InteractiveFly: GeneBrief

domino: Biological Overview | References

Phosphorylation of the human histone variant H2A.X and H2Av, its homolog in Drosophila melanogaster, occurs rapidly at sites of DNA double-strand breaks. Little is known about the function of this phosphorylation or its removal during DNA repair. The Drosophila Tip60 (dTip60) chromatin-remodeling complex acetylates nucleosomal phospho-H2Av and exchanges it with an unmodified H2Av. Both the histone acetyltransferase dTip60 as well as the adenosine triphosphatase Domino/p400 catalyze the exchange of phospho-H2Av. 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).

Chromatin regulation mediated by Domino and PcG-like factors controls E2F activity and cell growth

Regulation of chromatin structure is critical in many fundamental cellular processes. Previous studies have suggested that the Rb tumor suppressor may recruit multiple chromatin regulatory proteins to repress E2F, a key regulator of cell proliferation and differentiation. Taking advantage of the evolutionary conservation of the E2F pathway, a genome-wide RNAi screen was conducted in cultured Drosophila cells for genes required for repression of E2F activity. Among the genes identified are components of the putative Domino chromatin remodeling complex, as well as the Polycomb Group (PcG) protein-like fly tumor suppressor, L3mbt, and the related Scm-related gene containing four mbt domains (CG16975/dSfmbt). These factors are recruited to E2F-responsive promoters through physical association with E2F and are required for repression of endogenous E2F target genes. Surprisingly, their inhibitory activities on E2F appear to be independent of Rb. In Drosophila, domino mutation enhances cell proliferation induced by E2F overexpression and suppresses a loss-of-function cyclin E mutation. These findings suggest that potential chromatin regulation mediated by Domino and PcG-like factors plays an important role in controlling E2F activity and cell growth (Lu, 2007).

This study identified the putative Dom/SWR1 chromatin remodeling complex and the PcG-like MBT domain-containing factors were identified as E2F repressors. These proteins are recruited to E2F target promoters through association with E2F and inhibit E2F in an apparently Rb-independent manner. Depletion of these genes resulted in derepression of some endogenous E2F target genes accompanied by changes in histone modification. More importantly, dom genetically interacts with the E2F pathway. These proteins show an extensive degree of evolutionary conservation, indicating the mechanism of E2F regulation provided by these factors may be well conserved (Lu, 2007).

Regulation of E2F is tightly linked to cell proliferation and differentiation. Existing evidence suggests that perturbation of the Dom and MBT proteins may cause dysregulation of these cellular processes. Apart from the fact that the heterozygous dom mutation modifies cell growth in an E2F-transgenic or a cycE hypomorphic background, fly mutants homozygous for several dom alleles show enlarged lymph glands apparently because of excessive proliferation of prehemocytes. In human, the Dom complex subunit YL1 possesses growth suppressive activity (Horikawa, 1995), and the Dom homolog p400 is an essential target for the viral oncoprotein E1A-mediated transformation (Fuchs, 2001). Indeed, overexpression of E1A disrupts the association of E2F with the Dom complex in mammalian cells. Furthermore, mutations in the fly tumor suppressor gene l3mbt result in overgrowth of the larval brain lobes and epithelial imaginal discs, and failure of neural differentiation (Wismar, 1995). This is intriguing, because in mammalian cells, many E2F-regulated genes are repressed during quiescence and differentiation, and mammalian MBT proteins are found in an inhibitory E2F complex purified from quiescent cells (Lu, 2007).

Although the mechanism of Rb-mediated repression on E2F is complex, these studies indicate that Dom and MBT possess Rb-independent activities. In support of this view, recent studies suggest that the C. elegans Dom and Rb homologs share redundant functions in vulva development, a process controlled by the E2F pathway (Ceol, 2004). In addition, these proteins may participate in distinct E2F complexes. Mammalian MBT orthologs have been identified from Rb-independent complexes (Ogawa, 2002), and they can associate with E2F forms lacking the Rb-binding motif, such as E2F6 and a C-terminal truncated E2F3 mutant. Interestingly, L3mbt is shown to interact with dREAM, a dE2F2-Rb complex (Georlette, 2007), even though it is not a stoichiometric subunit (Korenjak, 2004; Lewis, 2004). But unlike L3mbt, RNAi of dE2F2 and several other components of the core dREAM complex had no effect on the E2F reporter. This observation may hence indicate the existence of multiple L3mbt-containing complexes or hint at a potential collaboration among different E2F regulatory activities. So far, there is no evidence linking Dom and CG16975 to Rb. It is likely that both Rb-mediated and -independent chromatin modulations play critical roles in E2F regulation and cell proliferation. Future biochemical and genetic studies may shed light on these potentially independent and collaborative relations (Lu, 2007).

Drosophila Reptin and other TIP60 complex components promote generation of silent chromatin

Histone acetyltransferase (HAT) complexes have been linked to activation of transcription. Reptin is a subunit of different chromatin-remodeling complexes, including the TIP60 HAT complex, which includes Domino as a subunit. In Drosophila, Reptin also copurifies with the Polycomb group (PcG) complex PRC1, which maintains genes in a transcriptionally silent state. Genetic interactions have been demonstrated between reptin mutant flies and PcG mutants, resulting in misexpression of the homeotic gene Scr. Genetic interactions are not restricted to PRC1 components, but are also observed with another PcG gene. In reptin homozygous mutant cells, a Polycomb response-element-linked reporter gene is derepressed, whereas endogenous homeotic gene expression is not. Furthermore, reptin mutants suppress position-effect variegation (PEV), a phenomenon resulting from spreading of heterochromatin. These features are shared with three other components of TIP60 complexes, namely Enhancer of Polycomb, Domino, and dMRG15. It is concluded that Drosophila Reptin participates in epigenetic processes leading to a repressive chromatin state as part of the fly TIP60 HAT complex rather than through the PRC1 complex. This shows that the TIP60 complex can promote the generation of silent chromatin (Qi, 2006).

A fundamental regulatory step in transcription and other DNA-dependent processes in eukaryotes is the control of chromatin structure, which regulates access of proteins to DNA. Histone acetylation and the protein complexes that mediate this modification have been linked to activation of transcription. It is believed that lysine acetylation of histone N termini results in less compact chromatin by neutralizing the positive charge of histones and that the acetyl groups are recognized by regulatory proteins that promote transcription. However, it is becoming clear that histone acetyltransferases (HATs) can have functions other than facilitating transcription. For example, the TIP60 HAT complex has been implicated in DNA repair in yeast, flies, and mammals. This study investigated the role of Drosophila Reptin and other TIP60 components in chromatin regulation in vivo (Qi, 2006).

The Reptin protein, also known as TIP48, TIP49b, or RUVBL2, is related to bacterial RuvB, an ATP-dependent DNA helicase that promotes branch migration in Holliday junctions. Reptin, and the related Pontin (TIP49, TIP49a, or RUVBL1) protein, possess intrinsic ATPase and helicase activities and can heterodimerize. In yeast, both Reptin and Pontin are part of the INO80 chromatin-remodeling complex, as well as the Swr1 complex that can exchange histone H2A with the variant histone H2A.Z. Reptin and Pontin appear to play antagonistic roles in development by regulating Wnt signaling (Bauer, 2000) and heart growth in zebrafish embryos. Mammalian Reptin and Pontin are present in TIP60 HAT complexes, which are involved in induction of apoptosis in response to DNA damage and which interact with the c-Myc protein to promote its oncogenic activity (Qi, 2006 and references therein).

TIP60 is a HAT of the MYST family (Utley, 2003). The homologous yeast protein Esa1 is the catalytic subunit of the nucleosome acetyltransferase of H4 (NuA4) complex, which acetylates lysines in histone H4 and H2A (Doyon, 2004). In Drosophila, the TIP60 complex acetylates the phosphorylated variant histone H2Av after DNA double-strand breaks and exchanges it with unmodified H2Av. The composition of TIP60 and NuA4 complexes has recently been determined. TIP60 (yeast Esa1), ING3 (Yng2), and Enhancer of Polycomb (EPC1, yeast Epl1) form a core complex that is sufficient for acetylation of histones in nucleosomes. Mammalian and Drosophila TIP60 complexes contain four subunits not present in yeast NuA4: Brd8, Reptin, Pontin, and Domino (also known as p400), the homolog of yeast Swr1 (Qi, 2006).

Polycomb group (PcG) proteins are evolutionarily conserved chromatin regulators that maintain appropriate expression patterns of developmental control genes, such as the Hox genes. PcG proteins are generally repressors that maintain the off state of genes and exist in at least two distinct protein complexes. The Esc-E(z) complex is a histone methyltransferase that includes the catalytic subunit Enhancer of zeste [E(z)], as well as the Extra sex combs (Esc) and Suppressor of zeste 12 [Su(z)12] subunits. Another complex purified from Drosophila embryos, Polycomb repressive complex 1 (PRC1) has a mass of >1 MDa. In addition to genetically identified PcG proteins, it includes TFIID subunits, the Reptin protein, and other polypeptides. The PRC1 complex can block chromatin remodeling by the SWI/SNF complex in vitro. A core PRC1 complex consisting of Polycomb (Pc), Posterior sex combs (Psc), Polyhomeotic (Ph), and dRING1/Sex combs extra (Sce) is sufficient for the in vitro activities of PRC1. Recently, it was shown that dRing1/Sce as well as its mammalian orthologs are E3 ubiquitin ligases that monoubiquitylate histone H2A (Qi, 2006 and references therein).

This study investigates the role of Drosophila Reptin in chromatin regulation. Reptin is shown to interact genetically with PcG gene products and suppresses position-effect variegation (PEV), properties shared by other Drosophila TIP60 complex components. It is suggested that the fly TIP60 complex regulates epigenetic processes leading to a repressive chromatin state. This is a novel activity of a HAT complex that has previously been implicated in transcription activation and DNA repair (Qi, 2006).

It is proposed that Reptin acts as a subunit of the TIP60 HAT complex to generate a repressive chromatin state. This is a novel activity of a HAT complex previously shown to promote transcription. This study shows that Reptin copurifes with the Polycomb complex PRC1. This prompted an investigation of whether the biochemical interaction with PRC1 was accompanied by a genetic interaction. It was shown that Reptin and PRC1 components genetically interact to regulate expression of the Hox gene Scr. However, Reptin also interacts with a PcG gene product not associated with the PRC1 complex, Pcl. Although no interactions were detected between reptin heterozygous mutants and several PREs tested, a PRE from the Ubx gene is derepressed in reptin homozygous mutant cells. This shows that Reptin contributes an essential function to the activity of this PRE. However, unlike most PcG genes, reptin homozygous mutants do not derepress endogenous Hox gene expression. It appears that repression of endogenous Hox genes is more complex and not as sensitive to the loss of Reptin as the Ubx PRE. In contrast to most PcG genes, reptin mutants suppress PEV. Interestingly, derepression of the Ubx PRE also occurs in embryos mutant for other suppressors of PEV, indicating that this PRE may be highly sensitive to the chromatin environment in its vicinity. Since reptin mutants suppress PEV and fail to derepress endogenous Hox gene expression, reptin is not considered a bona fide PcG gene, and it is found unlikely that Reptin protein contributes an essential function to the PRC1 complex. In fact, the biochemical activities ascribed to PRC1 can be reconstituted either with recombinant dRing1/Sce or with four core components whose activity can be further enhanced by the DNA-binding proteins Zeste and GAGA (Qi, 2006).

Given that Reptin is present in TIP60 complexes in mammals and recently was shown to be a component of a Drosophila TIP60 complex, the possibility is considered that the genetic interactions observed with PcG genes are due to the presence of Reptin in the fly TIP60 complex. The products of two previously characterized Drosophila genes, E(Pc) and domino, are also present in the TIP60 complex. Strikingly, E(Pc) and domino mutants share with reptin the ability to genetically interact with PcG genes and suppress PEV. E(Pc) is an unusual PcG gene that has very minor effects on Hox gene expression, and unlike most PcG genes, modifies PEV. In both yeast and humans, E(Pc) homologs form a core complex with Esa1 (TIP60) and Yng2 (ING3) that is sufficient for the nucleosomal acetylation of histones H4 and H2A by the NuA4 complex (Boudreault, 2003; Doyon, 2004). That such an integral NuA4/TIP60 complex component displays phenotypes similar to reptin mutants suggests that Reptin functions through the fly TIP60 complex (Qi, 2006).

Domino protein is similar to p400 and to SRCAP in mammals and to Swr1 in yeast (Eissenberg, 2005). Swr1 has recently been shown to exchange the variant histone H2A.Z (Htz1 in yeast) for H2A in nucleosomes (Krogan, 2003; Kobor, 2004; Mizuguchi, 2004). Intriguingly, an involvement of Htz1 (H2A.Z) in controlling the spreading of silenced chromatin has recently been demonstrated in yeast. Exchange of variant histones may be a conserved feature of chromatin regulation since a recent report demonstrates that Drosophila H2Av behaves genetically as a PcG gene and suppresses PEV (Swaminathan, 2005). Domino exchanges phosphorylated and acetylated H2Av for unmodified H2Av after DNA damage (Kusch, 2004). However, no change was found in binding of H2Av to polytene chromosomes prepared from domino mutant larvae (Qi, 2006).

A P-element insertion was identified in the gene encoding one additional TIP60 complex component, the chromodomain-containing protein MRG15. Human MRG15 (MORF-related gene on chromosome 15) has been implicated in cellular senescence and regulation of the B-myb promoter. Both human and yeast (Eaf3/Alp13) MRG15 have been found in Sin3/HDAC complexes in addition to the TIP60 (NuA4) complex, where it directs the histone deacetylase to coding regions through interaction of its chromodomain with methylated histone H3 lysine 36. This study found that MRG15 mutant flies interact with PcG genes and suppress PEV, just as other TIP60 complex components do. This is taken as further support of the conclusion that Reptin's effects on chromatin processes are mediated through its association with the fly TIP60 complex (Qi, 2006).

What is the basis for the genetic interaction between TIP60 components and PcG genes? One possibility is that the TIP60 complex regulates PcG expression. However, no reduction was observed in Pc expression in reptin mutant embryos. Another possibility is that the enzymatic activities of the TIP60 complex cooperate with PcG genes to mediate transcriptional silencing. Since binding of Pc to polytene chromosomes is abolished in H2Av mutant animals (Swaminathan, 2005), TIP60 complex-mediated histone variant exchange might cause the genetic interaction with PRC1. However, this study found that binding of PcG proteins to polytene chromosomes is unaffected in domino mutant larvae. It is possible that PRC1-mediated H2A ubiquitylation helps to recruit the TIP60 complex, whose histone acetylation or histone exchange activity assists in transcriptional repression. Alternatively, histone acetylation or exchange facilitates binding of the PRC1 complex to PREs. A similar mechanism has been invoked for the cooperation of the Esc-E(z) complex and PRC1, where Esc-E(z) trimethylates histone H3 lysine 27, which is recognized by the chromodomain of Polycomb (Qi, 2006).

This study has shown that the Drosophila TIP60 complex plays a role in epigenetic gene silencing in vivo. A similar case has been described for the yeast HAT complex SAGA (Spt-Ada-Gen5-acetyltransferase) that is required for both activation and repression of the ARG1 gene. Two other yeast HATs, Sas2 and Sas3, also promote gene silencing. Interestingly, the Drosophila HAT Chameau suppresses PEV and cooperates with PcG genes as well. TIP60, Sas2, Sas3, and Chameau are HATs that belong to the MYST family. Therefore, MYST family HATs in both yeast and flies can control epigenetic inheritance of silent chromatin (Qi, 2006).

p21 transcription is regulated by differential localization of histone H2A.Z directed by p53

In yeast cells, H2A.Z regulates transcription and is globally associated within a few nucleosomes of the initiator regions of numerous promoters. H2A.Z is deposited at these loci by an ATP-dependent complex, Swr1.com. H2A.Z suppresses the p53 --> p21 transcription and senescence responses. Upon DNA damage, H2A.Z is first evicted from the p21 promoter, followed by the recruitment of the Tip60 histone acetyltransferase to activate p21 transcription. p400, a human Swr1 homolog, is required for the localization of H2A.Z, and largely colocalizes with H2A.Z at multiple promoters investigated. Notably, the presence of sequence-specific transcription factors, such as p53 and Myc, provides positioning cues that direct the location of H2A.Z-containing nucleosomes within these promoters. Collectively, this study strongly suggests that certain sequence-specific transcription factors regulate transcription, in part, by preferentially positioning histone variant H2A.Z within chromatin. This H2A.Z-centered process is part of an epigenetic process for modulating gene expression (Gévry, 2007).

Eukaryotic DNA is condensed many fold (e.g., 10,000) into chromatin, the basic unit of which contains 146 base pairs (bp) of DNA and an octamer of histone proteins (H2A, H2B, H3, and H4). Due to the high level of compaction, chromatin typically represses certain cellular DNA transactions, including transcription. For successful transcription, it is argued that nucleosomes need to be remodeled or evicted from promoter regions for the transcriptional machinery to be efficiently recruited to a target gene (Gévry, 2007).

The incorporation of histone variants into specific nucleosomes within a promoter region constitutes a mechanism by which promoter region chromatin can become more permissive to transcription initiation and elongation following receipt of a proper physiological cue. One such histone variant is H2A.Z. In Saccharomyces cerevisiae, it can elicit positive effects on gene expression. In addition, H2A.Z regulates genes that are proximal to telomeres and acts as a 'buffer' to antagonize the spread of heterochromatin into euchromatic regions (Meneghini, 2003). Furthermore, recent reports (Guillemette, 2005; Li, 2005; Raisner, 2005; Zhang, 2005) have shown that H2A.Z is preferentially localized within a few nucleosomes of the initiator regions of multiple promoters in the yeast genome. Interestingly, these H2A.Z-rich loci are largely devoid of transcriptional activity, which suggests that the variant histone prepares genes for activation (Guillemette, 2005) and/or operates as a transcriptional repressor. Finally, yeast H2A.Z has been shown to regulate nucleosome positioning, which provides mechanistic insight into how its presence can alter promoter transcriptional state (Gévry, 2007).

An ATP-dependent chromatin remodeling complex that specifically loads H2A.Z onto chromatin and exchanges it with H2A exists in yeast (Krogan, 2003; Kobor, 2004; Mizuguchi, 2004). This complex, in which the catalytic subunit is Swr1, also shares essential subunits with the NuA4 histone acetyltransferase complex (Krogan, 2003; Kobor, 2004). In addition to their importance in gene regulation, the Swr1 complex, H2A.Z, and NuA4 are all involved in the regulation of yeast chromosome stability (Krogan, 2004). This is noteworthy because, in mammalian cells, depletion of H2A.Z causes major nuclear and chromosomal abnormalities (Rangasamy, 2004) as witnessed by a high incidence of lagging chromosomes and chromatin bridges (Gevry, 2007).

There are two homologs of Swr1 in human cells: p400/Domino (referred to as p400), and SRCAP. There are also three uncharacterized p400-type SWI2-SNF2 molecules, including hIno80. Members of this family of SWI2/SNF2 chromatin remodeling enzymes each contain a spacer region inserted into the SWI2/SNF2 homology region (Gevry, 2007).

p400 was originally isolated as an E1A-associated protein, and it was also shown to interact with p53, Myc, and SV40 large T antigen. It is also required for E1A to induce p53-mediated apoptosis. SRCAP has been isolated as a CREB-binding protein. While one report shows that both p400 and SRCAP constitute part of the same complex, a recent study shows that SRCAP and p400 exist in distinct complexes with H2A.Z (Jin, 2005; Ruhl, 2006). Recently an SRCAP-containing complex was purified, and it was shown to have the ability to exchange H2A-H2B for H2A.Z-H2B in reconstituted mononucleosomes (Ruhl, 2006). It remains to be determined whether mammalian homolog(s) of Swr1, such as p400 and SRCAP, also catalyze H2A.Z deposition in vivo (Gevry, 2007).

Depletion of p400 elevates p21 synthesis to initiate premature senescence in primary human fibroblasts (Chan, 2005). Senescence has been observed in tissue culture cells as a stable form of cell growth arrest provoked by diverse stresses. Recently, oncogene-induced senescence was shown to occur in various precancerous lesions both in humans and mice, further suggesting that senescence acts as a defense mechanism against malignant cell development. Importantly, the action of p400 at p21 depends on the function of p53, a key regulator of p21 transcription (Gevry, 2007).

Given the possibility of a link between p400 and H2A.Z, it was asked whether H2A.Z is also an important regulator of p21 expression. The results of this effort show that H2A.Z depletion induces p21 expression in a p53-dependent fashion, as well as the premature senescence of primary diploid fibroblasts. Similar to senescence induced by p400 depletion, inactivating p53 or p21 blocked the emergence of certain senescent phenotypes following H2A.Z depletion. In a normal setting, H2A.Z is highly enriched at discrete p53-binding sites that lie within the p21 promoter. This distinctive localization pattern depends on the presence of p53, and was detected at other p53 target gene promoters as well. The presence of p400 is required to localize H2A.Z at those loci, and purified recombinant p400 from insect cells can carry out in vitro exchange of H2A.Z-H2B dimers into chromatin. H2A.Z and p400 localization at the p53-binding sites in p21 is severely diminished following p21 induction, and this process is not dependent on active p21 transcription per se. After H2A.Z and p400 eviction from the p53-binding sites in p21, it was observed that the Tip60 histone acetyltransferase isrecruited to the distal p53-binding site in the promoter to positively regulate p21 expression. Finally, overexpression of Myc, a known suppressor of p21 synthesis, significantly increases H2A.Z localization at the Myc-binding site in the TATA initiator region of the p21 promoter. This observation is consistent with the view that Myc represses p21 expression by preferentially recruiting H2A.Z-containing nucleosome(s) to this element (Gevry, 2007).

Search PubMed for articles about Drosophila Domino

Bauer, A., et al. (2000). Pontin52 and reptin52 function as antagonistic regulators of beta-catenin signalling activity. EMBO J. 19(22): 6121-30. Medline abstract: 11080158

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date revised: 30 March 2008

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