatin: more or less stable? Nucleic Acids Res., doi:10.1093/nar/gks865
10 Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F. and Richmond, T. J. (1997)
Crystal structure of the nucleosome core particle at 2.8 Å resolution. Nature 389,
251–260
11 Fan, J. Y., Rangasamy, D., Luger, K. and Tremethick, D. J. (2004) H2A.Z alters the
nucleosome surface to promote HP1α-mediated chromatin fiber folding. Mol. Cell 16,
655–661
12 Zhou, J., Fan, J. Y., Rangasamy, D. and Tremethick, D. J. (2007) The nucleosome surface
regulates chromatin compaction and couples it with transcriptional repression. Nat.
Struct. Mol. Biol. 14, 1070–1076
13 Wyrick, J. J., Kyriss, M. N. and Davis, W. B. (2012) Ascending the nucleosome face:
recognition and function of structured domains in the histone H2A-H2B dimer. Biochim.
Biophys. Acta 1819, 892–901
14 Roussel, L., Erard, M., Cayrol, C. and Girard, J. P. (2008) Molecular mimicry between
IL-33 and KSHV for attachment to chromatin through the H2A-H2B acidic pocket. EMBO
Rep. 9, 1006–1012
15 Ausio, J. and Abbott, D. W. (2002) The many tales of a tail: carboxyl-terminal tail
heterogeneity specializes histone H2A variants for defined chromatin function.
Biochemistry 41, 5945–5949
16 Chakravarthy, S., Patel, A. and Bowman, G. D. (2012) The basic linker of macroH2A
stabilizes DNA at the entry/exit site of the nucleosome. Nucleic Acids Res. 40,
8285–8295
17 Chang, E. Y., Ferreira, H., Somers, J., Nusinow, D. A., Owen-Hughes, T. and Narlikar,
G. J. (2008) MacroH2A allows ATP-dependent chromatin remodeling by SWI/SNF and
ACF complexes but specifically reduces recruitment of SWI/SNF. Biochemistry 47,
13726–13732
c The Authors Journal compilation c 2013 Biochemical Society
18 Bao, Y., Konesky, K., Park, Y. J., Rosu, S., Dyer, P. N., Rangasamy, D., Tremethick, D. J.,
Laybourn, P. J. and Luger, K. (2004) Nucleosomes containing the histone variant
H2A.Bbd organize only 118 base pairs of DNA. EMBO J. 23, 3314–3324
19 van Attikum, H. and Gasser, S. M. (2005) The histone code at DNA breaks: a guide to
repair? Nat. Rev. Mol. Cell Biol. 6, 757–765
20 Clarkson, M. J., Wells, J. R., Gibson, F., Saint, R. and Tremethick, D. J. (1999) Regions
of variant histone His2AvD required for Drosophila development. Nature 399, 694–697
21 Bonisch, C., Schneider, K., Punzeler, S., Wiedemann, S. M., Bielmeier, C., Bocola, M.,
Eberl, H. C., Kuegel, W., Neumann, J., Kremmer, E. et al. (2012) H2A.Z.2.2 is an
alternatively spliced histone H2A.Z variant that causes severe nucleosome
destabilization. Nucleic Acids Res. 40, 5951–5964
22 Wratting, D., Thistlethwaite, A., Harris, M., Zeef, L. A. and Millar, C. B. (2012) A
conserved function for the H2A.Z C terminus. J. Biol. Chem. 287, 19148–19157
23 Kim, H. S., Vanoosthuyse, V., Fillingham, J., Roguev, A., Watt, S., Kislinger, T., Treyer, A.,
Carpenter, L. R., Bennett, C. S., Emili, A. et al. (2009) An acetylated form of histone
H2A.Z regulates chromosome architecture in Schizosaccharomyces pombe . Nat. Struct.
Mol. Biol. 16, 1286–1293
24 Gardner, J. M., Smoyer, C. J., Stensrud, E. S., Alexander, R., Gogol, M., Wiegraebe, W.
and Jaspersen, S. L. (2011) Targeting of the SUN protein Mps3 to the inner nuclear
membrane by the histone variant H2A.Z. J. Cell Biol. 193, 489–507
25 Faast, R., Thonglairoam, V., Schulz, T. C., Beall, J., Wells, J. R., Taylor, H., Matthaei, K.,
Rathjen, P. D., Tremethick, D. J. and Lyons, I. (2001) Histone variant H2A.Z is required
for early mammalian development. Curr. Biol. 11, 1183–1187
26 Kustatscher, G., Hothorn, M., Pugieux, C., Scheffzek, K. and Ladurner, A. G. (2005)
Splicing regulates NAD metabolite binding to histone macroH2A. Nat. Struct. Mol. Biol.
12, 624–625
27 Timinszky, G., Till, S., Hassa, P. O., Hothorn, M., Kustatscher, G., Nijmeijer, B.,
Colombelli, J., Altmeyer, M., Stelzer, E. H., Scheffzek, K. et al. (2009) A
macrodomain-containing histone rearranges chromatin upon sensing PARP1 activation.
Nat. Struct. Mol. Biol. 16, 923–929
28 Rasmussen, T. P., Huang, T., Mastrangelo, M. A., Loring, J., Panning, B. and Jaenisch,
R. (1999) Messenger RNAs encoding mouse histone macroH2A1 isoforms are
expressed at similar levels in male and female cells and result from alternative splicing.
Nucleic Acids Res. 27, 3685–3689
29 Pehrson, J. R., Costanzi, C. and Dharia, C. (1997) Developmental and tissue expression
patterns of histone macroH2A1 subtypes. J. Cell. Biochem. 65, 107–113
30 Novikov, L., Park, J. W., Chen, H., Klerman, H., Jalloh, A. S. and Gamble, M. J. (2011)
QKI-mediated alternative splicing of the histone variant MacroH2A1 regulates cancer cell
proliferation. Mol. Cell. Biol. 31, 4244–4255
31 Moosmann, A., Campsteijn, C., Jansen, P. W., Nasrallah, C., Raasholm, M.,
Stunnenberg, H. G. and Thompson, E. M. (2011) Histone variant innovation in a rapidly
evolving chordate lineage. BMC Evol. Biol. 11, 208
32 Buchanan, L., Durand-Dubief, M., Roguev, A., Sakalar, C., Wilhelm, B., Stralfors, A.,
Shevchenko, A., Aasland, R., Ekwall, K. and Francis Stewart, A. (2009) The
Schizosaccharomyces pombe JmjC-protein, Msc1, prevents H2A.Z localization in
centromeric and subtelomeric chromatin domains. PLoS Genet. 5, e1000726
33 Ishibashi, T., Dryhurst, D., Rose, K. L., Shabanowitz, J., Hunt, D. F. and Ausio, J. (2009)
Acetylation of vertebrate H2A.Z and its effect on the structure of the nucleosome.
Biochemistry 48, 5007–5017
34 Thambirajah, A. A., Li, A., Ishibashi, T. and Ausio, J. (2009) New developments in
post-translational modifications and functions of histone H2A variants. Biochem. Cell
Biol. 87, 7–17
35 Updike, D. L. and Mango, S. E. (2006) Temporal regulation of foregut development by
HTZ-1/H2A.Z and PHA-4/FoxA. PLoS Genet. 2, e161
36 van Daal, A. and Elgin, S. C. (1992) A histone variant, H2AvD, is essential in Drosophila
melanogaster . Mol. Biol. Cell 3, 593–602
37 Liu, X., Li, B. and Gorovsky, M. A. (1996) Essential and nonessential histone H2A
variants in Tetrahymena thermophila . Mol. Cell. Biol. 16, 4305–4311
38 Buschbeck, M., Uribesalgo, I., Wibowo, I., Rue, P., Martin, D., Gutierrez, A., Morey, L.,
Guigo, R., Lopez-Schier, H. and Di Croce, L. (2009) The histone variant macroH2A is an
epigenetic regulator of key developmental genes. Nat. Struct. Mol. Biol. 16, 1074–1079
39 Boulard, M., Storck, S., Cong, R., Pinto, R., Delage, H. and Bouvet, P. (2010) Histone
variant macroH2A1 deletion in mice causes female-specific steatosis. Epigenet.
Chromatin. 3, 8
40 Millar, C. B. and Grunstein, M. (2006) Genome-wide patterns of histone modifications in
yeast. Nat. Rev. Mol. Cell Biol. 7, 657–666
41 Rando, O. J. and Chang, H. Y. (2009) Genome-wide views of chromatin structure. Annu.
Rev. Biochem. 78, 245–271
42 Guillemette, B., Bataille, A. R., Gevry, N., Adam, M., Blanchette, M., Robert, F. and
Gaudreau, L. (2005) Variant histone H2A.Z is globally localized to the promoters of
inactive yeast genes and regulates nucleosome positioning. PLoS Biol. 3, e384
Organizing the genome with H2A histone variants
43 Li, B., Pattenden, S. G., Lee, D., Gutierrez, J., Chen, J., Seidel, C., Gerton, J. and
Workman, J. L. (2005) Preferential occupancy of histone variant H2AZ at inactive
promoters influences local histone modifications and chromatin remodeling. Proc. Natl.
Acad. Sci. U.S.A. 102, 18385–18390
44 Hou, H., Wang, Y., Kallgren, S. P., Thompson, J., Yates, 3rd, J. R. and Jia, S. (2010)
Histone variant H2A.Z regulates centromere silencing and chromosome segregation in
fission yeast. J. Biol. Chem. 285, 1909–1918
45 Zofall, M., Fischer, T., Zhang, K., Zhou, M., Cui, B., Veenstra, T. D. and Grewal, S. I.
(2009) Histone H2A.Z cooperates with RNAi and heterochromatin factors to suppress
antisense RNAs. Nature 461, 419–422
46 Millar, C. B., Xu, F., Zhang, K. and Grunstein, M. (2006) Acetylation of H2AZ Lys 14 is
associated with genome-wide gene activity in yeast. Genes Dev. 20, 711–722
47 Mavrich, T. N., Jiang, C., Ioshikhes, I. P., Li, X., Venters, B. J., Zanton, S. J., Tomsho,
L. P., Qi, J., Glaser, R. L., Schuster, S. C. et al. (2008) Nucleosome organization in the
Drosophila genome. Nature 453, 358–362
48 Zhang, H., Roberts, D. N. and Cairns, B. R. (2005) Genome-wide dynamics of Htz1, a
histone H2A variant that poises repressed/basal promoters for activation through histone
loss. Cell 123, 219–231
49 Zilberman, D., Coleman-Derr, D., Ballinger, T. and Henikoff, S. (2008) Histone H2A.Z
and DNA methylation are mutually antagonistic chromatin marks. Nature 456, 125–129
50 Barski, A., Cuddapah, S., Cui, K., Roh, T. Y., Schones, D. E., Wang, Z., Wei, G.,
Chepelev, I. and Zhao, K. (2007) High-resolution profiling of histone methylations in the
human genome. Cell 129, 823–837
51 Creyghton, M. P., Markoulaki, S., Levine, S. S., Hanna, J., Lodato, M. A., Sha, K., Young,
R. A., Jaenisch, R. and Boyer, L. A. (2008) H2AZ is enriched at polycomb complex target
genes in ES cells and is necessary for lineage commitment. Cell 135, 649–661
52 Hardy, S., Jacques, P. E., Gevry, N., Forest, A., Fortin, M. E., Laflamme, L., Gaudreau, L.
and Robert, F. (2009) The euchromatic and heterochromatic landscapes are shaped by
antagonizing effects of transcription on H2A.Z deposition. PLoS Genet. 5, e1000687
53 Whittle, C. M., McClinic, K. N., Ercan, S., Zhang, X., Green, R. D., Kelly, W. G. and Lieb,
J. D. (2008) The genomic distribution and function of histone variant HTZ-1 during C.
elegans embryogenesis. PLoS Genet. 4, e1000187
54 Raisner, R. M., Hartley, P. D., Meneghini, M. D., Bao, M. Z., Liu, C. L., Schreiber, S. L.,
Rando, O. J. and Madhani, H. D. (2005) Histone variant H2A.Z marks the 5 ends of both
active and inactive genes in euchromatin. Cell 123, 233–248
55 Albert, I., Mavrich, T. N., Tomsho, L. P., Qi, J., Zanton, S. J., Schuster, S. C. and Pugh,
B. F. (2007) Translational and rotational settings of H2A.Z nucleosomes across the
Saccharomyces cerevisiae genome. Nature 446, 572–576
56 Lantermann, A. B., Straub, T., Stralfors, A., Yuan, G. C., Ekwall, K. and Korber, P. (2010)
Schizosaccharomyces pombe genome-wide nucleosome mapping reveals positioning
mechanisms distinct from those of Saccharomyces cerevisiae . Nat. Struct. Mol. Biol.
17, 251–257
57 Soboleva, T. A., Nekrasov, M., Pahwa, A., Williams, R., Huttley, G. A. and Tremethick,
D. J. (2012) A unique H2A histone variant occupies the transcriptional start site of active
genes. Nat. Struct. Mol. Biol. 19, 25–30
58 Ku, M., Jaffe, J. D., Koche, R. P., Rheinbay, E., Endoh, M., Koseki, H., Carr, S. A. and
Bernstein, B. E. (2012) H2A.Z landscapes and dual modifications in pluripotent and
multipotent stem cells underlie complex genome regulatory functions. Genome Biol. 13,
R85
59 Nekrasov, M., Amrichova, J., Parker, B. J., Soboleva, T. A., Jack, C., Williams, R., Huttley,
G. A. and Tremethick, D. J. (2012) Histone H2A.Z inheritance during the cell cycle and its
impact on promoter organization and dynamics. Nat. Struct. Mol. Biol. 19, 1076–1083
60 Jin, C., Zang, C., Wei, G., Cui, K., Peng, W., Zhao, K. and Felsenfeld, G. (2009)
H3.3/H2A.Z double variant-containing nucleosomes mark ‘nucleosome-free regions’ of
active promoters and other regulatory regions. Nat. Genet. 41, 941–945
61 Gevry, N., Chan, H. M., Laflamme, L., Livingston, D. M. and Gaudreau, L. (2007) p21
transcription is regulated by differential localization of histone H2A.Z. Genes Dev. 21,
1869–1881
62 Chauhan, S. and Boyd, D. D. (2011) Regulation of u-PAR gene expression by H2A.Z is
modulated by the MEK-ERK/AP-1 pathway. Nucleic Acids Res. 40, 600–613
63 Gallant-Behm, C. L., Ramsey, M. R., Bensard, C. L., Nojek, I., Tran, J., Liu, M., Ellisen,
L. W. and Espinosa, J. M. (2012) Np63α represses anti-proliferative genes via H2A.Z
deposition. Genes Dev. 26, 2325–2336
64 Santisteban, M. S., Kalashnikova, T. and Smith, M. M. (2000) Histone H2A.Z regulates
transcription and is partially redundant with nucleosome remodeling complexes. Cell
103, 411–422
65 Adam, M., Robert, F., Larochelle, M. and Gaudreau, L. (2001) H2A.Z is required for
global chromatin integrity and for recruitment of RNA polymerase II under specific
conditions. Mol. Cell. Biol. 21, 6270–6279
66 Halley, J. E., Kaplan, T., Wang, A. Y., Kobor, M. S. and Rine, J. (2010) Roles for H2A.Z
and its acetylation in GAL1 transcription and gene induction, but not
GAL1-transcriptional memory. PLoS Biol. 8, e1000401
577
67 Meneghini, M. D., Wu, M. and Madhani, H. D. (2003) Conserved histone variant H2A.Z
protects euchromatin from the ectopic spread of silent heterochromatin. Cell 112,
725–736
68 Bruce, K., Myers, F. A., Mantouvalou, E., Lefevre, P., Greaves, I., Bonifer, C., Tremethick,
D. J., Thorne, A. W. and Crane-Robinson, C. (2005) The replacement histone H2A.Z in a
hyperacetylated form is a feature of active genes in the chicken. Nucleic Acids Res. 33,
5633–5639
69 Valdes-Mora, F., Song, J. Z., Statham, A. L., Strbenac, D., Robinson, M. D., Nair, S. S.,
Patterson, K. I., Tremethick, D. J., Stirzaker, C. and Clark, S. J. (2011) Acetylation of
H2A.Z is a key epigenetic modification associated with gene deregulation and epigenetic
remodeling in cancer. Genome Res. 22, 307–321
70 Zemach, A., McDaniel, I. E., Silva, P. and Zilberman, D. (2010) Genome-wide
evolutionary analysis of eukaryotic DNA methylation. Science 328, 916–919
71 Xiao, S., Xie, D., Cao, X., Yu, P., Xing, X., Chen, C. C., Musselman, M., Xie, M., West, F.
D., Lewin, H. A. et al. (2012) Comparative epigenomic annotation of regulatory DNA.
Cell 149, 1381–1392
72 Rangasamy, D., Berven, L., Ridgway, P. and Tremethick, D. J. (2003) Pericentric
heterochromatin becomes enriched with H2A.Z during early mammalian development.
EMBO J. 22, 1599–1607
73 Greaves, I. K., Rangasamy, D., Devoy, M., Marshall Graves, J. A. and Tremethick, D. J.
(2006) The X and Y chromosomes assemble into H2A.Z-containing facultative
heterochromatin following meiosis. Mol. Cell. Biol. 26, 5394–5405
74 Hammoud, S. S., Nix, D. A., Zhang, H., Purwar, J., Carrell, D. T. and Cairns, B. R. (2009)
Distinctive chromatin in human sperm packages genes for embryo development. Nature
460, 473–478
75 Zhang, Z. and Pugh, B. F. (2011) Genomic organization of H2Av containing
nucleosomes in Drosophila heterochromatin. PLoS ONE 6, e20511
76 Carr, A. M., Dorrington, S. M., Hindley, J., Phear, G. A., Aves, S. J. and Nurse, P. (1994)
Analysis of a histone H2A variant from fission yeast: evidence for a role in chromosome
stability. Mol. Gen. Genet. 245, 628–635
77 Krogan, N. J., Baetz, K., Keogh, M. C., Datta, N., Sawa, C., Kwok, T. C., Thompson, N. J.,
Davey, M. G., Pootoolal, J., Hughes, T. R. et al. (2004) Regulation of chromosome
stability by the histone H2A variant Htz1, the Swr1 chromatin remodeling complex, and
the histone acetyltransferase NuA4. Proc. Natl. Acad. Sci. U.S.A. 101, 13513–13518
78 Rangasamy, D., Greaves, I. and Tremethick, D. J. (2004) RNA interference demonstrates
a novel role for H2A.Z in chromosome segregation. Nat. Struct. Mol. Biol. 11, 650–655
79 van Attikum, H., Fritsch, O. and Gasser, S. M. (2007) Distinct roles for SWR1 and INO80
chromatin remodeling complexes at chromosomal double-strand breaks. EMBO J. 26,
4113–4125
80 Kalocsay, M., Hiller, N. J. and Jentsch, S. (2009) Chromosome-wide Rad51 spreading
and SUMO-H2A.Z-dependent chromosome fixation in response to a persistent DNA
double-strand break. Mol. Cell 33, 335–343
81 Dion, M. F., Kaplan, T., Kim, M., Buratowski, S., Friedman, N. and Rando, O. J. (2007)
Dynamics of replication-independent histone turnover in budding yeast. Science 315,
1405–1408
82 Costanzi, C. and Pehrson, J. R. (1998) Histone macroH2A1 is concentrated in the
inactive X chromosome of female mammals. Nature 393, 599–601
83 Chadwick, B. P. and Willard, H. F. (2001) Histone H2A variants and the inactive X
chromosome: identification of a second macroH2A variant. Hum. Mol. Genet. 10,
1101–1113
84 Hoyer-Fender, S., Costanzi, C. and Pehrson, J. R. (2000) Histone macroH2A1.2 is
concentrated in the XY-body by the early pachytene stage of spermatogenesis. Exp. Cell
Res. 258, 254–260
85 Zhang, R., Poustovoitov, M. V., Ye, X., Santos, H. A., Chen, W., Daganzo, S. M.,
Erzberger, J. P., Serebriiskii, I. G., Canutescu, A. A., Dunbrack, R. L. et al. (2005)
Formation of macroh2a-containing senescence-associated heterochromatin foci and
senescence driven by ASF1a and HIRA. Dev. Cell 8, 19–30
86 Mietton, F., Sengupta, A. K., Molla, A., Picchi, G., Barral, S., Heliot, L., Grange, T., Wutz,
A. and Dimitrov, S. (2009) Weak but uniform enrichment of the histone variant
macroH2A1 along the inactive X chromosome. Mol. Cell. Biol. 29, 150–156
87 Changolkar, L. N., Singh, G., Cui, K., Berletch, J. B., Zhao, K., Disteche, C. M. and
Pehrson, J. R. (2010) Genome-wide distribution of macroH2A1 histone variants in
mouse liver chromatin. Mol. Cell. Biol. 30, 5473–5483
88 Tanasijevic, B. and Rasmussen, T. P. (2011) X chromosome inactivation and
differentiation occur readily in ES cells doubly-deficient for macroH2A1 and
macroH2A2. PLoS ONE 6, e21512
89 Changolkar, L. N., Costanzi, C., Leu, N. A., Chen, D., McLaughlin, K. J. and Pehrson,
J. R. (2007) Developmental changes in histone macroH2A1-mediated gene regulation.
Mol. Cell. Biol. 27, 2758–2764
90 Gamble, M. J., Frizzell, K. M., Yang, C., Krishnakumar, R. and Kraus, W. L. (2010) The
histone variant macroH2A1 marks repressed autosomal chromatin, but protects a subset
of its target genes from silencing. Genes Dev. 24, 21–32
c The Authors Journal compilation c 2013 Biochemical Society
578
C. B. Millar
91 Creppe, C., Janich, P., Cantarino, N., Noguera, M., Valero, V., Musulen, E., Douet, J.,
Posavec, M., Martin-Caballero, J., Sumoy, L. et al. (2012) MacroH2A1 regulates the
balance between self-renewal and differentiation commitment in embryonic and adult
stem cells. Mol. Cell. Biol. 32, 1442–1452
92 Tolstorukov, M. Y., Goldman, J. A., Gilbert, C., Ogryzko, V., Kingston, R. E. and Park,
P. J. (2012) Histone variant H2A.Bbd is associated with active transcription and mRNA
processing in human cells. Mol. Cell 47, 596–607
93 Pasque, V., Radzisheuskaya, A., Gillich, A., Halley-Stott, R. P., Panamarova, M.,
Zernicka-Goetz, M., Surani, M. A. and Silva, J. C. (2012) Histone variant macroH2A
marks embryonic differentiation in vivo and acts as an epigenetic barrier to induced
pluripotency. J. Cell Sci., doi:10.1242/jcs.113019
94 Buschbeck, M. and Di Croce, L. (2010) Approaching the molecular and physiological
function of macroH2A variants. Epigenetics 5, 118–123
95 Gamble, M. J. and Kraus, W. L. (2010) Multiple facets of the unique histone variant
macroH2A: from genomics to cell biology. Cell Cycle 9, 2568–2574
96 Costanzi, C. and Pehrson, J. R. (2001) MACROH2A2, a new member of the MARCOH2A
core histone family. J. Biol. Chem. 276, 21776–21784
97 Bernstein, E., Muratore-Schroeder, T. L., Diaz, R. L., Chow, J. C., Changolkar, L. N.,
Shabanowitz, J., Heard, E., Pehrson, J. R., Hunt, D. F. and Allis, C. D. (2008) A
phosphorylated subpopulation of the histone variant macroH2A1 is excluded from the
inactive X chromosome and enriched during mitosis. Proc. Natl. Acad. Sci. U.S.A. 105,
1533–1538
98 Chu, F., Nusinow, D. A., Chalkley, R. J., Plath, K., Panning, B. and Burlingame, A. L.
(2006) Mapping post-translational modifications of the histone variant MacroH2A1
using tandem mass spectrometry. Mol. Cell. Proteomics 5, 194–203
99 Ogawa, Y., Ono, T., Wakata, Y., Okawa, K., Tagami, H. and Shibahara, K. I. (2005) Histone
variant macroH2A1.2 is mono-ubiquitinated at its histone domain. Biochem. Biophys.
Res. Commun. 336, 204–209
100 Redon, C., Pilch, D., Rogakou, E., Sedelnikova, O., Newrock, K. and Bonner, W. (2002)
Histone H2A variants H2AX and H2AZ. Curr. Opin. Genet. Dev. 12, 162–169
101 Rossetto, D., Truman, A. W., Kron, S. J. and Cote, J. (2010) Epigenetic modifications in
double-strand break DNA damage signaling and repair. Clin. Cancer Res. 16,
4543–4552
102 Rozenzhak, S., Mejia-Ramirez, E., Williams, J. S., Schaffer, L., Hammond, J. A., Head,
S. R. and Russell, P. (2010) Rad3 decorates critical chromosomal domains with γ H2A to
protect genome integrity during S-phase in fission yeast. PLoS Genet. 6, e1001032
103 Szilard, R. K., Jacques, P. E., Laramee, L., Cheng, B., Galicia, S., Bataille, A. R., Yeung,
M., Mendez, M., Bergeron, M., Robert, F. and Durocher, D. (2010) Systematic
identification of fragile sites via genome-wide location analysis of γ -H2AX. Nat. Struct.
Mol. Biol. 17, 299–305
104 Kitada, T., Schleker, T., Sperling, A. S., Xie, W., Gasser, S. M. and Grunstein, M. (2011)
γ H2A is a component of yeast heterochromatin required for telomere elongation. Cell
Cycle 10, 293–300
105 Fernandez-Capetillo, O., Mahadevaiah, S. K., Celeste, A., Romanienko, P. J.,
Camerini-Otero, R. D., Bonner, W. M., Manova, K., Burgoyne, P. and Nussenzweig, A.
(2003) H2AX is required for chromatin remodeling and inactivation of sex chromosomes
in male mouse meiosis. Dev. Cell 4, 497–508
106 Celeste, A., Petersen, S., Romanienko, P. J., Fernandez-Capetillo, O., Chen, H. T.,
Sedelnikova, O. A., Reina-San-Martin, B., Coppola, V., Meffre, E., Difilippantonio, M. J.
et al. (2002) Genomic instability in mice lacking histone H2AX. Science 296, 922–927
107 Seo, J., Kim, S. C., Lee, H. S., Kim, J. K., Shon, H. J., Salleh, N. L., Desai, K. V., Lee,
J. H., Kang, E. S., Kim, J. S. and Choi, J. K. (2012) Genome-wide profiles of H2AX and
γ -H2AX differentiate endogenous and exogenous DNA damage hotspots in human
cells. Nucleic Acids Res. 40, 5965–5974
108 Eirin-Lopez, J. M., Ishibashi, T. and Ausio, J. (2008) H2A.Bbd: a quickly evolving
hypervariable mammalian histone that destabilizes nucleosomes in an
acetylation-independent way. FASEB J. 22, 316–326
109 Viens, A., Mechold, U., Brouillard, F., Gilbert, C., Leclerc, P. and Ogryzko, V. (2006)
Analysis of human histone H2AZ deposition in vivo argues against its direct role in
epigenetic templating mechanisms. Mol. Cell. Biol. 26, 5325–5335
110 Luk, E., Ranjan, A., Fitzgerald, P. C., Mizuguchi, G., Huang, Y., Wei, D. and Wu, C.
(2010) Stepwise histone replacement by SWR1 requires dual activation with histone
H2A.Z and canonical nucleosome. Cell 143, 725–736
111 Weber, C. M., Henikoff, J. G. and Henikoff, S. (2010) H2A.Z nucleosomes enriched over
active genes are homotypic. Nat. Struct. Mol. Biol. 17, 1500–1507
112 Chakravarthy, S. and Luger, K. (2006) The histone variant macro-H2A preferentially
forms ‘hybrid nucleosomes’. J. Biol. Chem. 281, 25522–25531
113 Park, Y. J. and Luger, K. (2008) Histone chaperones in nucleosome eviction and histone
exchange. Curr. Opin. Struct. Biol. 18, 282–289
114 Keck, K. M. and Pemberton, L. F. (2011) Histone chaperones link histone nuclear import
and chromatin assembly. Biochim. Biophys. Acta 1819, 277–289
c The Authors Journal compilation c 2013 Biochemical Society
115 Elsasser, S. J. and D’Arcy, S. (2012) Towards a mechanism for histone chaperones.
Biochim. Biophys. Acta 1819, 211–221
116 Hota, S. K. and Bartholomew, B. (2011) Diversity of operation in ATP-dependent
chromatin remodelers. Biochim. Biophys. Acta 1809, 476–487
117 Clapier, C. R. and Cairns, B. R. (2009) The biology of chromatin remodeling complexes.
Annu. Rev. Biochem. 78, 273–304
118 Hondele, M. and Ladurner, A. G. (2011) The chaperone-histone partnership: for the
greater good of histone traffic and chromatin plasticity. Curr. Opin. Struct. Biol. 21,
698–708
119 Zhou, Z., Feng, H., Hansen, D. F., Kato, H., Luk, E., Freedberg, D. I., Kay, L. E., Wu, C.
and Bai, Y. (2008) NMR structure of chaperone Chz1 complexed with histones
H2A.Z-H2B. Nat. Struct. Mol. Biol. 15, 868–869
120 Ramos, I., Martin-Benito, J., Finn, R., Bretana, L., Aloria, K., Arizmendi, J. M., Ausio, J.,
Muga, A., Valpuesta, J. M. and Prado, A. (2010) Nucleoplasmin binds histone H2A-H2B
dimers through its distal face. J. Biol. Chem. 285, 33771–33778
121 Luk, E., Vu, N. D., Patteson, K., Mizuguchi, G., Wu, W. H., Ranjan, A., Backus, J., Sen,
S., Lewis, M., Bai, Y. and Wu, C. (2007) Chz1, a nuclear chaperone for histone H2AZ.
Mol. Cell 25, 357–368
122 Angelov, D., Bondarenko, V. A., Almagro, S., Menoni, H., Mongelard, F., Hans, F.,
Mietton, F., Studitsky, V. M., Hamiche, A., Dimitrov, S. and Bouvet, P. (2006) Nucleolin is
a histone chaperone with FACT-like activity and assists remodeling of nucleosomes.
EMBO J. 25, 1669–1679
123 Park, Y. J., Chodaparambil, J. V., Bao, Y., McBryant, S. J. and Luger, K. (2005)
Nucleosome assembly protein 1 exchanges histone H2A-H2B dimers and assists
nucleosome sliding. J. Biol. Chem. 280, 1817–1825
124 Flaus, A., Martin, D. M., Barton, G. J. and Owen-Hughes, T. (2006) Identification of
multiple distinct Snf2 subfamilies with conserved structural motifs. Nucleic Acids Res.
34, 2887–2905
125 Mizuguchi, G., Shen, X., Landry, J., Wu, W. H., Sen, S. and Wu, C. (2004) ATP-driven
exchange of histone H2AZ variant catalyzed by SWR1 chromatin remodeling complex.
Science 303, 343–348
126 Papamichos-Chronakis, M., Watanabe, S., Rando, O. J. and Peterson, C. L. (2011)
Global regulation of H2A.Z localization by the INO80 chromatin-remodeling enzyme is
essential for genome integrity. Cell 144, 200–213
127 Bruno, M., Flaus, A., Stockdale, C., Rencurel, C., Ferreira, H. and Owen-Hughes, T.
(2003) Histone H2A/H2B dimer exchange by ATP-dependent chromatin remodeling
activities. Mol. Cell 12, 1599–1606
128 Lu, P. Y., Levesque, N. and Kobor, M. S. (2009) NuA4 and SWR1-C: two
chromatin-modifying complexes with overlapping functions and components. Biochem.
Cell Biol. 87, 799–815
129 Cuadrado, A., Corrado, N., Perdiguero, E., Lafarga, V., Munoz-Canoves, P. and Nebreda,
A. R. (2010) Essential role of p18Hamlet/SRCAP-mediated histone H2A.Z chromatin
incorporation in muscle differentiation. EMBO J. 29, 2014–2025
130 Cai, Y., Jin, J., Gottschalk, A. J., Yao, T., Conaway, J. W. and Conaway, R. C. (2006)
Purification and assay of the human INO80 and SRCAP chromatin remodeling
complexes. Methods 40, 312–317
131 Ruhl, D. D., Jin, J., Cai, Y., Swanson, S., Florens, L., Washburn, M. P., Conaway, R. C.,
Conaway, J. W. and Chrivia, J. C. (2006) Purification of a human SRCAP complex that
remodels chromatin by incorporating the histone variant H2A.Z into nucleosomes.
Biochemistry 45, 5671–5677
132 Choi, J., Heo, K. and An, W. (2009) Cooperative action of TIP48 and TIP49 in H2A.Z
exchange catalyzed by acetylation of nucleosomal H2A. Nucleic Acids Res. 37,
5993–6007
133 Wu, W. H., Alami, S., Luk, E., Wu, C. H., Sen, S., Mizuguchi, G., Wei, D. and Wu, C.
(2005) Swc2 is a widely conserved H2AZ-binding module essential for ATP-dependent
histone exchange. Nat. Struct. Mol. Biol. 12, 1064–1071
134 Jensen, K., Santisteban, M. S., Urekar, C. and Smith, M. M. (2011) Histone H2A.Z acid
patch residues required for deposition and function. Mol. Genet. Genomics 285,
287–296
135 Venters, B. J. and Pugh, B. F. (2009) A canonical promoter organization of the
transcription machinery and its regulators in the Saccharomyces genome. Genome Res.
19, 360–371
136 Wong, M. M., Cox, L. K. and Chrivia, J. C. (2007) The chromatin remodeling protein,
SRCAP, is critical for deposition of the histone variant H2A.Z at promoters. J. Biol.
Chem. 282, 26132–26139
137 Hartley, P. D. and Madhani, H. D. (2009) Mechanisms that specify promoter nucleosome
location and identity. Cell 137, 445–458
138 Gevry, N., Hardy, S., Jacques, P. E., Laflamme, L., Svotelis, A., Robert, F. and Gaudreau,
L. (2009) Histone H2A.Z is essential for estrogen receptor signaling. Genes Dev. 23,
1522–1533
Organizing the genome with H2A histone variants
139 Altaf, M., Auger, A., Monnet-Saksouk, J., Brodeur, J., Piquet, S., Cramet, M., Bouchard,
N., Lacoste, N., Utley, R. T., Gaudreau, L. and Cote, J. (2010) NuA4-dependent
acetylation of nucleosomal histones H4 and H2A directly stimulates incorporation of
H2A.Z by the SWR1 complex. J. Biol. Chem. 285, 15966–15977
140 Hardy, S. and Robert, F. (2010) Random deposition of histone variants: a cellular mistake
or a novel regulatory mechanism? Epigenetics 5, 368–372
141 Yen, K., Vinayachandran, V., Batta, K., Koerber, R. T. and Pugh, B. F. (2012)
Genome-wide nucleosome specificity and directionality of chromatin remodelers. Cell
149, 1461–1473
142 Jin, J., Cai, Y., Yao, T., Gottschalk, A. J., Florens, L., Swanson, S. K., Gutierrez, J. L.,
Coleman, M. K., Workman, J. L., Mushegian, A. et al. (2005) A mammalian chromatin
remodeling complex with similarities to the yeast INO80 complex. J. Biol. Chem. 280,
41207–41212
143 Durand-Dubief, M., Will, W. R., Petrini, E., Theodorou, D., Harris, R. R., Crawford, M. R.,
Paszkiewicz, K., Krueger, F., Correra, R. M., Vetter, A. T. et al. (2012) SWI/SNF-like
chromatin remodeling factor Fun30 supports point centromere function in S. cerevisiae .
PLoS Genet. 8, e1002974
144 Stralfors, A., Walfridsson, J., Bhuiyan, H. and Ekwall, K. (2011) The FUN30 chromatin
remodeler, Fft3, protects centromeric and subtelomeric domains from euchromatin
formation. PLoS Genet. 7, e1001334
145 Csankovszki, G., Panning, B., Bates, B., Pehrson, J. R. and Jaenisch, R. (1999)
Conditional deletion of Xist disrupts histone macroH2A localization but not maintenance
of X inactivation. Nat. Genet. 22, 323–324
146 Ratnakumar, K., Duarte, L. F., LeRoy, G., Hasson, D., Smeets, D., Vardabasso, C.,
Bonisch, C., Zeng, T., Xiang, B., Zhang, D. Y. et al. (2012) ATRX-mediated chromatin
association of histone variant macroH2A1 regulates α-globin expression. Genes Dev.
26, 433–438
147 Chadwick, B. P., Valley, C. M. and Willard, H. F. (2001) Histone variant macroH2A
contains two distinct macrochromatin domains capable of directing macroH2A to the
inactive X chromosome. Nucleic Acids Res. 29, 2699–2705
148 Nusinow, D. A., Sharp, J. A., Morris, A., Salas, S., Plath, K. and Panning, B. (2007) The
histone domain of macroH2A1 contains several dispersed elements that are each
sufficient to direct enrichment on the inactive X chromosome. J. Mol. Biol. 371,
11–18
149 Hernandez-Munoz, I., Lund, A. H., van der Stoop, P., Boutsma, E., Muijrers, I.,
Verhoeven, E., Nusinow, D. A., Panning, B., Marahrens, Y. and van Lohuizen, M. (2005)
Stable X chromosome inactivation involves the PRC1 Polycomb complex and requires
histone MACROH2A1 and the CULLIN3/SPOP ubiquitin E3 ligase. Proc. Natl. Acad. Sci.
U.S.A. 102, 7635–7640
150 Nashun, B., Yukawa, M., Liu, H., Akiyama, T. and Aoki, F. (2010) Changes in the nuclear
deposition of histone H2A variants during pre-implantation development in mice.
Development 137, 3785–3794
151 Fujimoto, S., Seebart, C., Guastafierro, T., Prenni, J., Caiafa, P. and Zlatanova, J. (2012)
Proteome analysis of protein partners to nucleosomes containing canonical H2A or the
variant histones H2A.Z or H2A.X. Biol. Chem. 393, 47–61
152 Kusch, T., Florens, L., Macdonald, W. H., Swanson, S. K., Glaser, R. L., Yates, 3rd, J. R.,
Abmayr, S. M., Washburn, M. P. and Workman, J. L. (2004) Acetylation by Tip60 is
required for selective histone variant exchange at DNA lesions. Science 306, 2084–2087
579
153 Okuwaki, M., Kato, K., Shimahara, H., Tate, S. and Nagata, K. (2005) Assembly and
disassembly of nucleosome core particles containing histone variants by human
nucleosome assembly protein I. Mol. Cell. Biol. 25, 10639–10651
154 Boskovic, A., Bender, A., Gall, L., Ziegler-Birling, C., Beaujean, N. and Torres-Padilla,
M. E. (2012) Analysis of active chromatin modifications in early mammalian embryos
reveals uncoupling of H2A.Z acetylation and H3K36 trimethylation from embryonic
genome activation. Epigenetics 7, 747–757
155 Banaszynski, L. A., Allis, C. D. and Lewis, P. W. (2010) Histone variants in metazoan
development. Dev. Cell 19, 662–674
156 Hua, S., Kallen, C. B., Dhar, R., Baquero, M. T., Mason, C. E., Russell, B. A., Shah, P. K.,
Liu, J., Khramtsov, A., Tretiakova, M. S. et al. (2008) Genomic analysis of estrogen
cascade reveals histone variant H2A.Z associated with breast cancer progression. Mol.
Syst. Biol. 4, 188
157 Kapoor, A., Goldberg, M. S., Cumberland, L. K., Ratnakumar, K., Segura, M. F., Emanuel,
P. O., Menendez, S., Vardabasso, C., Leroy, G., Vidal, C. I. et al. (2010) The histone
variant macroH2A suppresses melanoma progression through regulation of CDK8.
Nature 468, 1105–1109
158 Sporn, J. C., Kustatscher, G., Hothorn, T., Collado, M., Serrano, M., Muley, T., Schnabel,
P. and Ladurner, A. G. (2009) Histone macroH2A isoforms predict the risk of lung cancer
recurrence. Oncogene 28, 3423–3428
159 Dryhurst, D., McMullen, B., Fazli, L., Rennie, P. S. and Ausio, J. (2012) Histone H2A.Z
prepares the prostate specific antigen (PSA) gene for androgen receptor-mediated
transcription and is upregulated in a model of prostate cancer progression. Cancer Lett.
315, 38–47
160 Hatch, C. L. and Bonner, W. M. (1990) The human histone H2A.Z gene. Sequence and
regulation. J. Biol. Chem. 265, 15211–15218
161 Dryhurst, D., Ishibashi, T., Rose, K. L., Eirin-Lopez, J. M., McDonald, D., Silva-Moreno,
B., Veldhoen, N., Helbing, C. C., Hendzel, M. J., Shabanowitz, J. et al. (2009)
Characterization of the histone H2A.Z-1 and H2A.Z-2 isoforms in vertebrates. BMC Biol.
7, 86
162 Matsuda, R., Hori, T., Kitamura, H., Takeuchi, K., Fukagawa, T. and Harata, M. (2010)
Identification and characterization of the two isoforms of the vertebrate H2A.Z histone
variant. Nucleic Acids Res. 38, 4263–4273
163 Mannironi, C., Bonner, W. M. and Hatch, C. L. (1989) H2A.X. a histone isoprotein with a
conserved C-terminal sequence, is encoded by a novel mRNA with both DNA replication
type and polyA 3 processing signals. Nucleic Acids Res. 17, 9113–9126
164 Nagata, T., Kato, T., Morita, T., Nozaki, M., Kubota, H., Yagi, H. and Matsushiro, A.
(1991) Polyadenylated and 3 processed mRNAs are transcribed from the mouse histone
H2A.X gene. Nucleic Acids Res. 19, 2441–2447
165 Bonner, W. M., Mannironi, C., Orr, A., Pilch, D. R. and Hatch, C. L. (1993) Histone
H2A.X gene transcription is regulated differently than transcription of other
replication-linked histone genes. Mol. Cell. Biol. 13, 984–992
166 Chadwick, B. P. and Willard, H. F. (2001) A novel chromatin protein, distantly related to
histone H2A, is largely excluded from the inactive X chromosome. J. Cell Biol. 152,
375–384
167 Ishibashi, T., Li, A., Eirin-Lopez, J. M., Zhao, M., Missiaen, K., Abbott, D. W., Meistrich,
M., Hendzel, M. J. and Ausio, J. (2010) H2A.Bbd: an X-chromosome-encoded histone
involved in mammalian spermiogenesis. Nucleic Acids Res. 38, 1780–1789
Received 31 October 2012/9 November 2012; accepted 12 November 2012
Published on the Internet 9 January 2013, doi:10.1042/BJ20121646
c The Authors Journal compilation c 2013 Biochemical Society