Tài liệu Báo cáo khoa học: Histones in functional diversification Core histone variants pdf

REVIEW ARTICLE Histones in functional diversification Core histone variants Rama-Haritha Pusarla and Purnima Bhargava Centre for Cellular & Molecular Biology, Tarnaka, Hyderabad, India Introduction Eukaryotic cells package their DNA in the form of chromatin to accommodate it in the small space provi- ded by their nuclei [1]. In spite of the 10 000-fold com- paction of DNA due to this packaging, minute details of a local structure regulate the accessibility of any small region. The folding of 147 bp of DNA over a histone octamer (two molecules each of the four core histones, H4, H3, H2A and H2B) surface gives a neat organization of the DNA into a chromatin fibre of 10 nm diameter. The primary structure of 10 nm chro- matin has a characteristic ‘beads on a string’ appear- ance. This uniformity of the nucleosomal chain might impose difficulties in region-specific, localized recogni- tion and in uncoiling of the structure; both essential for function. Thus, higher order folding of the chroma- tin into a 30 nm fibre and larger domains could be an attempt by the genome to demarcate itself into various regions of activities. Histones are abundant, basic, structural proteins that bring in variety and novelty to the complicated gene regulation mechanisms [1]. Apart from binding to DNA and giving chromatin its strength, stability and form, certain highly similar forms of histones, termed ‘histone variants’, have evolved to carry out many vital functions. Though the focus on histone variants appears to be very recent, they were known as early as 1969 when only standard biochemical methods of pro- tein fractionation could be applied to discover and iso- late new proteins [1]. Their incorporation into nucleosomes as a mode of marking chromatin regions is now shown to have high impact on gene regulation, DNA repair and meiotic events. They have been impli- cated in epigenetic inheritance mechanisms of chroma- tin markings [2,3] and shown to play significant roles in gene expression, antisilencing, heterochromatiniza- tion and the formation of specialised regions of the chromatin [4–7]. With the new revelations, other chro- matin regulatory mechanisms such as covalent histone Keywords chromatin; nucleosome; histones; gene expression; histone variants Correspondence P. Bhargava, Centre for Cellular & Molecular Biology, Uppal Road, Tarnaka, Hyderabad-500007, India Fax: +91 40 27160591 Tel: +91 40 27192603 E-mail: purnima@ccmb.res.in (Received 6 July 2005, accepted 22 August 2005) doi:10.1111/j.1742-4658.2005.04930.x Recent research suggests that minor changes in the primary sequence of the conserved histones may become major determinants for the chromatin structure regulating gene expression and other DNA-related processes. An analysis of the involvement of different core histone variants in different nuclear processes and the structure of different variant nucleosome cores shows that this may indeed be so. Histone variants may also be involved in demarcating functional regions of the chromatin. We discuss in this review why two of the four core histones show higher variation. A comparison of the status of variants in yeast with those from higher eukaryotes suggests that histone variants have evolved in synchrony with functional require- ment of the cell. Abbreviations Cid, centromere identifier; DSB, double strand break; IRIF, irradiation induced foci; MSCI, meiotic sex chromosome inactivation; NHEJ, nonhomologous end joining; RC, replication coupled; RI, replication independent. FEBS Journal 272 (2005) 5149–5168 ª 2005 FEBS 5149 modifications or ATP-dependent chromatin remodel- ling [8–10] are joined now by histone variants. This review focusses mainly on new advances in chromatin- related processes with reference to the ‘core histone variants’ and their contribution to chromatin structure. Other aspects, including the role of linker histone vari- ants, can be found in other recent reviews [11–13]. Variation in high conservation – the evolution of histone variants Histones are among the most conserved proteins in eukaryotes, and make the chromatin nonstatic and parent nucleosomes regulatory. Folding of chromatin domains is defined at a lower level by the compactness of the basic units, guided and determined by the his- tone–DNA as well as particle–particle interactions. High conservation of core histone structure and their contacts with each other and with DNA leaves little scope for any heterogeneity. Therefore, apart from try- ing to reshuffle or remove nucleosomes from the underlying DNA, eukaryotic cells have developed some very subtle and precise methods for breaking the monotony of the chromatin structure by adding a vari- ety of tags to their basic units, histones in the nucleo- somes. These taggings result in altered structures and interactions of the core particles, affecting the local chromatin structure. Tags in the form of covalent modifications of histone tails have been extensively studied over the past few years [14,15]. Histone codes of the genes generated by histone modifications along with other chromatin remodelling mechanisms have been proposed to be the major players in gene regula- tion mechanisms [16,17]. More recent research suggests that minor changes in the primary sequence of con- served histones also contribute to altering the chroma- tin structure [18–20]. The ‘bulk’ histones are encoded by genes belonging to multicopy, intronless families that are transcribed into nonpolyadenylated mRNA. Their highly conserved sequences suggest that they nonspecifically bind DNA from any source. A variation could be detrimental as it may restrict the required interactions. The variants are nonallelic isoforms of the major histones that display sequence variations, often at single residue, and occupy restricted and defined locations in chromatin. They are encoded by genes located outside the canonical histone gene cluster, mostly in single copies and with introns. They are constitutively expressed into polyadenylated mRNA, and as the cell ages they replace the bulk histones, suggesting that this exchange is an active pro- cess throughout the cell cycle and quiescence (old age) [21,22]. The variants have diverged from the normal histones early in the course of evolution, acquiring differential expression patterns. The structural hetero- geneity conferred by the variants to chromatin can potentially regulate various nuclear functions such as transcription, gene silencing, chromosome segregation, replication, repair and recombination. Such multiface- ted regulatory activities of the nucleosomes through variations in the subunits of the histone octamer would not have been possible with a strict conservation of histones at all the times and everywhere. Variants have provided an added advantage. Variants of H2A Histones are proposed to have evolved from a com- mon and simple ancestral archeal protein [23,24] and followed three evolutionary histories. H2A and H2B have diverged faster than H3 and H4. Different H2A variants have arisen in two single events, while variants of H3 have probably evolved through multiple inde- pendent events [25]. They have evolved slowly in such a way that they could not only fulfill the basic function of DNA compaction and maintain the higher order chromatin structure but also have gained functional specialization due to the acquired changes [23,26]. Var- iants of H2A show divergent functions in different contexts (Table 1). H2A has the largest macro hetero- geneous family of variants and all of them are found to have a crucial role in gene expression and nuclear dynamics [4]. Five human H2A genes encode proteins with sequences considerably different from the major H2A sequence (Fig. 1). Of these, H2A.X and H2A.Z were identified in the 1980s, two others (macroH2A1 and macroH2A2) in the 1990s, and finally H2A.Bbd in 2001 [27]. Homologues of H2A.X are found across all phyla, including fungi, animals, plants and the most primitive eukaryotes such as Giardia [23]. However, a comparative analysis of H2A.X from various organ- isms does not give a clear idea of the evolutionary links [23]. The sequence of mammalian H2A.X is nearly identical to the major vertebrate H2A comple- ment H2A.1 ⁄ 2 homologues [27] but the distance between the globular region and carboxyl terminus in H2A.X is increased. One of the best studied H2A variants, H2A.Z com- prises roughly 5–10% of cellular H2As and probably controls several major functions of the cell [28]. Highly conserved H2A.Z sequences have been given different names in different organisms. The H2A variants H2A.Z (mammals), H2A.F (birds), H2A.F ⁄ Z (sea urchin), H2Av (Drosophila), Htz1 (Saccharomyces cere- visiae) and hv1 (Tetrahymena) arose very early in evo- lution and are more closely related to each other than Histone variants in various functions R H. Pusarla and P. Bhargava 5150 FEBS Journal 272 (2005) 5149–5168 ª 2005 FEBS to major H2A from the same species [25]. The third H2A variant, macroH2A (mH2A), may have evolved comparatively recently. It is a 42 kDa protein [29], extremely divergent from major H2A, with 64% iden- tity at its N-terminus and an extensive 25 kDa non- histone region at the carboxyl end, which forms two third of the protein’s molecular mass. The H2A region of this variant is 50% identical to H2A.Z, both having homology with the corresponding region of conven- tional H2A. The nonhistone region, now termed as the ‘macrodomain’, contains a short, highly basic region and a putative leucine zipper domain (Fig. 1; amino acids 132–159 and 181–208, respectively, in rat liver protein). Macrodomains may be associated with differ- ent functions as they are found in diverse proteins such as those containing poly(ADP-ribose) polymerase activity and other single strand RNA viral proteins. They show structural similarity to the DNA binding domain of leucine aminopeptidases, suggesting that DNA binding activity is associated with macrodomains [30]. The exact functional status of the macrodomain in mH2A is not known. Variants of H3 Initial studies on histone H3 variants in mice have helped to classify them according to their relationship with DNA replication. The major, bulk histones are deposited over newly synthesized DNA during replica- tion in a replication-dependent chromatin assembly pathway, whereas the replacement histone variants undergo a replication-independent chromatin assembly [31]. A replication coupled (RC) ⁄ dependent assembly pathway involves a variety of components such as CAF-1, RCAF (histone chaperones) and proliferating cell nuclear antigen (PCNA), and deposits histones on replicating DNA during the S-phase [32–34]. The repli- cation-independent (RI) pathway occurs outside the Table 1. Functional diversity of histone variants. Histone Variant Functional associationMammals Yeast Drosophila H3 H3.1 – – S-phase subtypes H3.2 – – S-phase subtypes H3.3 H3.3 H3.3 Transcriptionally active regions Cenp-A Cse4 Cid Centromeric nucleosomes H2A H2A.Z Htz1 H2Av a Different functions in various organisms: maintenance of pericentric and telomeric heterochromatin, transcriptional activation and viability H2A.X H2A H2Av a Sex body in mammals, site of DNA double stranded breaks; condensation and silencing of male sex chromosome MacroH2A – – Inactivation of X-chromosome, interferes with both transcription factor binding and SWI ⁄ SNF remodelling H2A.Bbd – – Close spacing of nucleosomes a Drosophila melanogaster has a single H2A variant, H2Av, in addition to the major H2A. H2Av is not only a member of H2A.Z family, it also contains an SQ motif similar to mammalian H2A.X. It is phosphorylated at Ser137 and hence it is a functional homologue of H2A.X. Fig. 1. Schematic comparison of the organization of histone H2A variants. Solid blocks represent a-helical regions, the histone fold is consti- tuted by helices a1–a3, and the acidic patch of H2A.Z is shown by the overlined regions. The C-terminal SQ motif in H2A.X, and basic as well as leucine zipper regions of mH2A are indicated. R H. Pusarla and P. Bhargava Histone variants in various functions FEBS Journal 272 (2005) 5149–5168 ª 2005 FEBS 5151 S-phase or in nondividing cells that undergo continued gene expression. Of the three somatic H3 variants known, H3.1 and H3.2 were classified as ‘strictly repli- cation dependent’ and H3.3 as replication-independent [1]. The RI variant accumulates as the tissue matures. H3.1 and H3.2 are closely related, only differing in a Cys-to-Ser substitution at amino acid 96, and belong to the S-phase subtypes [35]. While only one type of histone H3, similar to H3.3 is expressed [36] in yeast, there are three variants of H3 in Drosophila; major H3, H3.3 and centromeric centromere identifier (Cid). H3.3 is almost identical to H3 and differs at only four positions; one in the N-terminal tail (A31) and three in the histone fold domain (S87, V89, M90) [37]. Centromere-specific H3 variants of all Drosophila species are documented to show adaptive evolution continuing for 25 million years [38]. Unlike H3.3, Cid is characteristically a structural component of the centromeres. It is very much diverged from H3, having homologies only in histone fold domains although con- served blocks are also seen in the N-terminal tail [38]. The evolutionary comparison of CenH3s from various Drosophila species suggests a unique packaging func- tion for the N-terminal tail at the cytological marker of centromeres, the primary constriction [38]. In com- parison, human centromeric H3-like protein, CENP-A, shows 62% identity with H3 in its carboxy terminal portion but there is no sequence similarity in the N-terminus, which varies from 20 to 200 amino acids in CENP-A as compared to 45 amino acids in the N-terminus of H3 [39]. The histone fold domain of CENP-A, the region required for localization of CENP-A to the centromere, has evolved more rapidly than that of H3 [23,39]. Variants of other histones It is evident from the above description that a variety of changes have evolved in the primary sequence of core histones. While no variants are known for H4, a few variants of H2B and H1 are known, which play important roles in spermatogenesis. How can small changes in the primary sequence of one of the histones introduce a change in the overall structure of the core particle? Can this change be tolerated? These could have been the major issues that guided the evolution of the variants. Variants of core histones in various nuclear processes Histone variants might act as ‘control panels’ in regu- lating all DNA-related processes. Minor histone variants are now becoming known as major players in chromatin metabolism. Cells exploit the intimacy of nuclear processes with the chromatin structure of genomic DNA for regulatory purposes by using chro- matin modifications and histone variants. Thus, func- tional requirements of a nuclear process in which chromatin may be involved would have established the suitability of variation in histones. Variants in DNA repair and recombination Transcription in both prokaryotes and eukaryotes is coupled to the repair process, in particular nucleotide excision repair, through factors that allow recruitment of the repair machinery by the transcription complex at the DNA damage site [40,41]. However, DNA may be damaged under various conditions and cells have several mechanisms for its repair [42]. Under nontran- scribing conditions, recognition of DNA damage and recruitment of the repair machinery may need other signalling mechanisms [43,44]. For example, during radiation-induced DNA damage or other events lead- ing to double stranded breaks (DSBs) in DNA, a his- tone variant present at the DNA damage point may act as a marker for the quick recruitment of a repair complex, thereby helping to maintain the eukaryotic genome [45]. H2A.X is randomly incorporated into nucleosomes and represents 10–15% of the total cellular H2A. Phosphorylation of H2A.X is suggested to mark the damaged DNA for recruitment of the repair machin- ery, although it is not clear how the damage is indica- ted in regions with bulk H2A. Nevertheless, immunocytochemical analyses have shown that not every contiguous H2A.X molecule is phosphorylated [46]. The carboxy terminus of H2A.X differs from that of bulk H2A in being longer and having a four amino acid sequence element SQEL at the extreme end of the protein (Fig. 1). Within this C-terminal motif, an aci- dic residue follows the two relatively invariant amino acids (SQ) while the last carboxy-terminal residue is hydrophobic [27]. The SQE motif is part of the com- mon consensus motif found in targets of the phospha- tidylinositol kinases. Indeed, three members of the phosphatidylinositol kinase family (ATM, ATR and DNA-PK) are now known to generate this terminally phosphorylated form called c-H2A.X. While H2A phosphorylation in yeast is shown to require both ATM ⁄ ATR homologues Mec1p and Tel1p in response to DSBs [47,48], ATM is required for H2A.X phos- phorylation in murine fibroblasts [49]. Recent evidence, however, shows that ATR is the kinase that phos- phorylates H2A.X and the tumour suppressor protein Histone variants in various functions R H. Pusarla and P. Bhargava 5152 FEBS Journal 272 (2005) 5149–5168 ª 2005 FEBS BRCA1 plays an important role in recruiting ATR to XY chromatin [50]. Phosphorylation at the conserved serine of the SQ motif (Ser129 in yeast and Ser139 in mammals) is now shown to regulate DNA DSB repair [45,46], meiotic recombination preceding synaptic crossover [51], apoptotic DNA digestion following caspase-activated DNase activity [46], V(D)J splicing [52] and class switch recombination [53] during the development of immunoglobulin variability. The presence of doubly charged, bulky phosphate in c-H2A.X may generate localized decondensation of chromatin domains with increased accessibility to var- ious effectors such as modulating enzymes or repair complexes, or simply mark spots for downstream events. In agreement with this, genomic DNA showed nuclease hypersensitivity in an S129E yeast H2A.X mutant that mimics the charged state of c-H2A.X [47]. Removal of the SQE motif leads to impaired nonho- mologous end joining (NHEJ) in S. cerevisiae, whereas phosphorylation of the serine residue in response to DNA fragmentation facilitates NHEJ by decondensing chromatin at the damaged DNA sites and making it accessible to repair factors [47]. Deficiency of H2A.X in mice leads to meiotic defects, such as retaining unprocessed double stranded breaks after asynapsis and increased predisposition to various tumours in the absence of p53 [54]. Thus the rapid observed colocali- zation of the p53 binding protein1 (53BP1) with c-H2A.X foci after introduction of DNA double strand breaks may have great clinical implications. Phosphorylated H2A.X ensures an error-free process by using the sister chromatid as a template in exclu- ding the error-prone repair (single-strand annealing) at chromosomal DSBs [55]. Furthermore, H2A.X phos- phorylation by primary DNA damage checkpoint kin- ases makes a large chromatin domain permissive for a de novo recruitment of cohesins required for cohesion of sister chromatids. Cohesins tether the broken DNA ends, making them a preferred substrate for repair and preventing the highly reactive DNA ends from aber- rant translocations and large interstitial deletions [56]. Several examples from various species, including Xenopus, Drosophila, mammals and S. cerevisiae, have shown that ionizing radiations and other agents that cause double-strand breaks result in rapid and massive phosphorylation of the histone variant H2A.X. Effi- cient, homologous recombinational repair of a chro- mosomal DSB is evidently found to require Ser139 of mammalian H2A.X. Recent studies with yeast have given better understanding of the involvement of H2A.X in the repair process. Yeast H2A phosphoryla- tion is not required for activation of S-phase DNA damage check points [48] or for the initial recruitment of several repair factors [57], which is followed by for- mation of large, irradiation-induced foci (IRIFs) con- taining a large number of repair factors. Formation of IRIFs that sequester multiple DNA DSBs [58,59] uses the SQ motif of H2A.X [57,60], suggesting that the phosphorylation may promote the spreading and sta- bilization of the repair factors through IRIFs. It is quite likely that some of the initially recruited repair factors bring in the specific kinases for the subsequent phosphoryation of H2A.X. The phosphorylation is seen to spread for approximately 25 kb on both the sides of a DSB, but is absent from approximately 1–2 kb immediately adjacent. This is probably due to the loss or exchange of H2A.X, brought about by the recruited chromatin modifying activities at DSBs, as discussed later. A mechanism that recruits and spreads the repair machinery from the foci having c-H2A.X at the dam- age point rather than globally recruiting it to other points having bulk H2A as well (probably via certain other mechanisms) may be advantageous for cells. It reduces the number of recruitment sites and therefore the total requirement of these repair factors. This may also be a mechanism of tethering the repair machinery to the DNA double strand breaks, analogous to the transcription-coupled nucleotide excision repair path- way, which uses a general transcription factor [40,41]. Phosphorylation at the SQ motif of the variant may be easier and more economical than developing a new method of marking the damage site with the bulk H2A. ATP-dependent chromatin remodelling and covalent histone modifications are two processes associated with the regulation of gene expression from a chromatin region. A close relationship between chromatin remod- elling and DNA repair reported recently [61] is an excellent example of the economy practiced by cells in general. It suggests that chromatin remodelling may not be a process related only to gene expression. Rather, the same proteins may be active in other DNA-related processes, coupling the two processes. An HMG-like subunit, Nhp10, of the yeast chromatin remodelling complex INO80, is shown to interact with c-H2A.X at DSBs to recruit the INO80 complex. Gen- etic evidence for the interaction of Nhp10 with mem- bers of the RAD52-dependent repair pathway suggests that INO80 may in turn recruit the repair machinery at the damage site through Nhp10 [62]. In Drosophila, the H2A variant H2Av, is a functional homologue of both H2A.X as well as H2A.Z in mammals [63]. The Drosophila Tip60 chromatin remodelling complex acetylates nucleosomal phospho-H2Av. At the same time, the ATPase activity of dTip60 exchanges the R H. Pusarla and P. Bhargava Histone variants in various functions FEBS Journal 272 (2005) 5149–5168 ª 2005 FEBS 5153 phospho-H2Av with the unmodified H2Av, presenting an example of two chromatin modifying activities within the same complex [64]. One of the histone acetyl transferase (HAT) complexes of yeast, NuA4, through one of its subunits (Arp4) is shown to associate specif- ically with the phospho-H2A peptide. Arp4, which is also a subunit of two further ATP-dependent chroma- tin remodelling complexes, INO80 and Swr1, is required for the recruitment of NuA4 to DSB, con- comitant with Ser129 phosphorylation of c-H2A.X. The other two remodellers also interact with P-Ser129, although after NuA4 recruitment [65]. Therefore, effi- cient DNA repair in yeast appears to require sequen- tial remodelling by three chromatin modifiers. These chromatin modifications may lead to the decondensa- tion of the chromatin required for DSB repair, as well as help remove the phosphorylated H2A.X and thereby avoiding a permanent marking of the damage spot. Variants in silencing and heterochromatinization Eukaryotic genomic DNA is organized into two char- acteristically different forms. Euchromatin is constitu- ted by the transcriptionally active, open and decondensed chromatin structure. In contrast, hetero- chromatin is considered transcriptionally inactive, with compact and highly condensed chromatin regions. Methylation of H3K9, recruitment of HP1 and other condensing proteins, and DNA methylation participate in the process of heterochromatinization. In addition, by virtue of their capacity to generate different nucleo- somal conformations, some histone variants are also known to associate with and promote the heterochro- matin formation [66]. For example, Drosophila H2Av is found to participate in heterochromatin formation by marking the region for subsequent acetylation at H4K12 and methylation at H3K9 with HP1 recruit- ment [67]. It shows a nonuniform pattern of wide dis- tribution in the genome and is present in thousands of euchromatic bands as well as the heterochromatic chromocentre of polytene chromosomes [28]. In mouse spermatocytes, c-H2A.X plays a crucial role in sex chromosome condensation and transcrip- tional inactivation under the process of meiotic sex chromosome inactivation (MSCI). It regulates chroma- tin remodelling and associated silencing of male sex chromosomes by initiating heterochromatinization in the sex body. Absence of H2A.X in mice results in infertility in the male but not in the female, and several sex body proteins such as XMR and macroH2A1 ⁄ 2 fail to localize to the sex chromosome [68]. The absence of condensed sex body and the failure of meiotic pairing by X and Y chromosomes in H2A.X deficiency suggests that H2A.X is more important for heterochromatinization in the male than the female. Mammalian H2A.Z is also found to be essential for establishing higher order chromatin structure at consti- tutive heterochromatic domains, probably by control- ling the localization of HP1a. It is localized along with HP1a on chromosome arms but not on centromeric regions [69]. Arrays of positioned nucleosomes con- taining H2A.Z over the defined sequence 208–12 DNA (12 repeats of 208 bp sea urchin 5S rDNA positioning sequence), organize into 30 nm fibres but do not con- dense into the next higher level of compaction [70], even at high Mg 2+ levels that are known to promote chromatin condensation. Another study has now established that the acidic patch of H2A.Z (described below) provides an altered nucleosome surface for localized compaction of chromatin fibre folding with- out crosslinking, and enhances the binding of HP1 to the condensed higher order chromatin structures [71]. Therefore, H2A.Z along with HP1 appears to regulate heterochromatin formation by preventing the further compaction of the 30 nm chromatin fibre. One of the H2A variants, macroH2A, with its two nonallelic forms mH2A1 and mH2A2, appears to be involved in X chromosome inactivation. It shows high- est expression in liver followed by testes [72], with one mH2A for every 30 nucleosomes in rat liver [29]. Its presence in the XY body of spermatocytes indicates its role in the spermatogenic process, which is consistent with its absence in invertebrates and evolution in verte- brates. It evidently associates with Barr bodies (the inactive X chromosomes) at levels higher than other chromatin proteins [73,74]. The inactive chromatin of the Barr body is characterized by denser chromatin domains and higher nucleosome density, and shows the presence of both H2A and mH2A [75]. Addition- ally, mH2A colocalizes on the uncoiled X chromo- some, with methylated H3-K4 at a potential activation boundary during metaphase [73], and with heterochro- matin protein M31 during meiotic prophase [76], thus suggesting that the association of macroH2A may not be specific to the Barr body. It brings about X-chro- mosome inactivation probably by stabilizing the bind- ing of Xist to the X chromosome through its nonhistone region [77]. Nucleosomes containing mH2A have altered struc- ture owing to the high a-helical content in their C-ter- minal nonhistone regions [78]. The unusual structure of mH2A with a large C-terminal tail may give a unique conformation to the nucleosome, as reflected by their low sedimentation coefficient despite a 25% increase in the mass. The core particles having mH2A Histone variants in various functions R H. Pusarla and P. Bhargava 5154 FEBS Journal 272 (2005) 5149–5168 ª 2005 FEBS show slower gel mobility but the same stability as that of native nucleosomes, suggesting an asymmetric and extended conformation. Presence of the nonhistone region may be responsible for the observed DNaseI hypersensitivity near the dyad axis and around entry ⁄ exit sites of DNA in the nucleosome [78]. Macro- H2A exerts its repressive action through control over transcription and chromatin remodelling. The presence of mH2A in a positioned nucleosome disrupts access for NF-jB, as well as remodelling and mobilization of variant nucleosomes by SW1 ⁄ SNF without affecting either its binding or ATPase activity [79]. A macro- H2A C-terminal region present near to a promoter reduces the transcriptional activity, probably by acting as a road-block to the passage of RNA polymerase [75]. Variants in gene expression Several core histone variants have been found to regu- late gene expression and antisilencing mechanisms in different ways. Active participation of the chromatin structure in the process of transcription on a tran- scribed gene demands a dynamic nature in the chroma- tin template requiring a constant reshuffling of the nucleosomes over this. A chromatin structure estab- lished due to deposition of the major histones in the S-phase of the cell cycle may not be fluid enough to give the required dynamism, as histones are strong DNA-binding proteins. Replacement or exchange of the major histones or their modified forms by their variants having different affinities and strength of binding to the DNA may provide a better alternative outside the S-phase. RC assembly usually results in a rigid chromatin structure over genes, which are deficient in modifica- tions that facilitate the mobility of nucleosomes. RI assembly delineates active regions making them relat- ively dynamic and variants mark these regions in addi- tion to giving them the required flexibility. The replacement variant H3.3 is found to account for  25% of total histone H3 in a Drosophila cell line, sufficient to deposit nucleosomes on all of the tran- scribed DNA [80]. It is also found deposited over act- ive rDNA arrays on the X chromosome, where it shows a constant turnover. The deposition of H3.3 is directly linked to active transcription at the hsp70 gene locus, as it stops replacing H3 after the induced gene is switched off [81]. Constitutive synthesis replenishes H3.3, which is shown to be short-lived compared to bulk H3. The changing of one amino acid from his- tone H3 to its H3.3 counterpart relieved the block to RI assembly and further deposition of H3 outside S phase [82]. Thus, while the N-terminal was required for RC deposition, specific residues in the histone fold could switch it to the RI deposition pathway, which seems to be restricted to H3.3 deposition and targeted to transcriptionally active chromatin. In mice, the transcript levels of both H3.1 and H3.2 decrease as cell division slows down during differenti- ation, whereas H3.3 continues to be synthesized and maintained throughout differentiation. Similarly, Droso- phila H3 is deposited only during S-phase, whereas H3.3 is deposited both during and outside of S-phase, suggesting that H3.3 might accumulate in nondividing cells [2]. Excess accumulation of H3.3 in nerve cells leads to further severity of Rett syndrome, a common mental disorder directly related to the loss of MeCP2, a methylated CpG binding protein. MeCP2 deficiency leads to the loss of silencing mechanisms involving H3K9 methylation and histone deacetylase activity. Acetylation of H3K9 is associated with active chroma- tin while H3K9 methylation marks inactive chromatin regions. Thus, the unintended activation due to H3.3 accumulation (associated with transcribed regions) and excess H3 acetylation (due to reduced deacetylation) might further aggravate the condition [83]. As compared to H3, H3.3 shows several fold enrichment of modifications found on active genes, which is a significant mark for active chromatin [80,84]. The chromatin modifiers introduce these act- ive modifications probably by associating with specific nucleosome assembly proteins. The stepwise assembly pathway of a nucleosome core particle proposes the association of histones H3 and H4 (two copies each) into a tetramer as the first step in assembly. The RC variant H3.1 and RI variant H3.3 form complexes with distinct histone chaperones [85]. A histone chap- erone, HIRA, which acts as a specific nucleosome assembly factor, deposits H3.3 in a replication-inde- pendent manner [86] while CAF-1 deposits the major variant H3.1. Isolation of the two complexes also suggested that histones H3 and H4 can exist and be deposited as dimers rather than tetramers [85]. Tran- scription-coupled deposition of H3.3 in an RI nucleo- some assembly pathway targets it to transcriptionally active loci throughout the cell cycle. Thus, modified histones such as methylated H3, which act as an epi- genetic mark for silencing, can be rapidly replaced by H3.3 in the RI pathway. A detailed account of deposition pathways for histone variants can be found in a recent review [6]. Histone replacement ⁄ exchange by RI assembly on transcribed templates suggests a possible mechanism for read-through of a nucleosomal template by the enzyme RNA polymerase. It was found in an in vitro R H. Pusarla and P. Bhargava Histone variants in various functions FEBS Journal 272 (2005) 5149–5168 ª 2005 FEBS 5155 study that RNA polymerase II (pol II) can transcribe through a nucleosome without completely displacing histones from it [87]. The protein complex facilitates chromatin transcription (FACT) facilitates read- through of the nucleosomal template by RNA polym- erase II during transcription elongation [88]. Associ- ated histone chaperone activity of FACT can help remove as well as redeposit an H2A-H2B dimer during the transcription [89]. Chromatin reassembly in yeast becomes dependent on the Hir⁄ Hpc (human HIRA homologue) pathway on the loss of yeast FACT activ- ity [90], suggesting that both chaperones may be work- ing on transcribed templates. Removal of H2A ⁄ H2B by FACT may facilitate access of H3 for exchange with H3.3 by HIRA in the next step. Nevertheless, a recent study reports the exchange of H2A.Z with bulk H2A on the c-myc gene during transcription [91]. These findings suggest that nucleosomes can indeed be shuffled during read-through by RNA pol II in vivo without displacing the histone octamer completely. In the budding yeast S. cerevisiae, H2A.Z is found to be important for both positive and negative gene regulation [92–95]. Loss of Htz1 in yeast cells leads to slow growth and formamide sensitivity at 28 °C and lethality at 37 °C [96]. The PHO5 promoter is found to be more open in the htz1 D⁄snf2D mutant [95], suggesting this H2A variant in yeast acts with chromatin modifiers such as SWI⁄ SNF and SAGA on this locus. Thus, it binds the PHO5 locus and regulates its expression. An important role for H2A.Z in both gene activation and silencing is also demonstrated by localization of H2A.Z containing transcriptionally activated gene domains near telom- eres as well as in regions flanking HMR loci. These regions prevent the ectopic spread of the repressor proteins Sir2 and Sir3 into the flanking euchromatin, as Sir proteins are found to extend beyond the nor- mal boundaries in htz1D cells [97]. Global sensitivity of chromatin to nucleases is affected in htz1D cells while H2A.Z is found to facilitate the recruitment of RNA pol II transcription machinery to gene promo- ters [92] and modulate its functional interactions with the regulatory components. This activator-like func- tion of H2A.Z resides in its C-terminal region, which is linked to its ability to preferentially localize to cer- tain intergenic DNA regions [98]. Thus, the associ- ation of H2A.Z with transcriptionally active chromatin may require the carboxy terminal and not the histone fold region, which is essential for viability [99,100]. The nucleosome core particles with variant H2A.Z also showed an altered surface harbouring a metal ion. This altered surface may act as an activating surface by participating in the recruitment of tran- scription factors and chromatin remodellers, and set the stage for gene activation upon a proper induction [98]. Thus, the variant may be required to mark and not maintain the transcriptionally active state. In a functional dynamic study, nucleosomes were found to show two types of large motions in space; a stretch- ing-compression along the dyad axis and the flipping, bending sideways motions with respect to the dyad axis, a result of the dynamism of the N-termini of H3 and the H2A.Z-H2B dimer. The nucleosomes with variant histones show comparatively weaker correla- tions between internal motions, resulting in the per- turbation of interactions between the contact regions of the variant histones with overlying DNA [19]. In agreement with this, H2A.Z-H2B dimers in the vari- ant nucleosomes dissociate with comparative ease, correlating with the observation that chromatin regions containing H2A.Z probably do not require SW1 ⁄ SNF remodelling complexes [95]. However, in a global analysis, a 13 protein complex, SWR-C, neces- sary for promoting gene expression near silent hetero- chromatic regions of yeast, is found to be required for the recruitment of Htz1 to chromatin also [101]. Incorporation of Htz1 is facilitated by one of the components of SWR-C, Swr1, an ATPase of Snf2 family, which acts as a histone exchanger and effi- ciently replaces H2A with H2A.Z in nucleosome arrays [94]. Genetic and biochemical approaches also demonstrated the requirement of Swr1p for the depos- ition of H2A.Z into euchromatic regions at several sites [102]. Both groups identified a bromodomain (which recognizes an acetyl group) containing protein Bdf1 that also interacts with transcription factor IID (TFIID, a basal transcription factor) as another com- ponent of the Swr1 complex. Higher acetylation levels in euchromatin may recruit a Bdf1-containing Swr1 complex that may finally replace H2A with H2A.Z. A genetic interaction between SWR-dependent H2A.Z recruitment at centromeres, the SWR1 complex and NuA4 (a histone H4 acetylase) is linked to chromo- somal stability [103], suggesting a direct role for H2A.Z in chromosomal segregation. Both NuA4 and SWR-C share some common subunits. Acetylation is a post- translational histone modification, which happens pre- dominantly in the N-terminal tail and changes its charge. H2A.Z acetylation is essential in Tetrahymena, and the replacement of all six lysines that can be acetylated with arginines is lethal. Nevertheless, retaining even a single such lysine can avert this leth- ality, suggesting that the function of H2A.Z is guided through a charge patch and not the histone code [104]. Histone variants in various functions R H. Pusarla and P. Bhargava 5156 FEBS Journal 272 (2005) 5149–5168 ª 2005 FEBS Variants in different chromatin structures Variations in core histones can give minor, localized alterations in nucleosomal conformation. Subtle chan- ges in one of the components can generate unique nucleosomal surfaces that may regulate interparticle interactions thereby bringing about changes in the three-dimensional folding of the chromatin fibre and establishing special chromatin structural regions. Fig- ure 2 illustrates the involvement of various H2A vari- ants in generating a variety of chromatin structures. Generation of the condensed chromatin domains (Fig. 2H), starting from fully extended and relaxed ‘beads on a string’ (Fig. 2C), requires compaction of the 10 nm fibre (Fig. 2B) followed by folding, conden- sation and superfolding through the 30 nm stage to higher order chromatin structure. The details of the nucleosome structure in Fig. 2A depict the positions where two of the core histones H3 and H2A can acquire changes. H2A variants can lead to inactive or condensed heterochromatin (Fig. 2D,E,G) as explained above. However, they can also be found in active, euchromatic regions as described in the following stud- ies. Thus, H2A.Z is one of the variants that has been found to induce both repressive and antisilencing effects. H2A.Z is essential for establishing the proper chro- matin structure required for early development in many organisms, including mice, Drosophila and Tetra- hymena [105–107]. Absence of H2A.Z in mammals leads to genome instability and defects in chromosome segregation [69]. During embryonic differentiation sta- ges, it is excluded from the nucleolus as well as the inactive X chromosome and made its first appearance A B C D E F G H Fig. 2. Involvement of H2A variants in the formation of different chromatin structures. (A) Nucleosome core structure details showing only H3 and H2A (H4 and H2B are omitted for clarity). The right half shows the normal histones, while possible positions of the variations in amino acids are marked with an asterisk in the left hand side counterparts. (B) Normal folding of the 10 nm fibre with canonical, bulk histones into the zig-zag fibre. (C) The extended 10 nm fibre with ‘beads on a string’ appearance. (D) H2A.X helps in higher order structure formation at the constitutive heterochromatin. (E) Shorter length and greater accessibility of DNA wrapped in nucleosomes due to H2A.Bbd. (F) The acidic patch of H2A.Z allows greater interaction with the N-terminal tail of H4 from the neighbouring nucleosome. (G) Longer C-termini of mH2A or CENP-A may interact with the nucleosomal DNA to make nucleosomes more rigid and help further condensation. (H) Condensed chromatin showing close contacts of core particles due to the dense packing. R H. Pusarla and P. Bhargava Histone variants in various functions FEBS Journal 272 (2005) 5149–5168 ª 2005 FEBS 5157 in the pericentric regions of nucleus, providing a poss- ible signal to distinguish constitutive and facultative heterochromatin [108]. Biophysical studies of chromatin fibres having H2A.Z suggested that it resists condensation when compared to its major H2A counterpart and the fibre assumes a relaxed conformation [70]. This proposes a mechanism under which chromatin is poised for tran- scriptional initiation by depositing variant nucleo- somes. Native gel electrophoresis did not distinguish between the core particles having major H2A.1 or the variant H2A.Z, which is only 59% identical to the conventional H2A [109]. However, sedimentation ana- lysis under changing ionic strength showed a substan- tial instability of the variant core particle, indicating a less tight binding of the H2A.Z-H2B dimer to the rest of the octamer [109]. A recent thermodynamic study has confirmed that the H2A.Z-H2B dimer has the least stable folding and that the canonical H2A-H2B dimer shows the most stable folding [110]. The 2.6 A ˚ resolu- tion crystal structure of the variant nucleosome core particle showed surprisingly small changes in the over- all structure of H2A.Z [111]. However, distinct and subtle destabilization of the interaction between the H2A.Z-H2B dimer and the (H3-H4) 2 tetramer is seen. The L1 loop domain of H2A (Fig. 2B), which ensures incorporation of only one type of molecule, is altered in H2A.Z. As a result, pairing of H2B with both H2A.Z and H2A within the same nucleosome core particle leads to steric imbalance that may favour binding to another H2A.Z. A unique feature of the acidic patch on the surface of normal H2A is extended by replacement of Asn and Lys with Asp and Ser in H2A.Z [111]. This enhanced charge patch at the C-ter- minus is required for higher order chromatin forma- tion and may offer a stronger docking domain for the H4 tail of a neighbouring nucleosome [71], thereby promoting interparticle folding in arrays (Fig. 2F). Functional evidence of the implicit repressive role of H2A.Z comes from a recent study demonstrating replacement of the H2A.Z-H2B dimer by the H2A-H2B dimer by transcribing RNA pol II [91]. While the acidic nature of the charged patch of H2A is increased in H2A.Z, it is decreased in a newly identified ‘Barr body deficient’ histone variant, H2A.Bbd. This is found to be 48% identical to (but shorter than) conventional H2A. Its distribution is similar to that of acetylated H4 and it is excluded from the inactive X chromosome, hence the name [112]. Its primary sequence in the docking domain differs con- siderably from H2A. It is conspicuous by the absence of lysines or any of the target residues for the post-translational modifications acetylation, phosphorylation and ubiquitination [15], but its hall- marks are the presence of a continuous stretch of six arginines in the N-terminus. H2A.Bbd organizes only 118 ± 2 bp into nucleo- somes as compared with 147 in canonical nucleosomes [113]. It gives arrays with shorter repeat length and higher nucleosome density, an organization that could repress transcription from a natural promoter in an activator-responsive manner (Fig. 2E). Within H2A.Bbd-containing nucleosome core particles, DNA ends are less tightly bound and interactions of H2A.Bbd-H2B with an (H3-H4) 2 tetramer are weak [113]. It is also found that the relaxed structure and altered conformation of the Bbd nucleosome is due to the changes in the H2A docking domain and not due to the absence of the C-terminal tail. Thus, H2A.Bbd has destabilizing effect on nucleosome structure under normal conditions but SWI ⁄ SNF and ACF complexes (ATP-dependent chromatin remodellers) failed to mobilize H2A.Bbd containing nucleosomes [114]. However, the lower stability of H2A.Bbd-containing nucleosomes may facilitate the exchange of the H2A.Bbd compared to H2A [115], probably promoting transcription through nucleosomes during the elonga- tion phase. Similar to H3.3, the third H3 variant in Drosophila, Cid, is deposited in an RI manner throughout the cell cycle. An open chromatin configuration at both cen- tromeres (due to the lack of H3K9 methylation in Cid) as well as active chromatin is proposed to be the com- mon basis of RI histone deposition at these sites [37]. Conserved blocks in the N-terminus and histone fold of Cid may mediate essential protein–protein interac- tions for recruitment of other centromeric proteins, neutralize phosphates in linker DNA and further help in higher order chromatin structure. Centromeric nucleosomes of mice also are characterized by the pres- ence of the centromeric H3 variant CENP-A [116]. It is required for the recruitment of components essential for kinetochore formation and chromosome segrega- tion; disturbance in these important activities due to targeted deletion of CENP-A in mice results in embryo- nic death [117]. CENP-A competes with H3 for H4 during nucleosome formation and can be reconstituted with DNA into nucleosomes with properties similar to those of bulk nucleosomes [118]. CENP-A and H4 subnucleosome tetramers are more compact and con- formationally rigid compared to normal tetramers [119]. This tetrameric compaction in the nucleosomes gives the centromeres a specialized, rigid structure: a competent configuration necessary at centromeres to withstand various mechanical and physical insults of pulls to the two poles during cell division. Histone variants in various functions R H. Pusarla and P. Bhargava 5158 FEBS Journal 272 (2005) 5149–5168 ª 2005 FEBS [...]... 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 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-terminal region in mH2A both result in inactive and compact... 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, making contacts not only with the DNA backbone but also offering involvement in nucleosome–nucleosome... important interactions in core particle assembly Therefore, H2B may not be preferred 5159 Histone variants in various functions A R.-H Pusarla and P Bhargava 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. .. 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 outside of the superhelix (Fig 3A) Thus, N-terminal tails are involved in deciding the DNA histone interactions, and to keep an intact nucleosome they need to be spared from the changes that could destroy these interactions Changes in C-termini instead may give nucleosomes... Biochem Sci 29, 127–135 11 Brown DT (2001) Histone variants: Are they functionally heterogeneous? Genome Biol 2, 1–6 12 Govin J, Caron C, Lestrat C, Rousseaux S & Khochbin S (2004) The role of histone in chromatin remodeling during mammalian spermiogenesis Eur J Biochem 271, 3459–3469 13 Khochbin S (2001) Histone H1 diversity: bridging regulatory signals to linker histone function Gene 271, 1–12 14 Berger... competent chromatin during nuclear development in Tetrahymena thermophila Genes Dev 7, 2641–2651 Kobor MS, Venkatasubrahmanyam S, Meneghini MD, Gin JW, Jennings JL, Link AJ, Madhani HD & Rine J (2004) A protein complex containing the conserved Swi2 ⁄ Snf2 related ATPase Swr1p deposits histone variant H2A.Z into euchromatin PLoS Biol 2, 587–598 Krogan NJ, Keogh M-C, Datta N, Sawa C, Ryan OW, Ding H et al... Compared to other core histones, H2A has a strategic placement in the nucleosome and contains the largest consensus C-terminal tail (Fig 2) This tail protrudes on the outside of nucleosome near the entry and exit sites of the DNA, and amino acids 105–117 link aN of the opposite H3 to the H3-H4 histone fold domains Preceding this, amino acids 92–108 of H2A form a folded docking domain with its a3 helix... nucleosome–nucleosome interactions (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 homology 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 segments of N-terminal tails... 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 C B 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... specializes histone H2A variants for defined chromatin function Biochemistry 41, 5945–5949 5 Kamakaka RT & Biggins S (2005) Histone variants: deviants? Genes Dev 19, 295–310 FEBS Journal 272 (2005) 5149–5168 ª 2005 FEBS Histone variants in various functions 6 Sarma K & Reinberg D (2005) Histone variants meet their match Nat Mol Cell Biol Rev 6, 139–149 7 Smith MM (2002) Centromeres and variant histones: . 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. other single strand RNA viral proteins. They show structural similarity to the DNA binding domain of leucine aminopeptidases, suggesting that DNA binding