Histone H2A variants H2AX and H2AZ

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Histone H2A variants H2AX and H2AZ Christophe Redon, Duane Pilch, Emmy Rogakou*, Olga Sedelnikova, Kenneth Newrock and William Bonner Two of the nucleosomal histone families, H3 and H2A, have highly conserved variants with specialized functions. Recent studies have begun to elucidate the roles of two of the H2A variants, H2AX and H2AZ. H2AX is phosphorylated on a serine four residues from the carboxyl terminus in response to the introduction of DNA double-strand breaks, whether these breaks are a result of environmental insult, metabolic mistake, or programmed process. H2AZ appears to alter nucleosome stability, is partially redundant with nucleosome remodeling complexes, and is involved in transcriptional control. Addresses Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA *MGC-Department of Cell Biology and Genetics, Center for Biomedical Genetics, Erasmus University 3000 DR Rotterdam, The Netherlands Current Opinion in Genetics & Development 2002, 12:162–169 0959-437X/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Abbreviations 53BP1 p53 binding protein 1 ATM ataxia telangiectasia mutated ATR ataxia telangiectasia and Rad3 related protein ChIP chromatin immunoprecipitation DNA-PK DNA-activated protein kinase DSB double-strand break H2A-Bbd histone H2A-Barr body deficient Nbs1 Nijmegen breakage syndrome 1 NHEJ non-homologous end joining PI3 kinase phosphatidyl inositol 3 kinase wt wild type

Introduction Eucaroytic cells contain genomes ranging in size from 12 × 106 base pairs in Saccharomyces cerevisiae to many times larger than the human of 6 × 109 base pairs. These sizes correspond to combined DNA fiber lengths of 4 mm in the former to >2 meters in the latter. This DNA is integrated into cells ~0.01 mm in diameter by histone binding and condensation into nucleosomes. The nucleosome comprises 145 bp DNA and eight histones, two from each of the four core histone families — H4, H3, H2B and H2A. A minimum of another 20 bp of DNA stretches between nucleosomes complexed with the linker histone H1. In mammals, the result is that the 4 meters of DNA in a G2 human cell exists as 180 mm of 30 nm diameter fibers and is further condensed to 120 µm of 700 nm diameter arms of mitotic chromosomes. Although researchers recognized that the complex modification patterns and sequence variations of the histone proteins gave multitudinous opportunities for the regulation of chromosome functions, only recently have the tools and

techniques become available to characterize these. Antibodies specific to site-specific modifications coupled with chromatin immunoprecipitation (ChIP) and PCR techniques have made it possible to study the effects of a particular histone modification on a particular gene region in chromatin. Most histone modifications take place on their amino- and carboxy-terminal tails, presumably because these are on the chromatin surface accessible to the prerequisite enzymes. Regions buried in the nucleosome may not be accessible to modifying enzymes and variations in those regions originate at the gene rather than protein level, leading to sequence variants. In this review, we concentrate on the variants of the histone H2A family, in particular H2AX and H2AZ. The term variant can confuse because some sequence differences may not confer a discernible differential function on a protein. Originally the term variant was an operational one, referring to related protein species that were resolvable by a method such as gel electrophoresis in the presence of Triton X-100™ [1]. However, the question was whether these sequence differences were the result of allowable evolutionary diversity for the same protein function, perhaps with a different pattern or timing of expression, or whether the differences conferred a unique function on the protein [2,3]. While the major interest at the protein level is in the conferring of novel function, the issue is complicated by the coexistence of both types of sequence diversity. With the sequencing of the human genome, the sequences of all the most abundant H2A species are known as well as those of some (and possibly all) of the rare species (Figure 1). The human genome contains 10 genes that encode for H2A peptides classified as H2A1 variants, six identical in sequence and four that vary in up to three of four positions. These peptides are not resolvable by gel electrophoresis in the presence of Triton X-100™. H2A gene family member O (Oh!) encodes a peptide in which leucine residue 51 has been replaced by methionine; this change confers an altered electrophoretic mobility to the protein which is named H2A2 [2]. The H2A1 and H2A2 variants comprise the bulk of the mammalian H2A, all migrate as a single band on SDS gels and do not have any known differential functions. These proteins have also been known as the major variants because of their abundance in mammals. There are five other human H2A genes that encode peptides the sequences of which differ considerably from the bulk H2A sequences (Figure 1). The proteins they encode are present in smaller amounts and have been known as minor variants because of their rarity. However, it is now becoming apparent that the minor variants may have major roles in chromatin metabolism. Two of the minor variants, H2AX and H2AZ [4], were identified in the 1980s, two, macroH2A1 [5]

Histone H2A variants H2AX and H2AZ Redon et al.

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Figure 1

A 1 C 1 D 1 E 1 G 1 I 1 L 1 M 1 N 1 O 2 P 1 X X Z Z M2 Macro2 M1 Macro1 B Bbd Consensus H2A variant

H2A gene

Consensus

Protein motif

sgrgkqggkarakaktRssrAglqFpVgrvhRllr-kgnyaeRvgagApVYlAAvlEYLtAeiLELAGNaa v20 v30 v40 v50 v60 v70 v10 SGRGKQGGKARAKAKTRSSRAGLQFPVGRVHRLLR-KGNYAERVGAGAPVYLAAVLEYLTAEILELAGNAA SGRGKQGGKARAKAKTRSSRAGLQFPVGRVHRLLR-KGNYAERVGAGAPVYLAAVLEYLTAEILELAGNAA SGRGKQGGKARAKAKTRSSRAGLQFPVGRVHRLLR-KGNYAERVGAGAPVYLAAVLEYLTAEILELAGNAA SGRGKQGGKARAKAKTRSSRAGLQFPVGRVHRLLR-KGNYAERVGAGAPVYLAAVLEYLTAEILELAGNAA SGRGKQGGKARAKAKTRSSRAGLQFPVGRVHRLLR-KGNYSERVGAGAPVYLAAVLEYLTAEILELAGNAA SGRGKQGGKARAKAKTRSSRAGLQFPVGRVHRLLR-KGNYAERVGAGAPVYLAAVLEYLTAEILELAGNAA SGRGKQGGKARAKAKSRSSRAGLQFPVGRVHRLLR-KGNYSERVGAGAPVYLAAVLEYLTAEILELAGNAA SGRGKQGGKARAKAKTRSSRAGLQFPVGRVHRLLR-KGNYSERVGAGAPVYLAAVLEYLTAEILELAGNAA SGRGKQGGKARAKAKTRSSRAGLQFPVGRVHRLLR-KGNYAERVGAGAPVYLAAVLEYLTAEILELAGNAA SGRGKQGGKARAKAKSRSSRAGLQFPVGRVHRLLR-KGNYAERVGAGAPVYMAAVLEYLTAEILELAGNAA SGRGKQGGKARAKAKTRSSRAGLQFPVGRVHRLLR-KGNYAERVGAGAPVYLAAVLEYLTAEILELAGNAA SGRGKTGGKARAKAKSRSSRAGLQFPVGRVHRLLR-KGHYAERVGAGAPVYLAAVLEYLTAEILELAGNAA AGGKAGKDSGKAKTKAVSRSQRAGLQFPVGRIHRHLKSRTTSHGRVGATAAVYSAAILEYLTAEVLELAGNAS --SGR-SGKKKMSKLSRSARAGVIFPVGRLMRYLK-KGTFKYRISVGAPVYMAAVIEYLAAEILELAGNAA --SSR-GGKKKSTKTSRSAKAGVIFPVGRMLRYIK-KGHPKYRIGVGAPVYMAAVLEYLTAEILELAGNAA PRRRRRRGSSGAGGRGRTCSRTVRAELSFSVSQVERSLR-EGHYAQRLSRTAPVYLAAVIEYLTAKVLELAGNEA sgrgkqggkarakaktRssrAglqFpVgrvhRllr-kgnyaeRvgagApVYlAAvlEYLtAeiLELAGNaa N - t a i l _ _ _ _ _ _ _ αα-N-αα _ _ αααα-1-ααα loop1 αααααααααααα-2-ααααααααααα Other H2A

A C D E G I L M N O P X Z M2 M1 B Consensus H2A variant

H2A gene

Consensus

Protein motif

rdnkktriiPrhlqlairnDeeLnkLlgkvTIaqggvlpniqavLlpKKteshhkakgk* v90 v100 v110 v120 v130 v140 v80 1 RDNKKTRIIPRHLQLAIRNDEELNKLLGKVTIAQGGVLPNIQAVLLPKKTESHHKAKGK* 1 RDNKKTRIIPRHLQLAIRNDEELNKLLGKVTIAQGGVLPNIQAVLLPKKTESHHKAKGK* 1 RDNKKTRIIPRHLQLAIRNDEELNKLLGKVTIAQGGVLPNIQAVLLPKKTESHHKAKGK* 1 RDNKKTRIIPRHLQLAIRNDEELNKLLGKVTIAQGGVLPNIQAVLLPKKTESHHKTK* 1 RDNKKTRIIPRHLQLAIRNDEELNKLLGKVTIAQGGVLPNIQAVLLPKKTESHHKAKGK* 1 RDNKKTRIIPRHLQLAIRNDEELNKLLGKVTIAQGGVLPNIQAVLLPKKTESHHKAKGK* 1 RDNKKTRIIPRHLQLAIRNDEELNKLLGRVTIAQGGVLPNIQAVLLPKKTESHHKAKGK* 1 RDNKKTRIIPRHLQLAIRNDEELNKLLGRVTIAQGGVLPNIQAVLLPKKTESHHKAKGK* 1 RDNKKTRIIPRHLQLAIRNDEELNKLLGKVTIAQGGVLPNIQAVLLPKKTESHHKAKGK* γ 2 RDNKKTRIIPRHLQLAIRNDEELNKLLGKVTIAQGGVLPNIQAVLLPKKTESHHKAKGK* 1 RDNKKTRIIPRHLQLAIRNDEELNKLLGKVTIAQGGVLPNIQAVLLPKKTESHHKAKGK* RDNKKTRIIPRHLQLAIRNDEELNKLLGGVTIAQGGVLPNIQAVLLPKKTSATVGPKAPSGGKKATQASQEY* X Z KDLKVKRITPRHLQLAIRGDEELDSLI-KATIAGGGVIPHIHKSLIGKKGQQKTV* Macro2 RDNKKARIAPRHILLAVANDEELNQLLKGVTIASGGVLPRIHPELLAKK(nonhistone portion) Macro1 RDNKKGRVTPRHILLAVANDEELNQLLKGVTIASGGVLPNIHPELLAKK(nonhistone portion) Bbd QNSGERNITPLLLDMVVHNDRLLSTLFNTTTISQVAPGED* rdnkktriiPrhlqlairnDeeLnkLlgkvTIaqggvlpniqavLlpKKteshhkakgk* α -2-αα _ _ _ ααα -3-αα αα -C-α C-tail/linker/H2AX tail* d o ck i n g H4 95-100

H3 43-60 Current Opinion in Genetics & Development

Sequence comparison of the known human histone H2A species. Sequences were obtained from GenBank® and aligned manually. H2A genes A, C, D, I, N, and P all encode the identical peptide (residues shown in grey); any residue differences are shown in black. In the consensus

sequence, residues conserved in all known human H2A sequences are shown in upper case, others in lower. Protein motifs and interaction domains are taken from [39]. The arrow labelled ‘γ’ points to the conserved serine four residues from the H2AX carboxyl terminus (see Figure 2).

and macroH2A2 [6,7], in the 1990s and one, H2A-Bbd [8], just recently. Several of the variants differ from the consensus sequence at many positions throughout the sequence, whereas one, H2AX, differs primarily in the carboxyl terminus. Two variants, H2AX and H2AZ, are highly conserved as unique H2A species from S. cerevisiae to human. The macroH2A species may have arisen more recently in evolution and appear to be involved in X-chromosome silencing. (The reader is

referred to the review by Cohen and Lee in this issue [pp 219–224] for coverage of that topic.) H2A-Bbd has just been reported [8]. We limit this review to describing recent developments in understanding the roles of H2AX and H2AZ.

H2AX has a conserved SQ(E,D)(I,L,F,Y)* motif It had been surmised on the basis of size and peptide mapping that H2AX contained a carboxy-terminal region

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Figure 2 Species Human Mouse Fruit fly Spruce Arabidopsis Chick pea Tet. Py. Tet. Th. Grey rot Aspergillus S. cere 1 S. cere 2 S. pombe 1 S. pombe 2 Giardia

Classification (mammal) (mammal) (insect) (plant) (plant) (plant) (protozoa) (protozoa) (fungi) (yeast) (yeast) (yeast) (yeast) (yeast) (protist)

v100 v110 v120 v130 v140 LGGVTIAQGGVLPNIQAVLLPKKTSATVGPKAPSGGKKATQASQEY* LGGVTIAQGGVLPNIQAVLLPKKSSATVGPKAPAVGKKASQASQEY* IKA-TIAGGGVIPHIHKSLIGKK-(4)EETVQDPQRKGNVILSQAY* LGAVTIANGGVLPNIHQVLLPKK-(8)----SGKDKGEIGSASQEF* LGSVTIANGGVLPNIHQTLLPSK-(8)----VGKNKGDIGSASQEF* LGSVTIANGGVLPNIHQTLLPKK-(9)-----VGKGKGIGSASQEF* MANTTIADGGVLPNINPMLLPSK-(9)-----SKKTESRGQASQDI* MANTTIADGGVLPNINPMLLPSK-(9)-----SKKTESRGQASQDL* LGHVTIAQGGVLPNIHQSLLPKK-(9)-----TAKTAGGKPASQEL* LGHVTIAQGGVLPNIHQNLLPKK-(12)-------TPKAGKGSQEL* LGNVTIAQGGVLPNIHQNLLPKK-(12)-------SAKATKASQEL* LGNVTIAQGGVLPNIHQNLLPKK-(12)-------SAKTAKASQEL* LGHVTIAQGGVVPNINAHLLPKT-(12)-------SGGTGKPSQEL* LGHVTIAQGGVVPNINAHLLPKQ-(13)--------SGKGKPSQEL* FANVTIREGGVARSAKEGREGKG-(16)-----------SHRSQDL*

H2A sequences with an S(–4)Q motif were obtained from GenBank® and aligned manually. The S(–4)Q motif including the second SQ or TQ motif in mammalian H2AX, the TI, and the GGV conserved motifs are shown in black, all other residues are in grey.

Current Opinion in Genetics & Development

longer than those of the bulk H2A species, but it was the isolation of the human H2AX cDNA that revealed a protein with a carboxy-terminal region highly homologous with the H2A species of S. cerevisiae and Schizosaccharomyces pombe [9]. This result suggested that the conserved motif centering on serine four residues from the carboxyl terminus may have a conserved function. As histone sequences have accumulated, it has become clear that H2AX homologs are found throughout evolution among animals, plants, fungi, and various protists including Giardia, an amitochondriate protist with a 12 Mb genome and considered to be among the most primitive of eukaryotes (Figure 2). In S. cerevisiae and S. pombe, the total H2A complement is accounted for by three genes [10]: two for H2AX homologs and one for an H2AZ. In Tetrahymena thermophila, there are also three genes, but in addition to one gene each for H2AX and H2AZ homologs, a third encodes an H2A1 homolog [11]. Whether the H2A1 species is solely a truncated H2AX remains to be seen, but as genome size becomes larger, the H2A1/2 homologs come to predominate the H2A complement. Several sequence characteristics of the H2AX family are apparent from Figure 2. The SQ motif is invariant and its position relative to the carboxyl terminus is invariant. The SQ is followed by an acidic residue and the carboxyl terminal residue is hydrophobic. Noticeable too is the increase in distance between the globular portion of H2AX and the carboxyl terminus through evolution, being 16 residues longer in mammals than in Giardia. Another observation of note is that in Drosophila melanogaster H2AvD, the H2AX carboxyl terminus is fused with the H2AZ globular region, suggesting that H2AX and H2AZ can have the same localization. It also appears that mammalian H2AX contains a second copy of the SQ motif: SQ in mouse and TQ in humans.

H2AX SQ motif is massively and rapidly phosphorylated in response to DNA DSBs In mammals, Xenopus laevis, D. melanogaster, and S. cerevisiae, the serine four residues from the carboxyl terminus of an H2A species becomes phosphorylated in response to ionizing

radiation and other agents that introduce DNA double-strand breaks (DSBs) [12,13]. In mammals, the second (S,T)Q motif also becomes phosphorylated to a lesser extent. For simplicity and clarity, because the H2A species with the SQ motif have different names and lengths in different organisms (Figure 2), this phosphorylated form has been denoted with a γ before the relevant H2A homolog. The phenomenon is characterized by the rapid phosphorylation of the γ-serine immediately after the introduction of DNA DSBs breaks into cells. The phosphorylation is apparent within a minute and reaches a maximum in 10 minutes. An antibody specific to the phosphorylated H2AX carboxy-terminal peptide reveals that γ-H2AX molecules form foci in interphase nuclei and bands on metaphase chromosomes [13] with the number of foci approximating the number of expected DNA DSBs. After 30 minutes, γ-H2AX is dephosphorylated with a half-life of ~2 h, a value similar to the kinetics of DNA DSB repair; immunocytochemical analyses indicate that the number of foci decrease. By directing DNA DSBs to particular nuclear volumes, it was shown that the foci form at the sites of the breaks. The response is highly amplified. In mammals, ~0.03% of the H2AX is phosphorylated per DNA DSB; corresponding to the amount of H2AX that would be randomly distributed along 2 Mb equivalents of chromatin [12]. However, immunocytochemical analyses indicate that regions ten times larger are involved, suggesting that not every contiguous H2AX molecule is phosphorylated. Proteins involved in DNA repair often form foci as revealed by specific antibodies. Proteins BRCA1, Nbs1, Rad50, and Rad51 form foci that colocalize with preexisting γ-H2AX foci [14••]. The H2AX phosphorylation motif is SQE, a common PI-3 kinase phosphorylation motif, and the PI-3 kinase inhibitor wortmannin was effective in preventing γ-H2AX foci as well as the foci of the other noted factors when added 5 minutes before irradiation but not when added 5 minutes after. In addition, the human astrocytoma cell line M059J, known to be deficient in both

Histone H2A variants H2AX and H2AZ Redon et al.

DNA-PK and ATM (ataxia telangiectasia mutated) activities, is also partially deficient in γ-H2AX formation after irradiation. DNA-PK knockout mouse cell lines and embryo fibroblasts form γ-H2AX in response to agents that introduce DSBs into DNA, but the ATM knockout cell lines are severely deficient in γ-H2AX formation [15••]. When an ATM knockout line was transiently transfected with an ATM gene, γ-H2AX was again formed in response to ionizing radiation. ATM immunoprecipitated from cell extracts was able to form γ-H2AX in vitro and ATM extracted from irradiated cells was considerably more active in this process [15••]. When DNA DSB damage is introduced into cells, a portion of the nuclear ATM is retained in foci that colocalize with γ-H2AX foci [16••]. Both the retained ATM and γ-H2AX foci are visible at 10 minutes, most intense at 30 minutes and noticeably less intense at 1 hour. Tumor suppressor p53 binding protein 1 (53BP1) also forms foci colocalized with γ-H2AX foci rapidly after introduction of DNA DSBs and is also phosphorylated by ATM [17,18].

γ-H2AX forms after accidental DNA DSBs Different kinases may respond to different stimuli. γ-H2AX is formed in response to replicational stress, induced by hydroxyurea or ultraviolet light [19••]. Only S-phase cells in a culture form γ-H2AX foci under these conditions, whereas all cells in the culture form γ-H2AX foci in response to ionizing radiation. ATM–/– cells show an undiminished γ-H2AX response to hydroxyurea and ultraviolet light. In contrast, ATR ‘kinase dead’ cells show greatly diminished responses to these two agents, but an undiminished response to ionizing radiation. Thus, it may be that ATM is primarily responsible for γ-H2AX phosphorylation after accidental DNA DSBs from environmental stress whereas ATR is primarily responsible for γ-H2AX formation after accidental DNA DSBs from metabolic stress; the two types of lesions may be expected to be quite different and specific sensors may be needed for each. For example, ionizing radiation induces ~100 base and sugar modifications and 20 single strand breaks for each DSB. This extent of chemical damage would not be expected during metabolic stress. On the other hand, both types of lesions may occur together; ionizing radiation during S-phase may cause replicational stress as well as environmental stress, both leading to DNA DSBs, albeit different types. In S. cerevisiae, H2A1 (HTA1) and H2A2 (HTA2), both with SQEY* motifs, are phosphorylated in the presence of agents that generate DNA DSBs, and the phosphorylation of this site depends on Mec1 and Tel 1, the homologs of DNA-PK and ATM [20•]. Elimination of the SQEL motif leads to impaired non-homologous end joining (NHEJ). However, the mutant yeast strains were not more sensitive to ionizing radiation, perhaps because homologous recombination is the preferred mode of DNA DSB repair in yeast. When serine 129 was substituted to glutamic acid to mimic constitutive phosphorylation, a decrease in chromatin

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compaction was observed. Thus, SQ phosphorylation may facilitate NHEJ by decondensing chromatin to render damaged DNA sites accessible to repair factors.

γ-H2AX forms after programmed DNA DSB In addition to the environmentally and metabolically caused breaks, programmed DNA DSBs are essential steps in several important eukaryotic processes, such as DNA rearrangement during immune system development in mammals, meiotic recombination during germ cell formation, and programmed cell death by apoptosis. The first process occurs by NHEJ, the second by homologous recombination, and the third by a caspase-activated DNase. Nevertheless, studies have demonstrated γ-H2AX formation in all three. In immune system development, a genomic library of variable regions are assembled in the B and T lymphocyte populations of an organism by the process of V(D)J recombination. The process involves directed DNA DSBs to remove a DNA segment and rejoin the remaining DNA to form a functioning gene. γ-H2AX is induced at sites of ongoing V(D)J recombination colocalizing with a DNA FISH probe specific to the V(D)J locus [21•]. This result also demonstrates that γ-H2AX foci can detect a single DSB per mammalian genome. During zygotene of the first meiotic prophase, the homologous chromosomes align themselves in a process known as homologous synapsis. Meiotic recombination in the mouse is initiated by Spo11-dependent DSBs that form during leptotene. γ-H2AX foci formation shows that initiation of recombination in the mouse precedes synapsis [22••]. The processing of early recombination intermediates occurs in a synapsis-dependent fashion with the loss of γ-H2AX staining being temporally and spatially correlated with synapsis, even when this synapsis is ‘non-homologous’. During apoptosis, an early event is the appearance of high molecular weight DNA fragments as a result of caspaseactivated DNase. γ-H2AX forms extensively in all apoptotic systems examined concurrent with the initial DNA fragmentation [23].

H2AX in vitro studies Frog oocytes and eggs contain a large store of histones sufficient for the fertilized egg to develop into a blastula without histone synthesis. The major form of stored H2A is H2AX [24], which can become phosphorylated during nucleosome assembly and replication of DNA added to a cell-free extract prepared from eggs [25]. γ-H2AX formation has been demonstrated on NotI-digested chromatin added to these extracts whereas added undigested nuclei do not induce γ-H2AX formation. However, if the extracts are depleted of Mre11 — a protein involved in the Mre11/Nbs1/Rad50 complex required for normal replication products — γ-H2AX accumulates [26•]. Adding the Mre11 complex back to the extract prevents γ-H2AX accumulation in a dose-dependent manner.

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Possible models The commonality of γ-H2AX formation in these processes is its tight correlation with the formation and presence of DNA DSBs irrespective of origin. As γ-H2AX forms at the sites of the breaks, a physical signal, such as a DNA end or a chromatin deformation near the site of the break, could initially present a high-affinity site for a PI3 kinase such as ATM or ATR. PI3 kinases recruited thus could then phosphorylate H2AX molecules starting near the break and progressing away from it. Comparing the stoichiometry of γ-H2AX formation with the immunocytochemistry suggests that every contiguous H2AX molecule is not phosphorylated but perhaps 10–20% of the H2AX expected to be in a region. PI3 kinase molecules attracted to the break site and then constrained to track along the 30 nm fiber could explain these findings. ATM molecules could accumulate and form visible foci as fast as the γ-H2AX foci become visible. As H2AX is a histone, it is securely anchored into the chromatin and marks the break site. Mobile factors such as 53BP1 are recruited to the focus either by affinity for the phosphorylated H2AX carboxyterminal tail or an altered chromatin conformation induced by the tail. Phosphatases could be present and remove the γ-phosphate. The γ-phosphate would be replenished by kinase molecules as long as the break is extant. When the break is repaired, kinase molecules can no longer be recruited and as the retained ones dissociate, phosphatases remove the γ-phosphate.

H2AZ is a necessary but not sufficient H2A The H2AZ protein was identified in mammalian cells as an H2A variant in 1980 by gel and tryptic peptide analysis [4] and shown to be present in nucleosomes [27]. The cDNA for a chicken variant H2AF was isolated in 1985 [28]; when the mammalian H2AZ cDNA sequence was reported [29], it was found that these two proteins were homologs — hence H2AF/Z. In those organisms where they have been determined, H2AZ protein levels are ~10% of the total H2A complement. H2AZ provides an essential function to chromatin but it also cannot completely replace the H2A1 species in S. cerevisiae and T. thermophila; a mixture of the two is necessary for chromatin to function optimally [30,31•]. H2AZ sequences are highly conserved throughout evolution, but have been given different names in different organisms. Studies on H2AZ importance to organism survival have been performed in S. cerevisiae (HTA3 or HTZ1 [10]), S. pombe (PHT1 [32]), T. thermophila (hv1 [11]), D. melanogaster (H2AvD [33,34]), and mice [35]. For simplicity, the name H2AZ will be used here. In mice, deletion of the H2AZ gene results in early embryonic lethality [35]. In D. melanogaster, embryos null for H2AZ could be rescued by injection of constructs carrying the H2AZ gene [34]. In T. thermophila, H2AZ is found in the transcriptionally active macronucleus and is absent from the micronucleus except when that nucleus is transcriptionally active during early conjugation [36], results suggesting that H2AZ may be involved in transcriptional functions. H2AZ is essential for Tetrahymena survival. In

S. pombe, cells lacking H2AZ grow slowly, exhibit an altered colony morphology, increased resistance to heat shock and show a significant decrease in the fidelity of segregation of an S. pombe minichromosome [32]. In S. cerevisiae, H2AZ deletion results in slow growth and formamide sensitivity at 28°C and lethality at 37°C [31•].

H2AZ essential motif Chimeric constructs injected into D. melanogaster H2AZ null embryos showed that the essential portion of H2AvD is the αC helix and H3/H4-binding domains (Figure 1) [33]. Similar chimeric constructs restore the wild-type phenotype to S. cerevisiae [31•]. However, other studies show that H2AZ may serve functions other than just providing an H2A–H2B dimer with an altered interface to the H3–H4 tetramer. In T. thermophila, changing the six lysine residues in the H2AZ amino-terminal to arginines is lethal, suggesting that one of the roles of H2AZ is to modulate an essential charge patch [37•]. A charge patch is a region of the histone that needs to be able to modulate a certain charge but, in contrast to histone code modifications, it does not matter exactly which amino acids are charged and uncharged. The arginine residues provide a charge patch that cannot be modulated and presumably prevents gene activation. Two studies have tried to determine if H2AZ is distributed randomly or localized to particular sequences. In D. melanogaster, when H2AZ was localized in polytene chromosomes by indirect immunofluorescence and in diploid chromosomes by chromatin immunoprecipitation [34,38], it was found to be widely but nonuniformly distributed in the genome and was not limited to sites of active transcription. H2AZ immunofluorescence did not parallel the concentration of DNA as did H2A1 immunofluorescence, indicating that the results did indeed reflect H2AZ concentrations rather than epitope accessibility. H2AZ was present in thousands of euchromatic bands and the heterochromatic chromocenter of polytene chromosomes. In ChIP studies, it was found to be associated with both transcribed and nontranscribed genes as well as noncoding euchromatic and heterochromatic sequences. Similar immunoprecipitation studies in S. cerevisiae demonstrated a nonuniform pattern of H2AZ cross-linking across the PHO5 and GAL1 loci in contrast to a relatively uniform pattern of H2A1 cross-linking across the loci [31•]. If H2AZ is not uniformly distributed, this raises the important question of how H2AZ1-containing nucleosomes are targeted to specific chromatin sequences. These preferences could be mediated by an inherent DNA sequence specificity of H2AZ-containing nucleosomes, their interactions with other DNA binding proteins, statistical positioning, epigenetic assembly, or a combination of mechanisms.

H2AZ structural and physical studies The crystal structure of H2AZ-containing nucleosomes has been determined at 2.6 Å [39••]. It shows a core particle similar to that with H2A but with some intriguing differences.

Histone H2A variants H2AX and H2AZ Redon et al.

The L1 loop domain, which helps mediate interactions between H2A molecules in the same nucleosome, is altered in such a way that may favor binding to another H2AZ, resulting in nucleosomes with two H2AZ molecules. Secondly the tetramer docking domain, where the H2AZ–H2B dimer interfaces with the H3–H4 tetramer, has a subtly different conformation and includes a metal binding site. Such differences appear to result in differences in nucleosome properties. Analytical ultracentrifugation of nucleosomes reconstituted with H2AZ show a larger decrease in S20,W with increasing ionic strength than do those with H2A1, suggesting that the former may have a decreased stability compared to the latter [40••]. In addition, H2AZ-containing nucleosomes when reconstituted onto DNA fragments containing 12 repeats of the 208 bp sea urchin Lytechinus variegatus 5S rRNA gene, had the same S20,W values as those with H2A1 in the absence of salt but sedimented more slowly in 0.15 M salt. These results provide in vitro support for the idea that H2AZ-containing chromatin has a decreased stability, possibly resulting in the preferential loss of H2AZ–H2B dimers, thereby affording greater access of the transcription machinery to the DNA.

H2AZ functional studies Several studies have focussed on H2AZ functions in S. cerevisiae. In silencing studies, strains were generated in which a mutant Sir1 protein can be recruited to HMR silencers but once there cannot silence [41•]. A screen for overexpressed proteins that could overcome this defect yielded H2AZ. Deletion of H2AZ by itself resulted in only a partial derepression of silencing at HMR (ADE2 reporter), but a profound loss of telomeric silencing of a URA3 gene. Such variability in silencing effects might result from a nonrandom distribution of H2AZ as indicated above. H2AZ was also found in a synthetic lethal screen to study the effect of H4 mutations that affected interactions between the H3–H4 tetramer and H2A–H2B dimer subunits [31•]. An H4 Y98H mutant grew at 28°C but not at 37°C, and had spt (suppressor of Ty) and sin (SWI/SNFindependent) phenotypes. A screen for gene-dosage suppressors of the temperature-sensitive phenotype resulted in H2AZ; however, overexpressed H2AZ did not suppress the latter two phenotypes. Strains lacking H2AZ had a temperature-sensitive phenotype and those lacking H2AZ and SNF2, a component of SWI/SNF, were severely defective even on rich medium and glucose. This was unexpected as histone mutants generally have a sin phenotype. Likewise, double mutants with deletions of H2AZ and components of the SAGA complex were also impaired for growth. Transcription of genes that were either independent of (CLN3 and CUP1) or highly dependent upon SWI/SNF (SUC2 and INO1) were also independent of H2AZ. However, genes whose transcription was partially dependent on both H2AZ and SWI/SNF (PHO5 and GAL1) yielded no transcripts in the htz1∆ snf2∆ double mutant. Although yeast with the htz1∆ sin1∆ or snf2∆ sin1∆ had greater PHO5 and GAL1 expressions than htz1∆ or

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snf2∆ alone, crossing sin1∆ into the htz1∆ snf2∆ double mutant did not restore any expression of these two genes. When the PHO5 gene is activated, a site in the promoter is made accessible to ClaI. This accessibility occurs in the wt, htz1∆, snf2∆, and the double, even though PHO5 transcripts are made only in the first three strains — indicating that the defect is downstream of this step. The ChIP assay with antibodies to RNA PolII and TBP showed that both proteins were cross-linked to increasing amounts of GAL1-10 promoter sequences over a period of 2 hours in the wt, but not in the htz1∆ strain [42•]. A nonrandom distribution of H2AZ in the chromatin would place genes in chromatin with different amounts of H2AZ. In regions with high H2AZ content, SWI/SNF remodeling complexes may not be as necessary because H2AZ–H2B dimers are more easily dissociated. This idea is consistent with mutations in other histones often making the yeast less dependent on the SWI/SNF complex because those nucleosomes may also be less stable. As H2AZ is a basic component of the chromatin structure with an optimum ratio to H2AX, either over, under or not expressing it might be expected to affect multiple processes including both gene silencing, gene activation, and the switching between the two processes.

Conclusions It is now accepted that histones not only package DNA but also monitor whether the package is intact, and when and where it is to be opened and closed. Although histones are not necessary to study the basic chemical pathways in transcription and replication in vitro, understanding histones is essential to understanding how those processes are harnessed in a eucaryotic cell. Insight into the role of histone H2AX in eucaryotes is likely to come from studies on organisms in which the H2AX has been either deleted or modified to remove the γ-serine residue. The H2AX–/– mouse is viable and will be a useful tool in furthering insight into the varied appearances of γ-H2AX, for example, the unexpected presence of γ-H2AX in the sex body after it has disappeared from paired autosomes during the first meiotic division of mouse spermatogenesis [22••]. In contrast, deletion of H2AZ results in an early embyronic lethal in the mouse and inviable ES cells [35]. However, further development in gene-targeting techniques in the areas of tissue and timespecific targeting [43] should lead to further insights into the roles of H2AZ in cellular metabolism.

Update Mice have been produced that lack H2AX; the mice survive but are impaired in several processes of which impairments in class switch recombination has been documented [44••]. Class switch recombination is a region-specific recombination that replaces one immunoglobulin heavy-chain constant region with another. B cells from H2AX–/– mice have 50–86% reduction in surface IgG1 levels compared to

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Chromosomes and expression mechanisms

their normal littermates. The results with CSR do not imply that H2AX–/– mice are not deficient in V(D)J recombination, but just that CSR is easier to study because it can be induced in culture from cells removed from mice.

antibody to γ-H2AX demonstrates foci formation in interphase nuclei. γ-H2AX is revealed in mitotic chromosomes and on one end of broken chromosome arms. In [14••], other repair factors — BRCA1, Nbs1, Rad50, and Rad51 — are shown to colocalize with γ-H2AX foci over time. A cell line defective in DNA-PK and ATM is also deficient in γ-H2AX formation. The laser scissors are used in [13,14••] to show that the foci form at the sites of breaks.

Further involvement of γ-H2AX formation in replication stress responses has been demonstrated in xeroderma pigmentosum variant (XPV) cells that lack a functional bypass DNA polymerase H [45]. γ-H2AX foci positive XPV cells increase in number over several hours after UV-irradiation, in contrast to the immediate increase in minutes after γ-irradiation. The results suggest that DNA DSBs form gradually during replication arrest in XPV cells, presumably either as a build-up of a normal repair intermediate or as an aberrant repair response.

15. Burma S, Chen BP, Murphy M, Kurimasa A, Chen DJ: ATM •• phosphorylates histone H2AX in response to DNA double-strand breaks. J Biol Chem 2001, 276:42462-42467. Demonstrates a role for ATM in γ-H2AX formation induced by ionizing radiation. Induction is shown to be almost absent in mouse Atm–/– cells, and almost normal in DNA-PK–/– cells (both by immunoblotting and by IHC). A plasmid with wt ATM gene restores ability of ATM–/– to phosphorylate H2AX. ATM is shown to phosphorylate H2AX in vitro.

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Histone H2A variants H2AX and H2AZ Redon et al.

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28. Harvey RP, Whiting JA, Coles LS, Krieg PA, Wells JR: H2A.F: an extremely variant histone H2A sequence expressed in the chicken embryo. Proc Natl Acad Sci USA 1983, 80:2819-2823. 29. Hatch CL, Bonner WM: Sequence of cDNAs for mammalian H2A.Z, an evolutionarily diverged but highly conserved basal histone H2A isoprotein species. Nucleic Acids Res 1988, 16:1113-1124. 30. Liu X, Bowen J, Gorovsky MA: Either of the major H2A genes but not an evolutionarily conserved H2A.F/Z variant of Tetrahymena thermophila can function as the sole H2A gene in the yeast Saccharomyces cerevisiae. Mol Cell Biol 1996, 16:2878-2887. 31. Santisteban MS, Kalashnikova T, Smith MM: Histone H2A.Z • regulates transcription and is partially redundant with nucleosome remodeling complexes. Cell 2000, 103:411-422. This paper, along with [41•,42•] are in-depth studies of gene regulation in yeast lacking H2AZ. Here, the authors show that lack of H2AZ has a variable effect on different genes but increases dependence on SNF/SWI remodeling in certain cases. The authors show that the lack of H2AZ increases dependence on SNF/SWI remodeling. They demonstrate that the PHO5 promoter can be cut with ClaI when the gene is activated by low PO4 in wt, htz1∆, snf2∆, and htz1∆ snf2∆ double mutant strains; however, PHO5 transcripts are made only in the first three strains. Thus the defect is downstream of (at least some) remodeling. In [41•], the authors also find variable effect of H2AZ deletion in the loss of silencing. In [42•], the authors demonstrate that yeast lacking H2AZ are defective in recruiting RNA Pol II to the GAL-10 promoter. 32. Carr AM, Dorrington SM, Hindley J, Phear GA, Aves SJ, Nurse P: Analysis of a histone H2A variant from fission yeast: evidence for a role in chromosome stability. Mol Gen Genet 1994, 245:628-635. 33. Clarkson MJ, Wells JR, Gibson F, Saint R, Tremethick DJ: Regions of variant histone His2AvD required for Drosophila development. Nature 1999, 399:694-697.

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Tetrahymena H2AZ. Using gene technology developed in their laboratory for Tetrahymena, the authors replaced some or all of the six lysine residues in the H2AZ tail with arginines and glutamines. Whereas Tetrahymena cannot survive with six replacement arginine residues, neutralizing one of the six arginine positive charges with an adjacent acidic residue, or replacing any of several arginine residues with a glutamine residue restored viability. That the effect is not position specific leads to the idea of the charge patch rather than a histone code. 38. Leach TJ, Mazzeo M, Chotkowski HL, Madigan JP, Wotring MG, Glaser RL: Histone H2A.Z is widely but nonrandomly distributed in chromosomes of Drosophila melanogaster. J Biol Chem 2000, 275:23267-23272. 39. Suto RK, Clarkson MJ, Tremethick DJ, Luger K: Crystal structure of a •• nucleosome core particle containing the variant histone H2A.Z. Nat Struct Biol 2000, 7:1121-1124. The X-ray crystal structure determined for H2AZ-containing nucleosomes shows their overall similarity to those containing bulk H2A, but also shows several subtle structural differences that may give possible insights into the important differences that make H2AZ essential. 40. Abbott DW, Ivanova VS, Wang X, Bonner WM, Ausio J: •• Characterization of the stability and folding of H2A.Z chromatin particles. Implications for transcriptional activation. J Biol Chem 2001, 276:41945-41949. This study demonstrates a difference in nucleosomes containing H2AZ. Analytical ultracentrifugation shows that nucleosomes reconstituted with recombinant H2AZ are less stable than those reconstituted with recombinant H2A1 or natural H2A. The lessened stability may be the result of the H2AZ–H2B dimer binding less tightly to the nucleosome. Analytical ultracentrifuge analysis of the H2A.Z 208–12 reconstituted oligonucleosome complexes in the absence of histone H1 shows that their NaCl-dependent folding ability is significantly reduced compared to control oligonucleosomes. 41. Dhillon N, Kamakaka RT: A histone variant, Htz1p, and a Sir1p-like • protein, Esc2p, mediate silencing at HMR. Mol Cell 2000, 6:769-780. See annotation [31•]. 42. Adam M, Robert F, Larochelle M, Gaudreau L: H2A.Z is required for • global chromatin integrity and for recruitment of RNA polymerase II under specific conditions. Mol Cell Biol 2001, 21:6270-6279. See annotation [31•]. 43. Metzger D, Chambon P: Site- and time specific gene targeting in the mouse. Methods 2001, 24:71-80.

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44. Petersen S, Casellas R, Reina-San-Martin B, Chen HT, •• Difilippantonio MJ, Wilson PC, Hanitsch L, Celeste A, Muramatsu M, Pilch DR et al.: AID is required to initiate Nbs1/γγ-H2AX focus formation and mutations at sites of class switching. Nature 2001, 414:660-665. This paper uses the newly-developed H2AX–/– mouse in the study of activation-induced cytidine deaminase (AID) involvement in class-switch recombination (CSR). Cytometry showed large decreases in expression of IgG1 (one of several immunoglobulins derived by CSR) on the surface of B-cell populations from H2AX–/– mice as well as a large decrease in Sµ–Sγ rearrangements in their DNA. The H2AX–/– mouse was generated using a vector in which a region coding for the H2A conserved core (amino acid residues 48–56) was replaced by a neo cassette. The authors conclude that H2AX promotes efficient CSR but is not essential for the reaction.

37. Ren Q, Gorovsky MA: Histone H2A.Z acetylation modulates an • essential charge patch. Mol Cell 2001, 7:1329-1335. Although most of the interest in H2AZ focuses on its core sequences, these authors have demonstrated the importance of the amino-terminal tail of

45. Limoli CL, Giedzinski E, Bonner WM, Cleaver JE: UV-induced replication arrest in the xeroderma pigmentosum variant leads to DNA double-strand breaks, gamma -H2AX formation, and Mre11 relocalization. Proc Natl Acad Sci USA 2002, 99:233-238.

34. van Daal A, Elgin SC: A histone variant, H2AvD, is essential in Drosophila melanogaster. Mol Biol Cell 1992, 3:593-602. 35. Faast R, Thonglairoam V, Schulz TC, Beall J, Wells JR, Taylor H, Matthaei K, Rathjen PD, Tremethick DJ, Lyons I: Histone variant H2A.Z is required for early mammalian development. Curr Biol 2001, 11:1183-1187.