Histones in functional diversification
Core histone variants
Rama-Haritha Pusarla and Purnima Bhargava
Centre for Cellular & Molecular Biology, Tarnaka, Hyderabad, India

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 requirement of the cell.

Eukaryotic cells package their DNA in the form of chromatin to accommodate it in the small space provided by their nuclei [1]. In spite of the 10 000-fold compaction 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 chromatin has a characteristic ‘beads on a string’ appearance. This uniformity of the nucleosomal chain might impose difficulties in region-specific, localized recognition and in uncoiling of the structure; both essential for function. Thus, higher order folding of the chromatin 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 protein fractionation could be applied to discover and isolate 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 implicated in epigenetic inheritance mechanisms of chromatin markings [2,3] and shown to play significant roles in gene expression, antisilencing, heterochromatinization and the formation of specialised regions of the chromatin [4–7]. With the new revelations, other chromatin regulatory mechanisms such as covalent histone

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.

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Histone variants in various functions

R.-H. Pusarla and P. Bhargava

modifications or ATP-dependent chromatin remodelling [8–10] are joined now by histone variants. This review focusses mainly on new advances in chromatinrelated processes with reference to the ‘core histone variants’ and their contribution to chromatin structure. Other aspects, including the role of linker histone variants, 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 histone–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 trying 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 variety of tags to their basic units, histones in the nucleosomes. 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 regulation mechanisms [16,17]. More recent research suggests that minor changes in the primary sequence of conserved histones also contribute to altering the chromatin 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 process 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 heterogeneity conferred by the variants to chromatin can potentially regulate various nuclear functions such as transcription, gene silencing, chromosome segregation, replication, repair and recombination. Such multifaceted 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 common 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 independent 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]. Variants of H2A show divergent functions in different contexts (Table 1). H2A has the largest macro heterogeneous 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 organisms 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 complement 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 comprises 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 cerevisiae) and hv1 (Tetrahymena) arose very early in evolution and are more closely related to each other than
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which forms two third of the protein’s molecular mass.Z is shown by the overlined regions.X. Variant Histone H3 Mammals H3. They show structural similarity to the DNA binding domain of leucine aminopeptidases. The third H2A variant.3 Cid H2Ava Functional association S-phase subtypes S-phase subtypes Transcriptionally active regions Centromeric nucleosomes Different functions in various organisms: maintenance of pericentric and telomeric heterochromatin.X MacroH2A H2A. Solid blocks represent a-helical regions. site of DNA double stranded breaks. The major.3 Cenp-A H2A.Bbd a H2A – – H2Ava – – Drosophila melanogaster has a single H2A variant. Fig. it also contains an SQ motif similar to mammalian H2A. amino acids 132–159 and 181–208. transcriptional activation and viability Sex body in mammals. It is phosphorylated at Ser137 and hence it is a functional homologue of H2A. The H2A region of this variant is 50% identical to H2A. A replication coupled (RC) ⁄ dependent assembly pathway involves a variety of components such as CAF-1. The replication-independent (RI) pathway occurs outside the 5151 .Z family.R. H2Av is not only a member of H2A. suggesting that FEBS Journal 272 (2005) 5149–5168 ª 2005 FEBS DNA binding activity is associated with macrodomains [30]. respectively. macroH2A (mH2A). The exact functional status of the macrodomain in mH2A is not known. to major H2A from the same species [25]. 1. RCAF (histone chaperones) and proliferating cell nuclear antigen (PCNA). condensation and silencing of male sex chromosome Inactivation of X-chromosome. Bhargava Histone variants in various functions Table 1. and the acidic patch of H2A. whereas the replacement histone variants undergo a replication-independent chromatin assembly [31]. 1. Macrodomains may be associated with different functions as they are found in diverse proteins such as those containing poly(ADP-ribose) polymerase activity and other single strand RNA viral proteins. bulk histones are deposited over newly synthesized DNA during replication in a replication-dependent chromatin assembly pathway. interferes with both transcription factor binding and SWI ⁄ SNF remodelling Close spacing of nucleosomes H2A H2A. The nonhistone region. in rat liver protein). Variants of H3 Initial studies on histone H3 variants in mice have helped to classify them according to their relationship with DNA replication. may have evolved comparatively recently. Schematic comparison of the organization of histone H2A variants.X. the histone fold is constituted by helices a1–a3. contains a short. H2Av. both having homology with the corresponding region of conventional H2A.X.Z Yeast – – H3. Functional diversity of histone variants.3 Cse4 Htz1 Drosophila – – H3. Pusarla and P. and basic as well as leucine zipper regions of mH2A are indicated.-H. in addition to the major H2A.2 H3.Z. extremely divergent from major H2A.1 H3. The C-terminal SQ motif in H2A. It is a 42 kDa protein [29]. highly basic region and a putative leucine zipper domain (Fig. with 64% identity at its N-terminus and an extensive 25 kDa nonhistone region at the carboxyl end. and deposits histones on replicating DNA during the S-phase [32–34]. now termed as the ‘macrodomain’.

however. It is very much diverged from H3. a histone variant present at the DNA damage point may act as a marker for the quick recruitment of a repair complex. However. similar to H3. in particular nucleotide excision repair. ATM is required for H2A. major H3.39]. Nevertheless.2 were classified as ‘strictly replication dependent’ and H3. shows 62% identity with H3 in its carboxy terminal portion but there is no sequence similarity in the N-terminus. Phosphorylation of H2A. an acidic residue follows the two relatively invariant amino acids (SQ) while the last carboxy-terminal residue is hydrophobic [27].Histone variants in various functions R. and belong to the S-phase subtypes [35]. V89. 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. The RI variant accumulates as the tissue matures. Pusarla and P. there are three variants of H3 in Drosophila. although it is not clear how the damage is indicated in regions with bulk H2A.X is randomly incorporated into nucleosomes and represents 10–15% of the total cellular H2A.X is suggested to mark the damaged DNA for recruitment of the repair machinery. functional requirements of a nuclear process in which chromatin may be involved would have established the suitability of variation in histones.1 and H3. The evolutionary comparison of CenH3s from various Drosophila species suggests a unique packaging function for the N-terminal tail at the cytological marker of centromeres. While only one type of histone H3. 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. recognition of DNA damage and recruitment of the repair machinery may need other signalling mechanisms [43.3 is expressed [36] in yeast. While H2A phosphorylation in yeast is shown to require both ATM ⁄ ATR homologues Mec1p and Tel1p in response to DSBs [47. which varies from 20 to 200 amino acids in CENP-A as compared to 45 amino acids in the N-terminus of H3 [39].1 and H3. Centromere-specific H3 variants of all Drosophila species are documented to show adaptive evolution continuing for 25 million years [38]. H3.48]. which play important roles in spermatogenesis.-H. Thus.3. only differing in a Cys-to-Ser substitution at amino acid 96. human centromeric H3-like protein. Within this C-terminal motif.44]. shows that ATR is the kinase that phosphorylates H2A. CENP-A. Indeed. has evolved more rapidly than that of H3 [23. having homologies only in histone fold domains although conserved blocks are also seen in the N-terminal tail [38]. Bhargava S-phase or in nondividing cells that undergo continued gene expression.3 as replication-independent [1]. variants are now becoming known as major players in chromatin metabolism.3 is almost identical to H3 and differs at only four positions. Minor histone 5152 . Under nontranscribing conditions. The SQE motif is part of the common consensus motif found in targets of the phosphatidylinositol kinases. through factors that allow recruitment of the repair machinery by the transcription complex at the DNA damage site [40. The carboxy terminus of H2A. the region required for localization of CENP-A to the centromere. For example. a few variants of H2B and H1 are known. Variants in DNA repair and recombination Transcription in both prokaryotes and eukaryotes is coupled to the repair process.41]. In comparison. DNA may be damaged under various conditions and cells have several mechanisms for its repair [42]. M90) [37]. Cells exploit the intimacy of nuclear processes with the chromatin structure of genomic DNA for regulatory purposes by using chromatin modifications and histone variants. thereby helping to maintain the eukaryotic genome [45].X molecule is phosphorylated [46]. H3.3 and centromeric centromere identifier (Cid). The histone fold domain of CENP-A. Of the three somatic H3 variants known. during radiation-induced DNA damage or other events leading to double stranded breaks (DSBs) in DNA. Cid is characteristically a structural component of the centromeres. While no variants are known for H4. three members of the phosphatidylinositol kinase family (ATM. immunocytochemical analyses have shown that not every contiguous H2A. ATR and DNA-PK) are now known to generate this terminally phosphorylated form called c-H2A. H3.2 are closely related.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. Unlike H3. the primary constriction [38]. H2A. one in the N-terminal tail (A31) and three in the histone fold domain (S87.X and the tumour suppressor protein FEBS Journal 272 (2005) 5149–5168 ª 2005 FEBS Variants of core histones in various nuclear processes Histone variants might act as ‘control panels’ in regulating all DNA-related processes.X.X phosphorylation in murine fibroblasts [49]. 1). H3. Recent evidence.

X mutant that mimics the charged state of c-H2A. which uses a general transcription factor [40. Phosphorylated H2A. In agreement with this.X [47]. H2A.X foci after introduction of DNA double strand breaks may have great clinical implications. Bhargava Histone variants in various functions BRCA1 plays an important role in recruiting ATR to XY chromatin [50]. including Xenopus. Deficiency of H2A.X at the damage point rather than globally recruiting it to other points having bulk H2A as well (probably via certain other mechanisms) may be advantageous for cells.X may generate localized decondensation of chromatin domains with increased accessibility to various effectors such as modulating enzymes or repair complexes. coupling the two processes. It is quite likely that some of the initially recruited repair factors bring in the specific kinases for the subsequent phosphoryation of H2A. Efficient.X as well as H2A. 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. Removal of the SQE motif leads to impaired nonhomologous end joining (NHEJ) in S. Recent studies with yeast have given better understanding of the involvement of H2A. the same proteins may be active in other DNA-related processes.R. Pusarla and P. Rather. cerevisiae. In Drosophila. or simply mark spots for downstream events.60]. This is probably due to the loss or exchange of H2A. the ATPase activity of dTip60 exchanges the 5153 .Z in mammals [63]. The Drosophila Tip60 chromatin remodelling complex acetylates nucleosomal phospho-H2Av. Yeast H2A phosphorylation is not required for activation of S-phase DNA damage check points [48] or for the initial recruitment FEBS Journal 272 (2005) 5149–5168 ª 2005 FEBS of several repair factors [57]. 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]. Furthermore.X in the repair process. A close relationship between chromatin remodelling and DNA repair reported recently [61] is an excellent example of the economy practiced by cells in general. 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. Nhp10. making them a preferred substrate for repair and preventing the highly reactive DNA ends from aberrant translocations and large interstitial deletions [56].46]. ATP-dependent chromatin remodelling and covalent histone modifications are two processes associated with the regulation of gene expression from a chromatin region.X [57. of the yeast chromatin remodelling complex INO80.X at DSBs to recruit the INO80 complex. is a functional homologue of both H2A. analogous to the transcription-coupled nucleotide excision repair pathway.X. Several examples from various species. The phosphorylation is seen to spread for approximately 25 kb on both the sides of a DSB. cerevisiae.X. brought about by the recruited chromatin modifying activities at DSBs. irradiation-induced foci (IRIFs) containing a large number of repair factors. A mechanism that recruits and spreads the repair machinery from the foci having c-H2A. such as retaining unprocessed double stranded breaks after asynapsis and increased predisposition to various tumours in the absence of p53 [54]. apoptotic DNA digestion following caspase-activated DNase activity [46]. Drosophila.X ensures an error-free process by using the sister chromatid as a template in excluding the error-prone repair (single-strand annealing) at chromosomal DSBs [55].X.-H. This may also be a mechanism of tethering the repair machinery to the DNA double strand breaks. homologous recombinational repair of a chromosomal DSB is evidently found to require Ser139 of mammalian H2A. is shown to interact with c-H2A.X phosphorylation by primary DNA damage checkpoint kinases makes a large chromatin domain permissive for a de novo recruitment of cohesins required for cohesion of sister chromatids.59] uses the SQ motif of H2A. mammals and S. suggesting that the phosphorylation may promote the spreading and stabilization of the repair factors through IRIFs. bulky phosphate in c-H2A. Genetic evidence for the interaction of Nhp10 with members of the RAD52-dependent repair pathway suggests that INO80 may in turn recruit the repair machinery at the damage site through Nhp10 [62]. meiotic recombination preceding synaptic crossover [51]. The presence of doubly charged. the H2A variant H2Av. It suggests that chromatin remodelling may not be a process related only to gene expression.X in mice leads to meiotic defects. genomic DNA showed nuclease hypersensitivity in an S129E yeast H2A. as discussed later. have shown that ionizing radiations and other agents that cause double-strand breaks result in rapid and massive phosphorylation of the histone variant H2A. At the same time. Cohesins tether the broken DNA ends.41]. Formation of IRIFs that sequester multiple DNA DSBs [58. V(D)J splicing [52] and class switch recombination [53] during the development of immunoglobulin variability. Thus the rapid observed colocalization of the p53 binding protein1 (53BP1) with c-H2A. but is absent from approximately 1–2 kb immediately adjacent. An HMG-like subunit.X. which is followed by formation of large. It reduces the number of recruitment sites and therefore the total requirement of these repair factors.

with methylated H3-K4 at a potential activation boundary during metaphase [73]. Another study has now established that the acidic patch of H2A. It brings about X-chromosome inactivation probably by stabilizing the binding of Xist to the X chromosome through its nonhistone region [77]. Therefore.X and thereby avoiding a permanent marking of the damage spot. The absence of condensed sex body and the failure of 5154 meiotic pairing by X and Y chromosomes in H2A. H2A. mH2A colocalizes on the uncoiled X chromosome. by virtue of their capacity to generate different nucleosomal conformations. c-H2A. and with heterochromatin protein M31 during meiotic prophase [76]. The unusual structure of mH2A with a large C-terminal tail may give a unique conformation to the nucleosome. The core particles having mH2A FEBS Journal 272 (2005) 5149–5168 ª 2005 FEBS . It evidently associates with Barr bodies (the inactive X chromosomes) at levels higher than other chromatin proteins [73. and enhances the binding of HP1 to the condensed higher order chromatin structures [71]. with compact and highly condensed chromatin regions. These chromatin modifications may lead to the decondensation of the chromatin required for DSB repair.74]. Mammalian H2A. Absence of H2A. concomitant with Ser129 phosphorylation of c-H2A. some histone variants are also known to associate with and promote the heterochromatin formation [66].Z (described below) provides an altered nucleosome surface for localized compaction of chromatin fibre folding without crosslinking. through one of its subunits (Arp4) is shown to associate specifically with the phospho-H2A peptide. In contrast. In addition. open and decondensed chromatin structure. Therefore. Arp4. and several sex body proteins such as XMR and macroH2A1 ⁄ 2 fail to localize to the sex chromosome [68].X in mice results in infertility in the male but not in the female.X. although after NuA4 recruitment [65]. It shows a nonuniform pattern of wide distribution in the genome and is present in thousands of euchromatic bands as well as the heterochromatic chromocentre of polytene chromosomes [28]. Nucleosomes containing mH2A have altered structure owing to the high a-helical content in their C-terminal nonhistone regions [78]. INO80 and Swr1. which is consistent with its absence in invertebrates and evolution in vertebrates.Z is also found to be essential for establishing higher order chromatin structure at constitutive heterochromatic domains. is required for the recruitment of NuA4 to DSB. In mouse spermatocytes. appears to be involved in X chromosome inactivation. heterochromatin is considered transcriptionally inactive. Variants in silencing and heterochromatinization Eukaryotic genomic DNA is organized into two characteristically different forms. with one mH2A for every 30 nucleosomes in rat liver [29]. The other two remodellers also interact with P-Ser129.X is more important for heterochromatinization in the male than the female. organize into 30 nm fibres but do not condense into the next higher level of compaction [70]. It is localized along with HP1a on chromosome arms but not on centromeric regions [69].-H.X plays a crucial role in sex chromosome condensation and transcriptional inactivation under the process of meiotic sex chromosome inactivation (MSCI). even at high Mg2+ levels that are known to promote chromatin condensation. For example.Z along with HP1 appears to regulate heterochromatin formation by preventing the further compaction of the 30 nm chromatin fibre. Arrays of positioned nucleosomes containing H2A. The inactive chromatin of the Barr body is characterized by denser chromatin domains and higher nucleosome density. Methylation of H3K9. with its two nonallelic forms mH2A1 and mH2A2. It regulates chromatin remodelling and associated silencing of male sex chromosomes by initiating heterochromatinization in the sex body. which is also a subunit of two further ATP-dependent chromatin remodelling complexes. Its presence in the XY body of spermatocytes indicates its role in the spermatogenic process. Euchromatin is constituted by the transcriptionally active. as reflected by their low sedimentation coefficient despite a 25% increase in the mass. Bhargava phospho-H2Av with the unmodified H2Av. It shows highest expression in liver followed by testes [72]. and DNA methylation participate in the process of heterochromatinization. One of the histone acetyl transferase (HAT) complexes of yeast. thus suggesting that the association of macroH2A may not be specific to the Barr body. Additionally. presenting an example of two chromatin modifying activities within the same complex [64]. as well as help remove the phosphorylated H2A.X deficiency suggests that H2A. macroH2A. Drosophila H2Av is found to participate in heterochromatin formation by marking the region for subsequent acetylation at H4K12 and methylation at H3K9 with HP1 recruitment [67]. efficient DNA repair in yeast appears to require sequential remodelling by three chromatin modifiers. Pusarla and P. probably by controlling the localization of HP1a. NuA4.Z over the defined sequence 208–12 DNA (12 repeats of 208 bp sea urchin 5S rDNA positioning sequence). recruitment of HP1 and other condensing proteins. and shows the presence of both H2A and mH2A [75]. One of the H2A variants.Histone variants in various functions R.

HIRA. Constitutive synthesis replenishes H3.-H. 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. H3. It is also found deposited over active rDNA arrays on the X chromosome. modified histones such as methylated H3. Similarly. Transcription-coupled deposition of H3. It was found in an in vitro 5155 .3 might accumulate in nondividing cells [2].1. which act as an epigenetic mark for silencing. 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]. sufficient to deposit nucleosomes on all of the transcribed DNA [80].3 deposition and targeted to transcriptionally active chromatin.3 shows several fold enrichment of modifications found on active genes. The RC variant H3. The deposition of H3. which acts as a specific nucleosome assembly factor.3 in the RI pathway. Thus. A detailed account of deposition pathways for histone variants can be found in a recent review [6]. deposits H3. a methylated CpG binding protein. The replacement variant H3. Drosophila H3 is deposited only during S-phase. as it stops replacing H3 after the induced gene is switched off [81]. A chromatin structure established 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. RC assembly usually results in a rigid chromatin structure over genes.1 and H3. Thus.3 is deposited both during and outside of S-phase. the transcript levels of both H3. Active participation of the chromatin structure in the process of transcription on a transcribed gene demands a dynamic nature in the chromatin template requiring a constant reshuffling of the nucleosomes over this. specific residues in the histone fold could switch it to the RI deposition pathway. The chromatin modifiers introduce these active modifications probably by associating with specific nucleosome assembly proteins. MeCP2 deficiency leads to the loss of silencing mechanisms involving H3K9 methylation and histone deacetylase activity.3 continues to be synthesized and maintained throughout differentiation.3 in a replication-independent manner [86] while CAF-1 deposits the major variant H3.3 accumulation (associated with transcribed regions) and excess H3 acetylation (due to reduced deacetylation) might further aggravate the condition [83]. RI assembly delineates active regions making them relatively dynamic and variants mark these regions in addition to giving them the required flexibility. Thus. whereas H3. Excess accumulation of H3. As compared to H3. a common mental disorder directly related to the loss of MeCP2.3 in nerve cells leads to further severity of Rett syndrome. which is a significant mark for active chromatin [80.3. 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. Isolation of the two complexes also suggested that histones H3 and H4 can exist and be deposited as dimers rather than tetramers [85]. suggesting an asymmetric and extended conformation. which are deficient in modifications that facilitate the mobility of nucleosomes. 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. as histones are strong DNA-binding proteins.3 is found to account for  25% of total histone H3 in a Drosophila cell line. while the N-terminal was required for RC deposition. which is shown to be short-lived compared to bulk H3. A histone chaperone. MacroH2A exerts its repressive action through control over transcription and chromatin remodelling.R. The presence of mH2A in a positioned nucleosome disrupts access for NF-jB.84]. In mice. Acetylation of H3K9 is associated with active chromatin while H3K9 methylation marks inactive chromatin regions. where it shows a constant turnover.3 in an RI nucleosome assembly pathway targets it to transcriptionally active loci throughout the cell cycle. Bhargava Histone variants in various functions show slower gel mobility but the same stability as that of native nucleosomes.2 decrease as cell division slows down during differentiation. the unintended activation due to H3. Variants in gene expression Several core histone variants have been found to regulate gene expression and antisilencing mechanisms in different ways. A macroH2A C-terminal region present near to a promoter reduces the transcriptional activity. which seems to be restricted to H3. probably by acting as a road-block to the passage of RNA polymerase [75]. suggesting that H3. Pusarla and P. as well as remodelling and mobilization of variant nucleosomes by SW1 ⁄ SNF without affecting either its binding or ATPase activity [79].3 counterpart relieved the block to RI assembly and further deposition of H3 outside S FEBS Journal 272 (2005) 5149–5168 ª 2005 FEBS phase [82].1 and RI variant H3. can be rapidly replaced by H3. The changing of one amino acid from histone H3 to its H3. whereas H3.3 form complexes with distinct histone chaperones [85].3 is directly linked to active transcription at the hsp70 gene locus.

Thus. a stretching-compression along the dyad axis and the flipping. H2A. Global sensitivity of chromatin to nucleases is affected in htz1D cells while H2A. FEBS Journal 272 (2005) 5149–5168 ª 2005 FEBS . Swr1. In agreement with this. The nucleosomes with variant histones show comparatively weaker correlations between internal motions. resulting in the perturbation of interactions between the contact regions of the variant histones with overlying DNA [19]. Loss of Htz1 in yeast cells leads to slow growth and formamide sensitivity at 28 °C and lethality at 37 °C [96]. Pusarla and P. the association of H2A. which happens predominantly in the N-terminal tail and changes its charge. This activator-like function of H2A.Z probably do not require SW1 ⁄ SNF remodelling complexes [95].-H.Z in chromosomal segregation. suggesting a direct role for H2A. Associated histone chaperone activity of FACT can help remove as well as redeposit an H2A-H2B dimer during the transcription [89]. Both groups identified a bromodomain (which recognizes an acetyl group) containing protein Bdf1 that also interacts with transcription factor IID (TFIID. suggesting that the function of H2A.Z into euchromatic regions at several sites [102]. Acetylation is a posttranslational histone modification.Z acetylation is essential in Tetrahymena.Z resides in its C-terminal region. Thus. a result of the dynamism of the N-termini of H3 and the H2A. retaining even a single such lysine can avert this lethality. Removal of H2A ⁄ H2B by FACT may facilitate access of H3 for exchange with H3. suggesting this H2A variant in yeast acts with chromatin modifiers such as SWI ⁄ SNF and SAGA on this locus.Z recruitment at centromeres. a basal transcription factor) as another component of the Swr1 complex. which is linked to its ability to preferentially localize to certain intergenic DNA regions [98]. and the replacement of all six lysines that can be acetylated with arginines is lethal. A genetic interaction between SWR-dependent H2A. as Sir proteins are found to extend beyond the normal boundaries in htz1D cells [97]. However.3 by HIRA in the next step. nucleosomes were found to show two types of large motions in space. it binds the PHO5 locus and regulates its expression. This altered surface may act as an activating 5156 surface by participating in the recruitment of transcription factors and chromatin remodellers. Chromatin reassembly in yeast becomes dependent on the Hir ⁄ Hpc (human HIRA homologue) pathway on the loss of yeast FACT activity [90]. An important role for H2A. an ATPase of Snf2 family. Nevertheless. in a global analysis.Histone variants in various functions R. The nucleosome core particles with variant H2A. the variant may be required to mark and not maintain the transcriptionally active state. the SWR1 complex and NuA4 (a histone H4 acetylase) is linked to chromosomal stability [103]. Incorporation of Htz1 is facilitated by one of the components of SWR-C. In a functional dynamic study.Z is guided through a charge patch and not the histone code [104]. These findings suggest that nucleosomes can indeed be shuffled during read-through by RNA pol II in vivo without displacing the histone octamer completely.Z containing transcriptionally activated gene domains near telomeres as well as in regions flanking HMR loci. which is essential for viability [99. Bhargava study that RNA polymerase II (pol II) can transcribe through a nucleosome without completely displacing histones from it [87]. cerevisiae. and set the stage for gene activation upon a proper induction [98].Z is found to be important for both positive and negative gene regulation [92–95].Z in both gene activation and silencing is also demonstrated by localization of H2A.Z in nucleosome arrays [94]. necessary for promoting gene expression near silent heterochromatic regions of yeast. The protein complex facilitates chromatin transcription (FACT) facilitates readthrough of the nucleosomal template by RNA polymerase II during transcription elongation [88]. a recent study reports the exchange of H2A. Both NuA4 and SWR-C share some common subunits. suggesting that both chaperones may be working on transcribed templates. Thus.Z with transcriptionally active chromatin may require the carboxy terminal and not the histone fold region. SWR-C.Z with bulk H2A on the c-myc gene during transcription [91]. In the budding yeast S.Z-H2B dimer. correlating with the observation that chromatin regions containing H2A.Z.Z is found to facilitate the recruitment of RNA pol II transcription machinery to gene promoters [92] and modulate its functional interactions with the regulatory components. bending sideways motions with respect to the dyad axis.100]. Genetic and biochemical approaches also demonstrated the requirement of Swr1p for the deposition of H2A. The PHO5 promoter is found to be more open in the htz1D ⁄ snf2D mutant [95]. H2A. H2A.Z also showed an altered surface harbouring a metal ion.Z-H2B dimers in the variant nucleosomes dissociate with comparative ease. Higher acetylation levels in euchromatin may recruit a Bdf1-containing Swr1 complex that may finally replace H2A with H2A. These regions prevent the ectopic spread of the repressor proteins Sir2 and Sir3 into the flanking euchromatin. Nevertheless. is found to be required for the recruitment of Htz1 to chromatin also [101]. a 13 protein complex. which acts as a histone exchanger and efficiently replaces H2A with H2A.

(F) The acidic patch of H2A. (D) H2A. 2B) followed by folding. (H) Condensed chromatin showing close contacts of core particles due to the dense packing. Involvement of H2A variants in the formation of different chromatin structures. Pusarla and P. 2D. 2C). FEBS Journal 272 (2005) 5149–5168 ª 2005 FEBS 5157 . condensation and superfolding through the 30 nm stage to higher order chromatin structure. The details of the nucleosome structure in Fig.Z allows greater interaction with the N-terminal tail of H4 from the neighbouring nucleosome. bulk histones into the zig-zag fibre. (A) Nucleosome core structure details showing only H3 and H2A (H4 and H2B are omitted for clarity). Generation of the condensed chromatin domains (Fig. However. Absence of H2A. starting from fully extended and relaxed ‘beads on a string’ (Fig. The right half shows the normal histones.Z in mammals leads to genome instability and defects in chromosome segregation [69]. Drosophila and Tetrahymena [105–107].Bbd. Bhargava Histone variants in various functions Variants in different chromatin structures Variations in core histones can give minor.X helps in higher order structure formation at the constitutive heterochromatin. it is excluded from the nucleolus as well as the inactive X chromosome and made its first appearance B C D A E F H G Fig. Subtle changes 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. During embryonic differentiation stages. they can also be found in active.E. euchromatic regions as described in the following studies. 2A depict the positions where two of the core histones H3 and H2A can acquire changes. H2A. including mice. H2A variants can lead to inactive or condensed heterochromatin (Fig. Figure 2 illustrates the involvement of various H2A variants in generating a variety of chromatin structures. requires compaction of the 10 nm fibre (Fig. (B) Normal folding of the 10 nm fibre with canonical.Z is one of the variants that has been found to induce both repressive and antisilencing effects.Z is essential for establishing the proper chromatin structure required for early development in many organisms. H2A.G) as explained above. (C) The extended 10 nm fibre with ‘beads on a string’ appearance. (G) Longer C-termini of mH2A or CENP-A may interact with the nucleosomal DNA to make nucleosomes more rigid and help further condensation. localized alterations in nucleosomal conformation. 2H). 2. (E) Shorter length and greater accessibility of DNA wrapped in nucleosomes due to H2A. while possible positions of the variations in amino acids are marked with an asterisk in the left hand side counterparts.-H.R. Thus.

pairing of H2B with both H2A.Bbd containing nucleosomes [114]. This tetrameric compaction in the nucleosomes gives the centromeres a specialized. 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. Functional evidence of the implicit repressive role of H2A. rigid structure: a competent configuration necessary at centromeres to withstand various mechanical and physical insults of pulls to the two poles during cell division.3. This enhanced charge patch at the C-terminus is required for higher order chromatin formation and may offer a stronger docking domain for the H4 tail of a neighbouring nucleosome [71].Bbd compared to H2A [115].Z. Biophysical studies of chromatin fibres having H2A. the third H3 variant in Drosophila. Within H2A. providing a possible signal to distinguish constitutive and facultative heterochromatin [108]. Its primary sequence in the docking domain differs considerably from H2A. 5158 phosphorylation and ubiquitination [15]. It is conspicuous by the absence of lysines or any of the target residues for the post-translational modifications acetylation.Bbd-containing nucleosomes may facilitate the exchange of the H2A. distinct and subtle destabilization of the interaction between the H2A.Bbd organizes only 118 ± 2 bp into nucleosomes as compared with 147 in canonical nucleosomes [113]. However.Z suggested that it resists condensation when compared to its major H2A counterpart and the fibre assumes a relaxed conformation [70]. Bhargava in the pericentric regions of nucleus.Z-H2B dimer has the least stable folding and that the canonical H2A-H2B dimer ˚ shows the most stable folding [110]. As a result.Z-H2B dimer to the rest of the octamer [109]. 2F).Z [111].6 A resolution crystal structure of the variant nucleosome core particle showed surprisingly small changes in the overall structure of H2A. DNA ends are less tightly bound and interactions of H2A.Z-H2B dimer by the H2A-H2B dimer by transcribing RNA pol II [91].Bbd-H2B with an (H3-H4)2 tetramer are weak [113]. 2B). thereby promoting interparticle folding in arrays (Fig. it is decreased in a newly identified ‘Barr body deficient’ histone variant. This proposes a mechanism under which chromatin is poised for transcriptional initiation by depositing variant nucleosomes. 2E).Bbd has destabilizing effect on nucleosome structure under normal conditions but SWI ⁄ SNF and ACF complexes (ATP-dependent chromatin remodellers) failed to mobilize H2A. H2A. CENP-A and H4 subnucleosome tetramers are more compact and conformationally rigid compared to normal tetramers [119]. which ensures incorporation of only one type of molecule. probably promoting transcription through nucleosomes during the elongation phase. While the acidic nature of the charged patch of H2A is increased in H2A. An open chromatin configuration at both centromeres (due to the lack of H3K9 methylation in Cid) as well as active chromatin is proposed to be the common basis of RI histone deposition at these sites [37]. which is only 59% identical to the conventional H2A [109]. FEBS Journal 272 (2005) 5149–5168 ª 2005 FEBS . A recent thermodynamic study has confirmed that the H2A. Similar to H3. Its distribution is similar to that of acetylated H4 and it is excluded from the inactive X chromosome.Z comes from a recent study demonstrating replacement of the H2A. 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]. but its hallmarks are the presence of a continuous stretch of six arginines in the N-terminus. sedimentation analysis under changing ionic strength showed a substantial instability of the variant core particle. the lower stability of H2A. neutralize phosphates in linker DNA and further help in higher order chromatin structure. Conserved blocks in the N-terminus and histone fold of Cid may mediate essential protein–protein interactions for recruitment of other centromeric proteins. Thus.Z and H2A within the same nucleosome core particle leads to steric imbalance that may favour binding to another H2A.Bbd. Pusarla and P. The 2. However. 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. hence the name [112].Histone variants in various functions R. However. disturbance in these important activities due to targeted deletion of CENP-A in mice results in embryonic death [117]. Centromeric nucleosomes of mice also are characterized by the presence of the centromeric H3 variant CENP-A [116]. indicating a less tight binding of the H2A. H2A. is altered in H2A. is deposited in an RI manner throughout the cell cycle. an organization that could repress transcription from a natural promoter in an activator-responsive manner (Fig.Z.Bbd-containing nucleosome core particles.-H. The L1 loop domain of H2A (Fig. It is required for the recruitment of components essential for kinetochore formation and chromosome segregation. This is found to be 48% identical to (but shorter than) conventional H2A.Z [111].Z-H2B dimer and the (H3-H4)2 tetramer is seen. It gives arrays with shorter repeat length and higher nucleosome density. Native gel electrophoresis did not distinguish between the core particles having major H2A.Z.Z. Cid. H2A.1 or the variant H2A.

showing the strong conservation of the centromeric features in both [123]. Why this heterogeneity? The answer probably lies in the arrangement of histones in the nucleosome core particle. are also required for proper CENP-A localization in human cells [127]. and to keep an intact nucleosome they need to be spared from the changes that could destroy these interactions. Thus the generation of functionally heterogeneous conditions may FEBS Journal 272 (2005) 5149–5168 ª 2005 FEBS become possible through variation in the histone primary structure that. Varying only one of the partners at a time can give an alteration in structure with the least perturbation. [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. as most of the sites of putative post-translational modifications are found in this region. 3A). it needs decondensation. The centromeric H3-like protein. In contrast. extreme condensation through strong interfibre as well as interparticle interactions. Recently Hayashi et al. in turn. creates precise structural changes in the nucleosomes. Changes in C-termini instead may give nucleosomes various properties without interfering with the basic scheme of their structure. Among the histone heterodimers of the core particle. expansion and uncoiling of the regions by weakening of the same interactions between its fibres or particles. Histone folds are arranged in a handshake manner to generate the octameric protein core. Thus. N-terminal tails are involved in deciding the DNA–histone interactions. Further studies will be necessary to reveal how Mis16 ⁄ Mis18 changes the chromatin environment at centromeres in order to allow CENP-A loading. However. Cse4p. while the N-terminal tails of all of the core histones protrude to surface of the nucleosome. Bhargava Histone variants in various functions Centromeric DNA is several hundreds of kilobases in higher organisms whereas a 125 bp unique region specifies the single nucleosome yeast centromere [120]. Individual nucleosomes contribute to these DNA–protein and protein–protein interactions through the N-terminal tail regions of their histones. While the random coil segments of N-terminal tails of both H3 and H2B pass between gyres of the DNA superhelix. it is not required for Cse4 deposition into the centromeric nucleosomes. In S.-H. is localized to the centromere and its proximal regions. Interaction of the H3-H4 tetramer with the H2A-H2B dimer is established through contacts made by H2B with H4 [128]. four amino acids of the H2A N-terminal tail. H2A has a rich family of variants and H3 is known to have a few distinctly important variants. The histone fold domain of Cse4 is sufficient for its localization to the centromere [124]. Rather. Therefore. bind to the minor groove on the outside of the superhelix (Fig. as revealed ˚ by the structure solved to 2. for interaction with kinetochore components. Why have variants evolved in H2A and H3? Variants are found for all histones (except H4) but with different propensities. In all of the above-mentioned nuclear processes chromatin acquires a variety of configurations. the chromatin structure needs superfolding of the fibres. All four core histones have a histone fold domain in their middle region and two unstructured tails of different lengths at both ends. N-terminal regions have significant homology even among the variants of histones. Similarly. making contacts not only with the DNA backbone but also offering involvement in nucleosome–nucleosome interactions (Fig. human Mis16-like proteins. It is conceivable from the previous sections that the functional diversification of chromatin is directly related to the structural variety brought about by the variants. due to the following reasons. RbAp46 and RbAp48. RSC. it helps remodel the associated regions for proper chromosome transmission [126].8 A resolution for crystals obtained under near physiological conditions [128]. 3A). one of the partners is usually found to be more varied. C-terminal tails usually harbour docking domains but greater variations in amino acid composition and domain length are also observed in this region. Pusarla and P. and in the H2A-H2B dimer H2A could be the better choice. Yeast ATP-dependent chromatin remodelling complex. an essential N-terminal domain (END) comprising 33 amino acids within the 130 amino acids long N-terminal tail is required [125]. for gene expression from active regions as well as site-specific DNA damage repair. H2B may not be preferred 5159 .R. cerevisiae. a chromosome missegregation mutant cse4-1 shows mitosis-specific arrest at elevated temperatures and the Cse4 gene was found to be essential for correct cell division [121]. is an integral component of the yeast centromere [122] and can substitute structurally and functionally for human CENP-A. However. which is one of the important interactions in core particle assembly. Most of the H2B and H1 variants are reported to participate in the spermatogenesis process. Generating two opposite end-results through the same set of interactions can be made possible by regulating the parameters that define these interactions. For inactivation of the X chromosome or heterochromatinization and the silencing of defined regions. close to the site of H2B interaction. While H4 is invariant.

Compared to other core histones. H4 makes contacts with the other three histones in the octamer. it appears that minor sequence variations in the C-terminal proximal histone fold region of H3 that guide it to actively transcribed chromatin regions can be tolerated easily. and it is not surprising that a larger number of H2A variants are known that impart different functional states to the nucleosomes carrying them. (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. 2). H2A replacement-dependent regulatory mechanisms may be energetically advantageous. 1). which are found in the N-terminal halves of the a2 helices. amino acids 92–108 of H2A form a folded docking domain with its a3 helix for H4 (Fig. Preceding this. Centromeric H3 with a very different N-terminal region or H3. The H2A-H2B dimer in the nucleosome is easily dissociable. out of the H3-H4 pair. Bhargava A B Fig. H2A has a strategic placement in the nucleosome and contains the largest consensus C-terminal tail (Fig. In contrast. They do not disturb incorporation of H3 into the nucleosome.3 of other eukaryotes. for variation. (B) Structure of the yeast nucleosome with both turns of the DNA.-H. and amino acids 105–117 link aN of the opposite H3 to the H3-H4 histone fold domains. Similarly. respectively. and readthrough of the nucleosome by RNA pol II was shown to result in the loss of a dimer from the core particle [130]. by altering its orientation in FEBS Journal 272 (2005) 5149–5168 ª 2005 FEBS .and N-terminal ends. showing histones of the lower half only partially.3 with only a slightly different C-terminal proximal histone fold region. C and N indicate the C. This tail protrudes on the outside of nucleosome near the entry and exit sites of the DNA. Due to these two carboxy-terminal regions of H2A. Therefore. Structural features of a nucleosome as revealed by the crystal structure analysis showing intranucleosomal interactions of histones. The location of these amino acids in the crystal structure of the yeast nucleosome core particle suggests that they may influence the interaction of the H3-H4 dimer with the H2A-H2B dimer. and variations in its sequence are least tolerated.Histone variants in various functions R. Pusarla and P. 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. No specific changes in structure are attributed [131] to the two different amino acids at positions 89 and 90 of yeast H3. Thus. are both deposited into open or transcriptionally active chromatin regions [37]. lighter shades are given to histones of the lower half. 3B). Most of these known variations map to the carboxy terminal domain of the protein (Fig. of the individual histones. This in turn also has wider possibilities for association with chromatin remodelling machineries. the H3 dimer occupies the dyad axis and has a central 5160 role in organizing the core particle. its exchange can offer a greater scope for heterogeneity to both the tetramer as well as the linker interface [129]. The tetramer of H3-H4 is formed by the interaction of two H3 molecules at the dyad axis via C-terminal halves of the their two a2 helices as well as the a3 helices (Fig. 3B). All four strands of DNA are shown in different shades for clarity. The C-terminal tail of H2A with the maximum number of variations known is highligted. those with open conformations could be better transcribed [132]. Small perturbations in H3 folding due to the presence of a probe at its unique and centrally placed cysteine (Cys96 or Cys110) in the a2 of the histone fold can generate different conformers of the nucleosome.

X ⁄ H2A. such as DNA compaction as well as decondensation. variations in three amino acids of the a2 in H3. Compared to mammals. The differences in the primary sequence of yeast histones from that of higher eukaryotes may generate different particle–particle interactions.R. The structural alterations in H3. 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.Z-H2B dimer in yeast [131]. 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 amino acid sequence of human H2A. a histone chaperone) was found to exchange the major H2A-H2B dimer as well as variant dimers from nucleosomes [141].3 probably facilitate loss ⁄ exchange of the H2A ⁄ B dimer during the transcription process. Hir1p and Hir2p. suggesting that yeast and human H2A may not have evolved through the same pathway. and the methylation at K4 is known to be associated with active chromatin.X of mammals [65]. Yeast chromatin shows a variable but discrete nucleosome repeat length with an increment of five or 10 bases. H2A and H3 (being more amenable to changes) have acquired several variations during evolution for a variety of opposing functions. Lower stability of yeast nucleosome core particles [131] and the presence of only one H3 variant. both H2A and H3 in yeast.3 [36]. Major H2A (90% of total H2A) itself functions like H2A. the presence of the H2A variant Htz1 is in agreement with all active status of yeast chromatin. it is probable that a compatible H3. which has sequence differences in all of the histones. Thus. though the crystal structure of yeast and Xenopus nucleosome core particles are threefold lower nucleosome density covering them [133]. The absence of the recently identified and universally present H3K4Me-specific demethylase in S. Interestingly. sequence differences of individual histones may be the cause of the observed crystal packing differences and destabilization of the yeast core particle [131]. Similarly. Absence or presence of a variant in yeast is well correlated with the requirements of a particular chromatin structure in this eukaryote.3 histone fold (positions 87. bulk forms. correlates well with the observation that whole of the yeast genome is active.3-H4 dimer could compensate for the altered orientation of the H2A. The yeast genome is reported to be largely active with no pseudogenes or repetitive DNA. Variations are not universal Yeast is considered a model eukaryote for many studies.Bbd. transcription of a gene is followed by replacement of the major H3 with the variant H3. Pusarla and P. It will indeed be interesting to find whether yeast and other eukaryotes followed same pathway of evolution from a common ancestor but diverged very early during evolution. Thus. Several reports now show that the differences may be deceptive and the higher aspects may have evolved from the basic features found in yeast. as demethylation of H3K4Me may be counterproductive. probably arising due to the presence of regions with closely spaced nucleosomes in its active chromatin [137. such that the active chromatin regions are enriched with this variant. H3. making them advantageous for active chromatin. Similar features can be generated due to nucleosomes having the histone variant H2A. two of the yeast cell cycle-regulated histone gene repressors. and it shows structurally distinct promoter and nonpromoter regions where promoters have a two. However. However.-H. are related more to the variant forms of higher organisms than to their canonical.3. As discussed above. although it differs in a number of features from higher eukaryotes. Yeast nucleosome assembly protein1 (Nap1.138] that show DNaseI hypersensitivity [139]. In higher eukaryotes. cerevisiae and Schizosaccharomyces pombe [136]. 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 chromosomelike Barr body of mammalian cells or a highly condensed heterochromatin. resulting in a different conformation compatible with active chromatin in higher eukaryotes. 89 and 90) of other organisms most probably influence its interaction with other histones. This may also be the reason that yeast chromatin has a FEBS Journal 272 (2005) 5149–5168 ª 2005 FEBS similarly folded 30 nm fibre [134] but still an ‘open’ higher order chromatin structure. cerevisiae [140] may be related to the maintenance of this all-active state of the yeast genome. Yeast H3 is probably not repressively methylated at K9. resulting in same overall nucleosome structure as in other eukaryotes. therefore. By analogy. On the other hand. fewer H2A variants in yeast are known. an H2A. Bhargava Histone variants in various functions space. along with chromatin assembly proteins CAF1 5161 . it is reported to have a higher order chromatin structure similar to that in higher eukaryotes [134]. No linker histone H1 was found in yeast for a long time.Bbd-like histone has not yet been reported in yeast. which is known to give nucleosomes with loosely bound DNA ends and arrays with shorter repeat lengths [113].X shows a C-terminal region highly homologous to H2A species of S.

In contrast. All of these reasons together might have resulted in extreme conservation of histones. variations in primary sequence or chain length give greater scope for changing the target interactions in both directions.Z. During the cell cycle. cerevisiae and Schizosaccharomyces pombe [142. 4. Finally. marking distinctions between facultative and constitutive heterochromatin by H2A. A C B D Fig. (C) Phosphorylation of Ser31 of mammalian H3. However.Z near the telomeres prevents the spread of silent zones (Fig. Their N-termini are required for interaction with neighbouring DNA while the C-termini provide docking domains for internucleosomal interactions. is a substrate for cyclin-cdk2 and blocks the S-phase [145]. and phosphorylation of mammalian H3. In yeast. the presence of mH2A near potential activation boundaries of decondensing X chromosome during metaphase. while the Xenopus homologue is an RI pathway-specific histone chaperone [86]. However. Thus variants and their modifications may regulate the timing of switching the chromatin domains open for replication. Pusarla and P. Hir proteins are also reported to be required for kinetochore function in both S.143].Histone variants in various functions R. 4).Z prevents the spread of silent chromatin into the neighbouring regions. 4C). An overview Studies with variants have given rise to several new ideas that highlight links and connectivities in all DNA-related processes. An additional N-terminal sequence in CENP-A or the extra C-terminal region in mH2A both result in inactive and compact chromatin regions (Fig. there is a spatial and temporal separation of replication and transcription. HIRA. (A) A typical chromosome showing its different regions. It can be noticed that variants and their covalently modified forms are involved in demarcating structurally as well as functionally different chromatin regions (Fig. Covalent modifications of charged residues in the N-termini and a perturbation of the C-terminus results in reduced interactions of histones with DNA as well as interparticle interactions. The basic chromatin structure and its fundamental units are universal. 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. are involved in chromatin formation and position-dependent gene silencing [142. global conservation of replication and its mechanisms in all eukaryotes demands that histone octamer deposition over DNA is also by similar mechanisms. 5162 FEBS Journal 272 (2005) 5149–5168 ª 2005 FEBS .-H. 4D).Bbd with a shorter C-terminal tail is localized to active chromatin regions. 2G).3 at S31 in the regions bordering centromeres [146] during metaphase (Fig. To form an octamer of the same organization. For example. Organization of DNA and histone octamers into nucleosomes is also the same in all organisms. the presence of H2A. conservation of histone fold regions needed for the handshake contacts is essential. (B) In yeast. 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. all may be signifying borders of active and inactive regions. H2A. Bhargava and Asf1. a need for variations for regulatory purposes would have also set in with evolution. H2A. The human homologue of Hir1p and Hir2p. 4B) while a single nucleosome with the H3 variant Cse4 is enough to mark the centromere region (Fig.3 surrounding the centromeric region. Histone variants may be involved in the demarcation of functional boundaries.144]. The assembly of variants into nucleosomes also shows a strong correlation with replication. (D) Centromeric nucleosome having the centromeric H3 variant.

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