Phosphorylation of the human histone variant H2A.X and H2Av, its homolog in Drosophila melanogaster, occurs rapidly at sites of DNA double-strand breaks. Little is known about the function of this phosphorylation or its removal during DNA repair. The Drosophila Tip60 (dTip60) chromatin-remodeling complex acetylates nucleosomal phospho-H2Av and exchanges it with an unmodified H2Av. Both the histone acetyltransferase dTip60 as well as the adenosine triphosphatase Domino/p400 catalyze the exchange of phospho-H2Av. Thus, these data reveal a previously unknown mechanism for selective histone exchange that uses the concerted action of two distinct chromatin-remodeling enzymes within the same multiprotein complex (Kusch, 2004).

DNA double-strand breaks (DSBs) are a deleterious type of DNA damage leading to chromosomal breakage. Cells have developed mechanisms to detect and repair DSBs, which must access nucleosomal DNA. Two classes of activities regulate the accessibility of DNA by either covalently modifying histones or using adenosine triphosphate (ATP) hydrolysis to catalyze histone mobilization. Current knowledge suggests that covalently modified histones can create specific interaction sites for regulatory proteins and complexes (Kusch, 2004).

Incorporation of histone variants into nucleosomes provides another mechanism for altering chromatin structure. Whereas the major histones are assembled into nucleosomes during DNA replication, histone variants can be incorporated into chromatin in a replication-independent manner. An example of such an activity is the yeast Swr1p ATPase complex, which catalyzes the exchange of H2A for the variant H2A.Z in nucleosomes (Kusch, 2004).

Histone modifications can mark distinct chromatin locations. H2A.X, an essential mammalian histone variant required for genomic stability, becomes phosphorylated at sites of DSBs by conserved DNA damage–recognizing factors. Like H2A.X, H2A and H2Av become phosphorylated at DSBs in yeast and flies, respectively. Because repair requires access to DNA, it has been suggested that this phosphorylation might attract chromatin-remodeling complexes to DSBs. The removal of phospho-H2A.X is replication-independent and could be catalyzed by the same complexes. DSBs accumulate upon inactivation of the human Tip60 complex, implicating it as one candidate for a chromatin-remodeling complex with a role in DNA repair (Kusch, 2004).

This study demonstrates that the Drosophila dTip60 multiprotein complex catalyzes exchange of phospho-H2Av with unmodified H2Av. This reaction is catalyzed by two chromatin-dependent enzymes within the dTip60 complex: the histone acetyltransferase dTip60 and the ATPase Domino. These factors sequentially acetylate and then replace nucleosomal phospho-H2Av with H2Av from within the dTip60 complex (Kusch, 2004).

The dTip60 complex was purified from Drosophila S2 cells. dPontin, the fly homolog of a subunit of the human Tip60 complex, was epitope-tagged with a hemagglutin (HA)-Flag tag at the C terminus. The dPontinHAFlag-associated proteins were isolated from nuclear extracts by sequential Flag- and HA-affinity purification followed by a glycerol gradient. Peak fractions of dPontin-HAFlag, dTip60, and Domino were identified by immunoblotting and assayed for histone acetyltransferase activity. Several polypeptides that copurified with dPontinHAFlag were identified by multidimensional protein identification technology (MudPIT). This study identified polypeptides with homology to all 16 subunits of the human Tip60 complex. This analysis also revealed a substantial number of tryptic peptides from histones H2Av and H2B but not from other histones (Kusch, 2004).

Antibodies against dTip60, dMrg15, dTra1, dGas41, dIng3, and E(Pc) as well as against Domino, H2Av, and H2B were used in immunoblotting of gradient peak fractions and anti-dTip60 immunoprecipitates from nuclear extracts to confirm that these proteins are part of the dTip60 complex. dPontin-HAFlag stably associated with all dTip60 complex subunits examined, including dReptin, the fly homolog of the human Tip60 complex component Tip49b. Histones H2Av and H2B stably associated with the dTip60 complex, whereas histone H2A and other histones were not detected (Kusch, 2004).

Tip60 complexes function in DSB repair and contain the ATPase Domino/P400 and H2Av/H2B heterodimers. Because H2Av becomes phosphorylated at sites of DSBs, whether dTip60 complex remodeled nucleosomes containing phospho-H2Av was tested. Recombinant Drosophila nucleosomes were assembled containing H2Av with a point mutation that mimicked phosphorylation at Ser¹³⁷ (Ser¹³⁷ to Glu¹³⁷; H2AvE). Upon incubation with the dTip60 complex, recombinant H2AvFlag/H2B heterodimers, acetyl-coenzyme A (acetyl-CoA), and ATP, a transfer of H2AvFlag to the nucleosomal arrays was observed. The transfer reaction proceeded rapidly (notable amounts of H2AvFlag were incorporated within 5 min) and depended on the presence of nucleosomes. Although relatively small amounts of H2AvFlag were transferred in the absence of ATP and/or acetyl-CoA, it was about seven times more efficient in the presence of both cofactors. Addition of a nonhydrolyzable ATP analog (gammaS-ATP) reduced the background activity of the complex. The dTip60 complex was highly selective for incorporation of H2Av into H2AvE-containing nucleosomal arrays. No H2AvEFlag was incorporated into nucleosomes containing H2Av, and no significant release of H2AvFlag was observed from nucleosomal arrays in the presence of H2AvEFlag/H2B heterodimers. Time course experiments revealed that the presence of acetyl-CoA enhanced the transfer speed and the quantity of H2Av incorporation. The incorporation rate of H2AvFlag into the nucleosomal arrays was unchanged when acetyl-CoA only was temporarily added to the exchange reactions and removed before the addition of heterodimers. This strongly suggests that the acetylation of the nucleosomal arrays by the dTip60 complex, but not of heterodimers, is crucial for optimal H2Av exchange (Kusch, 2004).

To examine the acetyltransferase specificity of the dTip60 complex, different combinations of recombinant histones as substrates in histone acetyltransferase (HAT) assays. In the presence of core histones, H2A, H2Av, and H2AvE were acetylated at equally low levels. However, in a nucleosomal context, acetylation of H2AvE was significantly increased over that observed for all other histones. This confirms that the dTip60 complex preferentially targets and acetylates phospho-H2Av in nucleosomes. In fact, Lys⁵ of histone H2Av is acetylated by the dTip60 complex. As individual monomeric histones, H2A, but not H2Av or H2AvE, was the preferred substrate of the dTip60 complex. By contrast, acetylation was about equal between H2A and H2Av when heterodimers with H2B were assayed, whereas acetylation of H2AvE was unchanged. Thus, dTip60 complex prefers H2Av-containing heterodimers over those containing H2AvE (Kusch, 2004).

Upon induction of DSBs, phospho-H2Av rapidly accumulates on chromatin with peak amounts after 10 to 15 min. During the course of DNA repair, this phosphorylation becomes undetectable within 180 min. The dTip60 complex acetylates and removes phospho-H2Av from nucleosomes in vitro. Thus, whether removal of phospho-H2Av during repair was dependent on dTip60 complex was tested in vivo. dTip60 or dMrg15 were depleted from S2 cells by RNA interference (RNAi). These cells were exposed to gamma irradiation to induce DSBs, and the nucleosomal histones were extracted after 0, 15, and 180 min. The amounts of H2Av and phospho-H2Av were compared by immunoblotting. In mock-treated cells, phospho-H2Av levels peaked after 15 min and were undetectable after 180 min. By contrast, phospho-H2Av levels remained high in cells depleted for either dTip60 or dMrg15. To confirm these findings in embryos, a null allele of dMrg15 was generated, and phospho-H2Av levels were tested after gamma irradiation. Again, the levels of phospho-H2Av remained higher in dMrg15 mutants than in wild-type embryos (Kusch, 2004).

Because the dTip60 complex acetylated nucleosomal phospho-H2Av in vitro, dependence of H2Av acetylation on dTip60 complex components was tested in vivo. Chromatin extracts were probed from gamma-irradiated double-stranded RNA (dsRNA)–treated S2 cells as well as dMrg15 mutant embryos with antibodies against H2A(acK5), which recognized H2Av(acK5). Transient acetylation of a protein band was detected that exhibits the migratory properties of phospho-H2Av. This acetylation was most prominent 15 min after gamma irradiation and was not detected in extracts of cells lacking dTip60 or dMrg15. Similar observations were made by immunolabeling dMrg15 mutant embryos. It is concluded that the dTip60 complex acetylates nucleosomal phospho-H2Av at Lys⁵ in a DSB-dependent manner (Kusch, 2004).

The Drosophila dTip60 complex is structurally homologous to its human counterpart. Both complexes share factors that are linked to cancer, transcription, and DNA repair, including Pontin, Reptin, Mrg15, Tra1, E(Pc), Gas41, and Tip60. The histone variant H2Av was detected within the Drosophila dTip60 complex. The human Tip60 complex is essential for DSB repair and regulation of apoptosis, two processes that have been linked to histone H2Av in flies. Also the yeast NuA4 complex appears to accumulate at DSBs (Kusch, 2004).

This study demonstrated that the Drosophila dTip60 complex acetylates nucleosomal phospho-H2Av and exchanges it with an unmodified H2Av. The histone-exchange reaction catalyzed by the ATPase Domino is enhanced by dTip60-mediated acetylation of nucleosomal phospho-H2Av. It appears likely that phospho-H2Av recruits the dTip60 complex to DSBs to facilitate chromatin remodeling during DNA repair. In yeast, the DNA damage–dependent H2A kinase Mec1 genetically interacts with subunits of the NuA4 complex, and cells missing NuA4 subunits are sensitive to DSB-inducing agents. The physiological roles of the dTip60-mediated phospho-H2Av removal at sites of DSBs could not be clearly separated from a potential function of this complex in DSB repair because of the intimate temporal link between DSB repair and phospho-H2Av clearance. However, the overexpression of phospho-H2Av did not induce G2/M arrest or affect DSB-dependent G2/M arrest, suggesting that this signal is not sufficient for damage checkpoint control (Kusch, 2004).

The loss of human Tip60 leads to the accumulation of DSBs and is linked to a growing number of cancer types. The histone variant H2A.X is essential for genomic stability and a candidate tumor suppressor. Thus, these findings help to understand the functional link between DNA damage–dependent H2A.X phosphorylation and the role of Tip60-type complexes during DSB repair in chromatin (Kusch, 2004).

Transcripts from the H2AvD gene are present in adult females and are abundant during the first 12 h of embryogenesis, peaking at ca. 6 h, the time of maximal DNA synthesis. The H2AvD transcripts thus show the same developmental profile over time as do the bulk of the histone transcripts, those from the histone genes at region 39D2-3 to 39E1-2, including the H2A.1 transcript. Because the H2A.1 mRNA and protein are generally synthesized predominantly during the S-phase of the cell cycle, it is not surprising that the Drosophila H2A.1 transcript is not abundant after 12 h, because mitotic divisions largely cease at about this time in D. melanogaster embryogenesis. Although one would not expect expression of the variant to be regulated within the cell cycle, the similarity of the developmental profiles of the H2A.1 and H2AvD transcripts suggests that synthesis of H2AvD, while constant throughout the cell cycle, may be related to the relative rate of cell division. The findings suggest a requirement for H2AvD as the genome is replicated. A developmental Western analysis using an antibody made to the unique C-terminal tail of the H2AvD protein (amino acids 126-140) shows that the H2AvD protein is present at all developmental stages at approximately constant levels, as are the major core histone proteins, This indicates that H2AvD is very stable, as is typical of core histones (van Daal, 1992).

Variant histones that differ in amino acid sequence from S-phase histones are widespread in eukaryotes, yet the structural changes they cause to nucleosomes and how those changes affect relevant cellular processes have not been determined. H2A.F/Z is a highly conserved family of H2A variants. H2Av, the H2A.F/Z variant of Drosophila melanogaster, was localized in polytene chromosomes by indirect immunofluorescence and in diploid chromosomes by chromatin immunoprecipitation. H2Av is widely distributed in the genome and not limited to sites of active transcription. H2Av is present in thousands of euchromatic bands and the heterochromatic chromocenter of polytene chromosomes, and the H2Av antibody precipitated both transcribed and nontranscribed genes as well as noncoding euchromatic and heterochromatic sequences. The distribution of H2Av was not uniform. The complex banding pattern of H2Av in polytene chromosomes did not parallel the concentration of DNA, as did the pattern of immunofluorescence using H2A antibodies, and the density of H2Av measured by immunoprecipitation varied between different sequences. Of the sequences assayed, H2Av was least abundant on 1.688 satellite sequences and most abundant on the hsp70 genes. Finally, transcription caused, to an equivalent extent, both H2Av and H2A to be less tightly associated with DNA (Leach, 2000).

H2AvD, a Drosophila melanogaster histone variant of the H2A.Z class, is encoded by a single copy gene in the 97CD region of the polytene chromosomes. Northern analysis shows that the transcript is expressed in adult females and is abundant throughout the first 12 h of embryogenesis but then decreases. The H2AvD protein is present at essentially constant levels in all developmental stages. Using D. melanogaster stocks with deletions in the 97CD region, the H2AvD gene was localized to the 97D1-9 interval. A lethal mutation in this interval, l(3)810, exhibits a 311-base pair deletion in the H2AvD gene, which removes the second exon. P-element mediated transformation using a 4.1-kilobase fragment containing the H2AvD gene rescues the lethal phenotype. H2AvD is therefore both essential and continuously present, suggesting a requirement for its utilization, either to provide an alternative capability for nucleosome assembly or to generate an alternative nucleosome structure (van Daal, 1992).

One way in which a distinct chromosomal domain could be established to carry out a specialized function is by the localized incorporation of specific histone variants into nucleosomes. H2AZ, one such variant of the histone protein H2A, is required for the survival of Drosophila melanogaster, Tetrahymena thermophila and mice. To search for the unique features of Drosophila H2AZ (His2AvD, also referred to as H2AvD) that are required for its essential function, amino-acid swap experiments were performed in which residues unique to Drosophila His2AvD were replaced with equivalently positioned Drosophila H2A.1 residues. Mutated His2AvD genes encoding modified versions of this histone were transformed into Drosophila and tested for their ability to rescue null-mutant lethality. The unique feature of His2AvD is shown not to reside in its histone fold but in its carboxy-terminal domain. This C-terminal region maps to a short alpha-helix in H2A that is buried deep inside the nucleosome core (Clarkson, 1999).

Amino-acid differences between Drosophila His2AvD and H2A span the whole sequence of His2AvD including the N and C termini (excluding the extra-long C-terminal tail of His2AvD, His2AvD has a net gain of five serines and three threonines compared to H2A.1). Clearly, some or all of these amino-acid differences must specify the unique function of His2AvD which cannot be provided by H2A. Therefore, to gain a molecular understanding of these special features that define H2AZ function, the amino acids unique to His2AvD were replaced, by changing the coding sequence, with H2A amino-acid residues. A deletion in the Drosophila His2AvD gene (His2AvD 810) is homozygous lethal. However, the precise developmental step at which lethality occurs has remained unknown. In this study, fly stocks containing the His2AvD null allele, His2AvD 810, were maintained against the TM6b balancer chromosome which carries the dominant markers Tubby and Humoral. Non-Tubby His2AvD 810 homozygotes undergo a protracted third instar and then die without entering pupation. Since His2AvD 810 message is maternally transcribed and loaded into the developing oocyte, the arrested development of the His2AvD null mutants during the third larval instar indicates that His2AvD derived from the maternal messenger RNA is depleted to subcritical levels at this stage. This null lethality can be rescued with a 4.0 kilobase (kb) genomic fragment containing the His2AvD gene. Lines rescued by the wild-type gene generate adult flies that are fertile (Clarkson, 1999).

Site-directed mutagenesis was used to change the amino-acid sequence of His2AvD. Specifically, 'cassettes' encoding amino-acid residues in the His2AvD-rescue DNA fragment were mutated to the equivalent H2A.1 residues. To change all the unique His2AvD amino acids, seven mutants were generated. To examine the functional contribution of the extended C-terminal domain of His2AvD, an additional mutant was constructed which removed this tail by introducing a stop codon at Gln 127. Whether these transgenes could rescue null lethality was tested by generating fly lines containing stably integrated wild-type and mutated His2AvD transgenes. These lines were then crossed to introduce each transgene into a homozygous His2AvD 810 null background. Rescue of the larval lethal phenotype of homozygous His2AvD 810 progeny was scored as the appearance of pupae and adults that lacked the dominant TM6b markers Tubby and Humoral, respectively. For analysis of results, rescue by the wild-type His2AvD gene was given a relative value of 100% (Clarkson, 1999).

All of the His2AvD transgenes containing different H2A-replacement cassettes could rescue null lethality up to pupation with the exception of M6. This non-rescuing transgene contains H2A sequences that lie at the C-terminal end of the protein and not in the histone fold or in the N-terminal tail. Specifically, transgene M6 has a cassette that encompasses the C-terminal alpha-helix of H2A that replaces identically located His2AvD residues. Since this transgene cannot compensate for His2AvD function, it is concluded that a small C-terminal region of His2AvD, which maps to the extra C-terminal alpha-helix of H2A and includes only six amino-acid differences (plus one amino-acid deletion) compared to H2A.1, is essential for H2AZ function as measured by survival of the fly. This is the first time that a small specific region of an individual histone, which lies outside the histone fold, has been shown to be essential for the survival of an organism (Clarkson, 1999). In contrast to the amino-acid segments required to reach the pupal stage, three additional regions of H2AZ (M1, M7 and, to a lesser extent, M4) appear to be important for His2AvD function during the pupal stages. Unlike transgene M6, transgenes M1, M4 and M7 do permit some survival to adulthood, but flies transformed with transgene M7 are particularly compromised and on average only about 9% of flies eclose. Cassette 7 lies immediately adjacent to cassette 6 in the C-terminal tail of His2AvD. Therefore, it is concluded that most of the C-terminal region of His2AvD is important for its function, although the terminal 14 amino acids of His2AvD are not important for survival (Clarkson, 1999).

Transgene M1 has part of the N-terminal tail of His2AvD swapped with the tail of H2A. This region of His2AvD would be expected to be important for its function because it is the site for post-translational modifications including acetylation and, like the tails of H3 and H4, it may be involved in unique interactions with regulatory proteins. These results raise the possibility that, during Drosophila development, different regions of His2AvD may carry out important functions at different times. However, other explanations exist and further characterization of these mutants during development is required to test this proposal (Clarkson, 1999).

To investigate whether the ability of these mutated His2AvD genes to rescue null lethality is further compromised at elevated temperatures, the above experiments were carried oit at 29°C instead of 25°C. The overall trend is very similar to the results obtained at non-elevated temperatures, although the rescue ability of some of the transgenes is affected more than others. One marked difference is that transformation with transgene 7 could not rescue null lethality at all. This observation may provide an opportunity to study His2AvD function in the adult fly, and during development, under normal and elevated temperature conditions. A second difference is that the rescue of His2AvD null lethality at high temperatures actually increases when the terminal 14 amino acids of His2AvD are removed. In other words, under conditions of moderate heat stress, removing part of the C-terminal tail increases the chance of survival of the adult fly (Clarkson, 1999).

Based on the crystal structure of the nucleosome, the nucleosome in which normal H2A is replaced with His2AvD was modelled. Since the essential C-terminal region of His2AvD critical for its function is buried inside the histone core, it is not, at least directly, involved in interactions with DNA. It is more likely that this region is involved in protein-protein interactions, perhaps influencing the stability of the core particle. This seems likely, as analysis of the crystal structure has led to the proposal that the C-terminal alpha-helix of normal H2A, which interacts with the C-terminal tail of H4, forms a docking domain with alpha-helix 3 of H2A, and this domain is important for interactions between the H2A-H2B dimer and the H3-H4 tetramer. Amino acids 105 to 117, the region of the tail that immediately follows this C-terminal alpha-helix of H2A, interacts with the N-terminal alpha-helix of histone H3. This interaction also links H2A with the H3-H4 tetramer. Therefore, the whole C-terminal region of H2A is involved in nucleosome stability and the amino-acid differences in His2AvD may alter this stability (Clarkson, 1999).

Any changes in the stability of the core particle would have a direct effect on the ability of remodelling machines and transcription factors to disrupt and access chromatin. Such changes in the stability or structure of the nucleosome could also potentially influence the formation of higher-order chromatin structures. For example, the C-terminal alpha-helix of H2A begins with glutamate, and this residue is part of an acidic patch which, in the nucleosome crystal, interacts with the positively charged N-terminal tail of H4, which protrudes from an adjacent nucleosome core particle (Clarkson, 1999).

In conclusion, these results identify regions that play key roles in the specificity of function of a variant histone and also highlight the multifunctional nature of a nucleosome. The hypothesis that the incorporation of His2AvD alters the stability of the nucleosome is currently being tested by in vitro chromatin reconstitution experiments (Clarkson, 1999).

It has been shown that the Drosophila H2Av variant is distributed in a nonrandom manner in third instar polytene chromosomes (van Daal, 1992; Leach, 2000). H2Av is present in the heterochromatic chromocenter and is associated with both transcribed and nontranscribed genes in polytene chromosome bands and interbands (Leach, 2000). To gain further insights into the function of H2Av, it was decided to test whether His2Av behaves genetically as a trithorax-Group (trxG) or Polycomb-Group (PcG) gene. In Drosophila, expression patterns of homeotic genes are maintained by the PcG and trxG proteins. Since H2Av is present in nontranscribed euchromatic regions (Leach, 2000), whether this histone variant is involved in Pc-mediated silencing was determined by examining whether mutations in the His2Av gene enhance the phenotype of Pc mutants. Adult flies from a strain heterozygous for Pc, Df(3R)Pc/+, show a partial transformation of the second leg into the first leg, visualized by the appearance of sex combs in the second leg of male flies. When flies are also heterozygous for a mutation in the His2Av gene, the frequency and severity of these transformations increase dramatically. Out of 100 flies of the genotype Df(3R)Pc+/+ His2Av⁰⁵¹⁴⁶ examined, 33% had extra sex combs in all four second and third legs and 40% had extra sex combs in the second legs and one of the third legs. Out of 220 flies of the genotype Df(3R)Pc+/+ His2Av⁸¹⁰ tested, 18% showed transformations of second into first leg and 72% showed transformation of both second and third legs into first. These results suggest that mutations in His2Av enhance the Pc phenotype and therefore His2Av might be classified as a PcG gene. To confirm this possibility, genetic interactions between His2Av and trxG mutants were examined. If His2Av is a PcG gene, mutations in His2Av should suppress the phenotype of trxG genes. The effect of His2Av⁰⁵¹⁴⁶ and His2Av⁸¹⁰ was examained on two different combinations of trG genes, ash1^VF101 trx^b11/++ and brm² trx^E2/++. Flies of the genotype ash1^VF101 trx^b11/++ show transformations of third leg into second leg by the appearance of an apical bristle on the third leg in 66% of 1000 flies examined. This frequency decreases to 37% in ash1^VF101 trx^b11+/++ His2Av⁰⁵¹⁴⁶ flies and to 29% in ash1^VF101 trx^b11+/++ His2Av⁸¹⁰ flies. Similarly, flies of the genotype brm², trx^E2/++ show a 43% frequency of haltere to wing or third leg to second leg transformations, and this frequency is reduced to 22% in brm² trx^E2+/++ His2Av⁰⁵¹⁴⁶ flies and to 21% in brm² trx^E2+/++ His2Av⁸¹⁰ flies. These data suggest that mutations in His2Av suppress the phenotype of trxG mutations and, together with the previously observed enhancement of the Pc phenotype, support the hypothesis that His2Av is a PcG gene (Swaminathan, 2005).

PcG gene products repress transcription of homeotic genes outside of their normal expression boundaries. If H2Av is a PcG protein, ectopic expression of homeotic genes in His2Av mutants can be expected. To test this possibility, the distribution of Antennapedia (Antp) protein was examined in flies homozygous for the His2Av⁸¹⁰ mutation. Antp localizes in the ventral ganglion of wild-type larvae in three bands of cells corresponding to the three thoracic segments. In the case of the His2Av⁸¹⁰ mutant, this pattern is altered and the Antp protein is present further posteriorly through the ventral ganglion. A second homeotic protein, Ultrabithorax (Ubx), is involved in the development of the third thoracic and first abdominal segments, and it is expressed posterior to the Antp expression in the ventral ganglion of wild-type larvae. This pattern is not disrupted in the His2Av⁸¹⁰ mutant; the band of Ubx expression appears to be similar in intensity and spatial distribution to that of wild-type larvae. These results suggest that H2Av might be required to maintain proper expression of homeotic genes in the anterior part of the animal, where Antp is expressed, but not in more posterior segments where Ubx expression occurs. The results also confirm the hypothesis suggesting that His2Av is a PcG gene (Swaminathan, 2005).

Recruitment of PcG complexes to silenced regions of the genome requires methylation of Lys 27 of histone H3 (Cao, 2002). To test whether H2Av replacement is required for Pc recruitment, the distribution of this protein in wild type versus His2Av mutants was compared. Pc localizes to ~100 sites on polytene chromosomes of wild-type-OR third instar larvae. In contrast, chromosomes from larvae homozygous for the His2Av⁸¹⁰ allele show a reduction in the number of Pc sites as well as in the amount of protein present at these sites. As a control, the Su(Hw) protein is present at similar levels in polytene chromosomes of wild-type and His2Av⁸¹⁰ flies. To test whether this decreased accumulation of Pc in polytene chromosomes is due to reduced synthesis of Pc protein or reduced recruitment of the protein to the chromosome, Western analyses of protein extracts obtained from wild-type and His2Av⁸¹⁰ mutant larvae were carried out. There is no significant difference in the levels of Pc protein between these two strains, suggesting that the observed effect is due to the inability of Pc to be recruited to the chromosomes in the absence of H2Av (Swaminathan, 2005).

Recent results suggest that H3 trimethylated at Lys 27 facilitates Pc binding to silenced regions and this modification is carried out by the Enhacer of zeste [E(z)] protein present in the ESC-E(z) complex. Since a reduction in Pc on polytene chromosomes was observed in His2Av mutants, whether recruitment of the ESC-E(z) complex is also impaired in these mutants was examined. In wild type, E(z) can be observed at multiple sites throughout the genome. The levels and localization of E(z) do not appear to be altered in the His2Av⁸¹⁰ mutant compared to wild type. Whether H3 Lys 27 methylation is affected by mutations in His2Av was examined. The levels and distribution of this modification appear to be the same in polytene chromosomes from wild-type and His2Av⁸¹⁰ mutant larvae. This result was confirmed by Western analysis, which shows equal levels of H3 trimethylated at Lys 27 in wild-type and His2Av⁸¹⁰ mutant larvae. These results suggest that H2Av is required upstream of Pc recruitment in the process of Pc-mediated silencing. Since neither recruitment of the E(z) complex nor H3 Lys 27 methylation seem to be affected in His2Av mutants, H2Av replacement might take place after H3 Lys 27 methylation and before Pc recruitment. Alternatively, Pc repression might require at least two parallel and independent pathways, one involving H2Av recruitment and a second one leading to H3 Lys 27 methylation, both of which might be required for proper Pc recruitment (Swaminathan, 2005).

Given the observed accumulation of H2Av in the centromeric heterochromatin (Leach, 2000), to test the possible involvement of H2Av in heterochromatic silencing it was determined whether mutations in the His2Av gene can act as modifiers of variegated phenotypes caused by the presence of a gene next to heterochromatin. The In(1)w^m4 allele is caused by an inversion that positions the white gene next to the centromeric heterochromatin of the X chromosome. This rearrangement results in the characteristic variegated phenotype. Mutations in the His2Av gene act as dominant suppressors of this phenotype, with flies of the genotype In(1)w^m4/In(1)w^m4; His2Av⁸¹⁰/+ showing a dramatic increase in eye pigmentation when compared to In(1)w^m4 alone. The presence of the H2Av histone variant in the centromeric heterochromatin and its requirement for the variegated phenotype of the In(1)w^m4 mutation suggest that H2Av plays an important role in the establishment and/or maintenance of heterochromatin (Swaminathan, 2005).

Formation of heterochromatin requires deacetylation of H3 Lys 9 followed by methylation of the same residue and recruitment of HP1. The heterochromatin of Drosophila chromosomes is enriched in dimethylated and trimethylated histone H3 in the Lys 9 residue. To analyze the possible role of H2Av in heterochromatin assembly, the localization was examined of H3 dimethylated at Lys 9 in polytene chromosomes from larvae carrying a mutation in the His2Av gene. Antibodies against histone H3 dimethylated in Lys 9 stain the pericentric heterochromatin in wild-type larvae. Interestingly, polytene chromosomes from His2Av⁸¹⁰ mutants show a decrease in the amount of methylated H3 Lys 9, whereas the presence of Su(Hw), used as a control, is the same in chromosomes from wild-type and His2Av⁸¹⁰ mutant larvae. Since modification of this residue is important for HP1 recruitment, whether localization of HP1 in heterochromatin is also affected by mutations in His2Av was examined. In wild-type larvae, HP1 localizes preferentially to the pericentric heterochromatin of the chromocenter, but accumulation of HP1 is dramatically reduced in the His2Av⁸¹⁰ mutant (Swaminathan, 2005).

To confirm these results, Western analyses of protein extracts obtained from wild-type and His2Av mutant larvae was carried out using antibodies against HP1 and histone H3 dimethylated in Lys 9. The results show little or no accumulation of histone H3 methylated in Lys 9, and lower levels of HP1 in the His2Av⁸¹⁰ mutant. Methylation of histone H3 at the Lys 9 residue is carried out by the Su(var)3-9 histone methyltransferase, and HP1 is encoded by the Su(var)2-5 gene. In order to ensure that the observed effects on the levels of HP1 or the methylation of H3 Lys 9 were not caused by alterations in transcription of Su(var)3-9 or Su(var)2-5 due to the His2Av mutation, quantitative RT-PCR analyses of RNA obtained from wild-type and His2Av⁸¹⁰ mutant third instar larvae were carried out . The results show that there are no significant changes in the levels of Su(var)3-9 or HP1 RNAs in His2Av⁸¹⁰ mutant larvae when compared to wild type. These results and those from immunocytochemistry analyses confirm a role for H2Av in the methylation of H3 Lys 9 and subsequent recruitment of HP1 (Swaminathan, 2005).

Based on the observed effects of His2Av mutations on H3 Lys 9 methylation and HP1 recruitment, it appears that the presence of H2Av in heterochromatin might be required prior to these two events. To confirm this hypothesis, the pattern of H2Av distribution on polytene chromosomes from larvae carrying mutations was examined in the Su(var)2-5 and Su(var)3-9 genes. In both cases, H2Av localization appears normal, suggesting that the presence of H2Av is required prior to H3 Lys 9 methylation and HP1 recruitment during the establishment of heterochromatin (Swaminathan, 2005).

An ectopic heterochromatin domain can be created by insertion into euchromatin of closely linked multiple copies of a P-element transposon containing the white gene. HP1 is recruited to this site, suggesting that ectopic heterochromatin formation by the transgene array follows the same pathway as normal constitutive heterochromatin. To test whether H2Av is also involved in ectopic heterochromatin formation or if its role is specific to centromeric heterochromatin, the presence of H2Av at the site of integration of transgene repeats was examined. In a strain carrying only one transgene insertion, the white gene present in the P transposon is expressed at normal levels, but in strains carrying an array of six closely linked transgenes, expression of the white gene shows a characteristic variegated phenotype. Mutations in His2Av suppress this variegated phenotype, showing a red pigmentation of the eye closer to that of wild-type flies. This result suggests a requirement for H2Av in the establishment of ectopic heterochromatin caused by transgene arrays (Swaminathan, 2005).

To further test this conclusion, it was determined whether H2Av is indeed present at the site of transgene insertion. For this, simultaneous fluorescence in situ hybridization (FISH) was performed using the white gene as a probe and immunolocalization was performed using antibodies against H2Av. The FISH signal marks the site of insertion of the transgene, which can then be compared to that of H2Av immunostaining. Analysis of polytene chromosomes from a fly strain carrying a single-copy transgene (strain 6-2) shows that the site of insertion is located in an interband, where the chromatin is decondensed. In this strain, H2Av is not present at the site of insertion, in agreement with the normal expression of the white gene observed in these flies. When the same experiment was performed with polytene chromosomes from a strain carrying an array of six transposons at the same chromosomal location (strain DX1), the site of insertion was found to be associated with a DAPI-staining band as well as H2Av. This finding confirms a role for H2Av in ectopic heterochromatin formation, and suggests that compaction of chromatin at an ectopic site as a consequence of the presence of a transgene array follows the same pathway as that used for the formation of centromeric heterochromatin (Swaminathan, 2005).

Histone H4 Lys 12 acetylation and heterochromatic silencing

The main covalent histone modification required for heterochromatin formation is the methylation of histone H3 at the Lys 9 residue. Based on the results described in this study, this process requires replacement of histone H2A for the H2Av variant. The presence of this variant might allow better access of Su(var)3-9 to the N-terminal tail of histone H3, but it is also possible that other steps not yet uncovered are required before modification of histone H3 can take place. Histone acetylation is usually thought to be involved in transcriptional activation, although there is also evidence for an involvement of this modification in silencing processes. In Drosophila, mutations in the chameau gene, a member of the MYST HAT family of histone acetyltransferases, dominantly suppress position effect variegation, and the Chameau protein is required for Pc-induced silencing (Grienenberger, 2002). In addition, histone H4 acetylated in Lys 12 has been found in pericentric heterochromatin in both Drosophila and plants. Therefore. whether H4 Lys 12 acetylation might have a role in heterochromatin formation was tested. H4 Lys 12 acetylation is enriched in the centromeric heterochromatin and in euchromatic DAPI-intense bands. This pattern of localization is disrupted by mutations in His2Av, with a significant reduction in the overall acetylation pattern and specifically in the heterochromatin region. This result suggests that acetylation of H4 Lys 12 might play a role also in the formation of heterochromatin at a step subsequent to H2Av deposition. The pattern of H4 Lys 12 acetylation appears normal in polytene chromosomes of larvae carrying mutations in the Su(var)3-9 and Su(var)2-5 genes, suggesting that this acetylation event takes place before H3 Lys 9 methylation. Western analysis performed with third instar larval extracts confirm the immunofluorescence results, indicating that there is reduced H4 Lys 12 acetylation in the His2Av⁸¹⁰ mutant, but the level remains unchanged in Su(var)3-9^evo/Su(var)3-9⁰⁶ and Su(var)2-5⁰⁴/Su(var)2-5⁰⁵ mutants (Swaminathan, 2005).

To confirm that acetylation of H4 Lys 12 is important for heterochromatin formation, whether this modification is also involved in the formation of ectopic heterochromatin by transgene arrays was tested. As seen for H2Av, chromosomes from a strain containing only one insertion of the transgene show no acetylation of H4 Lys 12 at the insertion site. Nevertheless, when six linked copies of the transgene are present at the same cytological location, a new band of H4 Lys 12 acetylation can be observed colocalizing with the new DAPI-positive band at the insertion site. Together, these results suggest that acetylation of H4 Lys 12 plays a key role in the formation of the heterochromatin domain at a step subsequent to H2Av replacement. This event might then help in the recruitment of an HDAC to deacetylate H3 Lys 9, which is then followed by methylation of this residue by Su(var)3-9 (Swaminathan, 2005).

The conserved histone variant H2A.Z fulfills many functions by being an integral part of the nucleosomes placed at specific regions of the genome. Telomeres cap natural ends of chromosomes to prevent their recognition as double-strand breaks. At yeast telomeres, H2A.Z prevents the spreading of silent chromatin into proximal euchromatin. A role for H2A.Z in capping, however, has not been reported in any organism. This study uncovered such a role for Drosophila H2A.Z. Loss of H2A.Z, through mutations in either its gene or the domino gene for the Swr1 chromatin-remodeling protein, suppressed the fusion of telomeres that lacked the protection of checkpoint proteins: ATM, ATR, and the Mre11-Rad50-NBS complex. Loss of H2A.Z partially restores the loading of the HOAP capping protein, possibly accounting for the partial restoration in capping. It is proposed that, in the absence of H2A.Z, checkpoint-defective telomeres adopt alternative structures, which are permissive for the loading of the capping machinery at Drosophila telomeres (Rong, 2008).

This study shows that loss of H2AvD in Drosophila suppresses fusion of telomeres that lack the protection of conserved checkpoint proteins: ATM, ATR, or MRN. Drosophila H2AvD encodes the functions for both H2A.X and H2A.Z variants that are translated from separate genes in other organisms. By using transgenes that either have or lack H2A.X function, it was established that H2AvD's role in regulating capping resides in its H2A.Z-homologous region. This conclusion is strengthened by the result from analyzing a domino mutation that behaved similarly to an h2AvD mutation. This represents a novel function of H2A.Z that has not been demonstrated in any other organism (Rong, 2008).

It is possible that the effect of h2AvD mutations on fusion frequencies is an indirect effect of transcriptional mis-regulation of genes controlling the repair and/or response to DSBs. This, however, is unlikely since cav mutant cells lacking the HOAP capping component are equally prone to telomere fusion with or without H2A.Z, suggesting that H2AvD mutant telomeres are not refractory to being repaired as DSBs. In addition, an h2AvD mutation was unable to suppress fusion in an atm cav h2AvD triple mutant, suggesting that cav is epistatic to h2AvD. In light of the observation that an h2AvD mutation can partially restore HOAP binding to atm atr double-mutant telomeres, it is suggested that loss of H2AvD might permit more efficient loading of capping proteins and, therefore, more efficient capping (Rong, 2008).

Another hypothesis considered is that H2AvD accumulates at checkpoint-defective telomeres, interfering with the binding of the capping machinery. However, evidence obtained from immuno-localization of H2AvD did not support this hypothesis. H2AvD has an interesting distribution on mitotic chromosomes in wild-type cells in that it is underrepresented in regions commonly considered heterochromatic. Telomeres are generally considered heterochromatic on the basis of their ability to silence nearby genes. However, recent results suggest that the heterochromatic features of Drosophila telomeres reside in the subtelomeric telomere-associated sequence (TAS) repeats and that the retro-transposon arrays at the extreme of chromosome ends possess certain euchromatic features (Biessmann, 2005). This is consistent with the fact that telomeric retro-transposons are actively transcribed to serve as transposition intermediates (Pardue, 2008). Therefore, H2AvD may not be excluded from wild-type telomeric regions, a suggestion supported by a recent genomewide localization study (Mavrich, 2008). Nevertheless, no elevated level of H2AvD was observed at checkpoint-defective telomeres even though these experiments were set up to favor detection of such enrichment. First, the atm atr double mutant - which has the strongest capping defects and on which the h2AvD mutation had the strongest suppressing effect - was included. Second, H2AvD enrichment would have been prominently detected on telomeres from the Y and fourth chromosomes as well as the short arm of the X chromosome on which H2AvD is normally underrepresented. Therefore, it is unlikely that H2AvD interferes with HOAP loading at checkpoint-defective telomeres and that the loss of such interference partly restores capping in h2AvD mutants (Rong, 2008).

Finally, the absence of H2A.Z might allow telomeres to adopt an alternative structure that is permissive to the loading of capping proteins. At S. cerevisiae telomeres, H2A.Z may demarcate the euchromatin-heterochromatin boundary. It may serve a similar function in Drosophila. Interestingly, recent results suggest that the heterochromatic features of Drosophila telomeres might reside in the subtelomeric TAS regions (Biessmann, 2005). It is possible that H2AvD prevents the spreading of TAS-associated heterochromatin into the transposon arrays. In the absence of H2AvD, Drosophila telomeres might adopt a heterochromatin-like structure, which facilitates the loading of capping proteins. This model is purely speculative due to the fact that the structure of Drosophila telomeres is poorly understood. In particular, the structural elements necessary for the loading of capping machinery remain undetermined. Nevertheless, due to the high degree of conservation in H2A.Z variants from different organisms, its role in regulating telomere capping uncovered in this study may also be conserved (Rong, 2008).

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