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 (see Tip60) 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).
According to the histone code hypothesis, histone variants and modified histones provide binding sites for proteins that change the chromatin state to either active or repressed. This study identified histone variants that regulate the targeting and enzymatic activity of poly(ADP-ribose) polymerase 1 (PARP1), a chromatin regulator in higher eukaryotes. PARP1 is targeted to chromatin by association with the histone H2A variant (H2Av)--the Drosophila homolog of the mammalian histone H2A variants H2Az/H2Ax--and subsequent phosphorylation of H2Av leads to PARP1 activation. This two-step mechanism of PARP1 activation controls transcription at specific loci in a signal-dependent manner. This study establishes the mechanism through which histone variants and changes in the histone modification code control chromatin-directed PARP1 activity and the transcriptional activation of target genes (Kotova, 2011).
The histone code hypothesis has long been accepted in the study of epigenetics, but there never has been a clear demonstration of the direct activation of an effector protein in response to changes in the histone environment. This study found that PARP1 activation and PARP1- mediated transcription depend on the regulation of a nucleosome's microenvironment, a mechanism that involves the phosphorylation of a histone variant (see Model of PARP1 protein regulation by histone H2Av variant and H2Av phosphorylation). This result supports the histone code hypothesis, and the underlying work also reveals a mechanism for PARP1 activation that is functionally important for the regulation of transcription, response to genotoxic stress, and silencing (Kotova, 2011).
Histone variant H2Av in Drosophila - and its homologs in Arabidopsis and Saccharomyces cerevisiae - localize in the promoter region of a subset of genes. These findings demonstrate that this localization is functionally significant. Specifically, H2Av is involved in the positioning and activation of the PARP1 protein. Nucleosomes containing H2Av form high-affinity sites at which the effector protein PARP1 binds with specific promoters. Thereafter, phosphorylation of H2Av alters the interaction of PARP1 with the nucleosomal histone H4, an event which, in turn, activates PARP1, leading to chromatin opening and facilitating transcription (Tulin, 2003; Petesch, 2008; Kim, 2004). Therefore, taken as a whole, the results of this study show that, by recruiting PARP1 protein, H2Av controls the chromatin state as well as transcription activation and genotoxic stress response (Kotova, 2011).
Having established that H2Av controls the PARP1 function both in vivo and in vitro, the causal underlying mechanism was examined. Nucleosomes containing H2Av have been reported previously to have a 'more open' and stable conformation, suggesting that the presence of H2Av may increase access to other core histones, i.e., those hidden in H2A-containing nucleosomes. Among the histones, PARP1 protein preferentially interacts with H3/H4 tetramers, possibly explaining the enrichment of PARP1 in the presence of H2Av-containing nucleosomes in vivo, as reported in this study. In other words, chromatin in 'more open; H2Av nucleosomes, with a high level of H3/H4 exposure, has a greater affinity for binding with PARP1 than does unexposed chromatin. Moreover, it was found that interaction with the N-terminal tail of the histone H4 triggers PARP1 protein activation. The SQ domain of histone H2Av, the phosphorylation of which controls PARP1 activation in vivo, as reported in this study, is positioned in close proximity to the N-tail of H4 in the nucleosome. Therefore, it is proposed that the histone-replacement machinery positions H2Av within the promoter region of specific genes, thereby creating nucleosomes with an 'open' configuration. Within these nucleosomes, exposed H3/H4 histones bind PARP1 protein and properly determine its localization in promoters. Phosphorylation of the H2Av C terminus then leads to exposure of the H4 histone N-tail, promoting its interaction with PARP1, and the activation of the PARP1 protein (Kotova, 2011).
Although these results establish a direct connection between PARP1, H2Av-containing nucleosomes, H2Av phosphorylation, and pADPr, a possible role for H2Av phosphorylation itself cannot be excluded, in regulating the activity of the PARP1 protein on a higher level of chromatin organization. This possibility is suggested by the observed difference between the in vivo and in vitro results. Although phosphorylation of H2Av was required to elicit PARP1 activation in vivo, purified nucleosomes containing either H2A or H2Av were able to elicit PARP1 activation in vitro. Mimicking the phosphorylation of H2Av (as in H2AvSE) resulted in a 26% increase in the activation of PARP1 in vitro. These observations suggest that although phospho-H2Av may act directly on PARP1, it also may mediate changes in the higher-order chromatin microenvironment (which could not be reproduced in vitro), leading to the disruption of PARP1 interaction with inhibitors and/or the interaction of PARP1 with activating epitopes in the context of the local chromatin. Alternatively, H2Av phosphorylation may be involved in a 'system restart' in vivo; i.e., phosphorylation of H2Av has been linked to the replacement of this histone in the local chromatin. Consequently, multiple repeated acts of transcriptional initiation, and therefore multiple acts of H2Av phosphorylation, may be required, for example, during heat-shock gene expression. Thus, in the absence of H2Av phosphorylation, H2Av replacement will be blocked, and transcriptional restart will be arrested (Kotova, 2011).
H2Av (H2Az/H2Ax) may have other roles in the nucleus in addition to the regulation of PARP1. For instance, although the yeast genome does not encode any obvious PARP1 homolog, yeasts have H2Az (HTZ1) and H2Ax homologs (14). Moreover, both yeast histones play essential roles. Histone H2Ax phosphorylation is involved in genotoxic stress response, but HTZ1 regulates chromatin remodeling, transcription, and transcriptional silencing in heterochromatin. Although PARP1 is a target that performs a critical role in higher-order chromatin, which otherwise cannot be accomplished by yeast, these observations suggest that the function of H2Av may not be restricted to PARP1 activation (Kotova, 2011).
Because PARP1 activation has been shown in this work to be mediated through H2Av phosphorylation, it was further asked what signaling pathway and kinases might be responsible for such H2Av phosphorylation. During genotoxic stress response, cell-cycle checkpoint kinases such as ataxia telangiectasia-mutated/ataxia telangiectasia and Rad- related (ATM/ATR) and DNA-PK kinase, are shown to phosphorylate the C-terminal tail of H2Ax. Although the possible roles of the Drosophila homolog of these enzymes in chromatin regulation and transcription cannot be excluded, kinases such as Jil-1 kinase, which functions inside the puffs of polytene chromosomes, seem to be more promising candidates for performing this function. Therefore, one of the future directions for investigating the mechanism of PARP1 regulation in chromatin is to identify the kinase enzyme responsible for triggering H2Av-mediated PARP1 activation (Kotova, 2011).
The current paradigm for the role of the PARP1 protein has two parts. The first part assigns to PARP1 the role of DNA repair and genotoxic stress response, and the second part assigns to PARP1 functional roles in the regulation of chromatin structure and transcription. In demonstrating that phosphorylation of H2Av (the H2Az/H2Ax homolog) controls the activity of the PARP1 protein in both pathways, a more universal mechanism has been established for PARP1 regulation. These findings also support the notion that PARP1 is not simply a component of either the DNA-repair or transcriptional complexes but instead is a universal regulator of high-order chromatin, which in eukaryotes needs management during both DNA repair and transcription. The activation of the PARP1 protein by histone H2Av phosphorylation ultimately leads to the loosening of compacted chromatin and opens access for either the DNA repair machinery or the transcriptional apparatus (Kotova, 2011).
H2Av is a versatile histone variant that plays both positive and negative roles in transcription, DNA repair, and chromatin structure in Drosophila. H2Av, and its broader homolog H2A.Z, tend to be enriched toward 5' ends of genes, and exist in both euchromatin and heterochromatin. Its organization around euchromatin genes and other features have been described in many eukaryotic model organisms. However, less is known about H2Av nucleosome organization in heterochromatin. This study reports the properties and organization of individual H2Av nucleosomes around genes and transposable elements located in Drosophila heterochromatic regions. The similarity and differences with that found in euchromatic regions are compared. This analyses suggests that nucleosomes are intrinsically positioned on inverted repeats of DNA transposable elements such as those related to the '1360' element, but are not intrinsically positioned on retrotransposon-related elements (Zhang, 2011).
It is concluded that H2Av nucleosomes are distributed throughout euchromatic and heterochromatic regions. The current findings indicate that the basic organization of the H2Av nucleosome is indistinguishable in these two regions. Moreover, H2Av nucleosomes generally adopt essentially the same positions relative to specific classes of genomic features (e.g., genes and transposons) in both types of environments, indicating that such features may dictate the positioning of resident nucleosomes. Retrotransposons appear to be more of an exception where positions relative to their start or end points are not intrinsic to the elements. Perhaps the local chromatin environment may influence the position of nucleosomes on these elements. Strikingly, whether it be genes, transposons, or replication origins, H2Av (and H2A.Z) nucleosomes seem to mark their boundaries, perhaps facilitating access of the relevant regulatory machinery (Zhang, 2011).
Nucleosome occupancy results in complex sequence variation rate heterogeneity by either increasing mutation rate or inhibiting DNA repair in yeast, fish, and human. H2A.Z nucleosome is extensively involved in gene transcription activation and regulation. To test whether H2A.Z nucleosome has the similar impact on sequence variability in the Drosophila genome, the H2A.Z nucleosome occupancy and sequence variation rate at gene ends and splicing sites was profiled. Consistent with previous studies, H2A.Z nucleosome positioning helps to demarcate the borders of exons. Nucleosome occupancy is anticorrelated with sequence divergence rate in the regions flanking transcription start sites and splicing sites. However, there is no rate heterogeneity between the linker DNA and H2A.Z nucleosomal DNA regardless of nucleosome occupancy, fuzziness, positioning in promoter, coding, and intergenic regions, young or old genes. But the rate at intergenic nucleosomes and the flanking linker regions is higher than that at the genic counterparts. Further analyses found that the high sequence divergence rate in the promoter regions that are usually nucleosome depleted regions may be likely resulted from the high mutation rate in the enriched tandem repeats. Interestingly, within nucleosomes spanning splicing sites, sequence variability of nucleosomal DNA significantly increases from the end within exons to the other end protruding into introns. The relaxed functional constraint in introns contributes to the high rate of nucleosomal DNA residing in introns while the strict functional constraint in exons maintains the low rate of nucleosomal DNA residing in exons. Taken together, H2A.Z nucleosome occupancy has no effect on sequence variability of Drosophila genome, which is likely determined by local sequence composition and the concomitant selection pressure (Tang, 2013).
Ataxia telangiectasia-mutated (ATM) and ataxia telangiectasia-related (ATR) kinases are conserved regulators of cellular responses to double strand breaks (DSBs). During meiosis, however, the functions of these kinases in DSB repair and the deoxyribonucleic acid (DNA) damage checkpoint are unclear. This paper shows that ATM and ATR have unique roles in the repair of meiotic DSBs in Drosophila. ATR mutant analysis indicated that it is required for checkpoint activity, whereas ATM may not be. Both kinases phosphorylate H2AV (γ-H2AV), and, using this as a reporter for ATM/ATR activity, it was found that the DSB repair response is surprisingly dynamic at the site of DNA damage. γ-H2AV is continuously exchanged, requiring new phosphorylation at the break site until repair is completed. However, most surprising is that the number of γ-H2AV foci is dramatically increased in the absence of ATM, but not ATR, suggesting that the number of DSBs is increased. Thus, it is concluded that ATM is primarily required for the meiotic DSB repair response, which includes functions in DNA damage repair and negative feedback control over the level of programmed DSBs during meiosis (Joyce, 2011).
ATR-dependent checkpoint activity in response to unrepaired DSBs causes oocyte development to proceed abnormally. A previous study noted that tefu mutants produced embryos with dorsal-ventral polarity defects, a possible indicator of elevated DSB repair checkpoint activity. Another reporter for this effect is Gurken (GRK), a TGF-α-related protein required for establishing dorsal-ventral polarity. When DSBs are not repaired, GRK localization is abnormal (Joyce, 2011).
At the restrictive temperature (25°C), tefu8 mutants are recessive lethal. To examine whether the meiotic DSB repair checkpoint was active in tefu8 mutants, homozygous females were raised at the permissive temperature (18°), shifted to the restrictive temperature, and whether there was a disruption of GRK localization was examined. GRK is normally concentrated in the cytoplasm of control oocytes. In 87% of similarly staged tefu8 mutant ovarioles, GRK expression was absent or much weaker than normal and mislocalized. Another characteristic feature of oocyte development is the assembly of the karyosome, in which the chromatin is condensed into a single round mass within the cell nucleus of stage 4 oocytes. In control oocytes, the karyosome appeared compact and spherical. However, in 80% of the tefu8 mutant oocytes, the karyosome appeared abnormally flattened or fragmented. Abnormal GRK localization and karyosome organization are ATR-dependent phenotypes that are typical of mutants unable to repair DSBs. ATM is required for the completion of meiotic recombination but is dispensable for the DSB repair checkpoint (Joyce, 2011).
MEI-W68 is the Drosophila homologue of Spo11, a conserved endonuclease that catalyzes meiotic DSB induction in eukaryotes. The GRK localization and karyosome morphology defects were suppressed in mei-W864572;tefu8 double mutants, indicating that the defects are a result of unrepaired meiotic DSBs. A double mutant genotype combination was tested with mei-41, the Drosophila homologue of ATR. The GRK mislocalization and karyosome defects in tefu8 mutants were suppressed in mei-41D3;tefu8 double mutants. These results show that loss of ATM function leads to activation of the ATR-dependent checkpoint response to unrepaired meiotic DSBs (Joyce, 2011).
Drosophila H2A variant, like mammalian H2AX, that is phosphorylated at the sites of DNA breaks. Antibodies to this phosphorylated protein (γ-H2AV) detect distinctive foci in the nucleus. To assay for DSB repair defects in tefu8 mutants, γ-H2AV staining was examined and compared with wild-type and mutants known to have DSB repair defects. Pachytene oocytes are arranged in order of developmental age within the germarium, which is divided into three regions. In wild-type females, a mean of 6.2 γ-H2AV foci was found in region 2a pachytene oocytes and was absent in region 3 oocytes. This is consistent with previous results suggesting that meiotic DSBs in wild-type oocytes are induced in region 2a and repaired before region 3 (Joyce, 2011).
Mutations in DSB repair genes such as spn-A (which encodes the Drosophila Rad51 homologue) exhibit an accumulation of γ-H2AV foci that persist throughout meiotic prophase, corresponding to unrepaired meiotic DSBs. A mean of 22.8 γ-H2AV foci was present in spn-A1 region 3 oocytes, which is similar to previous estimates for the total number of DSBs per nucleus. Similarly, γ-H2AV foci accumulated in region 3 oocytes of mei-41D3 mutants, indicating that ATR is required to repair meiotic DSBs in addition to its role in checkpoint activation. In tefu8 mutant germaria at the restrictive temperature, γ-H2AV staining persisted into region 3 oocytes, consistent with a DSB repair defect. However, in contrast to other repair mutants and wild type, the γ-H2AV staining in tefu8 mutants exhibited more robust and continuous labeling, colocalizing with most of the chromosomes rather than appearing as foci. All γ-H2AV staining was eliminated in mei-W864572;tefu8 double mutants, indicating that the abundant γ-H2AV staining in the tefu8 mutant is dependent on the induction of meiotic DSBs (Joyce, 2011).
The threadlike γ-H2AV labeling observed in tefu8 mutant oocytes could be a result of either unrestricted spreading of H2AV phosphorylation from the DSB sites or an increase in the number of programmed DSBs relative to wild type. These possibilities were investigated by examining the nurse cells in the germarium. Each pro-oocyte has 14 neighboring nurse cells that experience on average twofold less DSBs than the oocyte. At the restrictive temperature, tefu8 mutants exhibited distinct γ-H2AV foci in nurse cells, indicating that ATM-deficient cells can restrict their DSB response to the DSB sites, and the foci could be counted. The tefu8 mutant nurse cells had a mean of 9.3 γ-H2AV foci, which is >2.5 times greater than the 3.6 γ-H2AV foci per nurse cell nurse in wild typ. To estimate the total number of DSBs that occur in tefu8 mutant oocytes, a method was used that quantitatively measures the intensity of γ-H2AV fluorescence. In short, the intensity of a single γ-H2AV focus in adjacent nurse cells was compared with that of total fluorescence in oocytes. Based on this method, 25.2 γ-H2AV foci was found in spn-A region 3 oocytes, similar to the levels when counted manually. In tefu8 mutants, ~39.1 γ-H2AV foci (P = 0.0152) was estimated, a significant increase over spn-A that is consistent with the increase in γ-H2AV foci levels observed in nurse cells. Together, these results reveal a novel role for ATM in negatively regulating DSB formation during meiotic prophase (Joyce, 2011).
ATM and ATR have been implicated in the phosphorylation of H2AX at sites of chromosomal DSBs in somatic cells of mouse and humans. To investigate whether Drosophila ATM and ATR serve redundant roles in H2AV phosphorylation in response to meiotic DSBs, mei-41D3;tefu8 double mutant germaria were examined. At a permissive temperature (18°), mei-41D3;tefu8 displayed a γ-H2AV staining pattern similar in severity to mei-41D3 single mutants with a mean of 18.2 foci in region 3 oocytes. When shifted to the restrictive temperature (25°) for 24 h, no γ-H2AV staining was observed in the mei-41D3;tefu8 region 2a cysts, indicating that these mutants lost the ability to phosphorylate H2AV near newly generated DSBs. This is the first demonstration that ATM and ATR are redundant for the phosphorylation of H2AV in response to meiotic DSBs and is consistent with a study in somatic cells of other organisms (Joyce, 2011).
The absence of γ-H2AV staining from mei-41D3;tefu8 double mutant region 2a oocytes indicated that there was no phosphorylation in response to a DSB. However, γ-H2AV was also absent from older region 3 oocytes, indicating that γ-H2AV was lost from DSB sites after only 24 h at the restrictive temperature. That is, based on previous estimates for the timing of cyst progression (12-24 h per region), the region 3 oocytes were in region 2b (after DSB formation) at permissive temperature and would have had γ-H2AV staining before the shift to restrictive temperature. The loss of γ-H2AV staining upon shift to restrictive temperature indicates that there is a rapid turnover of the phosphorylation mark near meiotic DSBs. To confirm that the histone H2AV and DSBs were still present in region 3 nuclei, the mei-41D3;tefu8 double mutants were transferred from the restrictive temperature back to the permissive temperature and γ-H2AV staining was analyzed. After only 24 h at the permissive temperature, γ-H2AV staining returned to the double mutant oocytes, consistent with the presence of unrepaired DSBs and H2AV in region 3 oocytes. These findings indicate that γ-H2AV at meiotic DSB sites is continuously exchanged or dephosphorylated independent of repair and that rephosphorylation of H2AV is maintained by continuous ATM or ATR activity (Joyce, 2011).
The aforementioned results suggest that a component of the DSB repair response involves dynamic changes in chromatin structure, which may be important to maintain ATM/ATR activity until the DSB is repaired. To investigate the mechanism behind the repair-independent constitutive exchange of γ-H2AV, factors known to regulate H2AV exchange in other cell types were examined. In particular, the exchange of γ-H2AV with unphosphorylated H2AV in somatic cells is preceded by the acetylation of the histone by the Tip60 multiprotein complex. Whether the Tip60 complex component MRG15 is required for γ-H2AV exchange was determined by creating MRG15 mutant germline clones and analyzing H2AV levels throughout oogenesis. Strikingly, a complete absence of H2AV, both phosphorylated and unphosphorylated, was observed in MRG15j6A3 mutant cells throughout oogenesis. Mutant germline clones are generated in the premeiotic stem cells; therefore, these results indicate that MRG15 is required for the incorporation of H2AV into meiotic chromatin. With this function, MRG15 could also be required for a process that promotes γ-H2AV turnover during meiotic prophase by incorporating unphosphorylated H2AV into the nucleosomes after γ-H2AV has been removed (Joyce, 2011).
>In addition to the acetyltransferase Tip60, MRG15 has been found in another complex that includes the deacetylase Rpd3. Germline clones were made of Rpd304556, and it was found that, rather than loss of H2AV, there was abundant γ-H2AV foci and evidence of a repair defect. These results suggest that the Rpd3 complex is not required for H2AV exchange in the germline. Although the Tip60 complex is a strong candidate for this role, confirmation awaits the analysis of additional Tip60 complex components or the construction of Tip60 mutants (Joyce, 2011).
This evidence indicates that γ-H2AV is surprisingly dynamic, being constantly exchanged in a DSB-independent manner. A previous observation was confirmed and extended that in mutants with a defect in DSB repair, such as spn-A1, mei-41D3, and tefu8, γ-H2AV labeling persists until stage 5 and yet is never observed in more advanced stages of oogenesis. It was reasoned that this absence of γ-H2AV staining past stage 5 may reflect either a reduction in ATM/ATR activity, use of an alternative repair pathway, or loss of the H2AV substrate from the nucleosomes (Joyce, 2011).
To evaluate the presence of histone H2AV in nucleosomes during oogenesis, ovaries were stained with an H2AV antibody that recognizes both phosphorylated and unphosphorylated versions of the histone variant. As expected, H2AV labeling was abundant throughout the nucleus of all oocytes and nurse cells as well as mitotically dividing follicle cells from the germarium to stage 3 of oogenesis. Strikingly, at stage 4-5 of oogenesis, H2AV staining was drastically reduced in nurse cells and oocytes but not in follicle cells. This correlates well with the disappearance of γ-H2AV foci in both the oocyte and nurse cells at this stage in repair mutants. Indeed, the absence of H2AV at stage 5 was also found in spn-A1, mei-41D3, and tefu8 mutant ovarioles. Therefore, the loss of γ-H2AV signal at stage 5 of oogenesis is a result of the removal of H2AV. Similar results were observed with an H2AV:GFP fusion protein in oocytes, although the signal persisted longer in the nurse cells. These results have important implications for using γ-H2AV as a DSB reporter late in prophase, as it is impossible to determine whether ATM/ATR responds to DNA damage or whether that damage is repaired before the first meiotic division (Joyce, 2011).
This study has shown that the Drosophila ATM and ATR kinases have distinct roles in meiotic DSB repair, results that are consistent with the role of ATM in the mouse germline. Unlike ATR, however, ATM is dispensable for the meiotic DSB repair checkpoint, although it cannot be ruled out a minor role for ATM in the checkpoint because mei-41 mutants fail to completely suppress the effects of some DSB repair mutants. Interestingly, in Drosophila somatic cells, ATM is required for a checkpoint response only at low doses of radiation. Thus, the amount of damage may be high enough in meiotic cells such that ATR signaling is sufficient for the checkpoint response. An alternative is that the number of breaks is not as significant as how they are processed. DSBs experience rapid resection in meiosis to generate single-stranded DNA, which is necessary for ATR activation (Joyce, 2011).
ATM and ATR kinases clearly have common targets, such as the phosphorylation of H2AV. Using γ-H2AV as a reporter, a surprising dynamic component to this phosphorylation was found including at least two phases of H2AV clearance in the Drosophila female germline. First, γ-H2AV at meiotic DSB sites is rapidly exchanged with unphosphorylated H2AV. Because γ-H2AV is exchanged with H2AV independent of DSB repair, the removal of γ-H2AV from DSB sites after repair may only require the cessation of ATM and ATR activity. Second, most of the H2AV is removed between stages 5 and 6 of oogenesis (after pachytene) and occurs independently of the repair and phosphorylation state (Joyce, 2011).
The most surprising result of this study is that ATM negatively regulates meiotic DSB formation. Induction of DSBs is essential to generate crossovers. Approximately 20 DSBs occur per meiosis in Drosophila, but only six or seven become crossovers. Similarly, in yeast and mice, a surplus of DSBs is generated to produce crossovers. What remains unknown are the mechanisms that limit the number of DSBs to prevent excessive genomic damage. It is suggested that ATM is part of a negative feedback mechanism to limit the total number of DSBs. This mechanism of DSB regulation appears to be conserved, as DSB levels are also increased in mouse spermatocytes lacking ATM, which may explain circumstances in which crossovers are increased in the absence of ATM (Joyce, 2011).
Retrotransposon sequences are positioned throughout the genome of almost every eukaryote that has been sequenced. As mobilization of these elements can have detrimental effects on the transcriptional regulation and stability of an organism's genome, most organisms have evolved mechanisms to repress their movement. This study has identified a novel role for the Drosophila melanogaster Condensin II subunit, dCAP-D3 in preventing the mobilization of retrotransposons located in somatic cell euchromatin. dCAP-D3 regulates transcription of euchromatic gene clusters which contain or are proximal to retrotransposon sequence. ChIP experiments demonstrate that dCAP-D3 binds to these loci and is important for maintaining a repressed chromatin structure within the boundaries of the retrotransposon and for repressing retrotransposon transcription. dCAP-D3 prevents accumulation of double stranded DNA breaks within retrotransposon sequence, and decreased dCAP-D3 levels leads to a precise loss of retrotransposon sequence at some dCAP-D3 regulated gene clusters and a gain of sequence elsewhere in the genome. Homologous chromosomes exhibit high levels of pairing in Drosophila somatic cells, and FISH analyses demonstrate that retrotransposon-containing euchromatic loci are regions which are actually less paired than euchromatic regions devoid of retrotransposon sequences. Decreased dCAP-D3 expression increases pairing of homologous retrotransposon-containing loci in tissue culture cells. It is proposed that the combined effects of dCAP-D3 deficiency on double strand break levels, chromatin structure, transcription and pairing at retrotransposon-containing loci may lead to (1) higher levels of homologous recombination between repeats flanking retrotransposons in dCAP-D3 deficient cells and (2) increased retrotransposition. These findings identify a novel role for the anti-pairing activities of dCAP-D3/Condensin II and uncover a new way in which dCAP-D3/Condensin II influences local chromatin structure to help maintain genome stability (Schuster, 2013).
This study shows that decreased levels of dCAP-D3/Condensin II lead to retrotransposon mobilization within specific gene clusters shown to be transcriptionally regulated by dCAP-D3. In tissue culture cells, the results demonstrate that homologous retrotransposon containing clusters remain largely unpaired which is in striking contrast to homologous euchromatic loci that do not contain retrotransposon sequences. Interestingly, the mobilization events detected both in vivo and in vitro resulted in either the retention of a single LTR at the locus or a precise loss of retrotransposon sequence in one locus and a small increase in copy number elsewhere in the genome. A model puts forth the hypothesis that dCAP-D3/Condensin II mediated looping of chromatin at homologous, euchromatic, retrotransposon containing loci holds the regions at distances great enough to prevent recombination. In dCAP-D3 deficient cells, this rigid chromatin structure is not maintained, possibly leading to increased double strand breaks within retrotransposon sequence. This in turn would cause an opening of chromatin in the region and would give homologous retrotransposon containing loci more of an opportunity to pair. Repair mechanisms that would lead to a local loss of retrotransposon sequence at one of the loci and a gain of a copy elsewhere in the genome include repair by the single strand annealing pathway or unequal crossover events between the small repeats found before and after the retrotransposon sequence. While these types of recombination repair do explain the local loss of sequence, they do not explain the small increase in copy number seen in dCAP-D3 deficient cells. Therefore, it is also proposed that, as a result of the opening of the chromatin at these loci, transcription increases and allows retrotransposon encoded retrotransposase enzyme to be made and generate additional copies. These new retrotransposition events would allow both original copies to remain in their loci and new copies to be generated and insert elsewhere. Supporting evidence for a role of Condensin II in regulating homologous crossover events comes from a recent study in C. elegans that worms heterozygous for Condensin II subunits exhibit increases in double strand breaks, increases in crossover events, and increases in X chromosome axis length in meiotic tissue. The differential placement and number of double strand breaks in the C. elegans Condensin mutants were hypothesized to be caused by the changes in axial chromatin structure since axis lengths did not change in response to varying numbers of double strand breaks between mutants. Loss of Drosophila Condensin II subunits also lead to axial expansion. Interestingly, the mdg1-1403 retrotransposon locus appears expanded in the dCAP-D3 mutants, and it is possible that this local expansion and change in chromatin structure could be the cause of the repositioning of double stand breaks. Finally, the loss of Condensin II expression results in disorganization of chromosome territories and intermingling of chromosomes in Drosophila cells (Hartl, 2008a). Therefore, it is also possible that the frequency of recombination between retrotransposon sequences on different chromosomes could increase, leading to loss of the remaining retrotransposon copy on one of the homologs in cells deficient for dCAP-D3 (Schuster, 2013).
The minor, but significant increases in retrotransposon transcript levels in somatic tissues and cells expressing lower levels of dCAP-D3 suggest that dCAP-D3 regulates global retrotransposon transcript levels. Previous studies have shown that dCAP-D3 regulates transcription of many genes in Drosophila larvae and adults, but the mechanism remains unclear. Experiments in SG4 cells show that dCAP-D3 binds close to the junction between retrotransposon and neighboring DNA sequence. They also demonstrate that dCAP-D3 is necessary for maintaining basal transcription levels of retrotransposon-containing gene clusters prior to local loss of retrotransposon sequence. If dCAP-D3 acts to set up boundaries between a retrotransposon and neighboring DNA sequence, then binding sites located within the neighboring sequence could confer local specificity. In support of this, the data show an increased spreading of repressive H3K9me3 marks into the area surrounding retrotransposon mdg1-1403 in dCAP-D3 dsRNA treated cells. This data is also consistent with earlier findings that dCAP-D3 is a suppressor of Position Effect Variegation in somatic tissues (Longworth, 2008). Alternatively, the temporary increase in H3K9me3 at the locus prior to loss of retrotransposon sequence could be due to the increase in homolog pairing in dCAP-D3 knock down cells; silencing of extrachromosomal copies of genes proximal to transposons has been shown to increase when these regions pair. Transcription of genes surrounding mdg1-1403 increases above basal levels in dCAP-D3 dsRNA treated cells once the retrotransposon sequence is lost. Interestingly, even when dCAP-D3 expression levels return to normal, the increased transcription and increased levels of active H3K4me3 marks at the locus remain. It is also interesting to note that the band recognized by the mdg1-1403 probe in the dCAP-D3 mutant polytene chromatin squashes appeared longitudinally thicker and less condensed. This supports the model and suggests that the presence of the retrotransposon within the locus elicits a dCAP-D3-dependent structural configuration that is lost when the retrotransposon sequence is lost (Schuster, 2013).
Results presented in this study show that dCAP-D3 prevents increased γH2AX localization in retrotransposon sequence. Interestingly, human Brd4 isoform B was recently reported to bind to SMC2 and CAP-D3 proteins, and SMC2 was shown to be necessary for Brd4's ability to maintain a more condensed chromatin structure and inhibit DNA damage signaling following gamma irradiation. This suggests 1) that the functions of Condensin II in DNA damage repair may be conserved in human cells, and 2) that Condensin II's role in repair most likely requires its ability to maintain rigid chromosome structure and organization. Recently, a role for Condensins in organizing retrotransposons within the nucleus was reported in yeast. Retrotransposons cluster in yeast and it was demonstrated that the Non-Homologous End Joining (NHEJ) repair associated Ku proteins as well as Condensin were both necessary for the observed clustering. The reported association between DNA repair proteins and Condensin is intriguing and might suggest, if the interaction was conserved in flies, that Condensins play a role in the actual repair of double strand breaks at retrotransposon sequences. However, no mass clustering is seen of the mdg1-1403 retrotransposon in Drosophila cells and the current studies show in Drosophila that Condensin-associated mechanisms exist to prevent retrotransposons on homologous chromosomes from coming into close contact. Furthermore, sequencing results indicate that either single strand annealing or unequal crossover events have occurred in dCAP-D3 mutants, instead of NHEJ mediated repair. These discrepancies might be attributed to the high degree of homologous chromosome pairing throughout the cell cycle in Drosophila. In fact, single strand annealing (even over NHEJ) has been shown to be the dominant double strand break repair pathway at transposon containing loci in Drosophila when direct repeats flank a double strand break. Additionally, yeast only possess Condensin I and not Condensin II, so it is possible that Condensin II has diverged to have different functions or even to antagonize Condensin I function at retrotransposon sequences (Schuster, 2013).
Interestingly, ChIP for phosphorylated H2AX in human cells expressing SMC2 RNAi showed that double strand breaks occur frequently within LTR sequences and a type of non-LTR retrotransposon, SINES. Therefore, the ability of Condensin II to prevent double strand break accumulation and recombination within retrotransposon sequence may not be unique to Drosophila Condensin II. This has important implications for Condensin II as a possible tumor suppressor in human cells. Various types of tumor cells have been found to harbor mutations in Condensin II proteins including CAP-D3 (COSMIC database). While somatic homolog pairing is not as prevalent in human cells as in Drosophila, certain instances of abnormal pairing have been implicated in the generation of tumors. Further studies will be necessary to elucidate whether uncontrolled retrotransposon recombination and/or retrotransposition might play a role in the generation of genomic instability in human cells deficient for or expressing mutant Condensin II proteins (Schuster, 2013).
SWR1-type nucleosome remodeling factors replace histone H2A by variants to endow chromatin locally with specialized functionality. In Drosophila melanogaster a single H2A variant, H2A.V, combines functions of mammalian H2A.Z and H2A.X in transcription regulation and the DNA damage response. A major role in H2A.V incorporation for the only SWR1-like enzyme in flies, Domino, is assumed but not well documented in vivo. It is also unclear whether the two alternatively spliced isoforms, DOM-A and DOM-B, have redundant or specialized functions. Loss of both DOM isoforms compromises oogenesis, causing female sterility. This study systematically explored roles of the two DOM isoforms during oogenesis using a cell type-specific knockdown approach. Despite their ubiquitous expression, DOM-A and DOM-B have non-redundant functions in germline and soma for egg formation. It was shown that chromatin incorporation of H2A.V in germline and somatic cells depends on DOM-B, whereas global incorporation in endoreplicating germline nurse cells appears to be independent of DOM. By contrast, DOM-A promotes the removal of H2A.V from stage 5 nurse cells. Remarkably, therefore, the two DOM isoforms have distinct functions in cell type-specific development and H2A.V exchange (Börner, 2016).
In D. melanogaster the properties of the two ancient, ubiquitous histone H2A variants H2A.X and H2A.Z are combined in a single molecule, H2A.V. Given that H2A.V carries out functions as a DNA damage sensor and architectural element of active promoters, as well as having further roles in heterochromatin formation, this histone appears loaded with regulatory potential. Accordingly, placement of the variant, either randomly along with canonical H2A during replication or more specifically through nucleosome remodeling factors, becomes a crucial determinant in its function. Mechanistic detail for replacement of H2A-H2B dimers with variants comes from the analysis of the yeast SWR1 complex, which incorporates H2A.Z in a stepwise manner at strategic positions next to promoters (Börner, 2016).
So far, the published phenotypes associated with dom mutant alleles have not been systematically complemented. The comprehensive complementation analysis of this study shows that dom mutant phenotypes are indeed due to defects in the dom gene. Remarkably, dom lethality and sterility can be partially rescued by complementation with the orthologous human SRCAP gene, providing an impressive example of functional conservation of SWR1-like remodelers. The contributions of the two splice variants DOM-A and DOM-B had not been assessed. This study now demonstrates that both isoforms are essential for development, suggesting non-redundant functions. The DOM-A isoform contains a SANT domain followed by several poly-Q stretches, which are widely found in transcriptional regulators, where they may modulate protein interactions. By contrast, SANT domains are thought to function as histone tail interaction modules that couple binding to enzyme catalysis. Therefore, the SANT domain in DOM-A could mediate specificity towards H2A.V eviction depending on particular functional contexts. These features are also present in the C-terminus of p400 (EP400), the second human SWR1 ortholog, but are absent in either DOM-B or SRCAP. Remarkably, p400 interacts directly with TIP60 and the SANT domain of p400 inhibits TIP60 catalytic activity providing an interesting lead for further investigation of DOM isoforms and TIP60 interactions (Börner, 2016).
It is speculated that distinct functions of p400 and SRCAP in humans might be accommodated to some extent by the two DOM isoforms in flies. Accordingly, it will be interesting to explore whether the two isoforms reside in distinct complexes. Previous affinity purification of a TIP60-containing complex using a tagged pontin subunit apparently only identified DOM-A, but not DOM-B. Following up on the initial observation of early defects in GSCs and cyst differentiation upon loss of DOM (Yan, 2014), this study now finds that this phenotype is exclusively caused by loss of DOM-A. Interestingly, studies with human embryonic stem cells show that p400/TIP60 (KAT5) integrates pluripotency signals to regulate gene expression, suggesting similar roles for DOM-A in GSCs. This is in contrast to requirements for both isoforms for germline development outside of the germarium, highlighting a developmental specialization of the two DOM remodelers (Börner, 2016).
DOM is also involved in the differentiation and function of SSCs in the germarium. The data now document non-redundant requirements of both DOM isoforms in somatic cells for proper coordination of follicle cell proliferation with cyst differentiation. Failure to adjust these two processes leads to 16-cell cyst packaging defects that manifest as compound egg chambers. These rare phenotypes had previously only been described upon perturbation of some signaling pathways, such as Notch, or Polycomb regulation (Börner, 2016).
Because the phenotypes of DOM depletions resemble those of H2A.V depletion, the idea was favored that many of the cell-specification defects are due to compromised H2A.V incorporation, depriving key promoters of the H2A.Z-related architectural function. Alternatively, scaffolding activities might partially explain some roles of chromatin remodelers, as suggested for SRCAP. So far, knowledge of the mechanisms of H2A.V incorporation has been anecdotal. This comprehensive analysis revealed a specific involvement of DOM-B for the incorporation of H2A.V into chromatin at the global level. The N-termini of SWR1 and DOM-B harbor the HSA and ATPase spacer domains, with interaction surfaces for further complex subunits, and an additional H2A.Z-H2B dimer binding site. Given the requirement for both isoforms for cell specification during oogenesis, it is speculated that DOM-B might serve to incorporate bulk H2A.V into chromatin similar to SWR1, whereas DOM-A would be more involved in the regulatory refinement of location (Börner, 2016).
Although the failures in cell specification and egg morphogenesis are likely to be explained by loss of the H2A.Z-related features of H2A.V, ablation of DOM might also compromise the DNA damage response, which involves phosphorylation of H2A.V (γH2A.V). Conceivably, the role of γH2A.V as a DNA damage sensor might be best fulfilled by a broad distribution of H2A.V throughout the chromatin. Such an untargeted incorporation may be achieved by stochastic, chaperone-mediated incorporation during replication or by an untargeted activity of DOM-B. DOM-independent incorporation in endoreplicating polyploid nurse cells of stage 3 egg chambers is observed, where global H2A.V and γH2A.V signals did not depend on DOM. Immunofluorescence microscopy may lack the sensitivity to detect DOM-dependent incorporation of H2A.V at some specific sites. Nevertheless, DOM-independent incorporation of H2A.V might serve to cope with many naturally occurring DNA double-strand breaks during the massive endoreplication of nurse cells (Börner, 2016).
There is some evidence that nucleosome remodelers not only incorporate H2A variants but can also remove them. In yeast, the genome-wide distribution of H2A.Z appears to be established by the antagonistic functions of the SWR1 and Ino80 remodeling complexes, where Ino80 replaces stray H2A.Z-H2B with canonical H2A-H2B dimers. A recent study identified the vertebrate-specific histone chaperone ANP32E as part of a TIP60/p400 complex that facilitates the eviction of H2A.Z-H2B dimers from chromatin. Remarkably, in D. melanogaster a TIP60/DOM-A complex is involved in a similar reaction. The TIP60/DOM-A complex acetylates γH2A.V at lysine 5 to facilitate exchange of γH2A.V by unmodified H2A.V during the DNA damage response. Furthermore, it has been speculated that H2A.V and γH2A.V could be actively removed from nurse cells, since corresponding signals are absent from stage 5 onwards. This study now demonstrates that depletion of DOM-A and TIP60 leads to the persistence of H2A.V and γH2A.V in nurse cells of late egg chambers, clearly documenting the ability of the remodeler to remove bulk H2A.V and variants modified during DNA damage induction (Börner, 2016).
These findings highlight the specific requirements of DOM splice variants for the incorporation and removal of H2A.V during D. melanogaster oogenesis. It remains an interesting and challenging question how DOM-A and DOM-B complexes are targeted genome-wide and function in vivo to establish specific H2A.V patterns in different cell types during development (Börner, 2016).
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).
Following irradiation (IR), the DNA damage response (DDR) activates p53, which triggers death of cells in which repair cannot be completed. Lost tissue is then replaced and re-patterned through regeneration. The role of p53 in co-regulation of the DDR and tissue regeneration was examined following IR damage in Drosophila. After IR, p53 was found to be required for imaginal disc cells to repair DNA, and in its absence the damage marker, γ-H2AX is persistently expressed. p53 is also required for the compensatory proliferation and re-patterning of the damaged discs, and the results indicate that cell death is not required to trigger these processes. An IR-induced delay in developmental patterning in wing discs was identified that accompanies an animal-wide delay of the juvenile-adult transition; both of these delays require p53. In p53 mutants, the lack of developmental delays and of damage resolution leads to anueploidy and tissue defects, and ultimately to morphological abnormalities and adult inviability. It is proposed that p53 maintains plasticity of imaginal discs by co-regulating the maintenance of genome integrity and disc regeneration, and coordinating these processes with the physiology of the animal. These findings place p53 in a role as master coordinator of DNA and tissue repair following IR (Wells, 2011).
The results show that tissue regeneration subsequent to the DDR also requires p53. They add to previous work indicating that a continuum of events follows IR that culminates in regeneration of damaged tissue and survival of the animal. The process initiates with a stereotypical DDR in damaged imaginal disk cells within minutes of IR: damage is sensed and H2AX is phosphorylated, caspases are activated, and cell division in the disk transiently arrests. After approximately 5 h disk cells re-enter the cell cycle and continue to divide at apparently normal rates. Repair of DNA damage leads to loss of γ-H2AX, while ongoing apoptosis eliminates unrepaired cells. The results indicate that the high level of cell death significantly slows the net growth of wing disks, compelling continuous cell division. This is facilitated by a delay of pupariation; in parallel, the expression of late patterning genes is delayed in wing disks. Interestingly, it was found that after 40 Gy of IR, tissue damage was severe enough to require disk cell proliferation to continue not only during the extension of the larval developmental timer, but also into the pupal stage. Thus in contrast to what has been generally believed, disk regeneration is not restricted to the larval “growth phase” of development, but can continue in the early stages of pupal development. The ability of disks to continue regenerative growth after the hormonal cues that stimulate pupariation suggests that disk cell proliferation is only loosely regulated at the juvenile-adult transition (Wells, 2011).
In p53 mutants the events following IR are initially identical to wild-type, but subsequently show several differences. Cells lacking p53 recognize DNA damage, H2AX is phosphorylated, and the cell cycle checkpoint transiently arrests p53 mutant disk cells. p53 mutant cells also reenter the cell cycle with the same kinetics as controls. However, γ-H2AX persists at high levels in mutant disks, indicating that DNA damage is lingering, but the cells are unable to undergo apoptosis. Moreover, the mutant larvae do not significantly delay development, suggesting that p53 is required to regulate the developmental timer. Despite these differences, it was found that disk cells divide at a normal rate and thus the size of the wing disk initially increases after IR. Later, the persistence of damaged DNA coupled with cell division creates aneuploidy, which may contribute to a late wave of apoptosis that continues late into the pupal stage (Wells, 2011).
Cell division continues beyond the normal cessation time in both wild-type and p53 mutants. In wild-type these additional pupal cell divisions are productive. In contrast, the late pupal divisions of p53 mutant disk cells appear to be largely futile since cell death is still prevalent; surviving cells appear to be aneuploid. Perhaps as a result, wing morphogenesis is delayed in irradiated p53 mutants relative to controls, although the mechanisms that pack wing disk cells and re-shape the wing disk ultimately do occur. The lack of DNA repair impairs cell differentiation and/or function throughout the pupa and leads to defects that prevent most animals from eclosing. This is interesting in light of the finding that the persistent DNA damage in p53 mutants did not appear to interfere with cell division during the larval phase of regeneration (Wells, 2011).
p53 functions cell autonomously during disk regeneration, and conditional expression of p53 in the wing disk is sufficient to induce ectopic expression of Wg, compensatory tissue growth, and a systemic developmental delay, all common aspects of regeneration. These results suggest that p53 is activated and operates cell-autonomously in damaged cells to promote regeneration. However, p53 also regulates the larval developmental clock, with the result that it coordinates control of disk regeneration with the physiology of the whole animal. Collectively, the results indicate that p53 functions to ensure repair of damaged DNA, to regulate the developmental timing of the animal, and to coordinate disk and animal maturation via a patterning checkpoint that delays cell fate acquisition in the disk. This linkage provides a mechanism that coordinates the two processes in time and thus facilitates the survival of the animal after DNA and tissue damage (Wells, 2011).
In the absence of p53, DNA damage remains unrepaired, rendering cells incapable of completing the differentiation process. This is exacerbated by the absence of apoptosis immediately after IR in the p53 mutants, allowing cells with DNA damage to persist. Later, some of the persisting damaged cells in the mutants are eliminated by a late surge of disk cell death that continues into the pupal stage. However, although this rids the disk of many damaged cells, it is not induced within a time frame that allows replacement of lost tissue, leading to small pupal wing disk size and small adult wings. Shortly after the onset of the late wave of apoptosis in p53 mutant disks the larval–pupal transition is crossed and metamorphosis is initiated. Although p53 mutant pupal wing disk cells continue to proliferate long after their wildtype counterparts have exited the cell cycle, the results suggest that the juvenile-to-adult transition – the commitment to produce adult traits – prevents critical developmental and patterning cues or render cells incapable of responding to them. However, damaged, aneuploid cells can differentiate trichomes, and this study observed that some damaged cells did carry out aspects of trichome differentiation, including prehair formation. In addition, it is possible that most of the severely damaged cells in p53 mutants were ultimately eliminated (Wells, 2011).
The results are strikingly similar to observations made in mouse and human cells. Loss of the murine DNA damage checkpoint protein Hus1 in a p53-deficient background results in accumulation of damaged cells after IR and prevents the compensatory responses in mammary epithelium. In serial transplantation experiments, self-renewal of irradiated human hematopoietic stem cells (HSCs) is compromised when they are deficient for p53, and, like experiments with wing disk cells, γ-H2AX persisted in the HSCs. Collectively the data indicate that p53's role in Drosophila disk regeneration is analogous to its role in tissue remodeling and stem cell renewal in vertebrates, and suggest that these functions of p53 are conserved (Wells, 2011).
The argument can be made that Drosophila imaginal disks merely take advantage of and extend developmental programs to repair and re-pattern lost tissue. This requires that the appropriate hormonal milieu be maintained by prolonging the juvenile, larval stage. Animals were irradiated late during larval development but still within the disk growth period, and it was found that p53 function is required for the delay of the developmental timer that controls the juvenile-adult transition. Likewise, delay of the timer after RH-damage requires p53. There is a strong correlation between delay of the timer and continued proliferation of disks. Although this relationship remains mysterious it is generally thought that negative feedback from proliferating disks inhibits a neural or humoral target. In contrast to imaginal disks, the polyploid larval cells are relatively insensitive to IR. No induction of p53 activity was detected after either RH damage or IR in tissues known to play key roles in developmental timing, such as the fat body, Dilp-2 expressing neurons, the prothoracic gland, and the corpora allata. The p53 activity reporter contains 2 consensus p53 binding sites and is thus expected to report accurately in many tissues. Although more trivial possibilities cannot be excluded, the absence of induction in these tissues suggests that p53 function in imaginal disks is sufficient for the developmental delay induced upon IR as well as for the disk-autonomous responses. Thus, the data support the view that imaginal disks “signal” to the developmental clock to delay pupariation, and indicates that the putative signal requires p53 for its production (Wells, 2011).
Two independent lines of evidence argue that, in contrast to previous reports, cell proliferation occurs at the same rate during regeneration as it does under normal developmental conditions. First, it was found that the number and distribution of mitotic cells is similar in yw and in p53 mutant disks following IR at every examination from the cell cycle reentry at + 6 h until pupariation, despite the significant differences in the length of this period between the two genotypes. Second, RH-damage in clonal experiments showed that undamaged cells in the vicinity of RH-damaged cells proliferate at the same rate as cells in control disks without damage. These results agree with others, in which an IR dose-dependent lengthening of the developmental timer was observed that correlated with an increase in clone size in adult wings; it was concluded that the remaining cells “undergo additional divisions to compensate for this loss”. As a whole the data indicate that cell divisions occur at the normal rate, with additional divisions that occur during a p53-dependent slowing of the larval timer (Wells, 2011).
In addition, the results indicate that some aspects of disk patterning are delayed while the disk regenerates. This delay is also p53-dependent. One interpretation of these results is that the early, p53-dependent cell death program, by eliminating massive numbers of cells, directly delays ongoing the patterning process. However, the finding that dronc mutant animals, which are unable to induce cell death, exhibit the same regeneration responses as wild-type after IR argues against this idea. An alternative possibility arises from the observation that the disk patterning delay and the animal-wide delay are correlated in time, and thus could be inter-dependent. A third possibility is that p53 induces a disk-wide developmental checkpoint, directly dependent upon its role in the DDR but independent of the disk-produced “signal” that delays the larval timer, which couples regenerative growth to stage-appropriate cell fate specification. Further experiments are required to distinguish between these alternatives. Regardless of the mechanism, however, the finding that cell division proceeds at a similar rate during the delay of patterning regardless of p53 status implies that cell division and late patterning gene expression are independently regulated under these conditions (Wells, 2011).
It has been hypothesized that dying cells emit information that stimulates proliferation of surviving cells to regenerate the damaged tissue. The regulation of expression of pro-apoptotic genes and pathways such as Rpr, Hid, Eiger/TNF and JNK by p53, is consistent with the idea that p53 induces apoptosis, which in turn stimulates regeneration. However, the results indicate that this is not the case: the regeneration response is induced after IR even in cells rendered incapable of inducing apoptosis because of a null mutation in dronc. Since these dronc mutant cells remain wildtype for p53, these data support an apoptosis-independent role of p53 in provoking regeneration. Indeed, p53 is required for the tissue repair response even when RH genes are expressed. Thus, at a minimum, the data indicate that expression of pro-death genes and caspase activation are not sufficient to trigger regeneration (Wells, 2011).
Since regeneration does not occur in its absence, p53 appears to be upstream of the signal that triggers regeneration. Three scenarios are suggested for the regeneration trigger downstream of p53 activation. First, it was find that p53 functions cell-autonomously to promote the ectopic induction of Wg, an early event in regeneration induced by a variety of methods in numerous animals. Moreover, expression of p53 under conditional Gal4 control induces ectopic Wg in the wing disk. Thus, the re-organization of disk patterning of the damaged tissue by ectopic expression of Wg, which necessitates a developmental delay for the completion of patterning and growth, may serve to trigger regeneration. However, in contrast to previous models invoking Wg as a mitogen, the current findings indicate that cell proliferation continues at its developmentally programmed pace during this extended period of time (Wells, 2011).
Since JNK activation after tissue damage is p53-dependent, a second candidate for the regeneration trigger is the JNK signaling pathway. JNK is activated early after tissue damage and is important for wound healing. JNK signaling is also activated upon disruptions of Wg and Dpp in the wing disk, and can itself lead to activation of Wg and Dpp expression. JNK activity appears to be upstream of Wg expression, since hep null mutations, which eliminate JNK activity, prevent ectopic expression of Wg after RH damage. A third possibility is that regeneration is triggered via a distinct program of gene expression directed by p53, which is independent of JNK or Wg (Wells, 2011).
Overall, this work suggests that p53 acts as a master regulator of tissue plasticity through its roles in the DDR, in tissue repair, and in coordinating these events with the animal's physiology. In addition to its role in the initiation of regeneration, these results argue that p53 is responsible for regulating the expression of a signal(s) from disks that prolongs larval development to allow regeneration after either RH or IR damage. Studies that identify this signal, that determine from which tissue it arises, and that delineate the mechanism by which p53 controls each aspect of the regeneration process are important goals for the future (Wells, 2011).
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, ash1VF101 trxb11/++ and brm2 trxE2/++. Flies of the genotype ash1VF101 trxb11/++ 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 ash1VF101 trxb11+/++ His2Av05146 flies and to 29% in ash1VF101 trxb11+/++ His2Av810 flies. Similarly, flies of the genotype brm2, trxE2/++ show a 43% frequency of haltere to wing or third leg to second leg transformations, and this frequency is reduced to 22% in brm2 trxE2+/++ His2Av05146 flies and to 21% in brm2 trxE2+/++ 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)wm4 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)wm4/In(1)wm4; His2Av810/+ showing a dramatic increase in eye pigmentation when compared to In(1)wm4 alone. The presence of the H2Av histone variant in the centromeric heterochromatin and its requirement for the variegated phenotype of the In(1)wm4 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)3-9evo/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).
Many tissues are sustained by adult stem cells, which replace lost cells by differentiation and maintain their own population through self-renewal. The mechanisms through which adult stem cells maintain their identity are thus important for tissue homeostasis and repair throughout life. This study shows that a histone variant, His2Av, is required cell autonomously for maintenance of germline and cyst stem cells in the Drosophila testis. The ATP-dependent chromatin-remodeling factor Domino is also required in this tissue for adult stem cell maintenance possibly by regulating the incorporation of His2Av into chromatin. Interestingly, although expression of His2Av was ubiquitous, its function is dispensable for germline and cyst cell differentiation, suggesting a specific role for this non-canonical histone in maintaining the stem cell state in these lineages (Morillo Prado, 2003).
The results reveal that the histone variant His2Av is required cell autonomously for maintenance of two different adult stem cell types, GSCs and cyst stem cells (CySCs), in the Drosophila male gonad, but not for the differentiation of the progeny in these two stem cell lineages. The specific requirement for His2Av for adult stem cell maintenance suggests that His2Av may play critical role(s) in the mechanisms that maintain the ability of adult stem cells to self-renew rather than differentiate. His2Av function has been implicated in both transcriptional repression and transcriptional activation. His2Av could maintain adult stem cells by either favoring repression of pro-differentiation genes and/or activation of genes necessary for stem cell identity and function. In yeast, H2A.Z occupies transcriptionally inactive genes and intergenic regions, while in Drosophila, His2Av is required for the establishment of heterochromatin and transcriptional repression. Conversely, studies indicate that in Drosophila, yeast, and chicken, His2Av is enriched at nucleosomes downstream of the transcription start site of active or poised genes. Nucleosomes and histone dimers containing H2A.Z appear to be less stable than nucleosomes containing the canonical histone H2A. This lower stability may favor a more open chromatin, giving transcriptional activators or repressors better access to the DNA. Consistent with this model, a recent study showed that H2A.Z promotes self-renewal and pluripotency of murine embryonic stem cells (ESCs) by facilitating the binding of Oct4 to its target genes and the Polycomb repressive complex 2 to differentiation genes (Hu, 2013). However, in ESCs, unlike in Drosophila male GSCs and CySCs, His2A.Z function was also required for the expression of differentiation genes when ESCs were grown under conditions that induce differentiation (Morillo Prado, 2003).
It is proposed that the requirement of His2Av for adult stem cell maintenance, but not for differentiation, may reflect a subtle role for His2Av in maintaining expression of genes required for self-renewal versus differentiation. Adult stem cells lie at the cusp of two alternate fate choices, self-renewal and differentiation; the progeny of stem cell division are maintained in a state where they can execute either self-renewal or differentiation programs depending on local cues. The requirement for this balanced, bi-potential state may make adult stem cells more sensitive to the small alterations in the relative levels of key transcripts associated with the loss of His2Av function, tilting the balance from stem cell maintenance to onset of differentiation. Consistent with the model that His2Av may alter transcriptional levels subtly, H2A.Z was shown to be required to fine-tune the transcriptional state of hsp70 and a wide variety of other genes in response to temperature changes in Arabidopsis (Morillo Prado, 2003).
The ATP-dependent chromatin-remodeling factor Domino is required for GSC and CySC maintenance in the male germline, as previously shown for somatic follicle stem cells in the female gonad. The yeast Swr1 complex containing the homolog of Drosophila Domino exchanges His2A with Htz1 (the yeast His2A variant) and in Drosophila, Domino- containing Tip60 chromatin remodeling complex has been shown to exchange phospho-His2Av with unmodified His2Av in in vitro assays. The current studies indicate that Domino function is required in vivo in GSCs for the incorporation of His2Av into chromatin. Nuclei of domino mutant GSCs had lowered but still detectable levels of His2Av protein, possibly due to the weak domino allele used in this study. Alternatively, incorporation of His2Av in some regions of the chromatin may occur independently of Domino function, as has been reported in yeast, in which stress-responsive genes exhibit Swr1-independent incorporation of Htz in the coding region. Although ISWI, like His2Av, is required for GSC and CySC maintenance in the male germline, these proteins may function in parallel pathways to maintain adult stem cells in the testis. The ISWI containing nucleosome-remodeling factor (NURF) was shown to maintain GSCs and CySCs in the Drosophila testis by positively regulating the JAK-STAT signaling pathway; GSCs mutant for components of the NURF complex exhibited low levels of STAT92E protein. In contrast, as discussed below, His2Av may function independently of the JAK-STAT signaling pathway (Morillo Prado, 2003).
The results indicate that His2Av may function independently of the JAK-STAT signaling pathway to provide a chromatin environment that allows for stem cell maintenance. Expression of the His2Av and STAT92E proteins in GSCs was not dependent on each other. These studies indicate that His2Av may not be required for expression of at least one other key STAT-dependent gene in CySCs. Activation of the JAK-STAT signaling pathway in response to the Upd signal from the hub is important for CySC maintenance, possibly in part through STAT-dependent transcription of Zfh-1. However, CySCs lacking His2Av function still expressed Zfh-1. In GSCs, activation of the JAK-STAT pathway is important for maintaining hub-GSC adhesion and for centrosome orientation, both of which appeared unaffected in His2Av mutant GSCs. Loss of His2Av function did not strongly suppress the phenotype associated with ectopic overexpression of Upd in the testis, although a few His2Av mutant germ cells were able to initiate differentiation, possibly due to relatively lower levels of JAK-STAT activation in these cells. Even though loss of His2Av normally resulted in differentiation of GSCs and CySCs, the requirement for His2Av function can be overridden by high levels of activation of the JAK-STAT pathway, possibly maintaining somatic CySCs in a stem cell like state, which may fail to provide a microenvironment for germ cells to initiate differentiation (Morillo Prado, 2003).
Unlike other organisms that have evolved distinct H2A variants for different functions, Drosophila melanogaster has just one variant which is capable of filling many roles. This protein, H2A.V, combines the features of the conserved variants H2A.Z and H2A.X in transcriptional control/heterochromatin assembly and DNA damage response, respectively. This study shows that mutations in the gene encoding H2A.V affect chromatin compaction and perturb chromosome segregation in Drosophila mitotic cells. A microtubule (MT) regrowth assay after cold exposure revealed that loss of H2A.V impaired the formation of kinetochore-driven (k) fibers, which could account for defects in chromosome segregation. All defects were rescued by a transgene encoding H2A.V that lacked the H2A.x function in the DNA damage response, suggesting that the H2A.Z (but not H2A.X) functionality of H2A.V was required for chromosome segregation. Loss of H2A.V weakened HP1 localization, specifically at the pericentric heterochromatin of metaphase chromosomes. Interestingly, loss of HP1 yielded not only telomeric fusions but also mitotic defects similar to those seen in H2A.V null mutants, suggesting a role for HP1 in chromosome segregation. H2A.V precipitated HP1 from larval brain extracts indicating that both proteins were part of the same complex. Moreover, the overexpression of HP1 rescued chromosome missegregation and defects in the kinetochore-driven k-fiber regrowth of H2A.V mutants indicating that both phenotypes were influenced by unbalanced levels of HP1. Collectively, these results suggest that H2A.V and HP1 work in concert to ensure kinetochore-driven MT growth (Verní, 2015).
This study provides compelling evidence that H2A.V, the Drosophila histone H2A variant, plays an important and unanticipated role during Drosophila mitosis. The cytological characterization of H2A.V810 mutant larval brain chromosomes revealed that loss of H2A.V has an impact on chromosome organization and cell proliferation, which is consistent with previous results on a role of this histone variant in chromatin remodeling and heterochromatin organization. This study also demonstrates that a significant proportion of H2A.V mutant cells fails to complete mitosis and contains chromosomes that remain scattered across the spindle (pseudo anaphase or PA) due to failed metaphase plate alignment. Similar effects have been previously described in Drosophila S2 cells depleted by RNAi of either kinetochore proteins, augmin components or splicing factors. However unlike those S2 interfered cells, which exhibit PAs with long spindles, H2A.V810 mutant cells have PA (premature- or pseudo-anaphase) spindles that appear similar to wild type anaphases. The reason why H2A.V mutant cells are not elongated is unclear, but it may depend on the different cellular systems employed in the different studies. Intriguingly, the presence of PAs in H2A.V810 mutants indicates for the first time that Drosophila H2A.V is also necessary for chromosome segregation growth (Verní, 2015).
Interestingly, the results from both Dgt6 immunolocalization and spindle microtubule re-growth assay following cold-induced MT depolymerization in mitotic neuroblasts reveal that H2A.V might be involved in the organization of kinetochore-driven, k-fibers microtubule bundles that attach sister kinetochores to spindle poles. However, it is believed that defects in the organization of k-fibers are not a consequence of the reduction of Dgt6. Recent studies demonstrated that Wac, a newly discovered component of Augmin complex, is required for spindle formation in S2 cells but is dispensable for somatic mitosis. In fact, a wac deletion mutant was viable and displayed only weak defects in brain cell divisions, suggesting that the components of Augmin complex (including Dgt6) might have non essential roles in spindle assembly growth (Verní, 2015).
It has been previously reported that defective k-fiber formation and elongation disrupt chromosome segregation and spindle formation in Drosophila cells. The results, which are in line with this finding, indicate that a specific chromatin organization is also necessary to ensure a proper spindle assembly. It is speculated that the observed PAs are a result of improper organization of k-fibers, and that PAs fail to complete mitosis, thus reducing in part the frequency of anaphases in H2A.V810 mutants. It is also plausible that persistent chromosome misalignment leads to a mitotic arrest of these cells, which in turn could explain the presence of H2A.V810 mutant cells with overcondensed chromosomes. However, while in a previous study, the presence of PAs was always associated to a strong increase of mitotic index (MI), the current mutants the MI did not change. One explanation is that the reduction of anaphase frequency in H2A.V810 mutants (20%) is not as dramatic as that reported for Dgt6-depleted S2 cells (50%) and therefore it unlikely affects mitotic progression. An alternative explanation is that loss of H2A.V might affect the regulation of G2-M and/or M-A cell cycle checkpoints thus preventing a metaphase arrest. Further investigations are required to verify this hypothesis. It is worth noting that, although a role for H2A.Z in chromosome segregation has been previously documented in human and yeast cells, the current data provide the first evidence of an potential involvement of H2A.Z in the organization of k-fibers growth (Verní, 2015).
This study also provides unanticipated molecular evidence that H2A.V interacts directly or indirectly with HP1, confirming that both proteins are part of same complex. It is intriguing that the H2A.V-HP1 interaction depends on the HP1 CD domain, which also binds H3K9me2/3 and mediates heterochromatin formation. This supports the existence of a cascade of events that requires the recruitment of H2A.V and different histone modifications for the establishment of heterochromatin. Yet, the reason why depletion of H2A.V causes a direct loss of HP1 and particularly during mitosis is unclear. Nonetheless, as HP1 overexpression in H2A.V mutant cells prevents PA formation, it is speculated that a H2A.V-dependent stabilization/localization of HP1 at centromeric region is essential to ensure proper chromosomal behavior growth (Verní, 2015).
Previous studies have shown that H2A.Z alters the nucleosomal surface, thus enabling preferential binding of HP1a to condensed higher chromatin structures 44RIDcit0044. It is conceivable then that the H2A.V-HP1 interaction is favored by the condensation of pericentric chromatin fiber in metaphase. Alternatively, these interactions may be encouraged by metaphase-specific posttranslational modifications of H2A.V, HP1 or other interacting proteins. Indeed, it has been proposed the mechanism underpinning HP1 recruitment on mitotic chromosomes might be different from that in interphase. Still, little is known about the factors required for specific localization of HP1 at mitotic centromeres save for a few discoveries. Human HP1α binding to INCENP, for instance, has been demonstrated as necessary for HP1α targeting to mitotic centromeres. It is believed that H2A.V may play a similar role in mediating HP1 binding, but how this takes place remains to be seen growth (Verní, 2015).
This functional characterization of H2A.V has also unveiled the role of Drosophila HP1a in the assembly of the mitotic spindle. The results indicate that the loss of HP1 yields defects in the kinetochore-driven k-fiber organization, which can in turn compromise chromosome segregation thus generating PAs. Past studies have shown that HP1a contributes to chromosome segregation and centromere stability in a variety of organisms including mammals, but the mechanism is still not completely understood. HP1 is known to interact with components of the centromere and the kinetochore complex, providing targets to begin understanding how downregulation or mislocalization of HP1 result in mitotic defects. It has also been reported that in contrast to Swi6 in S. pombe, the correct localization of HP1 is not required for the recruitment of cohesins to centromeric regions in mammals. Yet, HP1a seems to help in protecting cohesins from degradation by recruiting the Shugoshin protein. This study has highlighted an additional function of HP1 during chromosome segregation, one which depends on interaction with H2A.V and is required to regulate k-fiber organization. These results thus provide further evidence of a functional versatility of HP1 that is likely conserved also in mammals growth (Verní, 2015).
Abbott, D. W., Ivanova, V. S., Wang, X., Bonner, W. M. and Ausio, J. (2001). Characterization of the stability and folding of H2A.Z chromatin particles: Implications for transcriptional activation. J. Biol. Chem. 276: 41945-41949. 11551971
Biessmann, H., et al. (2005). Two distinct domains in Drosophila melanogaster telomeres. Genetics 171: 1767-1777. PubMed Citation: 16143601
Börner, K. and Becker, P.B. (2016). Splice variants of the SWR1-type nucleosome remodeling factor Domino have distinct functions during Drosophila melanogaster oogenesis. Development 143: 3154-3167. PubMed ID: 27578180
Chan, H. M., Narita, M., Lowe, S. W. and Livingston, D. M. (2005). The p400 E1A-associated protein is a novel component of the p53 --> p21 senescence pathway. Genes Dev. 19: 196-201. PubMed citation: 15655109
Chen, P., Zhao, J., Wang, Y., Wang, M., Long, H., Liang, D., Huang, L., Wen, Z., Li, W., Li, X., Feng, H., Zhao, H., Zhu, P., Li, M., Wang, Q. F. and Li, G. (2013). H3.3 actively marks enhancers and primes gene transcription via opening higher-ordered chromatin. Genes Dev 27: 2109-2124. PubMed ID: 24065740
Clarkson, M. J., Wells, J. R., Gibson, F., Saint, R. and Tremethick, D. J. (1999). Regions of variant histone His2AvD required for Drosophila development. Nature 399: 694-697. 10385122
Dhillon, N. and Kamakaka, R. T. (2000). A histone variant, Htz1p and a Sir1p-like protein, Esc2p, mediate silencing at HMR. Mol. Cell 6: 769-780. 11090616
Faast, R., et al. (2001). Histone variant H2A.Z is required for early mammalian development. Curr. Biol. 11(15): 1183-7. 11516949
Fan, J. Y., Gordon, F., Luger, K., Hansen, J. C. and Tremethick, D. J. (2002). The essential histone variant H2A.Z regulates the equilibrium between different chromatin conformational states. Nat. Struct. Biol. 9: 172-176. 11850638
Fan, J. Y., Rangasamy, D., Luger, K., and Tremethick, D. J. (2004). H2A.Z alters the nucleosome surface to promote HP1alpha-mediated chromatin fiber folding. Mol. Cell. 16(4): 655-61. 15546624
Farris, S. D., Rubio, E. D., Moon, J. J., Gombert, W. M., Nelson, B. H. and Krumm, A. (2005). Transcription-induced chromatin remodeling at the c-myc gene involves the local exchange of histone H2A.Z. J Biol Chem. 280(26): 25298-303 15878876
Gévry, N., et al. (2007). p21 transcription is regulated by differential localization of histone H2A.Z. Genes Dev. 21(15): 1869-81. Medline abstract: 17671089
Gévry, N., et al. (2009). Histone H2A.Z is essential for estrogen receptor signaling. Genes Dev. 23(13): 1522-33. PubMed Citation: 19515975
Grienenberger, A., Miotto, B., Sagnier, T., Cavalli, G., Schramke, V., Geli, V., Mariol, M.C., Berenger, H., Graba, Y. and Pradel, J. (2002). The MYST domain acetyltransferase Chameau functions in epigenetic mechanisms of transcriptional repression. Curr. Biol. 12: 762-766. 12007422
Grigorian, M., DeBruhl, H. and Lipsick, J. S. (2017). The role of variant histone H2AV in D. melanogaster larval hematopoiesis. Development 144(8):1441-1449. PubMed ID: 28242611
Guillemette, B., Bataille, A.R., Gévry, N., Adam, M., Blanchette, M., Robert, F. and Gaudreau, L. (2005). Variant histone H2A.Z is globally localized to the promoters of inactive yeast genes and regulates nucleosome positioning. PLoS Biol. 3: e384. Medline abstract: 16248679
Hartley, P. D. and Madhani, H. D. (2009). Mechanisms that specify promoter nucleosome location and identity. Cell 137(3): 445-58. PubMed Citation: 19410542
Hu, G., Cui, K., Northrup, D., Liu, C., Wang, C., Tang, Q., Ge, K., Levens, D., Crane-Robinson, C. and Zhao, K. (2013). H2A.Z facilitates access of active and repressive complexes to chromatin in embryonic stem cell self-renewal and differentiation. Cell Stem Cell 12: 180-192. PubMed ID: 23260488
Jin, J., Cai, Y., Florens, L., Swanson, S. K., Kusch, T., Li, B., Workman, J. L., Washburn, M. P., Conaway, R. C. and Conaway, J. W. (2005). The mammalian YL1 protein is a shared subunit of the TRRAP/TIP60 histone acetyltransferase and SRCAP complexes. J. Biol. Chem. 280: 13665-13670. Medline abstract: 16230350
Joyce, E. F., et al. (2011). Drosophila ATM and ATR have distinct activities in the regulation of meiotic DNA damage and repair. J. Cell Biol. 195(3): 359-67. PubMed Citation: 22024169
Kim, M. Y., et al. (2004). NAD+-dependent modulation of chromatin structure and transcription by nucleosome binding properties of PARP-1. Cell 119: 803-814. PubMed Citation: 15607977
Kobor, M. S., Venkatasubrahmanyam, S., Meneghini, M. D., Gin, J. W., Jennings, J. L., Link, A. J., Madhani, H. D. and Rine, J. (2004). A protein complex containing the conserved Swi2/Snf2-related ATPase Swr1p deposits histone variant H2A.Z into euchromatin. PLoS Biol. 2: E131. 15045029
Kotova, E., et al. (2011). Drosophila histone H2A variant (H2Av) controls poly(ADP-ribose) polymerase 1 (PARP1) activation in chromatin. Proc. Natl. Acad. Sci. 108(15): 6205-10. PubMed Citation: 21444826
Krogan, N. J., Keogh, M. C., Datta, N., Sawa, C., Ryan, O. W., Ding, H., Haw, R. A., Pootoolal, J., Tong, A., Canadien, V., et al. (2003). A Snf2 family ATPase complex required for recruitment of the histone H2A variant Htz1. Mol. Cell 12: 1565-1576. 14690608
Kusch, T., et al. (2004). Acetylation by Tip60 is required for selective histone variant exchange at DNA lesions. Science 306(5704): 2084-7. Medline abstract: 15528408
Larochelle, M. and Gaudreau, L. (2003). H2A.Z has a function reminiscent of an activator required for preferential binding to intergenic DNA. EMBO J. 22: 4512-4522. 12941702
Law, C. and Cheung, P. (2015). Expression of non-acetylatable H2A.Z in myoblast cells blocks myoblast differentiation through disruption of MyoD expression. J Biol Chem. 290(21): 13234-49. PubMed ID: 25839232
Leach, T. J., Mazzeo, M., Chotkowski, H. L., Madigan, J. P., Wotring, M. G. and Glaser, R. L. (2000). Histone H2A.Z is widely but nonrandomly distributed in chromosomes of Drosophila melanogaster. J. Biol. Chem. 275: 23267-23272. 10801889
Li, B., Pattenden, S. G., Lee, D., Gutierrez, J., Chen, J., Seidel, C., Gerton, J. and Workman, J. L. (2005). Preferential occupancy of histone variant H2AZ at inactive promoters influences local histone modifications and chromatin remodeling. Proc. Natl. Acad. Sci. 102: 18385-18390. Medline abstract: 16344463
Li, Z., Thiel, K., Thul, P. J., Beller, M., Kuhnlein, R. P. and Welte, M. A. (2012). Lipid droplets control the maternal histone supply of Drosophila embryos. Curr Biol 22(22): 2104-2113. PubMed ID: 23084995
Matthaei, K., Rathjen, P. D., Tremethick, D. J. and Lyons, I. (2001). Histone variant H2A.Z is required for early mammalian development. Curr. Biol. 11: 1183-1187. 11516949
Mavrich, T. N., et al. (2008). Nucleosome organization in the Drosophila genome. Nature 453: 358-362. PubMed Citation: 18408708
Meneghini, M. D., Wu, M. and Madhani, H. D. (2003). Conserved histone variant H2A.Z protects euchromatin from the ectopic spread of silent heterochromatin. Cell 112: 725-736. 12628191
Mizuguchi, G., Shen, X., Landry, J., Wu, W.H., Sen, S. and Wu, C. (2004). ATP-driven exchange of histone H2AZ variant catalyzed by SWR1 chromatin remodeling complex. Science 303: 343-348. 14645854
Morillo Prado, J. R., Srinivasan, S. and Fuller, M. T. (2013). The histone variant His2Av is required for adult stem cell maintenance in the Drosophila testis. PLoS Genet 9: e1003903. PubMed ID: 24244183
Pardue, M. L., and Debaryshe, P. G. (2008). Drosophila telomeres: a variation on the telomerase theme. Fly 2: 1-10
Petesch, S. J. and Lis, J. T. (2008). Rapid, transcription-independent loss of nucleosomes over a large chromatin domain at Hsp70 loci. Cell 134: 74-84. PubMed Citation: 18614012
Raisner, R. M., Hartley, P. D., Meneghini, M. D., Bao, M. Z., Liu, C. L., Schreiber, S. L., Rando, O. J., and Madhani, H. D. (2005). Histone variant H2A.Z marks the 5' ends of both active and inactive genes in euchromatin. Cell 123: 233-248. Medline abstract: 16239142
Rangasamy, D., Berven, L., Ridgway, P. and Tremethick, D.J. (2003). Pericentric heterochromatin becomes enriched with H2A.Z during early mammalian development. EMBO J. 22: 1599-1607. 12660166
Rangasamy, D., Greaves, I. and Tremethick, D. J. (2004). RNA interference demonstrates a novel role for H2A.Z in chromosome segregation. Nat. Struct. Mol. Biol. 11(7): 650-5. 15195148
Ridgway, P., Brown, K. D., Rangasamy, D., Svensson, U. and Tremethick, D. J. (2004). Unique residues on the H2A.Z containing nucleosome surface are important for Xenopus laevis development. J. Biol. Chem. 279(42): 43815-20. 15299007
Rong, Y. S. (2008). Loss of the histone variant H2A.Z restores capping to checkpoint-defective telomeres in Drosophila. Genetics 180(4): 1869-75. PubMed Citation: 18845840
Ruhl, D. D., Jin, J., Cai, Y., Swanson, S., Florens, L., Washburn, M. P., Conaway, R. C., Conaway, J. W., and Chrivia, J. C. (2006). Purification of a human SRCAP complex that remodels chromatin by incorporating the histone variant H2A.Z into nucleosomes. Biochemistry 45: 5671-5677. Medline abstract: 16634648
Santisteban, M. S., Kalashnikova, T. and Smith, M. M. (2000). Histone H2A.Z regulates transcription and is partially redundant with nucleosome remodeling complexes. Cell 103: 411-422. 11081628
Schuster, A. T., Sarvepalli, K., Murphy, E. A. and Longworth, M. S. (2013). Condensin II Subunit dCAP-D3 Restricts Retrotransposon Mobilization in Drosophila Somatic Cells. PLoS Genet 9: e1003879. PubMed ID: 24204294
Stargell, L. A., Bowen, J., Dadd, C. A., Dedon, P. C., Davis, M., Cook, R. G., Allis, C. D. and Gorovsky, M. A. (1993). Temporal and spatial association of histone H2A variant hv1 with transcriptionally competent chromatin during nuclear development in Tetrahymena thermophila. Genes & Dev. 7: 2641-2651. 8276246
Stephenson, R. A., Thomalla, J. M., Chen, L., Kolkhof, P., White, R. P., Beller, M. and Welte, M. A. (2021). Sequestration to lipid droplets promotes histone availability by preventing turnover of excess histones. Development. PubMed ID: 34286844
Suto, R. K., Clarkson, M. J., Tremethick, D. J. and Luger, K. (2000). Crystal structure of a nucleosome core particle containing the variant histone H2A.Z. Nat. Struct. Biol. 7: 1121-1124. 11101893
Swaminathan J., Baxter, E. M. and Corces, V. G. (2005). The role of histone H2Av variant replacement and histone H4 acetylation in the establishment of Drosophila heterochromatin. Genes Dev. 19(1): 65-76. 15630020
Tang, Y., Dong, S., Cao, X., Zhou, Q., Ding, G. and Jiang, C. (2013). H2A.Z nucleosome positioning has no impact on genetic variation in Drosophila genome. PLoS One 8: e58295. PubMed ID: 23472174
Tulin, A. and Spradling, A. (2003). Chromatin loosening by poly(ADP)-ribose polymerase (PARP) at Drosophila puff loci. Science 299: 560-562. PubMed Citation: 12543974
van Daal, A., White, E. M., Gorovsky, M. A. and Elgin, S. C. (1988). Drosophila has a single copy of the gene encoding a highly conserved histone H2A variant of the H2A.F/Z type. Nucleic Acids Res. 16: 7487-7497. 3137528
van Daal, A. and Elgin, S. C. (1992). A histone variant, H2AvD, is essential in Drosophila melanogaster. Mol. Biol. Cell 3: 593-60. 1498368
Wallrath, L. L. and Elgin, S. C. (1995). Position effect variegation in Drosophila is associated with an altered chromatin structure. Genes Dev. 9: 1263-1277. 7758950
Wells, B. S. and Johnston, L. A. (2011). Maintenance of imaginal disc plasticity and regenerative potential in Drosophila by p53. Dev. Biol. 361(2): 263-76. PubMed Citation: 22036477
Yan, D., Neumuller, R. A., Buckner, M., Ayers, K., Li, H., Hu, Y., Yang-Zhou, D., Pan, L., Wang, X., Kelley, C., Vinayagam, A., Binari, R., Randklev, S., Perkins, L. A., Xie, T., Cooley, L. and Perrimon, N. (2014). A regulatory network of Drosophila germline stem cell self-renewal. Dev Cell 28: 459-473. PubMed ID: 24576427
Zhang, H., Roberts, D. N., and Cairns, B. R. (2005). Genome-wide dynamics of Htz1, a histone H2A variant that poises repressed/basal promoters for activation through histone loss. Cell 123: 219-231. Medline abstract: 16239141
Zhang, Z. and Pugh, B. F. (2011). Genomic organization of H2Av containing nucleosomes in Drosophila heterochromatin. PLoS One 6(6): e20511. PubMed Citation: 21738578
Zilberman, D., Coleman-Derr, D., Ballinger, T. and Henikoff, S. (2008). Histone H2A.Z and DNA methylation are mutually antagonistic chromatin marks. Nature 456(7218): 125-9. PubMed Citation: 18815594
date revised: 12 April 2022
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