InteractiveFly: GeneBrief

Nipped-A: Biological Overview | References

BIOLOGICAL OVERVIEW

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).

The Notch receptor controls many developmental processes through activation of specific target genes. One of the key paradigms for how Notch controls development is provided by its role in definition of the dorsal-ventral compartment boundary of the developing wing in Drosophila. Activation of Notch at the dorsal-ventral boundary results in expression of the vestigial and cut genes, which are required for margin development and wing growth. Notch activates vestigial through its boundary enhancer, and the vestigial protein activates cut through its margin enhancer (Gause, 2006 and references therein).

Nipped-A mutations were isolated in a genetic screen for factors that regulate activation of cut by the wing margin enhancer (Rollins, 1999); these mutations 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 (N^nd-1) mutation and decrease the severity of the vein-shortening phenotype caused by a gain-of-function Notch mutation (N^Ax-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 (Rollins, 1999). 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 (Myster, 2004). Tra1/TRRAP is a key component of the SAGA and Tip60 (NuA4) chromatin-remodeling complexes in yeast, flies, and humans. Drosophila Tra1 is a component of large multiprotein complexes that include Ada3, Ada2, Spt3 and associates with Gcn5 HAT activities. The human and yeast homologues of these factors are components of the SAGA-related GNAT complexes SAGA, PCAF, STAGA, and TFTC. SAGA-related complexes share a significant number of conserved subunits, and the SAGA, PCAF, and STAGA complexes have similar molecular masses of 1.8 to 2 MDa. The TFTC complex is 1.35 MDa, suggesting that higher eukaryotes might have more SAGA-related subcomplexes than yeast. Drosophila SAGA-related complexes elute from size exclusion columns in a comparable molecular-mass range. Moreover, size exclusion chromatography using a S400 column suggests that dGcn5, dAda3, dAda2b, and dSpt3 eluted in two peaks of 2 and 1.6 MDa. It therefore appears likely that Drosophila also might have different subtypes of SAGA-related complexes, as has been described for humans. The striking similarity in the sizes of Drosophila and human SAGA-type complexes suggests that their compositions might be very similar. This is supported by findings that Drosophila homologues of SAGA-specific human Tafs, such as dmTaf5, dmTaf9, and dmTaf10, associate with the dSAGA subunits dAda3, dAda2b, and dSpt3. In addition, dSAGA-type complexes interact with the acidic activator VP16 and the Drosophila p53 homologue. This suggests that these chromatin-remodeling complexes might have similar functions in the regulation of gene expression in flies and humans (Kusch, 2003).

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 (reviewed in reference Carrozza, 2003). Mammalian Tra1/TRRAP was first identified as a coactivator that interacts directly with the Myc and E2F activators (McMahon, 1998). Tra1/TRRAP is also a target of several other activators in yeast and mammalian cells, including Gal4 (Bhaumik, 2004), E1A (Deleu, 2001), VP16 (Memedula, 2003), nuclear receptors (Yanagisawa, 2002), and p53 (Ard, 2002). 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 (Park, 2001). The C terminus is also critical (Brown, 2001) 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 (Kurooka, 2000). 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).

A molecular genetic analysis of several Nipped-A mutations 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 in this study, combined with the finding that two Nipped-A mutants have point mutations in the Tra1/TRRAP gene (Myster, 2004), 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-A^NC106 for an exon that is not included in the rare variant is lethal and causes defects in Notch signaling. Nipped-A^NC106, however, had little effect on the N^Ax-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-A^NC105 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-A^NC105 lacks the ATM-PI3 kinase motif which, in mammalian cell culture experiments, is required for Tra1/TRRAP to associate with Gcn5 and Tip60 (Park, 2001). 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-A^NC105 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-A^NC105 on all three phenotypes. It is also unlikely that there is a linked second-site mutation that counteracts the effects of Nipped-A^NC105 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-A^NC194 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 mam^g2. Again, this differs from null alleles of Nipped-A, which affect all three phenotypes, suggesting that Nipped-A^NC194 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-A^NC194 on the mastermind phenotype. Similar to Nipped-A^NC105, heterozygous Nipped-A^NC194 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 (Kusch, 2003), 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-A^NC96 hypomorph, the N^nd-1 phenotype is more sensitive to the Nipped-A dosage than is mastermind. Moreover, the Nipped-A^NC194 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 (Kusch, 2004). The N^nd-1 and N^Ax-E2 phenotypes and the Mastermind phenotype are modified by domino mutations, although the effect on N^Ax-E2 is modest. These effects are similar to those of the Nipped-A^NC106 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 (Fryer, 2002). 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-A^NC96 and Nipped-A^NC186 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).

Search PubMed for articles about Drosophila Nipped-A

Ard, P. G., et al. (2002). Transcriptional regulation of the mdm2 oncogene by p53 requires TRRAP acetyltransferase complexes. Mol. Cell. Biol. 22: 5650-5661. PubMed ID: 12138177

Bhaumik, S. R., Raha, T. Aiello, D. P. and Green, M. R. (2004). In vivo target of a transcriptional activator revealed by fluorescence resonance energy transfer. Genes Dev. 18: 333-343. PubMed ID: 14871930

Brown, C. E., et al. (2001). Recruitment of HAT complexes by direct activator interactions with the ATM-related Tra1 subunit. Science 292: 2333-2337. PubMed ID: 11423663

Carrozza, M. J., Utley, R. T., Workman, J. L. and Cote, J. (2003). The diverse functions of histone acetyltransferase complexes. Trends Genet. 19: 321-329. PubMed ID: 12801725

Deleu, L., et al. (2001). Recruitment of TRRAP required for oncogenic transformation by E1A. Oncogene 20: 8270-8275. PubMed ID: 11781841

Fryer, C. J., et al. (2002). Mastermind mediates chromatin-specific transcription and turnover of the Notch enhancer complex. Genes Dev. 16: 1397-1411. PubMed ID: 12050117

Gause, M., Eissenberg, J. C., Macrae, A. F., Dorsett, M., Misulovin, Z., Dorsett, D. (2006). Nipped-A, the Tra1/TRRAP subunit of the Drosophila SAGA and Tip60 complexes, has multiple roles in Notch signaling during wing development. Mol. Cell. Biol. 26(6): 2347-59. PubMed ID: 16508010

Kurooka, H., and Honjo, T. (2000). Functional interaction between the mouse notch1 intracellular region and histone acetyltransferases PCAF and GCN5. J. Biol. Chem. 275: 17211-17220. PubMed ID: 10747963

Kusch, T., Guelman, S., Abmayr, S. M. and Workman, J. L. (2003). Two Drosophila Ada2 homologues function in different multiprotein complexes. Mol. Cell. Biol. 23: 3305-3319. PubMed ID: 12697829

Kusch, T., et al. (2004). Acetylation by Tip60 is required for selective histone variant exchange at DNA lesions. Science 306: 2084-2087. PubMed ID: 15528408

McMahon, S. B., et al. (1998). The novel ATM-related protein TRRAP is an essential cofactor for the c-Myc and E2F oncoproteins. Cell 94: 363-374. PubMed ID: 9708738

Memedula, S. and Belmont, A. S. (2003). Sequential recruitment of HAT and SWI/SNF components to condensed chromatin by VP16. Curr. Biol. 13: 241-246. PubMed ID: 12573221

Myster, S. H., et al. (2004). Genetic and bioinformatic analysis of 41C and the 2R heterochromatin of Drosophila melanogaster: a window on the heterochromatin-euchromatin junction. Genetics 166: 807-822. PubMed ID: 15020470

Park, J., Kunjibettu, S. McMahon, S. B. and Cole, M. D. (2001). The ATM-related domain of TRRAP is required for histone acetyltransferase recruitment and Myc-dependent oncogenesis. Genes Dev. 15: 1619-1624. PubMed ID: 11445536

Rollins, R. A., Morcillo, P. and Dorsett, D. (1999). Nipped-B, a Drosophila homologue of chromosomal adherins, participates in activation by remote enhancers in the cut and Ultrabithorax genes. Genetics 152: 577-593. PubMed ID: 10353901

Yanagisawa, J., et al. (2002). Nuclear receptor function requires a TFTC-type histone acetyl transferase complex. Mol. Cell 9: 553-562. PubMed ID: 11931763

date revised: 2 December 2007

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