Variants Of The Histone H2a

Variants of histone H2A are most common in higher eukaryotes. Thus far, five H2A-type histones have been described, of which two are found in all eukaryotes from yeast to mammals (Table 1). These are the histones H2A.X, and H2A.Z (Thatcher and Gorovsky 1994). While all other eukaryotes possess a canonical H2A, S. cerevisiae utilizes H2A.X as general, replication-dependent H2A form. Vertebrates possess an additional H2A variant named macroH2A, while the fifth known H2A variant H2ABBd (Barr body-deficient), is only conserved for mammals (Chow and Brown 2003; Gautier et al. 2004). Besides the most abundant canonical H2A, which is deposited into chromatin during DNA synthesis, other H2A variants also are synthesized outside of the S phase. Like specialized variants of H3, these proteins also are available for incorporation into chromatin independent of DNA replication.

3.1. Histone H2A.Z

H2A.Z is common for all eukaryotes from yeast to mammals, suggesting that this histone variant has important functions in chromatin-related processes. H2A.Z variants are known as the yeast Htz1, Plasmodium H2A.Z, Tetrahymena hv1, Drosophila H2Av, sea urchin H2AF.Z, avian H2AF, and the mammalian H2A.Z (also see Fig. 2; Table 1). While H2A.Z relatives only share about 60% homology to the canonical H2A, their amino acid sequences are highly conserved suggesting that this variant arose very early during evolution (Thatcher and Gorovsky 1994). H2A.Z contributes to an estimated 5-10% of all nucleosomal H2A variants (Redon et al. 2002). Although H2A.Z has been identified in the 1980s, its role within chromatin is not yet fully understood due to its complex biology.

The crystal structure of nucleosomes core particles containing H2A.Z has been resolved and indicates that H2A.Z confers unique structural features to a nucleosome compared to H2A (Suto et al. 2000). The most prominent difference exists in the region of interaction between H2A and the (H3 - H4)2 tetramer, which also is called docking-domain (amino acids 81-119; also see Fig. 2). Within the docking domain, three amino acid substitutions are mainly responsible for unique structural changes. These exchanges are believed to cause a partial destabilization of the H2A.Z-tetramer interaction. In addition, an extended acidic path on the surface of the H2A.Z-containing histone octamers might change the interaction with the N-terminal tail of H4 or generate a surface for the interaction with other non-histone proteins.

Various studies support the crystallographic analyses and confirmed that nucleo-somes containing H2A.Z are more salt-labile than canonical nucleosomes (Abbott et al. 2001; Flaus et al. 2004; Zhang et al. 2005). In fluorescence resonance energy transfer analyses, however, H2A.Z-containing nucleosomes exhibited slower dissociation kinetics compared to their canonical counterparts (Park et al. 2004). The different observations made in these studies could be due to the different sources of chromatin as well as the methodology used to compare the stability of H2A.Z-containing nucleosomes. Future studies will be necessary to determine whether the incorporation of H2A.Z into a nucleosome has positive or negative impact on its stability under in vivo conditions.

Histone H2a Structure

Figure 2. Histone H2A variants from yeast (Saccharomyces cerevisiae; S.c.), fruit fly (Drosophila melanogaster; D.m), and human (Homo sapiens; H.s). Two conserved domains distinguish H2A.Z-relatives (boxed regions; amino acid sequences in the top). H2A.X possesses a conserved C-terminal stretch of four amino acids. The serine (red) becomes phosphorylated at sites of DNA damage. H2ABbd ('Barr body-deficient') and marcoH2A are present in mammals

Figure 2. Histone H2A variants from yeast (Saccharomyces cerevisiae; S.c.), fruit fly (Drosophila melanogaster; D.m), and human (Homo sapiens; H.s). Two conserved domains distinguish H2A.Z-relatives (boxed regions; amino acid sequences in the top). H2A.X possesses a conserved C-terminal stretch of four amino acids. The serine (red) becomes phosphorylated at sites of DNA damage. H2ABbd ('Barr body-deficient') and marcoH2A are present in mammals

The genome-wide incorporation of H2A.Z into chromatin is not completely random as suggested by its replication-independent deposition. However, its deposition is not strictly associated to a specific cellular response and also seems to vary between different species. In Tetrahymena, the H2A.Z-varaint hv1 is predominantly associated with the transcriptionally active macronucleus (Stargell et al. 1993). This early findings suggested that H2A.Z mainly functions in transcriptional regulation in Tetrahymena. Genome-wide gene expression studies in S. cerevisiae supported this notion. Htz1 is required for the proper expression of about 200 genes (Mizuguchi et al. 2004; Kobor et al. 2004). Recent studies demonstrated that H2A.Z is deposited into the nucleosome directly upstream of promoters of the majority of gene promoters in yeast (Guillemette et al. 2005; Raisner et al. 2005; Zhang et al. 2005). The positioning of H2A.Z upstream of promoters is remarkably specific. In general, H2A.Z is present in two nucleosomes flanking a nucleosome-free region of about 22 base pairs. This region corresponds to the transcription initiation site of most genes. The 22 base pair sequence is sufficient to promote the deposition of the two H2A.Z-containing nucleosomes, but at the same time remains nucleosome-free (Raisner et al. 2005). These observations suggest that the promoter-proximal regions of most genes in yeast share a DNA sequence that mediates the deposition of H2A.Z. Indeed, the sequences contain a highly conserved binding site for the Myb-domain factor Reb1 flanked by an AT-rich tract, both of which are important for positioning of the two H2A.Z-containing nucleosomes. Taken together, the deposition of H2A.Z in promoter-proximal regions is likely to depend on a specific DNA sequence in yeast.

In mammalian cells, H2A.Z becomes removed from open reading frames upon transcriptional activation, suggesting that it might be inhibitory to transcription in higher eukaryotes (Farris et al. 2005). The Drosophila H2A.Z-relative H2Av also is involved in developmental gene silencing and functions as Polycomb Group protein (Swaminathan et al. 2005). Although yeast Htz1 also appears to be enriched upstream of inactive genes, it has not been clarified whether it becomes mobilized upon transcription activation or not (Li et al. 2005b; Zhang et al. 2005; Millar et al. 2006).

Besides its role in transcription, H2A.Z also might play an important role at the boundaries between euchromatin and heterochromatin. The loss of Htz1 causes ectopic spreading of the heterochromatic factors Sir2, Sir3, and Sir4 (Meneghini et al. 2003). In chicken erythrocytes, the 5' region of the P-globin locus is enriched for H2A.Z; However, H2A.Z is also more abundant at the heterochromatin-euchromatin boundary at nearby insulator (Bruce et al. 2005), suggesting that H2A.Z an insulator function of H2A.Z at the barrier between eu- and heterochromatin. In mammalian cell lines, H2A.Z is enriched in heterochromatic foci, and it must be assumed that H2A.Z has more functions in these cells than in the definition of eu- versus heterochromatin. Intriguingly, the RNAi-mediated knockdown of H2A.Z causes genomic instability and leads to chromosome breaks, and it likely acts as a guardian of genome integrity (Rangasamy et al. 2004). In yeast, the loss of Htz1 causes chromosome segregation defects (Krogan et al. 2004). Drosophila H2Av is not only present in euchromatin, but also is enriched in the chromocenters, pericentric chromatin and other heterochromatic regions (Leach et al. 2000). In flies, H2Av has important roles in heterochromatin formation as demonstrated by the effects of its mutation on Position Effect Variegation, and genome integrity control (Kusch et al. 2004; Swaminathan et al. 2005).

More recent studies indicate that H2A.Z deposition is dynamic and changes during development. In early mouse embryos, H2.A.Z is concentrated at pericentric heterochromatin, while it a little later becomes depleted from the inactive X chromosome. In cell lines from even later developmental stages, H2A.Z is not detectable at centromeres, but becomes enriched in other heterochromatin in the chromosome arms (Rangasamy et al. 2004). H2A.Z is also not detectable in totipotent cells prior to their differentiation, suggesting that the histone variant is not involved in the regulation of early developmental transcriptional programs (Rangasamy et al. 2003).

First studies in yeast demonstrated that H2A.Z is deposited by a complex containing 13 proteins including the ATPase Swr1p (Krogan et al. 2003; Kobor et al. 2004; Mizuguchi et al. 2004). This complex exchanges the canonical H2A from nucleosomes with H2A.Z in an ATP-dependent manner. While the Swr complex is mainly responsible for genome-wide deposition, residual chromosomal deposition independent of Swr1 has recently been reported (Wu et al. 2005). The subunits of this complex are conserved from yeast to mammals (Kusch et al. 2004; Cai et al. 2005). In mammals, the Swr1 homologue, SRCAP, catalyzes a similar histone exchange reaction. Intriguingly, mammals possess a second Swr1-type protein called p400. This factor is component of the Tip60 complex (Ikura et al. 2000), which is comprised of subunits of the Swr1 complex as well as the yeast NuA4 histone acetyltransferase complex. In Drosophila, a homologous complex has been demonstrated to be sufficient to catalyze the replication-independent incorporation of H2Av into nucleosomes (Kusch et al. 2004). Taken together, the Tip60 complex from higher eukaryotes is likely to combine the features of the Swr1 and NuA4 complexes.

Intriguingly, the acetyltransferase activity of the fly dTip60 complex is essential for efficient exchange, and recently it has been demonstrated that the NuA4 complex transiently acetylates Htz1 at lysine 14 (Millar et al. 2006). This acetylation depends on the transcriptional activity of the affected gene and is redundantly regulated by the NuA4 and SAGA histone acetyltransferase complexes. The latter is also highly conserved between yeast, flies and mammals, and future studies will reveal whether this complex also has a role in the posttranslational modification of H2A.Z in other organisms (Ogryzko et al. 1996; Kusch et al. 2003). In Tetrahymena, the deletion of all six lysines in the N-terminus of hv1 is lethal (Ren and Gorovsky 2001). In Drosophila, acetylation of lysine 5 of H2Av by the dTip60 complex is linked to its ATP-dependent exchange during DNA repair (Kusch et al. 2004).

The striking similarities between yeast and higher eukaryotes in H2A.Z metabolism suggest that the fundamental molecular mechanisms of chromatin regulation by this variant are mostly conserved. However, the readout of H2A.Z in both euchromatin and heterochromatin appears to have undergone some variation in different species. One must assume that both the targeted deposition as well as differential posttranslational modifications of nucleosomes containing H2A.Z are responsible for the dynamic and somewhat paradoxical roles of this variant in the definition of domains within eu- as well as heterochromatin. It is also likely that the role and regulation of H2A.Z incorporation into chromatin has changed during evolution. Further studies will be necessary to understand the function of H2A.Z-relatives in its entirety.

3.2. Histone H2A.X

H2A and its variant H2A.X are very similar in their amino acid sequences aside from their C-terminal region. Homologues of H2A.X are found in all eukaryotes including fungi, plants, protostomes, and deuterostomes (Malik and Henikoff 2003). Their similarity to the canonical H2A makes it difficult to trace their evolutionary links by comparative analyses. The C-terminal amino acid sequence contains a conserved serine residue (Fig. 2). This serine becomes phosphorylated in chromatin flanking sites of DNA lesions within minutes after the damage has occurred (reviewed in Li et al. 2005a). Phosphorylated H2A.X (also known as 7-H2A.X) functions in the control of DNA repair and has other functions in genome integrity control as outlined below. The incorporation of H2A.X into chromatin is rather random and is not restricted to certain chromatin domains. The general deposition of H2A.X also does not seem to occur outside of DNA replication as it has been reported for H2A.Z. Factors that specifically deposit H2A.X/H2B heterodimers have not been identified thus far.

The phosphatidylinositol 3 (PI3)-kinases, ATM, ATR, and DNA-PK, phospho-rylate the C-terminal serine of H2A.X within minutes after DNA damage occurs (reviewed in Sedelnikova et al. 2003). Members of this kinase family are found in all eukaryotes and they play highly conserved roles in the regulation of DNA damage response pathways. The addition of the bulky, negatively charged phosphate in 7-H2A.X might have some impact on the decondensation of the nucleosome, but it appears more likely that -y-H2A.X serves signaling purposes for a number of reasons. Only about 10% of all H2A.X-containing nucleosomes become phospho-rylated at sites of DNA damage. The phosphorylation spreads for several kilobases from the site of DNA damage, while 1-2 kilobases immediately adjacent to the site of the lesion are free of -y-H2A.X (Petersen et al. 2001). A mutation mimicking the phosphorylation of H2A.X (serine to glutamate) rescues DNA damage sensitivity of yeast cells lacking PI3 kinases (Downs et al. 2000). In mammalian cells, H2A.X is not essential for the initial formation of DNA repair foci, but for their stabilization (Fernandez-Capetillo et al. 2003a). In addition, -y-H2A.X recruits cohesins to DNA flanking sites of damage, presumably to tether the broken DNA ends or to allow repair by homologous recombination (Unal et al. 2004).

Knockout studies in mice revealed that the loss of H2A.X causes an increased frequency of chromosomal rearrangements, which often result in oncogenic transfor-

mations (Celeste et al. 2002). In addition, H2A.X seems to be important in functions associated with telomere maintenance and meiotic recombination (Fernandez-Capetillo et al. 2003b). In male mice, H2AX functions in the silencing of the sex chromosomes as well as in meiosis and its loss results in infertility (Fernandez-Capetillo et al. 2003c). Intriguingly, H2A.X is also necessary for the incorporation of the H2A variant macroH2A1.2 (see below) into the inactivated sex chromosomes. The underlying mechanism and the link between these H2A variants are not yet understood. H2A.X also mediates apoptosis and somatic recombination events (Petersen et al. 2001).

In yeast, H2A.X is dephosphorylated by the phosphatase PPH3p after it has been released from chromatin. The release of -y-H2A.X from repaired chromatin is independent of DNA replication, and it therefore must be assumed that a chromatin remodeling complex actively exchanges -y-H2A.X from nucleosomes. Thus far, several candidate remodeling complexes have been identified that specifically target nucleosomes containing -y-H2A.X.

A complex containing the ATPase Ino80p is recruited to sites of DNA double strand breaks and its deletion renders cells sensitive to genotoxic stresses (Downs et al. 2004; Morrison et al. 2004; van Attikum et al. 2004). Although Ino80 shares similarities in the ATPase domain with the H2A/H2A.Z-exchange factor Swr1p, it has not yet been demonstrated that the Ino80 complex indeed can catalyze the removal of -y-H2A.X from chromatin. Like for the INÛ80 complex, mutants for subunits of the SWR1 complex also exhibit DNA-damage sensitivity. Both complexes contain Arp4, a protein that preferentially interacts with -y-H2A.X over its unmodified counterpart (Downs et al. 2004), and it therefore cannot be excluded that both complexes function in -y-H2A.X turnover at sites of DNA damage. Arp4-relatives are also present in another complex with a conserved role in DNA repair: The Tip60-type complexes from yeast, flies, and mammals contain Arp4-relatives and their histone acetyltransferase activity is important for DNA double strand break repair (Ikura et al. 2000; Allard et al. 2004; Kusch et al. 2004). Indeed, the Drosophila dTip60 complex is capable of exchanging -y-H2Av with unmodified H2Av in vitro (Kusch et al. 2004). Mutations of subunits of the fly dTip60 complex severely impair the clearance of -y-H2Av from chromatin during DNA repair. Since the dTip60 complex is a combination of the yeast NuA4 and SWR1 complexes, it appears highly likely that the mechanisms of 7-H2A.X clearance are conserved in eukaryotes. The role of Ino80 in this process remains somewhat unclear. Recent studies in Drosophila suggest that Ino80 is involved in repression and might contribute to the generation of more condensed chromatin domains (Klymenko et al. 2006). Indeed, the loss of Ino80 complex subunits leads to a precocious decrease of -y-H2Av levels in Drosophila after DNA damage, suggesting that Ino80 might protect 7-H2A.X from premature release from damaged chromatin (T.K., unpublished). This might provide a mechanism by which damaged chromatin is remodeled to facilitate access of the damaged DNA to repair proteins without losing an important checkpoint for damage-dependent cell cycle arrest.

3.3. Histone MacroH2A

MacroH2A is an unusual vertebrate-specific H2A lineage. MacroH2A is enriched in the inactive female X chromosome ('Barr body') and in the inactivated male sex chromosomes during meiosis (Costanzi and Pehrson 1998; Hoyer-Fender et al. 2000). Its role in X chromosome inactivation might be a rather recent evolutionary event, since macroH2A is also present in birds, which do not regulate dosage compensation by inactivation of one sex chromosome (Ellegren 2002). Two distinct macroH2A variants have been identified in both mice and humans. Although they are closely related, their amino acid sequence deviates in the macroH2A-specific extended C-terminal stretch of about 200 amino acids. This C-terminus has similarity to nucleic acid binding domains, a putative leucine zipper domain, and can bind histone deacetylases (Chakravarthy et al. 2005). The C-terminal domain of macroH2A also bears strong homology to proteins with phosphoesterase activity found in RNA viruses, eubacteria, archea, and eukaryotes (Pehrson and Fuji 1998). This suggests that the C-terminal domain might carry out an enzymatic function; however, such an enzymatic activity and a potential substrate of macroH2A have not yet been described. Several reports support a role of macroH2A in transcriptional silencing (Angelov et al. 2003; Doyen et al. 2006). Nucleosomal arrays containing macroH2A are not accessible to transcription factors and are resistant to mobilization by chromatin remodeling complexes. In addition, macroH2A interferes with P300-dependent histone acetylation and RNA polymerase II passage through the nucleosomes in vitro. Taken together, macroH2A appears to confer a tighter packaging of nucleosomes. Little is known about the incorporation of macroH2A into chromosomes, and no macroH2A-specific deposition factors have been identified thus far.

3.4. Histone H2ABbd

H2A 'Barr body-deficient' (Bbd) is an evolutionary relatively 'young' histone variant sharing only about 48% amino acid sequence similarity to H2A. This histone variant appears to be specific for mammals (Chadwick and Willard 2001). As indicated by the name, the transcriptionally inactive and highly condensed X chromosome in female mammals (also known as 'Barr body') is depleted for H2ABbd, while this variant is detectable in autosomes and the active sex chromosomes. This observation suggested that H2ABbd is linked to transcriptionally active euchromatin. H2ABbd cofractionates in sedimentation centrifugation with hyper-acetylated histone H4, further corroborating that it associates with transcriptionally active euchromatin.

Nucleosomes core particles containing H2ABbd only have 118 base pairs of DNA incorporated compared to the canonical nucleosomes protecting about 147 base pairs from micrococcal nuclease (Bao et al. 2004). These nucleosomes are more flexible in structure and might facilitate passage of RNA polymerase II. However, the function of this histone variant in mammalian cells is not fully understood. As for macroH2A, it is also not yet known how and when H2ABbd becomes deposited into chromatin, and which chaperones associate with this H2A variant.

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