Targets of Activity

Pontin52 and reptin52 function as antagonistic regulators of beta-catenin signalling activity

In Wnt-stimulated cells, beta-catenin becomes stabilized in the cytoplasm, enters the nucleus and interacts with HMG box transcription factors of the lymphoid-enhancing factor-1 (LEF-1)/T-cell factor (TCF) family, thereby stimulating the transcription of specific target genes. Pontin52 has been identified as a nuclear protein interacting with beta-catenin and the TATA-box binding protein (TBP), suggesting its involvement in regulating beta-catenin-mediated transactivation. This study reports the identification of Reptin52 as an interacting partner of Pontin52. Highly homologous to Pontin52, Reptin52 likewise binds beta-catenin and TBP. Using reporter gene assays, it was shown that the two proteins antagonistically influence the transactivation potential of the beta-catenin-TCF complex. Furthermore, the evolutionary conservation of this mechanism is demonstrated in Drosophila: pontin and reptin are essential genes that act antagonistically in the control of Wingless signalling in vivo. These results indicate that the opposite action of Pontin52 and Reptin52 on beta-catenin-mediated transactivation constitutes an additional mechanism for the control of the canonical Wingless/Wnt pathway (Bauer, 2000; full text of article).

Myc interacts genetically with Tip48/Reptin and Tip49/Pontin to control growth and proliferation during Drosophila development

The transcription factor dMyc is the sole Drosophila ortholog of the vertebrate c-myc protooncogenes and a central regulator of growth and cell-cycle progression during normal development. The molecular basis of dMyc function was examined by analyzing its interaction with the putative transcriptional cofactors Tip48/Reptin (Rept) and Tip49/Pontin (Pont). Rept and Pont have conserved their ability to bind to Myc during evolution. All three proteins are required for tissue growth in vivo, because mitotic clones mutant for either dmyc, pont,or rept suffer from cell competition. Most importantly, pont shows a strong dominant genetic interaction with dmyc that is manifested in the duration of development, rates of survival and size of the adult animal and, in particular, of the eye. The molecular basis for these effects may be found in the repression of certain target genes, such as mfas, by dMyc:Pont complexes. These findings indicate that dMyc:Pont complexes play an essential role in the control of cellular growth and proliferation during normal development (Bellosta, 2005).

Myc proteins are essential regulators of growth, proliferation, and apoptosis in metazoans. These proteins act as transcription factors to control the expression of numerous target genes involved in growth, metabolism, and other processes. Less is known about the molecular mechanism that allows Myc to control the expression of these targets. In recent years, different modes of gene activation by Myc have been proposed, notably recruitment of chromatin remodelers, or RNA pol II kinases, but the physiological relevance of these different factors for Myc-dependent biological functions needs to be demonstrated. This study investigated the mechanisms of Myc-controlled growth and proliferation during normal development by using Drosophila as a model system. Initially, focus was placed on the interaction of Myc with two specific components of cofactor complexes, Tip48 and Tip49, because of the availability of null mutations in the corresponding genes [called reptin (rept) and pontin (pont) in flies, respectively] (Bellosta, 2005).

Tip48 and Tip49 are closely related proteins that show a high similarity to the bacterial ATP-dependent AAA+ super family DNA helicase RuvB. Orthologs of Tip48 and Tip49 have been identified in plants, yeast, and animals. Different observations strongly suggest that one major function of the Tip proteins resides in the control of transcription. Initially, vertebrate Tip49 was found to be a Tata-binding protein-interacting protein; later Tip48 and Tip49 were also shown to interact physically with the different transcription factors ß-catenin, E2F1 (only Tip49), raising the possibility that the Tip proteins could bridge basic transcription machinery and sequence-specific activators. Both proteins were also purified as part of several multiprotein complexes involved in transcriptional regulation: the Ino80 chromatin remodeling complex in yeast, Polycomb repressive complex 1 in Drosophila (only Tip48), the Tip60 HAT complex in vertebrates, and the Uri complex regulating nutrition-dependent gene expression in yeast and in vertebrates. Interestingly, three other proteins that were found to bind the N terminus of c-Myc share residence with Tip48 and Tip49 in the Ino80 (BAF53 and ß-actin) or Tip60 complex (transformation/transcription-domain-associated protein, BAF53, and ß-actin). Further support for an involvement of Tip48 and Tip49 in transcription is provided by the observations that both proteins colocalize with c-Myc on the nucleolin promoter and that elimination of Tip48 or Tip49 function in yeast rapidly affects the expression of a large number of targets. Such a transcriptional role is also consistent with the described genetic interactions between a tip48 mutation and ß-catenin in zebrafish and interactions of tip48 and tip49 with a ß-catenin-reporter system in Drosophila (Bauer, 2000; Rottbauer, 2002); in both of these in vivo interactions, Tip48 behaved as a negative component and Tip49 behaved as a positive component of the Wg signaling cascade. Similar opposing activities were also documented in a human cell line by assaying the ability of the ß-catenin–T cell factor complex to activate a reporter gene. A potential role for Tip49 in Myc-dependent functions was addressed in a recent study that examined the consequences of coexpressing wild-type or putative dominant-negative forms of Tip49 with c-Myc. Neither form had any effect on control cells, but both enhanced the apoptosis caused by overexpressed c-Myc, and they reduced the ability of c-Myc in combination with activated Ras to transform rat embryo fibroblasts, which indicates that, upon forced overexpression, Myc might require Tip49 activity (Dugan, 2002; Bellosta, 2005 and references therein).

The present study shows that the physical interaction between Myc and Pont/Rept is conserved in flies, that pont/rept are essential for tissue growth in vivo, and that dmyc and pont show a strong genetic interaction. The gene mfas was identified as a transcriptional target that is repressed by dMyc:Pont complexes. These studies provide the first evidence that Pont and Rept are essential cofactors for the normal functions of Myc in vivo (Bellosta, 2005).

This study provides evidence that Tip49/Pont (and possibly Tip48/Rept) is an essential partner for Myc during normal development and that it plays an important role in the control of Myc-dependent transcription, growth, and proliferation. These conclusions are supported by four lines of evidence (Bellosta, 2005).

(1) dMyc physically interacts with Rept and Pont in vitro, in cells, and in larvae. Although ternary complexes containing dMyc, Rept, and Pont can exist, evidence is provided that dMyc can associate with Pont in the absence of Rept, although it is unclear whether such complexes lacking Rept have any physiological role in vivo. The stronger genetic interaction with pont raises the possibility that some of dMyc's functions might be mediated by such complexes, but the large degree of overlap between the targets of Pont and Rept and the fact that in most biochemically purified complexes Tip48 is accompanied by Tip49 suggest that most often these two proteins function together (Bellosta, 2005).

(2) Flies lacking zygotic pont or rept gene products arrest their growth early during larval development, and mitotic clones homozygous mutant for pont or rept suffer from the same type of cell competition as do dmyc clones. These characteristics indicate a requirement for Pont/Rept for cellular proliferation and growth, which is consistent with their functioning as cofactors for dMyc (Bellosta, 2005).

(3) pont shows a strong dominant genetic interaction with dmyc. The causes for this interaction are likely to be defects in cellular growth and proliferation. The control of growth is most sensitive to variations in dMyc levels, because the moderate reduction of dMyc activity achieved in hypomorphic dmyc alleles already results in a decrease in cell size but not cell numbers. Removal of one copy of the pont gene exacerbates the growth defect and results in a reduction of cell numbers. No indication was found that apoptosis contributes to this reduction in cell number and, therefore, it is concluded that defects in cell number primarily reflect a proliferation defect. It is important to stress that none of these defects are seen in flies that are heterozygous for pont but wild-type for dmyc, arguing strongly against purely additive effect of the pont and dmyc mutations. Although the possibility that Pont and dMyc act in parallel growth-controlling pathways cannot be strictly ruled out, such a dominant genetic interaction is indicative of close functional connections. No dominant effect of the pont mutation on dMyc overexpression phenotypes has been observed, suggesting that Pont is not limiting in situations of mildly increased dMyc levels. However, by using a vertebrate tissue culture system (Rat1 cells), it has been demonstrated that dominant-negative Pont/Tip49 inhibits the ability of human c-Myc to transform Rat1 fibroblasts in conjunction with activated Ras. Overexpression of a dominant-negative protein mutant potentially allows a stronger reduction of Pont/Tip49 activity than can be obtained in a heterozygous pont-/+ situation, and, thus, these experiments further reinforce the observation of a genetic interaction between myc and pont (Bellosta, 2005).

(4) It has been shown that the expression of several genes, including mfas, is increased upon down-regulation of dmyc, pont, or rept in S2 cells and in dmyc/pont double-mutant eye imaginal discs in vivo. Chromatin immunoprecipitation experiments further suggest that mfas is a direct transcriptional target of Pont and dMyc (Bellosta, 2005).

Taken together, these data strongly argue that dMyc:Pont complexes are essential regulators of proliferation and growth in vivo and that they act at least partly by repressing the expression of target genes, such as mfas. A similar repressive function has recently also been found for Xenopus Pont and Rept; it was proposed that the well characterized repression of the transactivator Miz1 by c-Myc is mediated by Pont and Rept. Although it is tempting to speculate that Drosophila Pont functions analogously, no fly homolog of Miz1 has been identified. In addition, it is currently not know which of the reported Pont-containing complexes is responsible for the observed effect (Bellosta, 2005).

The function of Rept is less clear, because a rept mutant shows only a weak interaction and only with one dmyc allele. In contrast, overexpression of Rept strongly enhances the dmyc/pont mutant phenotypes. This observation could indicate that Rept also acts as antagonist of Pont and of dMyc:Pont complexes, analogously to what has been proposed for the interaction between Rept/Pont and ß-Catenin. Alternatively, overexpression of Rept functions in a dominant-negative fashion, possibly by titrating Pont and/or other factors away from the multiprotein complexes in which they normally reside; in addition, Rept might be relatively more abundant than Pont such that heterozygosity for rept does not show any effects in most situations. Although either explanation currently cannot be ruled out, identification of mfas as a common target for dMyc, Pont, and Rept is more consistent with the latter possibility (Bellosta, 2005).

In conclusion, it has been shown that Pont, and possibly Rept, assists dMyc in the control of cellular proliferation and growth during normal development, presumably in part by repressing the expression of certain target genes (Bellosta, 2005).

Protein Interactions

Acetylation by Tip60 is required for selective histone variant exchange at DNA lesions

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Nipped-A, the Tra1/TRRAP subunit of the Drosophila SAGA and Tip60 complexes, has multiple roles in Notch signaling during wing development

The Notch receptor controls development by activating transcription of specific target genes in response to extracellular signals. The factors that control assembly of the Notch activator complex on target genes and its ability to activate transcription are not fully known. This study shows, through genetic and molecular analysis, that the Drosophila Nipped-A protein is required for activity of Notch and its coactivator protein, Mastermind, during wing development. Nipped-A and Mastermind also colocalize extensively on salivary gland polytene chromosomes, and reducing Nipped-A activity decreases mastermind binding. Nipped-A is the fly homologue of the yeast Tra1 and human TRRAP proteins and is a key component of both the SAGA and Tip60 (NuA4) chromatin-modifying complexes. Like Nipped-A, the Ada2b component of SAGA and the Domino subunit of Tip60 are also required for Mastermind function during wing development. Based on these results, it is proposed that Nipped-A, through the action of the SAGA and Tip60 complexes, facilitates assembly of the Notch activator complex and target gene transcription (Gause, 2006).

Nipped-A mutations were isolated in a genetic screen for factors that regulate activation of cut by the wing margin enhancer, and it was found that they reduce Notch activity both at the wing margin and in the developing wing veins. Heterozygous Nipped-A mutations increase the severity of the mutant wing margin and blade reduction phenotype caused by the weak loss-of-function Notch (Nnd-1) mutation and decrease the severity of the vein-shortening phenotype caused by a gain-of-function Notch mutation (NAx-E2)(Gause, 2006).

Other genetic data also indicate that Nipped-A is important for Notch signaling. Mastermind is a coactivator protein required for transcriptional activation by Notch, and heterozygous Nipped-A mutations dramatically increase the weak wing-nicking phenotype caused by heterozygous mastermind mutations. The vestigial gene is directly activated by Notch, and flies heterozygous for both Nipped-A and vestigial mutations display wing margin defects. The Notch intracellular fragment binds to the Suppressor of Hairless [Su(H)] protein on target genes, and a Nipped-A Su(H) double mutant displays a dominant wing-nicking phenotype. Together, the effects that the Nipped-A dosage has on the mutant phenotypes displayed by Notch, mastermind, and vestigial mutants indicate that Nipped-A encodes a factor critical for Notch activity in the developing wing (Gause, 2006).

Two Nipped-A mutants have point mutations in the gene encoding the Drosophila homologue of the yeast Tra1 and mammalian TRRAP proteins. Tra1/TRRAP is a key component of the SAGA and Tip60 (NuA4) chromatin-remodeling complexes in yeast, flies, and humans (Gause, 2006).

Tra1/TRRAP is a direct target of transcriptional activators and helps them recruit the SAGA and Tip60 chromatin modification complexes to aid in gene activation. Mammalian Tra1/TRRAP was first identified as a coactivator that interacts directly with the Myc and E2F activators. Tra1/TRRAP is also a target of several other activators in yeast and mammalian cells, including Gal4, E1A, VP16, nuclear receptors, and p53. Tra1/TRRAP contains an ATM-phosphatidylinositol-3 (PI-3) kinase-like domain near the C terminus that is important for recruitment of histone acetyltransferase (HAT) activity in mammalian cells. The C terminus is also critical for interaction of yeast Tra1 with acidic activators (Gause, 2006 and references therein).

There is evidence that SAGA, which contains Tra1/TRRAP and the Gcn5/PCAF HAT, may be involved in transcriptional activation by the Notch complex. Several components of the Notch activator complex are known and functionally identical in worms, flies, and mammals. Upon binding of ligands such as Serrate or Delta to the extracellular EGF repeats of Notch, an intracellular fragment of Notch (NICD) is proteolytically released, allowing it to enter the nucleus, where it interacts with a DNA-bound CSL [CBF1/Su(H)/Lag-1] protein. NICD helps recruit the Mastermind coactivator. An N-terminal region of Mastermind interacts with both the CSL protein and an ankyrin repeat domain of NICD. The p300/CBP (CREB-binding protein) HAT coactivator is recruited by interactions with both the NICD ankyrin repeats and a specific region in the N-terminal half of Mastermind. The Gcn5/PCAF HAT is also recruited by the Notch activator complex in cultured mouse cells; this requires the ankyrin repeat region of NICD. The NICD ankyrin repeats bind other proteins, such as Mastermind and CBP, and thus it is possible that these proteins are also required to recruit Gcn5/PCAF. Because Tra1/TRRAP is the SAGA subunit targeted by several transcriptional activators, it is a distinct possibility that it is required for recruitment of Gcn5/PCAF by the Notch activator complex (Gause, 2006).

This study presents a molecular genetic analysis of several Nipped-A mutations that provides new insights into the roles of the Tra1/TRRAP protein and its complexes in Notch signaling. Reducing the Nipped-A gene dosage by half reduces both Mastermind and Notch activities during wing development and, surprisingly, certain mutant alleles can replace one copy of wild-type Nipped-A. These data also show that other subunits of the SAGA and Tip60 complexes that contain Nipped-A are required for Mastermind and Notch function in wing development and that Nipped-A is required for binding of Mastermind to chromosomes. Taken together, the results indicate that Nipped-A plays multiple roles in Notch signaling (Gause, 2006).

The evidence provided here, combined with the finding that two Nipped-A mutants have point mutations in the Tra1/TRRAP gene, demonstrates conclusively that Nipped-A encodes Tra1/TRRAP. All EMS-induced Nipped-A alleles sequenced to date have point mutations in the Tra1/TRRAP gene that affect the protein coding sequence or, in one case, the 3' UTR. A seventh allele generated by gamma rays, Nipped-A323, does not produce Tra1/TRRAP mRNA. Additional Nipped-A mutant alleles have been sequenced, and all contain point mutations that alter the protein coding sequence (Gause, 2006).

The results show that the major Nipped-A transcript differs from a previously reported splicing pattern, which appears to be a rare variant. Antibodies against a polypeptide encoded largely by the rare exons detect a weak Tra1/TRRAP signal in Western blot assays of concentrated nuclear extracts or purified complexes, confirming that the variant produces Tra1/TRRAP protein in vivo. The rare transcript does not, however, support at least one essential function of Nipped-A and Notch signaling in the wing margin because mutation of a splice site in Nipped-ANC106 for an exon that is not included in the rare variant is lethal and causes defects in Notch signaling. Nipped-ANC106, however, had little effect on the NAx-E2 wing vein phenotype, raising the possibility that the alternatively spliced product can support Notch function in developing wing veins (Gause, 2006).

An unexpected finding is that the Nipped-ANC105 allele, which encodes the N-terminal 2,048 residues of Tra1/TRRAP, suffices to replace one wild-type copy of Nipped-A to support Notch and Mastermind function in vivo. This was unexpected because the protein encoded by Nipped-ANC105 lacks the ATM-PI3 kinase motif which, in mammalian cell culture experiments, is required for Tra1/TRRAP to associate with Gcn5 and Tip60. One possible explanation is that the C terminus of the Nipped-A protein is not required for Notch and Mastermind function and that the truncated protein can replace the full-length protein. Because the effects of the Nipped-A mutations on Notch functions in wing development could only be studied in the presence of a wild-type allele, it is also possible that a truncated protein somehow increases the activity of the remaining full-length Nipped-A protein. The truncated protein could not be detected in Western blot assays of extracts or by immunostaining, suggesting that if this is the case, only a small amount of the mutant protein is sufficient. It is considered improbable that linked second-site mutations are masking effects of Nipped-ANC105 on both Notch mutant phenotypes and the mastermind phenotype. Many mutations have effects similar to Nipped-A, and few have opposing effects, and it would likely require multiple mutations to counteract the effects of Nipped-ANC105 on all three phenotypes. It is also unlikely that there is a linked second-site mutation that counteracts the effects of Nipped-ANC105 by increasing the expression of wild-type Nipped-A, because mutant embryos and larvae show the expected decrease in full-length Nipped-A protein (Gause, 2006).

The Nipped-ANC194 allele, which encodes residues 1 to 1500, had a significant effect on both of the Notch mutant phenotypes but did not increase the severity of the wing-nicking phenotype displayed by mamg2. Again, this differs from null alleles of Nipped-A, which affect all three phenotypes, suggesting that Nipped-ANC194 retains sufficient activity to replace one copy of the wild type in support of Mastermind activity. Again, one possible explanation is that Nipped-A residues 1 to 1500 are sufficient to support Mastermind function, although it is conceivable that the truncated protein somehow increases the activity of the remaining wild-type Nipped-A protein. It was not possible to detect this truncated protein, suggesting that if a truncated protein is responsible, only low levels are required. Despite extensive screens with a deficiency collection and candidate genes, no mutations that suppress mastermind mutant phenotypes have been mapped to chromosome 2. Thus, it is unlikely that a linked second-site mutation masks an effect of Nipped-ANC194 on the mastermind phenotype. Similar to Nipped-ANC105, heterozygous Nipped-ANC194 mutants display the expected reduced levels of full-length protein, although the possibility cannot be excluded of a subtle increase in the expression of the wild-type Nipped-A allele that is sufficient to rescue the mastermind phenotype but not the Notch mutant phenotypes (Gause, 2006).

Isolation and analysis of additional Nipped-A truncation alleles and development of more sensitive biochemical assays will lead to a fuller understanding of how Nipped-A alleles encoding truncated proteins support Notch signaling (Gause, 2006).

The experiments presented in this study indicate that the roles of Nipped-A in supporting Mastermind function likely involve both the SAGA and Tip60 complexes. The Ada2b protein is specific to SAGA, and Ada2b mutations affect the mastermind phenotype but not the two Notch mutant phenotypes. It is thought unlikely that the effect of the Ada2b mutations is more specific than Nipped-A mutations because the mastermind phenotype is more sensitive. As shown by the Nipped-ANC96 hypomorph, the Nnd-1 phenotype is more sensitive to the Nipped-A dosage than is mastermind. Moreover, the Nipped-ANC194 allele has a specificity opposite that of the Ada2b mutations and affects the Notch mutant phenotypes but not the mastermind phenotype. Combined, the contrasts in the effects of Ada2b and various Nipped-A mutations show that Nipped-A and its complexes play multiple roles in Notch signaling. They suggest that the SAGA complex, or at least the Ada2b subunit, is more specific for Mastermind function and that Nipped-A has additional functions (Gause, 2006).

Another possibility raised by the specificity of the effects of Ada2b mutations for effects on Mastermind activity in wing margin development is that Mastermind may have functions in margin development independent of Notch. For example, Mastermind could conceivably function as a coactivator for other activator proteins in addition to Notch. This possibility is consistent with the binding of Mastermind to several sites in polytene chromosomes, including the ecdysone-dependent puffs (Gause, 2006).

The Domino protein, a putative ATPase remodeling enzyme, is a subunit of the Tip60 complex. The Nnd-1 and NAx-E2 phenotypes and the Mastermind phenotype are modified by domino mutations, although the effect on NAx-E2 is modest. These effects are similar to those of the Nipped-ANC106 allele and thus suggest that the Tip60 complex also supports Mastermind function and Notch signaling during wing development. It is possible, however, that Domino functions independently of Tip60 and Nipped-A because the human Domino homologue SRCAP interacts directly with the CBP HAT enzyme that interacts with Mastermind. Nevertheless, the likely involvement of the Tip60 complex raises the possibility that histone exchange could facilitate transcriptional activation by Notch because, in addition to acetylating histone H4, Tip60 exchanges histone H2 variants during DNA repair (Gause, 2006).

As revealed by immunostaining of salivary gland polytene chromosomes, at least one function of Nipped-A is to regulate the binding of Mastermind to chromosomes. The reduction in binding of Mastermind to polytene chromosomes caused by the hypomorphic Nipped-ANC96 and Nipped-ANC186 alleles is dramatic. Supporting the idea that Nipped-A directly regulates Mastermind binding, virtually all sites on polytene chromosomes that bind Mastermind also bind Nipped-A. A few possible explanations for these results are envisioned. The SAGA and Tip60 complexes that contain Nipped-A could acetylate Mastermind, proteins in the Notch activator complex, and/or possibly histones to facilitate binding of the Notch activator complex to chromatin. These modifications could be made by Gcn5 and/or Tip60, which acetylate histones H3 and H4, respectively. Alternatively, Nipped-A or its complexes could bind to chromosomes cooperatively with Mastermind. This would be consistent with the published observation that the ankyrin repeats of the NICD fragment of Notch, which help recruit Mastermind to the Notch activator complex, are also required to recruit Gcn5/PCAF SAGA subunit in transfected mouse cells. Both the Ada2b component of SAGA and the Domino subunit of Tip60 affect Mastermind function, so it is likely that Nipped-A supports Mastermind function in more than one way (Gause, 2006).

Because the evidence suggests that Nipped-A supports Mastermind function through both the SAGA and Tip60 chromatin-modifying complexes, it is theorized that, in addition to controlling the binding of Mastermind to chromosomes, Nipped-A could also cooperate with Mastermind to recruit these complexes to facilitate transcriptional activation through chromatin modification (Gause, 2006).

The data indicate that the SAGA complex, or at least its Ada2b subunit, is not required for some functions of Nipped-A in Notch signaling. Unlike Nipped-A and domino mutations, Ada2b mutations did not affect Notch mutant phenotypes, while they did enhance the phenotype caused by a mastermind mutation. It is postulated, therefore, that the Tip60 complex is also required for functions of Nipped-A beyond controlling the binding of Mastermind to chromosomes. The Tip60 complex could affect the expression of Notch activator complex components, or it could modify proteins in the Notch activator complex. It is also possible that Tip60 modifies chromatin to either aid binding of the Su(H) protein to the Notch target genes or, as mentioned above, to aid transcriptional activation by the Notch activator complex. In any case, the evidence indicates that two subunits of Tip60, Nipped-A and Domino, play more than one role in Notch signaling during wing development (Gause, 2006).

Mutations in the extra sex combs and Enhancer of Polycomb genes increase homologous recombination in somatic cells of Drosophila melanogaster

Heterozygous mutant alleles of E(Pc) and esc increase homologous recombination from an allelic template in somatic cells in a P-element-induced double-strand break repair assay. Flies heterozygous for mutant alleles of these genes showed increased genome stability and decreased levels of apoptosis in imaginal discs and a concomitant increase in survival following ionizing radiation. It is proposed that this was caused by a genomewide increase in homologous recombination in somatic cells. A double mutant of E(Pc) and esc had no additive effect, showing that these genes act in the same pathway. Finally, it was found that a heterozygous deficiency for the histone deacetylase, Rpd3, masked the radiation-resistant phenotype of both esc and E(Pc) mutants. These findings provide evidence for a gene dosage-dependent interaction between the Esc/E(z) complex and the Tip60 histone acetyltransferase complex. It is proposed that esc and E(Pc) mutants enhance homologous recombination by modulating the histone acetylation status of histone H4 at the double-strand break (Holmes, 2006; full text of article).

Eukaryotes use both homologous recombination and nonhomologous end-joining to repair DSBs; survival is compromised severely when both are inactivated. In a homologous recombination assay, every cell in the adult head had a double-stranded break (DSB) generated at the white locus by excision of the P element. This was true for flies heterozygous for either the esc6 or the E(Pc)1 mutation and for wild-type flies. These breaks were repaired either by homologous recombination using the allelic white gene or by nonhomologous end-joining. Heterozygous mutants of any of three genes, E(Pc), Pcl, or esc, increased the likelihood that homologous recombination was used to repair a DSB made by P-element excision in cells of the developing eye-antennal imaginal disc. Thus, although nonhomologous end-joining was not measured directly, it was possible to conclude that the increase in pigmentation indicated an increase in the repair of DSBs by homologous recombination at the expense of nonhomologous end-joining and that heterozygosity for null alleles of either gene changed the balance between these two pathways. Further studies will be needed to determine if there is a defect in nonhomologous end-joining that is compensated by an increase in homologous recombination, or if nonhomologous end-joining is unaffected and homologous recombination is enhanced (Holmes, 2006).

The repair bias toward homologous recombination was observed when either paired or unpaired allelic templates were used. This suggested that the effects of the esc and E(Pc) mutants were not related to any chromosome-pairing-dependent activities of these genes (Holmes, 2006).

Alterations in DSB repair capacity or DSB pathway choice were not restricted to DSBs made by P-element excision at the white locus. Heterozygous mutants of either esc or E(Pc) showed a significant increase in genome integrity and a significant decrease in apoptosis following exposure to ionizing radiation. Not surprisingly, these animals were somewhat resistant to high doses of ionizing radiation (Holmes, 2006).

The increase in homologous recombination and the increase in genome integrity seen in esc6 heterozygous animals was suppressed by a P{esc+} transgene. This demonstrated that the effects were specific to the esc mutations and were not caused by genetic background effects. While the effect of the E(Pc)1 mutant was not suppressed with a transgene, it was observed that a large deficiency that included the E(Pc) gene had a similar effect to that of E(Pc)1 in the homologous recombination assay (Holmes, 2006).

Notably, the increase in homologous recombination was not seen in animals heterozygous for either single mutant of Psc. Furthermore, Psc, Pc, or Scm heterozygous animals are not resistant to ionizing radiation. The esc, Pcl, E(Pc), and Psc genes produce proteins that are localized to three different complexes. The proteins produced from the esc and Pcl genes are members of the esc/E(z) histone H3 methyltransferase complex, the E(Pc) protein is found in the Tip60/NuA4 histone acetyltransferase complex, and the Psc, Pc, and Scm proteins are components of the PRC1 complex. Interestingly, the esc and E(Pc) mutants alter the choice of repair pathway by a common mechanism since the esc6/E(Pc)1 double mutant lacked an additive effect in the homologous recombination assay over either single mutant. This suggests that the effects that were observed on the choice of repair pathway are not linked to the methylation activity of the Esc/E(z) complex or to the methyl-H3-binding activity of PCR1, but rather are linked to some other function that connects the Esc/E(z) and Tip60/NuA4 complexes (Holmes, 2006).

Since the Esc/E(z) complex often associates with a histone chaperone and the Rpd3 histone deacetylase and the E(Pc) protein is a component of a histone acetyl transferase known to be required for DSB repair in yeast, the hypothesis was tested that histone acetylation forms a link between these two complexes, and it was found that a heterozygous mutation for Rpd3 blocked the ability of heterozygous esc6 or E(Pc)1 alleles to confer resistance to ionizing radiation. This suggests that the Rpd3 gene product acts upstream of the esc and E(Pc) proteins in a pathway that influences the choice of which mechanism is used to repair DSBs (Holmes, 2006).

In yeast, the acetylation status of histone H4 plays a crucial role in determining whether a DSB is repaired by nonhomologous end-joining or by homologous recombination, and this role is distinct from its role in regulation of gene expression. Recent work in yeast and Drosophila shows that the NuA4/Tip60 histone acetyltransferase complex [which includes the yeast E(Pc) ortholog] is recruited to DSB sites and acetylates the tail lysines of histone H2A and H4. Histone acetylation is thought to neutralize the positively charged histone tail, thereby reducing the affinity between DNA and histones and loosening the compaction of the chromatin. The homologous recombination or nonhomologous end-joining repair machinery can then gain access to the damage and facilitate repair (Holmes, 2006).

Furthermore, nonhomologous end-joining requires the acetylation of all four lysine residues on the H4 histone tail, whereas homologous recombination requires only partial acetylation of these lysines. Likewise, mutations in the nonessential NuA4 subunit, Yng2, result in global hypoacetylation of histone H4 and are synthetic lethal with the YKU70 gene, further supporting the argument that nonhomologous end-joining requires complete acetylation of histone H4. Finally, yeast with hypoacetylated histone H4 not only are proficient in homologous recombination but also show enhanced recombination in a sister-chromatid exchange assay. These data suggest a compensatory relationship between nonhomologous end-joining and homologous recombination in Saccharomyces cerevisiae (Holmes, 2006).

The yeast data, together with the current results, suggest a model for the gene dose-dependent interaction among Rpd3, Esc, and E(Pc) in Drosophila. It is proposed that, similar to S. cerevisiae, nonhomologous end-joining in Drosophila somatic cells is dependent upon full acetylation of histone H4 tails, but that homologous recombination is not. In this model, a heterozygous E(Pc) mutation would cause a decrease in E(Pc) protein levels, resulting in a decrease in activity of the Tip60 histone acetyltransferase at the DSB and a shift toward homologous recombination. Likewise, a heterozygous esc mutation would result in less of the esc/E(z) complex. Since the Rpd3 protein is found in several different complexes aside from the Esc/E(z) complex, a decrease in the amount of Esc/E(z) complex would result in more free Rpd3 protein in the cell. The excess free Rpd3 protein could deacetylate more of the histone H4 at the DSB and thus shift DSB repair toward homologous recombination. In either instance, a decrease in the levels of Rpd3 protein in the presence of mutations in either esc or E(Pc) might be expected to result in more complete histone H4 acetylation and to restore the normal balance between homologous recombination and nonhomologous end-joining (Holmes, 2006).

It is becoming increasingly clear that PcG proteins have functions beyond the regulation of homeotic genes. Many recent studies have identified deregulation of different PcG proteins (Ezh2, Pcl, Bmi-1) in tumorigenesis, suggesting increased proliferation as a possible mechanism. If Esc or E(Pc) proteins were highly expressed in mammalian cancer, one might expect more frequent nonhomologous end-joining and, consequently, genome instability. Conversely, loss of one copy of either gene may provide a survival advantage under challenge with ionizing radiation or radiomimetic drugs. It is thus possible that the increased proliferation of tumor cells with mutations in these genes is, in part, not a direct result of increased expression of these genes, but rather a secondary effect of genome instability caused by decreases in gene conversion and increases in error-prone nonhomologous end-joining (Holmes, 2006).

Regulation of microtubule assembly and organization in mitosis by Pontin and Reptin

To identify novel proteins important for microtubule assembly in mitosis, a centrosome-based complementation assay was used to enrich for proteins with mitotic functions. An RNA interference (RNAi)-based screen of these proteins uncover 13 novel mitotic regulators. In-depth analyses was carried out of one of these proteins, Pontin, which is known to have several functions in interphase, including chromatin remodeling, DNA repair, and transcription. Reduction of Pontin by RNAi resulted in defects in spindle assembly in Drosophila S2 cells and in several mammalian tissue culture cell lines. Further characterization of Pontin in Xenopus egg extracts demonstrates that Pontin interacts with the γ tubulin ring complex (γ-TuRC). Because depletion of Pontin leads to defects in the assembly and organization of microtubule arrays in egg extracts, these studies suggest that Pontin has a mitosis-specific function in regulating microtubule assembly (Ducat, 2008).

This study identified both Pontin and Reptin, two proteins with well-established functions in chromatin remodeling, as being present in the centrosome-complementing fraction. Using Xenopus egg extract, both Pontin and Reptin were shown to interact with γTuRC, and they are required for the egg extract to nucleate and organize robust microtubule arrays. This suggests that both proteins have a mitosis-specific function in regulating the microtubule cytoskeleton. Consistent with this, reduction of Pontin in tissue culture cells resulted in increased spindle defects. However, it is thought likely that not all of the mitotic defects observed in Pontin RNAi-treated cells were solely due to deregulation of microtubules in mitosis. For example, the mitotic cell death phenotype observed cannot be simply explained by spindle defects alone, because defective spindle assembly often triggers the spindle checkpoint, typically leading to a more prolonged mitotic arrest in the cell lines used. Mitotic death (also referred to as mitotic catastrophe) can result from imbalanced transcription of apoptotic regulators or from irresolvable DNA damage. It has been shown that reduction of RanBP1, a regulator of the Ran system, also causes mitotic cell death, likely due to combined defects in mitotic spindle assembly and spindle checkpoint signaling. Because Pontin is a component of many interphase complexes, including those involved in chromatin function, it is suggested that the mitotic cell death phenotype could be caused by a combination of cellular defects, including defects in spindle formation and chromatin organization due to lack of Pontin. In this context, it will be interesting to analyze whether Pontin reduction results in DNA damage or disorganization of centromeric DNA (which could affect kinetochore functions), and whether this contributes to the observed mitotic cell death (Ducat, 2008).

Interestingly, whereas RNAi-mediated depletion of Pontin in Drosophila and in different human cell lines caused defective spindle assembly and mitotic cell death, similar reduction of Reptin did not affect mitosis. However, Reptin depletion was able to enhance the mitotic defects observed after Pontin knockdown. One possible explanation is that Reptin functions together with Pontin to regulate microtubule assembly, but the different functions that Pontin and Reptin perform in interphase could result in different mitotic phenotypes. Consistent with this, although Pontin and Reptin are related ATPases and they are found together in several chromatin remodeling complexes, they do not always function in the same manner in interphase. In fact, expression levels of these proteins are not always similar, and certain protein complexes contain only one of these proteins. Similarly, it has been shown that overexpression of Pontin in tissue culture cells can displace Reptin from the transcription factor c-Myc. Furthermore, Pontin and Reptin oppose one another to control transcription in the β-catenin-TCF pathway in both Drosophila and zebrafish. Therefore, knockdown of Reptin alone might affect Pontin-independent pathways in interphase that obscure any mitotic phenotype or preclude entry into mitosis. Alternatively, whereas both Pontin and Reptin are needed for the full rescue of microtubule assembly in Xenopus egg extracts, it is possible that the mitotic function of Reptin in assisting Pontin assembly is masked by other proteins in tissue culture cells. Currently, these two possibilities cannot be distinguished (Ducat, 2008).

How might Pontin and Reptin regulate microtubule assembly? Both Pontin and Reptin are members of the AAA+ family of ATPases, which are typically involved in regulating protein-protein and protein-DNA interactions in many cellular contexts. Consistent with a general role in assembly of diverse cellular complexes, Pontin and Reptin have been shown to transiently associate with U3 box C/D pre-small nucleolar ribonucleoproteins and the Ino80 chromatin remodeling complex in a manner that is essential for the full assembly of each complex. Interestingly, Pontin and Reptin are also rapidly up-regulated in response to flagellar reassembly in Chlamydomonas, a process that strongly increases expression of many microtubule-related factors, heat-shock proteins, and tubulin chaperones. In this context, it is tempting to speculate that Pontin, and possibly Reptin, may function as chaperones to facilitate microtubule assembly by transiently interacting with γTuRC. Such a chaperone function could facilitate the localization of γTuRC to both spindle poles and along spindle microtubules (Ducat, 2008).

reptin: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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