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R E V I E W A R T I C L E

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 ofchromatin 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 toDNA 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 [47]. 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: [email protected]

(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 indemarcating 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) 51495168 2005 FEBS 5149

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modifications or ATP-dependent chromatin remodel-

ling [810] 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 [1113].

Variation in high conservation theevolution 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-

toneDNA as well as particleparticle 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 havebeen 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 [1820].

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 throughvariations 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.Zwere 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.12 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 510% 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.FZ (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) 51495168 2005 FEBS

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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 proteins 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 132159 and 181208, 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 [3234]. 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 regionsCenp-A Cse4 Cid Centromeric nucleosomes

H2A H2A.Z Htz1 H2Ava Different functions in various organisms: maintenance of

pericentric and telomeric heterochromatin, transcriptional

activation and viability

H2A.X H2A H2Ava 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 SWISNF remodelling

H2A.Bbd Close spacing of nucleosomes

aDrosophila 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 a1a3, 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.

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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 belongto 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 ofCENP-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 variousnuclear 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 thesuitability 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 1015% 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

ATMATR 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



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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 isquite 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

12 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 covalenthistone 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



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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 macroH2A12

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 withHP1a on chromosome arms but not on centromeric

regions [69]. Arrays of positioned nucleosomes con-

taining H2A.Z over the defined sequence 20812 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 Mg2+ 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 (theinactive 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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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

entryexit sites of DNA in the nucleosome [78]. Macro-

H2A exerts its repressive action through control overtranscription 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 SW1SNF 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 ofbinding 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 replacementexchange 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



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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 helpremove as well as redeposit an H2A-H2B dimer during

the transcription [89]. Chromatin reassembly in yeast

becomes dependent on the HirHpc (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 H2AH2B

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 [9295]. 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 htz1D snf2D mutant

[95], suggesting this H2A variant in yeast acts with

chromatin modifiers such as SWISNF 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 containingtranscriptionally 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

SW1SNF 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].



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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 thethree-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 beenfound 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 [105107]. 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.



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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 amechanism 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 extendedby 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 couldrepress 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 SWISNF 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 foldof Cid may mediate essential proteinprotein 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.



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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-likeprotein, 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 (Cnp1spCENP-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 Mis16Mis18

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 DNAprotein

and proteinprotein 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 andH3?

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 nucleosomenucleosome 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 DNAhistone 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



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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

105117 link aN of the opposite H3 to the H3-H4 his-

tone fold domains. Preceding this, amino acids 92108of 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 ortranscriptionally 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 a2 of

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

A B

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.



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space. Thus, it is probable that a compatible H3.3-H4

dimer could compensate for the altered orientation of

the H2A.XH2A.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 lossexchange of the H2AB dimer during

the transcription process, making them advantageousfor 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 aspectsmay 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 particlepar-

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 chromatinstructure 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



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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-specificproteins. 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 andtemporal 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 allDNA-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.



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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 requiredfor 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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