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REVIEW ARTICLE
Histones in functional diversification
Core histone variants
Rama-Haritha Pusarla and Purnima Bhargava
Centre for Cellular & Molecular Biology, Tarnaka, Hyderabad, India
Introduction
Eukaryotic cells package their DNA in the form of
chromatin to accommodate it in the small space provi-
ded by their nuclei [1]. In spite of the 10 000-fold com-
paction of DNA due to this packaging, minute details
of a local structure regulate the accessibility of any
small region. The folding of 147 bp of DNA over a
histone octamer (two molecules each of the four core
histones, H4, H3, H2A and H2B) surface gives a neat
organization of the DNA into a chromatin fibre of
10 nm diameter. The primary structure of 10 nm chro-
matin has a characteristic ‘beads on a string’ appear-
ance. This uniformity of the nucleosomal chain might
impose difficulties in region-specific, localized recogni-
tion and in uncoiling of the structure; both essential
for function. Thus, higher order folding of the chroma-
tin into a 30 nm fibre and larger domains could be an
attempt by the genome to demarcate itself into various
regions of activities.
Histones are abundant, basic, structural proteins
that bring in variety and novelty to the complicated
gene regulation mechanisms [1]. Apart from binding to
DNA and giving chromatin its strength, stability and
form, certain highly similar forms of histones, termed
‘histone variants’, have evolved to carry out many vital
functions. Though the focus on histone variants

appears to be very recent, they were known as early as
1969 when only standard biochemical methods of pro-
tein fractionation could be applied to discover and iso-
late new proteins [1]. Their incorporation into
nucleosomes as a mode of marking chromatin regions
is now shown to have high impact on gene regulation,
DNA repair and meiotic events. They have been impli-
cated in epigenetic inheritance mechanisms of chroma-
tin markings [2,3] and shown to play significant roles
in gene expression, antisilencing, heterochromatiniza-
tion and the formation of specialised regions of the
chromatin [4–7]. With the new revelations, other chro-
matin regulatory mechanisms such as covalent histone
Keywords
chromatin; nucleosome; histones; gene
expression; histone variants
Correspondence
P. Bhargava, Centre for Cellular & Molecular
Biology, Uppal Road, Tarnaka,
Hyderabad-500007, India
Fax: +91 40 27160591
Tel: +91 40 27192603
E-mail:
(Received 6 July 2005, accepted 22 August
2005)
doi:10.1111/j.1742-4658.2005.04930.x
Recent research suggests that minor changes in the primary sequence of
the conserved histones may become major determinants for the chromatin
structure regulating gene expression and other DNA-related processes. An
analysis of the involvement of different core histone variants in different

nuclear processes and the structure of different variant nucleosome cores
shows that this may indeed be so. Histone variants may also be involved in
demarcating functional regions of the chromatin. We discuss in this review
why two of the four core histones show higher variation. A comparison of
the status of variants in yeast with those from higher eukaryotes suggests
that histone variants have evolved in synchrony with functional require-
ment of the cell.
Abbreviations
Cid, centromere identifier; DSB, double strand break; IRIF, irradiation induced foci; MSCI, meiotic sex chromosome inactivation;
NHEJ, nonhomologous end joining; RC, replication coupled; RI, replication independent.
FEBS Journal 272 (2005) 5149–5168 ª 2005 FEBS 5149
modifications or ATP-dependent chromatin remodel-
ling [8–10] are joined now by histone variants. This
review focusses mainly on new advances in chromatin-
related processes with reference to the ‘core histone
variants’ and their contribution to chromatin structure.
Other aspects, including the role of linker histone vari-
ants, can be found in other recent reviews [11–13].
Variation in high conservation – the
evolution of histone variants
Histones are among the most conserved proteins in
eukaryotes, and make the chromatin nonstatic and
parent nucleosomes regulatory. Folding of chromatin
domains is defined at a lower level by the compactness
of the basic units, guided and determined by the his-
tone–DNA as well as particle–particle interactions.
High conservation of core histone structure and their
contacts with each other and with DNA leaves little
scope for any heterogeneity. Therefore, apart from try-
ing to reshuffle or remove nucleosomes from the

underlying DNA, eukaryotic cells have developed
some very subtle and precise methods for breaking the
monotony of the chromatin structure by adding a vari-
ety of tags to their basic units, histones in the nucleo-
somes. These taggings result in altered structures and
interactions of the core particles, affecting the local
chromatin structure. Tags in the form of covalent
modifications of histone tails have been extensively
studied over the past few years [14,15]. Histone codes
of the genes generated by histone modifications along
with other chromatin remodelling mechanisms have
been proposed to be the major players in gene regula-
tion mechanisms [16,17]. More recent research suggests
that minor changes in the primary sequence of con-
served histones also contribute to altering the chroma-
tin structure [18–20].
The ‘bulk’ histones are encoded by genes belonging
to multicopy, intronless families that are transcribed
into nonpolyadenylated mRNA. Their highly conserved
sequences suggest that they nonspecifically bind DNA
from any source. A variation could be detrimental as it
may restrict the required interactions. The variants are
nonallelic isoforms of the major histones that display
sequence variations, often at single residue, and occupy
restricted and defined locations in chromatin. They are
encoded by genes located outside the canonical histone
gene cluster, mostly in single copies and with introns.
They are constitutively expressed into polyadenylated
mRNA, and as the cell ages they replace the bulk
histones, suggesting that this exchange is an active pro-

cess throughout the cell cycle and quiescence (old age)
[21,22]. The variants have diverged from the normal
histones early in the course of evolution, acquiring
differential expression patterns. The structural hetero-
geneity conferred by the variants to chromatin can
potentially regulate various nuclear functions such as
transcription, gene silencing, chromosome segregation,
replication, repair and recombination. Such multiface-
ted regulatory activities of the nucleosomes through
variations in the subunits of the histone octamer would
not have been possible with a strict conservation of
histones at all the times and everywhere. Variants have
provided an added advantage.
Variants of H2A
Histones are proposed to have evolved from a com-
mon and simple ancestral archeal protein [23,24] and
followed three evolutionary histories. H2A and H2B
have diverged faster than H3 and H4. Different H2A
variants have arisen in two single events, while variants
of H3 have probably evolved through multiple inde-
pendent events [25]. They have evolved slowly in such
a way that they could not only fulfill the basic function
of DNA compaction and maintain the higher order
chromatin structure but also have gained functional
specialization due to the acquired changes [23,26]. Var-
iants of H2A show divergent functions in different
contexts (Table 1). H2A has the largest macro hetero-
geneous family of variants and all of them are found
to have a crucial role in gene expression and nuclear
dynamics [4]. Five human H2A genes encode proteins

with sequences considerably different from the major
H2A sequence (Fig. 1). Of these, H2A.X and H2A.Z
were identified in the 1980s, two others (macroH2A1
and macroH2A2) in the 1990s, and finally H2A.Bbd in
2001 [27]. Homologues of H2A.X are found across all
phyla, including fungi, animals, plants and the most
primitive eukaryotes such as Giardia [23]. However, a
comparative analysis of H2A.X from various organ-
isms does not give a clear idea of the evolutionary
links [23]. The sequence of mammalian H2A.X is
nearly identical to the major vertebrate H2A comple-
ment H2A.1 ⁄ 2 homologues [27] but the distance
between the globular region and carboxyl terminus in
H2A.X is increased.
One of the best studied H2A variants, H2A.Z com-
prises roughly 5–10% of cellular H2As and probably
controls several major functions of the cell [28]. Highly
conserved H2A.Z sequences have been given different
names in different organisms. The H2A variants
H2A.Z (mammals), H2A.F (birds), H2A.F ⁄ Z (sea
urchin), H2Av (Drosophila), Htz1 (Saccharomyces cere-
visiae) and hv1 (Tetrahymena) arose very early in evo-
lution and are more closely related to each other than
Histone variants in various functions R H. Pusarla and P. Bhargava
5150 FEBS Journal 272 (2005) 5149–5168 ª 2005 FEBS
to major H2A from the same species [25]. The third
H2A variant, macroH2A (mH2A), may have evolved
comparatively recently. It is a 42 kDa protein [29],
extremely divergent from major H2A, with 64% iden-
tity at its N-terminus and an extensive 25 kDa non-

histone region at the carboxyl end, which forms two
third of the protein’s molecular mass. The H2A region
of this variant is 50% identical to H2A.Z, both having
homology with the corresponding region of conven-
tional H2A. The nonhistone region, now termed as the
‘macrodomain’, contains a short, highly basic region
and a putative leucine zipper domain (Fig. 1; amino
acids 132–159 and 181–208, respectively, in rat liver
protein). Macrodomains may be associated with differ-
ent functions as they are found in diverse proteins such
as those containing poly(ADP-ribose) polymerase
activity and other single strand RNA viral proteins.
They show structural similarity to the DNA binding
domain of leucine aminopeptidases, suggesting that
DNA binding activity is associated with macrodomains
[30]. The exact functional status of the macrodomain
in mH2A is not known.
Variants of H3
Initial studies on histone H3 variants in mice have
helped to classify them according to their relationship
with DNA replication. The major, bulk histones are
deposited over newly synthesized DNA during replica-
tion in a replication-dependent chromatin assembly
pathway, whereas the replacement histone variants
undergo a replication-independent chromatin assembly
[31]. A replication coupled (RC) ⁄ dependent assembly
pathway involves a variety of components such as
CAF-1, RCAF (histone chaperones) and proliferating
cell nuclear antigen (PCNA), and deposits histones on
replicating DNA during the S-phase [32–34]. The repli-

cation-independent (RI) pathway occurs outside the
Table 1. Functional diversity of histone variants.
Histone
Variant
Functional associationMammals Yeast Drosophila
H3 H3.1 – – S-phase subtypes
H3.2 – – S-phase subtypes
H3.3 H3.3 H3.3 Transcriptionally active regions
Cenp-A Cse4 Cid Centromeric nucleosomes
H2A H2A.Z Htz1 H2Av
a
Different functions in various organisms: maintenance of
pericentric and telomeric heterochromatin, transcriptional
activation and viability
H2A.X H2A H2Av
a
Sex body in mammals, site of DNA double stranded breaks;
condensation and silencing of male sex chromosome
MacroH2A – – Inactivation of X-chromosome, interferes with both transcription
factor binding and SWI ⁄ SNF remodelling
H2A.Bbd – – Close spacing of nucleosomes
a
Drosophila melanogaster has a single H2A variant, H2Av, in addition to the major H2A. H2Av is not only a member of H2A.Z family, it also
contains an SQ motif similar to mammalian H2A.X. It is phosphorylated at Ser137 and hence it is a functional homologue of H2A.X.
Fig. 1. Schematic comparison of the organization of histone H2A variants. Solid blocks represent a-helical regions, the histone fold is consti-
tuted by helices a1–a3, and the acidic patch of H2A.Z is shown by the overlined regions. The C-terminal SQ motif in H2A.X, and basic as
well as leucine zipper regions of mH2A are indicated.
R H. Pusarla and P. Bhargava Histone variants in various functions
FEBS Journal 272 (2005) 5149–5168 ª 2005 FEBS 5151
S-phase or in nondividing cells that undergo continued

gene expression. Of the three somatic H3 variants
known, H3.1 and H3.2 were classified as ‘strictly repli-
cation dependent’ and H3.3 as replication-independent
[1]. The RI variant accumulates as the tissue matures.
H3.1 and H3.2 are closely related, only differing in a
Cys-to-Ser substitution at amino acid 96, and belong
to the S-phase subtypes [35]. While only one type of
histone H3, similar to H3.3 is expressed [36] in yeast,
there are three variants of H3 in Drosophila; major
H3, H3.3 and centromeric centromere identifier (Cid).
H3.3 is almost identical to H3 and differs at only four
positions; one in the N-terminal tail (A31) and three in
the histone fold domain (S87, V89, M90) [37].
Centromere-specific H3 variants of all Drosophila
species are documented to show adaptive evolution
continuing for 25 million years [38]. Unlike H3.3, Cid
is characteristically a structural component of the
centromeres. It is very much diverged from H3, having
homologies only in histone fold domains although con-
served blocks are also seen in the N-terminal tail [38].
The evolutionary comparison of CenH3s from various
Drosophila species suggests a unique packaging func-
tion for the N-terminal tail at the cytological marker
of centromeres, the primary constriction [38]. In com-
parison, human centromeric H3-like protein, CENP-A,
shows 62% identity with H3 in its carboxy terminal
portion but there is no sequence similarity in the
N-terminus, which varies from 20 to 200 amino acids
in CENP-A as compared to 45 amino acids in the
N-terminus of H3 [39]. The histone fold domain of

CENP-A, the region required for localization of
CENP-A to the centromere, has evolved more rapidly
than that of H3 [23,39].
Variants of other histones
It is evident from the above description that a variety
of changes have evolved in the primary sequence of
core histones. While no variants are known for H4, a
few variants of H2B and H1 are known, which play
important roles in spermatogenesis. How can small
changes in the primary sequence of one of the histones
introduce a change in the overall structure of the core
particle? Can this change be tolerated? These could
have been the major issues that guided the evolution
of the variants.
Variants of core histones in various
nuclear processes
Histone variants might act as ‘control panels’ in regu-
lating all DNA-related processes. Minor histone
variants are now becoming known as major players in
chromatin metabolism. Cells exploit the intimacy of
nuclear processes with the chromatin structure of
genomic DNA for regulatory purposes by using chro-
matin modifications and histone variants. Thus, func-
tional requirements of a nuclear process in which
chromatin may be involved would have established the
suitability of variation in histones.
Variants in DNA repair and recombination
Transcription in both prokaryotes and eukaryotes is
coupled to the repair process, in particular nucleotide
excision repair, through factors that allow recruitment

of the repair machinery by the transcription complex
at the DNA damage site [40,41]. However, DNA may
be damaged under various conditions and cells have
several mechanisms for its repair [42]. Under nontran-
scribing conditions, recognition of DNA damage and
recruitment of the repair machinery may need other
signalling mechanisms [43,44]. For example, during
radiation-induced DNA damage or other events lead-
ing to double stranded breaks (DSBs) in DNA, a his-
tone variant present at the DNA damage point may
act as a marker for the quick recruitment of a repair
complex, thereby helping to maintain the eukaryotic
genome [45].
H2A.X is randomly incorporated into nucleosomes
and represents 10–15% of the total cellular H2A.
Phosphorylation of H2A.X is suggested to mark the
damaged DNA for recruitment of the repair machin-
ery, although it is not clear how the damage is indica-
ted in regions with bulk H2A. Nevertheless,
immunocytochemical analyses have shown that not
every contiguous H2A.X molecule is phosphorylated
[46]. The carboxy terminus of H2A.X differs from that
of bulk H2A in being longer and having a four amino
acid sequence element SQEL at the extreme end of the
protein (Fig. 1). Within this C-terminal motif, an aci-
dic residue follows the two relatively invariant amino
acids (SQ) while the last carboxy-terminal residue is
hydrophobic [27]. The SQE motif is part of the com-
mon consensus motif found in targets of the phospha-
tidylinositol kinases. Indeed, three members of the

phosphatidylinositol kinase family (ATM, ATR and
DNA-PK) are now known to generate this terminally
phosphorylated form called c-H2A.X. While H2A
phosphorylation in yeast is shown to require both
ATM ⁄ ATR homologues Mec1p and Tel1p in response
to DSBs [47,48], ATM is required for H2A.X phos-
phorylation in murine fibroblasts [49]. Recent evidence,
however, shows that ATR is the kinase that phos-
phorylates H2A.X and the tumour suppressor protein
Histone variants in various functions R H. Pusarla and P. Bhargava
5152 FEBS Journal 272 (2005) 5149–5168 ª 2005 FEBS
BRCA1 plays an important role in recruiting ATR to
XY chromatin [50]. Phosphorylation at the conserved
serine of the SQ motif (Ser129 in yeast and Ser139 in
mammals) is now shown to regulate DNA DSB repair
[45,46], meiotic recombination preceding synaptic
crossover [51], apoptotic DNA digestion following
caspase-activated DNase activity [46], V(D)J splicing
[52] and class switch recombination [53] during the
development of immunoglobulin variability.
The presence of doubly charged, bulky phosphate in
c-H2A.X may generate localized decondensation of
chromatin domains with increased accessibility to var-
ious effectors such as modulating enzymes or repair
complexes, or simply mark spots for downstream
events. In agreement with this, genomic DNA showed
nuclease hypersensitivity in an S129E yeast H2A.X
mutant that mimics the charged state of c-H2A.X [47].
Removal of the SQE motif leads to impaired nonho-
mologous end joining (NHEJ) in S. cerevisiae, whereas

phosphorylation of the serine residue in response to
DNA fragmentation facilitates NHEJ by decondensing
chromatin at the damaged DNA sites and making it
accessible to repair factors [47]. Deficiency of H2A.X
in mice leads to meiotic defects, such as retaining
unprocessed double stranded breaks after asynapsis
and increased predisposition to various tumours in the
absence of p53 [54]. Thus the rapid observed colocali-
zation of the p53 binding protein1 (53BP1) with
c-H2A.X foci after introduction of DNA double
strand breaks may have great clinical implications.
Phosphorylated H2A.X ensures an error-free process
by using the sister chromatid as a template in exclu-
ding the error-prone repair (single-strand annealing) at
chromosomal DSBs [55]. Furthermore, H2A.X phos-
phorylation by primary DNA damage checkpoint kin-
ases makes a large chromatin domain permissive for a
de novo recruitment of cohesins required for cohesion
of sister chromatids. Cohesins tether the broken DNA
ends, making them a preferred substrate for repair and
preventing the highly reactive DNA ends from aber-
rant translocations and large interstitial deletions [56].
Several examples from various species, including
Xenopus, Drosophila, mammals and S. cerevisiae, have
shown that ionizing radiations and other agents that
cause double-strand breaks result in rapid and massive
phosphorylation of the histone variant H2A.X. Effi-
cient, homologous recombinational repair of a chro-
mosomal DSB is evidently found to require Ser139 of
mammalian H2A.X. Recent studies with yeast have

given better understanding of the involvement of
H2A.X in the repair process. Yeast H2A phosphoryla-
tion is not required for activation of S-phase DNA
damage check points [48] or for the initial recruitment
of several repair factors [57], which is followed by for-
mation of large, irradiation-induced foci (IRIFs) con-
taining a large number of repair factors. Formation of
IRIFs that sequester multiple DNA DSBs [58,59] uses
the SQ motif of H2A.X [57,60], suggesting that the
phosphorylation may promote the spreading and sta-
bilization of the repair factors through IRIFs. It is
quite likely that some of the initially recruited repair
factors bring in the specific kinases for the subsequent
phosphoryation of H2A.X. The phosphorylation is
seen to spread for approximately 25 kb on both the
sides of a DSB, but is absent from approximately
1–2 kb immediately adjacent. This is probably due to
the loss or exchange of H2A.X, brought about by the
recruited chromatin modifying activities at DSBs, as
discussed later.
A mechanism that recruits and spreads the repair
machinery from the foci having c-H2A.X at the dam-
age point rather than globally recruiting it to other
points having bulk H2A as well (probably via certain
other mechanisms) may be advantageous for cells. It
reduces the number of recruitment sites and therefore
the total requirement of these repair factors. This may
also be a mechanism of tethering the repair machinery
to the DNA double strand breaks, analogous to the
transcription-coupled nucleotide excision repair path-

way, which uses a general transcription factor [40,41].
Phosphorylation at the SQ motif of the variant may be
easier and more economical than developing a new
method of marking the damage site with the bulk
H2A.
ATP-dependent chromatin remodelling and covalent
histone modifications are two processes associated with
the regulation of gene expression from a chromatin
region. A close relationship between chromatin remod-
elling and DNA repair reported recently [61] is an
excellent example of the economy practiced by cells in
general. It suggests that chromatin remodelling may
not be a process related only to gene expression.
Rather, the same proteins may be active in other
DNA-related processes, coupling the two processes.
An HMG-like subunit, Nhp10, of the yeast chromatin
remodelling complex INO80, is shown to interact with
c-H2A.X at DSBs to recruit the INO80 complex. Gen-
etic evidence for the interaction of Nhp10 with mem-
bers of the RAD52-dependent repair pathway suggests
that INO80 may in turn recruit the repair machinery
at the damage site through Nhp10 [62]. In Drosophila,
the H2A variant H2Av, is a functional homologue of
both H2A.X as well as H2A.Z in mammals [63]. The
Drosophila Tip60 chromatin remodelling complex
acetylates nucleosomal phospho-H2Av. At the same
time, the ATPase activity of dTip60 exchanges the
R H. Pusarla and P. Bhargava Histone variants in various functions
FEBS Journal 272 (2005) 5149–5168 ª 2005 FEBS 5153
phospho-H2Av with the unmodified H2Av, presenting

an example of two chromatin modifying activities
within the same complex [64]. One of the histone acetyl
transferase (HAT) complexes of yeast, NuA4, through
one of its subunits (Arp4) is shown to associate specif-
ically with the phospho-H2A peptide. Arp4, which is
also a subunit of two further ATP-dependent chroma-
tin remodelling complexes, INO80 and Swr1, is
required for the recruitment of NuA4 to DSB, con-
comitant with Ser129 phosphorylation of c-H2A.X.
The other two remodellers also interact with P-Ser129,
although after NuA4 recruitment [65]. Therefore, effi-
cient DNA repair in yeast appears to require sequen-
tial remodelling by three chromatin modifiers. These
chromatin modifications may lead to the decondensa-
tion of the chromatin required for DSB repair, as well
as help remove the phosphorylated H2A.X and
thereby avoiding a permanent marking of the damage
spot.
Variants in silencing and heterochromatinization
Eukaryotic genomic DNA is organized into two char-
acteristically different forms. Euchromatin is constitu-
ted by the transcriptionally active, open and
decondensed chromatin structure. In contrast, hetero-
chromatin is considered transcriptionally inactive, with
compact and highly condensed chromatin regions.
Methylation of H3K9, recruitment of HP1 and other
condensing proteins, and DNA methylation participate
in the process of heterochromatinization. In addition,
by virtue of their capacity to generate different nucleo-
somal conformations, some histone variants are also

known to associate with and promote the heterochro-
matin formation [66]. For example, Drosophila H2Av
is found to participate in heterochromatin formation
by marking the region for subsequent acetylation at
H4K12 and methylation at H3K9 with HP1 recruit-
ment [67]. It shows a nonuniform pattern of wide dis-
tribution in the genome and is present in thousands of
euchromatic bands as well as the heterochromatic
chromocentre of polytene chromosomes [28].
In mouse spermatocytes, c-H2A.X plays a crucial
role in sex chromosome condensation and transcrip-
tional inactivation under the process of meiotic sex
chromosome inactivation (MSCI). It regulates chroma-
tin remodelling and associated silencing of male sex
chromosomes by initiating heterochromatinization in
the sex body. Absence of H2A.X in mice results in
infertility in the male but not in the female, and several
sex body proteins such as XMR and macroH2A1 ⁄ 2
fail to localize to the sex chromosome [68]. The
absence of condensed sex body and the failure of
meiotic pairing by X and Y chromosomes in H2A.X
deficiency suggests that H2A.X is more important for
heterochromatinization in the male than the female.
Mammalian H2A.Z is also found to be essential for
establishing higher order chromatin structure at consti-
tutive heterochromatic domains, probably by control-
ling the localization of HP1a. It is localized along with
HP1a on chromosome arms but not on centromeric
regions [69]. Arrays of positioned nucleosomes con-
taining H2A.Z over the defined sequence 208–12 DNA

(12 repeats of 208 bp sea urchin 5S rDNA positioning
sequence), organize into 30 nm fibres but do not con-
dense into the next higher level of compaction [70],
even at high Mg
2+
levels that are known to promote
chromatin condensation. Another study has now
established that the acidic patch of H2A.Z (described
below) provides an altered nucleosome surface for
localized compaction of chromatin fibre folding with-
out crosslinking, and enhances the binding of HP1 to
the condensed higher order chromatin structures [71].
Therefore, H2A.Z along with HP1 appears to regulate
heterochromatin formation by preventing the further
compaction of the 30 nm chromatin fibre.
One of the H2A variants, macroH2A, with its two
nonallelic forms mH2A1 and mH2A2, appears to be
involved in X chromosome inactivation. It shows high-
est expression in liver followed by testes [72], with one
mH2A for every 30 nucleosomes in rat liver [29]. Its
presence in the XY body of spermatocytes indicates its
role in the spermatogenic process, which is consistent
with its absence in invertebrates and evolution in verte-
brates. It evidently associates with Barr bodies (the
inactive X chromosomes) at levels higher than other
chromatin proteins [73,74]. The inactive chromatin of
the Barr body is characterized by denser chromatin
domains and higher nucleosome density, and shows
the presence of both H2A and mH2A [75]. Addition-
ally, mH2A colocalizes on the uncoiled X chromo-

some, with methylated H3-K4 at a potential activation
boundary during metaphase [73], and with heterochro-
matin protein M31 during meiotic prophase [76], thus
suggesting that the association of macroH2A may not
be specific to the Barr body. It brings about X-chro-
mosome inactivation probably by stabilizing the bind-
ing of Xist to the X chromosome through its
nonhistone region [77].
Nucleosomes containing mH2A have altered struc-
ture owing to the high a-helical content in their C-ter-
minal nonhistone regions [78]. The unusual structure
of mH2A with a large C-terminal tail may give a
unique conformation to the nucleosome, as reflected
by their low sedimentation coefficient despite a 25%
increase in the mass. The core particles having mH2A
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5154 FEBS Journal 272 (2005) 5149–5168 ª 2005 FEBS
show slower gel mobility but the same stability as that
of native nucleosomes, suggesting an asymmetric and
extended conformation. Presence of the nonhistone
region may be responsible for the observed DNaseI
hypersensitivity near the dyad axis and around
entry ⁄ exit sites of DNA in the nucleosome [78]. Macro-
H2A exerts its repressive action through control over
transcription and chromatin remodelling. The presence
of mH2A in a positioned nucleosome disrupts access
for NF-jB, as well as remodelling and mobilization of
variant nucleosomes by SW1 ⁄ SNF without affecting
either its binding or ATPase activity [79]. A macro-
H2A C-terminal region present near to a promoter

reduces the transcriptional activity, probably by acting
as a road-block to the passage of RNA polymerase
[75].
Variants in gene expression
Several core histone variants have been found to regu-
late gene expression and antisilencing mechanisms in
different ways. Active participation of the chromatin
structure in the process of transcription on a tran-
scribed gene demands a dynamic nature in the chroma-
tin template requiring a constant reshuffling of the
nucleosomes over this. A chromatin structure estab-
lished due to deposition of the major histones in the
S-phase of the cell cycle may not be fluid enough to
give the required dynamism, as histones are strong
DNA-binding proteins. Replacement or exchange of
the major histones or their modified forms by their
variants having different affinities and strength of
binding to the DNA may provide a better alternative
outside the S-phase.
RC assembly usually results in a rigid chromatin
structure over genes, which are deficient in modifica-
tions that facilitate the mobility of nucleosomes. RI
assembly delineates active regions making them relat-
ively dynamic and variants mark these regions in addi-
tion to giving them the required flexibility. The
replacement variant H3.3 is found to account for
 25% of total histone H3 in a Drosophila cell line,
sufficient to deposit nucleosomes on all of the tran-
scribed DNA [80]. It is also found deposited over act-
ive rDNA arrays on the X chromosome, where it

shows a constant turnover. The deposition of H3.3 is
directly linked to active transcription at the hsp70 gene
locus, as it stops replacing H3 after the induced gene is
switched off [81]. Constitutive synthesis replenishes
H3.3, which is shown to be short-lived compared to
bulk H3. The changing of one amino acid from his-
tone H3 to its H3.3 counterpart relieved the block to
RI assembly and further deposition of H3 outside S
phase [82]. Thus, while the N-terminal was required
for RC deposition, specific residues in the histone fold
could switch it to the RI deposition pathway, which
seems to be restricted to H3.3 deposition and targeted
to transcriptionally active chromatin.
In mice, the transcript levels of both H3.1 and H3.2
decrease as cell division slows down during differenti-
ation, whereas H3.3 continues to be synthesized and
maintained throughout differentiation. Similarly, Droso-
phila H3 is deposited only during S-phase, whereas
H3.3 is deposited both during and outside of S-phase,
suggesting that H3.3 might accumulate in nondividing
cells [2]. Excess accumulation of H3.3 in nerve cells
leads to further severity of Rett syndrome, a common
mental disorder directly related to the loss of MeCP2,
a methylated CpG binding protein. MeCP2 deficiency
leads to the loss of silencing mechanisms involving
H3K9 methylation and histone deacetylase activity.
Acetylation of H3K9 is associated with active chroma-
tin while H3K9 methylation marks inactive chromatin
regions. Thus, the unintended activation due to H3.3
accumulation (associated with transcribed regions) and

excess H3 acetylation (due to reduced deacetylation)
might further aggravate the condition [83].
As compared to H3, H3.3 shows several fold
enrichment of modifications found on active genes,
which is a significant mark for active chromatin
[80,84]. The chromatin modifiers introduce these act-
ive modifications probably by associating with specific
nucleosome assembly proteins. The stepwise assembly
pathway of a nucleosome core particle proposes the
association of histones H3 and H4 (two copies each)
into a tetramer as the first step in assembly. The RC
variant H3.1 and RI variant H3.3 form complexes
with distinct histone chaperones [85]. A histone chap-
erone, HIRA, which acts as a specific nucleosome
assembly factor, deposits H3.3 in a replication-inde-
pendent manner [86] while CAF-1 deposits the major
variant H3.1. Isolation of the two complexes also
suggested that histones H3 and H4 can exist and be
deposited as dimers rather than tetramers [85]. Tran-
scription-coupled deposition of H3.3 in an RI nucleo-
some assembly pathway targets it to transcriptionally
active loci throughout the cell cycle. Thus, modified
histones such as methylated H3, which act as an epi-
genetic mark for silencing, can be rapidly replaced by
H3.3 in the RI pathway. A detailed account of
deposition pathways for histone variants can be
found in a recent review [6].
Histone replacement ⁄ exchange by RI assembly on
transcribed templates suggests a possible mechanism
for read-through of a nucleosomal template by the

enzyme RNA polymerase. It was found in an in vitro
R H. Pusarla and P. Bhargava Histone variants in various functions
FEBS Journal 272 (2005) 5149–5168 ª 2005 FEBS 5155
study that RNA polymerase II (pol II) can transcribe
through a nucleosome without completely displacing
histones from it [87]. The protein complex facilitates
chromatin transcription (FACT) facilitates read-
through of the nucleosomal template by RNA polym-
erase II during transcription elongation [88]. Associ-
ated histone chaperone activity of FACT can help
remove as well as redeposit an H2A-H2B dimer during
the transcription [89]. Chromatin reassembly in yeast
becomes dependent on the Hir⁄ Hpc (human HIRA
homologue) pathway on the loss of yeast FACT activ-
ity [90], suggesting that both chaperones may be work-
ing on transcribed templates. Removal of H2A ⁄ H2B
by FACT may facilitate access of H3 for exchange
with H3.3 by HIRA in the next step. Nevertheless, a
recent study reports the exchange of H2A.Z with bulk
H2A on the c-myc gene during transcription [91].
These findings suggest that nucleosomes can indeed be
shuffled during read-through by RNA pol II in vivo
without displacing the histone octamer completely.
In the budding yeast S. cerevisiae, H2A.Z is found
to be important for both positive and negative gene
regulation [92–95]. Loss of Htz1 in yeast cells leads
to slow growth and formamide sensitivity at 28 °C
and lethality at 37 °C [96]. The PHO5 promoter is
found to be more open in the htz1 D⁄snf2D mutant
[95], suggesting this H2A variant in yeast acts with

chromatin modifiers such as SWI⁄ SNF and SAGA
on this locus. Thus, it binds the PHO5 locus and
regulates its expression. An important role for
H2A.Z in both gene activation and silencing is also
demonstrated by localization of H2A.Z containing
transcriptionally activated gene domains near telom-
eres as well as in regions flanking HMR loci. These
regions prevent the ectopic spread of the repressor
proteins Sir2 and Sir3 into the flanking euchromatin,
as Sir proteins are found to extend beyond the nor-
mal boundaries in htz1D cells [97]. Global sensitivity
of chromatin to nucleases is affected in htz1D cells
while H2A.Z is found to facilitate the recruitment of
RNA pol II transcription machinery to gene promo-
ters [92] and modulate its functional interactions with
the regulatory components. This activator-like func-
tion of H2A.Z resides in its C-terminal region, which
is linked to its ability to preferentially localize to cer-
tain intergenic DNA regions [98]. Thus, the associ-
ation of H2A.Z with transcriptionally active
chromatin may require the carboxy terminal and not
the histone fold region, which is essential for viability
[99,100].
The nucleosome core particles with variant H2A.Z
also showed an altered surface harbouring a metal
ion. This altered surface may act as an activating
surface by participating in the recruitment of tran-
scription factors and chromatin remodellers, and set
the stage for gene activation upon a proper induction
[98]. Thus, the variant may be required to mark and

not maintain the transcriptionally active state. In a
functional dynamic study, nucleosomes were found to
show two types of large motions in space; a stretch-
ing-compression along the dyad axis and the flipping,
bending sideways motions with respect to the dyad
axis, a result of the dynamism of the N-termini of H3
and the H2A.Z-H2B dimer. The nucleosomes with
variant histones show comparatively weaker correla-
tions between internal motions, resulting in the per-
turbation of interactions between the contact regions
of the variant histones with overlying DNA [19]. In
agreement with this, H2A.Z-H2B dimers in the vari-
ant nucleosomes dissociate with comparative ease,
correlating with the observation that chromatin
regions containing H2A.Z probably do not require
SW1 ⁄ SNF remodelling complexes [95]. However, in a
global analysis, a 13 protein complex, SWR-C, neces-
sary for promoting gene expression near silent hetero-
chromatic regions of yeast, is found to be required
for the recruitment of Htz1 to chromatin also [101].
Incorporation of Htz1 is facilitated by one of the
components of SWR-C, Swr1, an ATPase of Snf2
family, which acts as a histone exchanger and effi-
ciently replaces H2A with H2A.Z in nucleosome
arrays [94]. Genetic and biochemical approaches also
demonstrated the requirement of Swr1p for the depos-
ition of H2A.Z into euchromatic regions at several
sites [102]. Both groups identified a bromodomain
(which recognizes an acetyl group) containing protein
Bdf1 that also interacts with transcription factor IID

(TFIID, a basal transcription factor) as another com-
ponent of the Swr1 complex. Higher acetylation levels
in euchromatin may recruit a Bdf1-containing Swr1
complex that may finally replace H2A with H2A.Z. A
genetic interaction between SWR-dependent H2A.Z
recruitment at centromeres, the SWR1 complex and
NuA4 (a histone H4 acetylase) is linked to chromo-
somal stability [103], suggesting a direct role for H2A.Z
in chromosomal segregation. Both NuA4 and SWR-C
share some common subunits. Acetylation is a post-
translational histone modification, which happens pre-
dominantly in the N-terminal tail and changes its
charge. H2A.Z acetylation is essential in Tetrahymena,
and the replacement of all six lysines that can be
acetylated with arginines is lethal. Nevertheless,
retaining even a single such lysine can avert this leth-
ality, suggesting that the function of H2A.Z is guided
through a charge patch and not the histone code
[104].
Histone variants in various functions R H. Pusarla and P. Bhargava
5156 FEBS Journal 272 (2005) 5149–5168 ª 2005 FEBS
Variants in different chromatin structures
Variations in core histones can give minor, localized
alterations in nucleosomal conformation. Subtle chan-
ges in one of the components can generate unique
nucleosomal surfaces that may regulate interparticle
interactions thereby bringing about changes in the
three-dimensional folding of the chromatin fibre and
establishing special chromatin structural regions. Fig-
ure 2 illustrates the involvement of various H2A vari-

ants in generating a variety of chromatin structures.
Generation of the condensed chromatin domains
(Fig. 2H), starting from fully extended and relaxed
‘beads on a string’ (Fig. 2C), requires compaction of
the 10 nm fibre (Fig. 2B) followed by folding, conden-
sation and superfolding through the 30 nm stage to
higher order chromatin structure. The details of the
nucleosome structure in Fig. 2A depict the positions
where two of the core histones H3 and H2A can
acquire changes. H2A variants can lead to inactive or
condensed heterochromatin (Fig. 2D,E,G) as explained
above. However, they can also be found in active,
euchromatic regions as described in the following stud-
ies. Thus, H2A.Z is one of the variants that has been
found to induce both repressive and antisilencing
effects.
H2A.Z is essential for establishing the proper chro-
matin structure required for early development in
many organisms, including mice, Drosophila and Tetra-
hymena [105–107]. Absence of H2A.Z in mammals
leads to genome instability and defects in chromosome
segregation [69]. During embryonic differentiation sta-
ges, it is excluded from the nucleolus as well as the
inactive X chromosome and made its first appearance
A
B
C
D
E
F

G
H
Fig. 2. Involvement of H2A variants in the formation of different chromatin structures. (A) Nucleosome core structure details showing only
H3 and H2A (H4 and H2B are omitted for clarity). The right half shows the normal histones, while possible positions of the variations in
amino acids are marked with an asterisk in the left hand side counterparts. (B) Normal folding of the 10 nm fibre with canonical, bulk
histones into the zig-zag fibre. (C) The extended 10 nm fibre with ‘beads on a string’ appearance. (D) H2A.X helps in higher order structure
formation at the constitutive heterochromatin. (E) Shorter length and greater accessibility of DNA wrapped in nucleosomes due to H2A.Bbd.
(F) The acidic patch of H2A.Z allows greater interaction with the N-terminal tail of H4 from the neighbouring nucleosome. (G) Longer
C-termini of mH2A or CENP-A may interact with the nucleosomal DNA to make nucleosomes more rigid and help further condensation.
(H) Condensed chromatin showing close contacts of core particles due to the dense packing.
R H. Pusarla and P. Bhargava Histone variants in various functions
FEBS Journal 272 (2005) 5149–5168 ª 2005 FEBS 5157
in the pericentric regions of nucleus, providing a poss-
ible signal to distinguish constitutive and facultative
heterochromatin [108].
Biophysical studies of chromatin fibres having
H2A.Z suggested that it resists condensation when
compared to its major H2A counterpart and the fibre
assumes a relaxed conformation [70]. This proposes a
mechanism under which chromatin is poised for tran-
scriptional initiation by depositing variant nucleo-
somes. Native gel electrophoresis did not distinguish
between the core particles having major H2A.1 or the
variant H2A.Z, which is only 59% identical to the
conventional H2A [109]. However, sedimentation ana-
lysis under changing ionic strength showed a substan-
tial instability of the variant core particle, indicating a
less tight binding of the H2A.Z-H2B dimer to the rest
of the octamer [109]. A recent thermodynamic study
has confirmed that the H2A.Z-H2B dimer has the least

stable folding and that the canonical H2A-H2B dimer
shows the most stable folding [110]. The 2.6 A
˚
resolu-
tion crystal structure of the variant nucleosome core
particle showed surprisingly small changes in the over-
all structure of H2A.Z [111]. However, distinct and
subtle destabilization of the interaction between the
H2A.Z-H2B dimer and the (H3-H4)
2
tetramer is seen.
The L1 loop domain of H2A (Fig. 2B), which ensures
incorporation of only one type of molecule, is altered
in H2A.Z. As a result, pairing of H2B with both
H2A.Z and H2A within the same nucleosome core
particle leads to steric imbalance that may favour
binding to another H2A.Z. A unique feature of the
acidic patch on the surface of normal H2A is extended
by replacement of Asn and Lys with Asp and Ser in
H2A.Z [111]. This enhanced charge patch at the C-ter-
minus is required for higher order chromatin forma-
tion and may offer a stronger docking domain for the
H4 tail of a neighbouring nucleosome [71], thereby
promoting interparticle folding in arrays (Fig. 2F).
Functional evidence of the implicit repressive role of
H2A.Z comes from a recent study demonstrating
replacement of the H2A.Z-H2B dimer by the
H2A-H2B dimer by transcribing RNA pol II [91].
While the acidic nature of the charged patch of
H2A is increased in H2A.Z, it is decreased in a newly

identified ‘Barr body deficient’ histone variant,
H2A.Bbd. This is found to be 48% identical to (but
shorter than) conventional H2A. Its distribution is
similar to that of acetylated H4 and it is excluded from
the inactive X chromosome, hence the name [112]. Its
primary sequence in the docking domain differs con-
siderably from H2A. It is conspicuous by the absence
of lysines or any of the target residues for
the post-translational modifications acetylation,
phosphorylation and ubiquitination [15], but its hall-
marks are the presence of a continuous stretch of six
arginines in the N-terminus.
H2A.Bbd organizes only 118 ± 2 bp into nucleo-
somes as compared with 147 in canonical nucleosomes
[113]. It gives arrays with shorter repeat length and
higher nucleosome density, an organization that could
repress transcription from a natural promoter in
an activator-responsive manner (Fig. 2E). Within
H2A.Bbd-containing nucleosome core particles, DNA
ends are less tightly bound and interactions of
H2A.Bbd-H2B with an (H3-H4)
2
tetramer are weak
[113]. It is also found that the relaxed structure and
altered conformation of the Bbd nucleosome is due to
the changes in the H2A docking domain and not due
to the absence of the C-terminal tail. Thus, H2A.Bbd
has destabilizing effect on nucleosome structure under
normal conditions but SWI ⁄ SNF and ACF complexes
(ATP-dependent chromatin remodellers) failed to

mobilize H2A.Bbd containing nucleosomes [114].
However, the lower stability of H2A.Bbd-containing
nucleosomes may facilitate the exchange of the
H2A.Bbd compared to H2A [115], probably promoting
transcription through nucleosomes during the elonga-
tion phase.
Similar to H3.3, the third H3 variant in Drosophila,
Cid, is deposited in an RI manner throughout the cell
cycle. An open chromatin configuration at both cen-
tromeres (due to the lack of H3K9 methylation in Cid)
as well as active chromatin is proposed to be the com-
mon basis of RI histone deposition at these sites [37].
Conserved blocks in the N-terminus and histone fold
of Cid may mediate essential protein–protein interac-
tions for recruitment of other centromeric proteins,
neutralize phosphates in linker DNA and further help
in higher order chromatin structure. Centromeric
nucleosomes of mice also are characterized by the pres-
ence of the centromeric H3 variant CENP-A [116]. It
is required for the recruitment of components essential
for kinetochore formation and chromosome segrega-
tion; disturbance in these important activities due to
targeted deletion of CENP-A in mice results in embryo-
nic death [117]. CENP-A competes with H3 for H4
during nucleosome formation and can be reconstituted
with DNA into nucleosomes with properties similar to
those of bulk nucleosomes [118]. CENP-A and H4
subnucleosome tetramers are more compact and con-
formationally rigid compared to normal tetramers
[119]. This tetrameric compaction in the nucleosomes

gives the centromeres a specialized, rigid structure: a
competent configuration necessary at centromeres to
withstand various mechanical and physical insults of
pulls to the two poles during cell division.
Histone variants in various functions R H. Pusarla and P. Bhargava
5158 FEBS Journal 272 (2005) 5149–5168 ª 2005 FEBS
Centromeric DNA is several hundreds of kilobases
in higher organisms whereas a 125 bp unique region
specifies the single nucleosome yeast centromere [120].
In S. cerevisiae, a chromosome missegregation mutant
cse4-1 shows mitosis-specific arrest at elevated temper-
atures and the Cse4 gene was found to be essential for
correct cell division [121]. The centromeric H3-like
protein, Cse4p, is an integral component of the yeast
centromere [122] and can substitute structurally and
functionally for human CENP-A, showing the strong
conservation of the centromeric features in both [123].
The histone fold domain of Cse4 is sufficient for its
localization to the centromere [124]. However, for
interaction with kinetochore components, an essential
N-terminal domain (END) comprising 33 amino acids
within the 130 amino acids long N-terminal tail is
required [125]. Yeast ATP-dependent chromatin
remodelling complex, RSC, is localized to the centro-
mere and its proximal regions. However, it is not
required for Cse4 deposition into the centromeric
nucleosomes. Rather, it helps remodel the associated
regions for proper chromosome transmission [126].
Recently Hayashi et al. [127] have found that two
mutants of the fission yeast mis16 and mis18 fail to

maintain inner centromere histones in a deacetylated
state and do not recruit CENP-A (Cnp1 ⁄ spCENP-A)
to centromeres. Similarly, human Mis16-like proteins,
RbAp46 and RbAp48, are also required for proper
CENP-A localization in human cells [127]. Further
studies will be necessary to reveal how Mis16 ⁄ Mis18
changes the chromatin environment at centromeres in
order to allow CENP-A loading.
In all of the above-mentioned nuclear processes
chromatin acquires a variety of configurations. For
inactivation of the X chromosome or heterochromati-
nization and the silencing of defined regions, the chro-
matin structure needs superfolding of the fibres,
extreme condensation through strong interfibre as well
as interparticle interactions. In contrast, for gene
expression from active regions as well as site-specific
DNA damage repair, it needs decondensation, expan-
sion and uncoiling of the regions by weakening of the
same interactions between its fibres or particles. Indi-
vidual nucleosomes contribute to these DNA–protein
and protein–protein interactions through the N-ter-
minal tail regions of their histones. Generating two
opposite end-results through the same set of inter-
actions can be made possible by regulating the para-
meters that define these interactions. It is conceivable
from the previous sections that the functional diversifi-
cation of chromatin is directly related to the structural
variety brought about by the variants. Thus the gen-
eration of functionally heterogeneous conditions may
become possible through variation in the histone pri-

mary structure that, in turn, creates precise structural
changes in the nucleosomes.
Why have variants evolved in H2A and
H3?
Variants are found for all histones (except H4) but
with different propensities. Most of the H2B and H1
variants are reported to participate in the spermato-
genesis process. While H4 is invariant, H2A has a rich
family of variants and H3 is known to have a few dis-
tinctly important variants. Why this heterogeneity?
The answer probably lies in the arrangement of
histones in the nucleosome core particle, as revealed
by the structure solved to 2.8 A
˚
resolution for crystals
obtained under near physiological conditions [128]. All
four core histones have a histone fold domain in their
middle region and two unstructured tails of different
lengths at both ends. Histone folds are arranged in a
handshake manner to generate the octameric protein
core, while the N-terminal tails of all of the core
histones protrude to surface of the nucleosome, mak-
ing contacts not only with the DNA backbone but also
offering involvement in nucleosome–nucleosome inter-
actions (Fig. 3A). C-terminal tails usually harbour
docking domains but greater variations in amino acid
composition and domain length are also observed in
this region. N-terminal regions have significant homol-
ogy even among the variants of histones, as most of
the sites of putative post-translational modifications

are found in this region. While the random coil seg-
ments of N-terminal tails of both H3 and H2B pass
between gyres of the DNA superhelix, four amino
acids of the H2A N-terminal tail, close to the site of
H2B interaction, bind to the minor groove on the out-
side of the superhelix (Fig. 3A). Thus, N-terminal tails
are involved in deciding the DNA–histone interactions,
and to keep an intact nucleosome they need to be
spared from the changes that could destroy these inter-
actions. Changes in C-termini instead may give nucleo-
somes various properties without interfering with the
basic scheme of their structure.
Among the histone heterodimers of the core particle,
one of the partners is usually found to be more varied.
Varying only one of the partners at a time can give an
alteration in structure with the least perturbation, and
in the H2A-H2B dimer H2A could be the better
choice, due to the following reasons. Interaction of the
H3-H4 tetramer with the H2A-H2B dimer is esta-
blished through contacts made by H2B with H4 [128],
which is one of the important interactions in core par-
ticle assembly. Therefore, H2B may not be preferred
R H. Pusarla and P. Bhargava Histone variants in various functions
FEBS Journal 272 (2005) 5149–5168 ª 2005 FEBS 5159
for variation. Compared to other core histones, H2A
has a strategic placement in the nucleosome and con-
tains the largest consensus C-terminal tail (Fig. 2).
This tail protrudes on the outside of nucleosome near
the entry and exit sites of the DNA, and amino acids
105–117 link aN of the opposite H3 to the H3-H4 his-

tone fold domains. Preceding this, amino acids 92–108
of H2A form a folded docking domain with its a3
helix for H4 (Fig. 3B). Due to these two carboxy-ter-
minal regions of H2A, its exchange can offer a greater
scope for heterogeneity to both the tetramer as well as
the linker interface [129]. This in turn also has wider
possibilities for association with chromatin remodelling
machineries. The H2A-H2B dimer in the nucleosome
is easily dissociable, and readthrough of the nucleo-
some by RNA pol II was shown to result in the loss
of a dimer from the core particle [130]. Therefore,
H2A replacement-dependent regulatory mechanisms
may be energetically advantageous, and it is not sur-
prising that a larger number of H2A variants are
known that impart different functional states to the
nucleosomes carrying them. Most of these known vari-
ations map to the carboxy terminal domain of the pro-
tein (Fig. 1).
Similarly, out of the H3-H4 pair, H4 makes contacts
with the other three histones in the octamer, and varia-
tions in its sequence are least tolerated. In contrast,
the H3 dimer occupies the dyad axis and has a central
role in organizing the core particle. The tetramer of
H3-H4 is formed by the interaction of two H3 mole-
cules at the dyad axis via C-terminal halves of the their
two a2 helices as well as the a3 helices (Fig. 3B). Cen-
tromeric H3 with a very different N-terminal region or
H3.3 with only a slightly different C-terminal proximal
histone fold region, are both deposited into open or
transcriptionally active chromatin regions [37]. Thus, it

appears that minor sequence variations in the C-ter-
minal proximal histone fold region of H3 that guide it
to actively transcribed chromatin regions can be toler-
ated easily. They do not disturb incorporation of H3
into the nucleosome, as shown by the similar overall
crystal structure of Xenopus and yeast nucleosomes
[131] with the latter having an H3 more akin to H3.3
of other eukaryotes. Small perturbations in H3 folding
due to the presence of a probe at its unique and cen-
trally placed cysteine (Cys96 or Cys110) in the a2of
the histone fold can generate different conformers of
the nucleosome; those with open conformations could
be better transcribed [132]. No specific changes in
structure are attributed [131] to the two different
amino acids at positions 89 and 90 of yeast H3, which
are found in the N-terminal halves of the a2 helices.
The location of these amino acids in the crystal struc-
ture of the yeast nucleosome core particle suggests that
they may influence the interaction of the H3-H4 dimer
with the H2A-H2B dimer, by altering its orientation in
AB
Fig. 3. Structural features of a nucleosome as revealed by the crystal structure analysis showing intranucleosomal interactions of histones.
(A) Half of a nucleosome (with one superhelical turn of 73 bp DNA) showing all domains of the four core histones and seven helical turns of
the DNA. The C-terminal tail of H2A with the maximum number of variations known is highligted. C and N indicate the C- and N-terminal
ends, respectively, of the individual histones. (B) Structure of the yeast nucleosome with both turns of the DNA, showing histones of the
lower half only partially. All four strands of DNA are shown in different shades for clarity; lighter shades are given to histones of the lower
half.
Histone variants in various functions R H. Pusarla and P. Bhargava
5160 FEBS Journal 272 (2005) 5149–5168 ª 2005 FEBS
space. Thus, it is probable that a compatible H3.3-H4

dimer could compensate for the altered orientation of
the H2A.X ⁄ H2A.Z-H2B dimer in yeast [131], resulting
in same overall nucleosome structure as in other euk-
aryotes. The structural alterations in H3.3 probably
facilitate loss ⁄ exchange of the H2A ⁄ B dimer during
the transcription process, making them advantageous
for active chromatin. By analogy, variations in three
amino acids of the a2 in H3.3 histone fold (positions
87, 89 and 90) of other organisms most probably influ-
ence its interaction with other histones, resulting in a
different conformation compatible with active chroma-
tin in higher eukaryotes.
As discussed above, therefore, H2A and H3 (being
more amenable to changes) have acquired several
variations during evolution for a variety of opposing
functions, such as DNA compaction as well as
decondensation. Interestingly, both H2A and H3 in
yeast, which has sequence differences in all of the hi-
stones, are related more to the variant forms of
higher organisms than to their canonical, bulk forms.
It will indeed be interesting to find whether yeast
and other eukaryotes followed same pathway of evo-
lution from a common ancestor but diverged very
early during evolution.
Variations are not universal
Yeast is considered a model eukaryote for many stud-
ies, although it differs in a number of features from
higher eukaryotes. Several reports now show that the
differences may be deceptive and the higher aspects
may have evolved from the basic features found in

yeast. The yeast genome is reported to be largely active
with no pseudogenes or repetitive DNA, and it shows
structurally distinct promoter and nonpromoter
regions where promoters have a two- to threefold
lower nucleosome density covering them [133]. No lin-
ker histone H1 was found in yeast for a long time.
However, it is reported to have a higher order chro-
matin structure similar to that in higher eukaryotes [134].
Identification of a gene coding for a putative histone
H1 of yeast [135] suggests that this H1-like protein
may be involved in forming a higher order chromatin
similar to that in other metazoans. The differences in
the primary sequence of yeast histones from that of
higher eukaryotes may generate different particle–par-
ticle interactions. Thus, though the crystal structure of
yeast and Xenopus nucleosome core particles are sim-
ilar, sequence differences of individual histones may be
the cause of the observed crystal packing differences
and destabilization of the yeast core particle [131]. This
may also be the reason that yeast chromatin has a
similarly folded 30 nm fibre [134] but still an ‘open’
higher order chromatin structure.
Compared to mammals, fewer H2A variants in yeast
are known. Major H2A (90% of total H2A) itself
functions like H2A.X of mammals [65]. The amino
acid sequence of human H2A.X shows a C-terminal
region highly homologous to H2A species of S. cere-
visiae and Schizosaccharomyces pombe [136], suggesting
that yeast and human H2A may not have evolved
through the same pathway. Similarly, the presence of

the H2A variant Htz1 is in agreement with all active
status of yeast chromatin. The acidic patch of Htz1
probably helps to give a relaxed conformation to the
30 nm fibre that resists further condensation in the
absence of proper H1. There is no sex chromosome-
like Barr body of mammalian cells or a highly con-
densed heterochromatin, and that may be the reason
why the variant macroH2A known to be involved in
condensation of chromatin is not yet documented for
yeast or other invertebrates. Yeast chromatin shows a
variable but discrete nucleosome repeat length with an
increment of five or 10 bases, probably arising due to
the presence of regions with closely spaced nucleo-
somes in its active chromatin [137,138] that show
DNaseI hypersensitivity [139]. Similar features can be
generated due to nucleosomes having the histone vari-
ant H2A.Bbd, which is known to give nucleosomes
with loosely bound DNA ends and arrays with shorter
repeat lengths [113]. However, an H2A.Bbd-like his-
tone has not yet been reported in yeast.
Absence or presence of a variant in yeast is well cor-
related with the requirements of a particular chromatin
structure in this eukaryote. In higher eukaryotes, tran-
scription of a gene is followed by replacement of the
major H3 with the variant H3.3, such that the active
chromatin regions are enriched with this variant.
Lower stability of yeast nucleosome core particles [131]
and the presence of only one H3 variant, H3.3 [36],
correlates well with the observation that whole of the
yeast genome is active. Yeast H3 is probably not

repressively methylated at K9, and the methylation at
K4 is known to be associated with active chromatin.
The absence of the recently identified and universally
present H3K4Me-specific demethylase in S. cerevisiae
[140] may be related to the maintenance of this all-act-
ive state of the yeast genome, as demethylation of
H3K4Me may be counterproductive.
Yeast nucleosome assembly protein1 (Nap1; a his-
tone chaperone) was found to exchange the major
H2A-H2B dimer as well as variant dimers from nucle-
osomes [141]. On the other hand, two of the yeast cell
cycle-regulated histone gene repressors, Hir1p and
Hir2p, along with chromatin assembly proteins CAF1
R H. Pusarla and P. Bhargava Histone variants in various functions
FEBS Journal 272 (2005) 5149–5168 ª 2005 FEBS 5161
and Asf1, are involved in chromatin formation and
position-dependent gene silencing [142,143]. Hir pro-
teins are also reported to be required for kinetochore
function in both S. cerevisiae and Schizosaccharomyces
pombe [142,144]. It is not yet clear whether CAF-1 and
Hir proteins are the specific chaperones for Cse4 or
whether they also assemble other centromere-specific
proteins. The human homologue of Hir1p and Hir2p,
HIRA, is a substrate for cyclin-cdk2 and blocks the
S-phase [145], while the Xenopus homologue is an RI
pathway-specific histone chaperone [86]. Involvement
of the members of same protein family from different
sources in various activities suggests a simultaneous
evolution of functional diversification of histones as
well as their chaperones.

It can be noticed that variants and their covalently
modified forms are involved in demarcating structur-
ally as well as functionally different chromatin regions
(Fig. 4). For example, marking distinctions between
facultative and constitutive heterochromatin by
H2A.Z, the presence of mH2A near potential activa-
tion boundaries of decondensing X chromosome dur-
ing metaphase, and phosphorylation of mammalian
H3.3 at S31 in the regions bordering centromeres [146]
during metaphase (Fig. 4C); all may be signifying bor-
ders of active and inactive regions. In yeast, the pres-
ence of H2A.Z near the telomeres prevents the spread
of silent zones (Fig. 4B) while a single nucleosome
with the H3 variant Cse4 is enough to mark the cen-
tromere region (Fig. 4D). The assembly of variants
into nucleosomes also shows a strong correlation with
replication. During the cell cycle, there is a spatial and
temporal separation of replication and transcription.
Thus variants and their modifications may regulate the
timing of switching the chromatin domains open for
replication.
An overview
Studies with variants have given rise to several new
ideas that highlight links and connectivities in all
DNA-related processes. The basic chromatin structure
and its fundamental units are universal. Organization
of DNA and histone octamers into nucleosomes is
also the same in all organisms. Finally, global conser-
vation of replication and its mechanisms in all eukar-
yotes demands that histone octamer deposition over

DNA is also by similar mechanisms. All of these rea-
sons together might have resulted in extreme conser-
vation of histones. However, a need for variations for
regulatory purposes would have also set in with evo-
lution. To form an octamer of the same organization,
conservation of histone fold regions needed for the
handshake contacts is essential. Their N-termini are
required for interaction with neighbouring DNA
while the C-termini provide docking domains for
internucleosomal interactions. Covalent modifications
of charged residues in the N-termini and a perturba-
tion of the C-terminus results in reduced interactions
of histones with DNA as well as interparticle inter-
actions. However, variations in primary sequence or
chain length give greater scope for changing the
target interactions in both directions. An additional
N-terminal sequence in CENP-A or the extra C-ter-
minal region in mH2A both result in inactive and
compact chromatin regions (Fig. 2G). In contrast,
H2A.Bbd with a shorter C-terminal tail is localized
to active chromatin regions.
A
B
C
D
Fig. 4. Histone variants may be involved in the demarcation of functional boundaries. (A) A typical chromosome showing its different
regions. (B) In yeast, H2A.Z prevents the spread of silent chromatin into the neighbouring regions. (C) Phosphorylation of Ser31 of mamma-
lian H3.3 surrounding the centromeric region. (D) Centromeric nucleosome having the centromeric H3 variant.
Histone variants in various functions R H. Pusarla and P. Bhargava
5162 FEBS Journal 272 (2005) 5149–5168 ª 2005 FEBS

One important as well as interesting question is why
the RI assembly process has evolved in the first place,
when histones are already deposited on the DNA dur-
ing cell-cycle replication. There may be two possibilit-
ies. Either for immediate chromatin repair during
transcription or to provide a remedial measure for
removing the repressive methylated histones required
for activating the silenced chromatin. Multiple rounds
of replacement of histones that carry epigenetic tags
can only be possible through the RI assembly. The
two kinds of nucleosome-assembly pathways also open
up new vistas. Due to the replacement mechanism, a
new possibility of a dimeric unit (rather than the well
known tetramer nucleation pathway) forming the basic
unit of nucleosome assembly has come into focus [3].
In addition to the structural and regulatory roles men-
tioned above, variants give one more advantage over
histone modifications. The idea that the variants can
provide tools for epigenetic inheritance, not provided
by modifications, is enforced by the presence of dis-
tinct chaperones for them.
From the above account it is clear that histone vari-
ants have evolved for some very special functions.
They confer variety and increase the leverage for regu-
lation to otherwise uniform chromatin structure. They
can act in tandem or as an alternative to histone modi-
fication for the generation of special chromatin
regions. Clearly, they are connected with every DNA-
related activity of the cell. How and why different vari-
ants are targeted to different specific regions and give

stably modified structures will be questions for future
research.
Acknowledgements
We thank Durgadas Kasbekar for critical editing of
the manuscript.
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