InteractiveFly: GeneBrief

domino: Biological Overview | References

BIOLOGICAL OVERVIEW

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).

Splice variants of the SWR1-type nucleosome remodeling factor Domino have distinct functions during Drosophila melanogaster oogenesis

SWR1-type nucleosome remodeling factors replace histone H2A by variants to endow chromatin locally with specialized functionality. In Drosophila melanogaster a single H2A variant, H2A.V, combines functions of mammalian H2A.Z and H2A.X in transcription regulation and the DNA damage response. A major role in H2A.V incorporation for the only SWR1-like enzyme in flies, Domino, is assumed but not well documented in vivo. It is also unclear whether the two alternatively spliced isoforms, DOM-A and DOM-B, have redundant or specialized functions. Loss of both DOM isoforms compromises oogenesis, causing female sterility. This study systematically explored roles of the two DOM isoforms during oogenesis using a cell type-specific knockdown approach. Despite their ubiquitous expression, DOM-A and DOM-B have non-redundant functions in germline and soma for egg formation. It was shown that chromatin incorporation of H2A.V in germline and somatic cells depends on DOM-B, whereas global incorporation in endoreplicating germline nurse cells appears to be independent of DOM. By contrast, DOM-A promotes the removal of H2A.V from stage 5 nurse cells. Remarkably, therefore, the two DOM isoforms have distinct functions in cell type-specific development and H2A.V exchange (Börner, 2016).

In D. melanogaster the properties of the two ancient, ubiquitous histone H2A variants H2A.X and H2A.Z are combined in a single molecule, H2A.V. Given that H2A.V carries out functions as a DNA damage sensor and architectural element of active promoters, as well as having further roles in heterochromatin formation, this histone appears loaded with regulatory potential. Accordingly, placement of the variant, either randomly along with canonical H2A during replication or more specifically through nucleosome remodeling factors, becomes a crucial determinant in its function. Mechanistic detail for replacement of H2A-H2B dimers with variants comes from the analysis of the yeast SWR1 complex, which incorporates H2A.Z in a stepwise manner at strategic positions next to promoters (Börner, 2016).

So far, the published phenotypes associated with dom mutant alleles have not been systematically complemented. The comprehensive complementation analysis of this study shows that dom mutant phenotypes are indeed due to defects in the dom gene. Remarkably, dom lethality and sterility can be partially rescued by complementation with the orthologous human SRCAP gene, providing an impressive example of functional conservation of SWR1-like remodelers. The contributions of the two splice variants DOM-A and DOM-B had not been assessed. This study now demonstrates that both isoforms are essential for development, suggesting non-redundant functions. The DOM-A isoform contains a SANT domain followed by several poly-Q stretches, which are widely found in transcriptional regulators, where they may modulate protein interactions. By contrast, SANT domains are thought to function as histone tail interaction modules that couple binding to enzyme catalysis. Therefore, the SANT domain in DOM-A could mediate specificity towards H2A.V eviction depending on particular functional contexts. These features are also present in the C-terminus of p400 (EP400), the second human SWR1 ortholog, but are absent in either DOM-B or SRCAP. Remarkably, p400 interacts directly with TIP60 and the SANT domain of p400 inhibits TIP60 catalytic activity providing an interesting lead for further investigation of DOM isoforms and TIP60 interactions (Börner, 2016).

It is speculated that distinct functions of p400 and SRCAP in humans might be accommodated to some extent by the two DOM isoforms in flies. Accordingly, it will be interesting to explore whether the two isoforms reside in distinct complexes. Previous affinity purification of a TIP60-containing complex using a tagged pontin subunit apparently only identified DOM-A, but not DOM-B. Following up on the initial observation of early defects in GSCs and cyst differentiation upon loss of DOM (Yan, 2014), this study now finds that this phenotype is exclusively caused by loss of DOM-A. Interestingly, studies with human embryonic stem cells show that p400/TIP60 (KAT5) integrates pluripotency signals to regulate gene expression, suggesting similar roles for DOM-A in GSCs. This is in contrast to requirements for both isoforms for germline development outside of the germarium, highlighting a developmental specialization of the two DOM remodelers (Börner, 2016).

DOM is also involved in the differentiation and function of SSCs in the germarium. The data now document non-redundant requirements of both DOM isoforms in somatic cells for proper coordination of follicle cell proliferation with cyst differentiation. Failure to adjust these two processes leads to 16-cell cyst packaging defects that manifest as compound egg chambers. These rare phenotypes had previously only been described upon perturbation of some signaling pathways, such as Notch, or Polycomb regulation (Börner, 2016).

Because the phenotypes of DOM depletions resemble those of H2A.V depletion, the idea was favored that many of the cell-specification defects are due to compromised H2A.V incorporation, depriving key promoters of the H2A.Z-related architectural function. Alternatively, scaffolding activities might partially explain some roles of chromatin remodelers, as suggested for SRCAP. So far, knowledge of the mechanisms of H2A.V incorporation has been anecdotal. This comprehensive analysis revealed a specific involvement of DOM-B for the incorporation of H2A.V into chromatin at the global level. The N-termini of SWR1 and DOM-B harbor the HSA and ATPase spacer domains, with interaction surfaces for further complex subunits, and an additional H2A.Z-H2B dimer binding site. Given the requirement for both isoforms for cell specification during oogenesis, it is speculated that DOM-B might serve to incorporate bulk H2A.V into chromatin similar to SWR1, whereas DOM-A would be more involved in the regulatory refinement of location (Börner, 2016).

Although the failures in cell specification and egg morphogenesis are likely to be explained by loss of the H2A.Z-related features of H2A.V, ablation of DOM might also compromise the DNA damage response, which involves phosphorylation of H2A.V (γH2A.V). Conceivably, the role of γH2A.V as a DNA damage sensor might be best fulfilled by a broad distribution of H2A.V throughout the chromatin. Such an untargeted incorporation may be achieved by stochastic, chaperone-mediated incorporation during replication or by an untargeted activity of DOM-B. DOM-independent incorporation in endoreplicating polyploid nurse cells of stage 3 egg chambers is observed, where global H2A.V and γH2A.V signals did not depend on DOM. Immunofluorescence microscopy may lack the sensitivity to detect DOM-dependent incorporation of H2A.V at some specific sites. Nevertheless, DOM-independent incorporation of H2A.V might serve to cope with many naturally occurring DNA double-strand breaks during the massive endoreplication of nurse cells (Börner, 2016).

There is some evidence that nucleosome remodelers not only incorporate H2A variants but can also remove them. In yeast, the genome-wide distribution of H2A.Z appears to be established by the antagonistic functions of the SWR1 and Ino80 remodeling complexes, where Ino80 replaces stray H2A.Z-H2B with canonical H2A-H2B dimers. A recent study identified the vertebrate-specific histone chaperone ANP32E as part of a TIP60/p400 complex that facilitates the eviction of H2A.Z-H2B dimers from chromatin. Remarkably, in D. melanogaster a TIP60/DOM-A complex is involved in a similar reaction. The TIP60/DOM-A complex acetylates γH2A.V at lysine 5 to facilitate exchange of γH2A.V by unmodified H2A.V during the DNA damage response. Furthermore, it has been speculated that H2A.V and γH2A.V could be actively removed from nurse cells, since corresponding signals are absent from stage 5 onwards. This study now demonstrates that depletion of DOM-A and TIP60 leads to the persistence of H2A.V and γH2A.V in nurse cells of late egg chambers, clearly documenting the ability of the remodeler to remove bulk H2A.V and variants modified during DNA damage induction (Börner, 2016).

These findings highlight the specific requirements of DOM splice variants for the incorporation and removal of H2A.V during D. melanogaster oogenesis. It remains an interesting and challenging question how DOM-A and DOM-B complexes are targeted genome-wide and function in vivo to establish specific H2A.V patterns in different cell types during development (Börner, 2016).

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).

The histone variant His2Av is required for adult stem cell maintenance in the Drosophila testis

Many tissues are sustained by adult stem cells, which replace lost cells by differentiation and maintain their own population through self-renewal. The mechanisms through which adult stem cells maintain their identity are thus important for tissue homeostasis and repair throughout life. This study shows that a histone variant, His2Av, is required cell autonomously for maintenance of germline and cyst stem cells in the Drosophila testis. The ATP-dependent chromatin-remodeling factor Domino is also required in this tissue for adult stem cell maintenance possibly by regulating the incorporation of His2Av into chromatin. Interestingly, although expression of His2Av was ubiquitous, its function is dispensable for germline and cyst cell differentiation, suggesting a specific role for this non-canonical histone in maintaining the stem cell state in these lineages (Morillo Prado, 2003).

The results reveal that the histone variant His2Av is required cell autonomously for maintenance of two different adult stem cell types, GSCs and cyst stem cells (CySCs), in the Drosophila male gonad, but not for the differentiation of the progeny in these two stem cell lineages. The specific requirement for His2Av for adult stem cell maintenance suggests that His2Av may play critical role(s) in the mechanisms that maintain the ability of adult stem cells to self-renew rather than differentiate. His2Av function has been implicated in both transcriptional repression and transcriptional activation. His2Av could maintain adult stem cells by either favoring repression of pro-differentiation genes and/or activation of genes necessary for stem cell identity and function. In yeast, H2A.Z occupies transcriptionally inactive genes and intergenic regions, while in Drosophila, His2Av is required for the establishment of heterochromatin and transcriptional repression. Conversely, studies indicate that in Drosophila, yeast, and chicken, His2Av is enriched at nucleosomes downstream of the transcription start site of active or poised genes. Nucleosomes and histone dimers containing H2A.Z appear to be less stable than nucleosomes containing the canonical histone H2A. This lower stability may favor a more open chromatin, giving transcriptional activators or repressors better access to the DNA. Consistent with this model, a recent study showed that H2A.Z promotes self-renewal and pluripotency of murine embryonic stem cells (ESCs) by facilitating the binding of Oct4 to its target genes and the Polycomb repressive complex 2 to differentiation genes (Hu, 2013). However, in ESCs, unlike in Drosophila male GSCs and CySCs, His2A.Z function was also required for the expression of differentiation genes when ESCs were grown under conditions that induce differentiation (Morillo Prado, 2003).

It is proposed that the requirement of His2Av for adult stem cell maintenance, but not for differentiation, may reflect a subtle role for His2Av in maintaining expression of genes required for self-renewal versus differentiation. Adult stem cells lie at the cusp of two alternate fate choices, self-renewal and differentiation; the progeny of stem cell division are maintained in a state where they can execute either self-renewal or differentiation programs depending on local cues. The requirement for this balanced, bi-potential state may make adult stem cells more sensitive to the small alterations in the relative levels of key transcripts associated with the loss of His2Av function, tilting the balance from stem cell maintenance to onset of differentiation. Consistent with the model that His2Av may alter transcriptional levels subtly, H2A.Z was shown to be required to fine-tune the transcriptional state of hsp70 and a wide variety of other genes in response to temperature changes in Arabidopsis (Morillo Prado, 2003).

The ATP-dependent chromatin-remodeling factor Domino is required for GSC and CySC maintenance in the male germline, as previously shown for somatic follicle stem cells in the female gonad. The yeast Swr1 complex containing the homolog of Drosophila Domino exchanges His2A with Htz1 (the yeast His2A variant) and in Drosophila, Domino- containing Tip60 chromatin remodeling complex has been shown to exchange phospho-His2Av with unmodified His2Av in in vitro assays. The current studies indicate that Domino function is required in vivo in GSCs for the incorporation of His2Av into chromatin. Nuclei of domino mutant GSCs had lowered but still detectable levels of His2Av protein, possibly due to the weak domino allele used in this study. Alternatively, incorporation of His2Av in some regions of the chromatin may occur independently of Domino function, as has been reported in yeast, in which stress-responsive genes exhibit Swr1-independent incorporation of Htz in the coding region. Although ISWI, like His2Av, is required for GSC and CySC maintenance in the male germline, these proteins may function in parallel pathways to maintain adult stem cells in the testis. The ISWI containing nucleosome-remodeling factor (NURF) was shown to maintain GSCs and CySCs in the Drosophila testis by positively regulating the JAK-STAT signaling pathway; GSCs mutant for components of the NURF complex exhibited low levels of STAT92E protein. In contrast, as discussed below, His2Av may function independently of the JAK-STAT signaling pathway (Morillo Prado, 2003).

The results indicate that His2Av may function independently of the JAK-STAT signaling pathway to provide a chromatin environment that allows for stem cell maintenance. Expression of the His2Av and STAT92E proteins in GSCs was not dependent on each other. These studies indicate that His2Av may not be required for expression of at least one other key STAT-dependent gene in CySCs. Activation of the JAK-STAT signaling pathway in response to the Upd signal from the hub is important for CySC maintenance, possibly in part through STAT-dependent transcription of Zfh-1. However, CySCs lacking His2Av function still expressed Zfh-1. In GSCs, activation of the JAK-STAT pathway is important for maintaining hub-GSC adhesion and for centrosome orientation, both of which appeared unaffected in His2Av mutant GSCs. Loss of His2Av function did not strongly suppress the phenotype associated with ectopic overexpression of Upd in the testis, although a few His2Av mutant germ cells were able to initiate differentiation, possibly due to relatively lower levels of JAK-STAT activation in these cells. Even though loss of His2Av normally resulted in differentiation of GSCs and CySCs, the requirement for His2Av function can be overridden by high levels of activation of the JAK-STAT pathway, possibly maintaining somatic CySCs in a stem cell like state, which may fail to provide a microenvironment for germ cells to initiate differentiation (Morillo Prado, 2003).

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).

The chromatin remodeling factor Bap55 functions through the TIP60 complex to regulate olfactory projection neuron dendrite targeting

The Drosophila olfactory system exhibits very precise and stereotyped wiring that is specified predominantly by genetic programming. Dendrites of olfactory projection neurons (PNs) pattern the developing antennal lobe before olfactory receptor neuron axon arrival, indicating an intrinsic wiring mechanism for PN dendrites. These wiring decisions are likely determined through a transcriptional program. This study found that loss of Brahma associated protein 55 kD (Bap55) results in a highly specific PN mistargeting phenotype. In Bap55 mutants, PNs that normally target to the DL1 glomerulus mistarget to the DA4l glomerulus with 100% penetrance. Loss of Bap55 also causes derepression of a GAL4 whose expression is normally restricted to a small subset of PNs. Bap55 is a member of both the Brahma (BRM) and the Tat interactive protein 60 kD (TIP60) ATP-dependent chromatin remodeling complexes. The Bap55 mutant phenotype is partially recapitulated by Domino and Enhancer of Polycomb mutants, members of the TIP60 complex. However, distinct phenotypes are seen in Brahma and Snf5-related 1 mutants, members of the BRM complex. The Bap55 mutant phenotype can be rescued by postmitotic expression of Bap55, or its human homologs BAF53a and BAF53b. These results suggest that Bap55 functions through the TIP60 chromatin remodeling complex to regulate dendrite wiring specificity in PNs. The specificity of the mutant phenotypes suggests a position for the TIP60 complex at the top of a regulatory hierarchy that orchestrates dendrite targeting decisions (Tea, 2011).

The stereotyped organization of the Drosophila olfactory system makes it an attractive model to study wiring specificity. The first olfactory processing center is the antennal lobe, a bilaterally symmetric structure at the anterior of the Drosophila brain. It is composed of approximately 50 glomeruli in a three-dimensional organization. Each olfactory projection neuron (PN) targets its dendrites to one of those glomeruli to make synaptic connections with a specific class of olfactory receptor neurons. Each PN sends its axon stereotypically to higher brain centers (Tea, 2011).

During development, the dendrites of PNs pattern the antennal lobe prior to axons of olfactory receptor neurons. The specificity of PN dendrite targeting is largely genetically pre-determined by the cell-autonomous action of transcription factors, several of which have been previously described. Furthermore, chromatin remodeling factors have been shown to play an important role in PN wiring (Tea, 2010), although very little is currently known about their specific functions. This study reports a genetic screen for additional factors that regulate PN dendrite wiring specificity; Brahma associated protein 55 kD (Bap55) was identified as a regulator of PN dendrite wiring specificity as part of the TIP60 chromatin remodeling complex (Tea, 2011).

Bap55 is an actin-related protein, the majority of which physically associates with the Brahma (BRM) chromatin remodeling complex in Drosophila embryo extracts. There are two distinct BRM complexes: BAP (Brahma associated proteins; homologous to yeast SWI/SNF) and PBAP (Polybromo-associated BAP; homologous to yeast RSC), both of which contain Brahma, Bap55, and Snf5-Related 1 (Snr1). The human homologs of the BAP and PBAP complexes are called the BAF (Brg1 associated factors) and PBAF (Polybromo-associated BAF) complexes, respectively. The BRM/BAF complexes are members of the SWI/SNF family of ATP-dependent chromatin-remodeling complexes, and have been shown to both activate and repress gene transcription, in some cases, of the same gene (Tea, 2011).

In experiments purifying proteins in complex with tagged Drosophila Pontin in S2 cells, Bap55 was also co-purified as a part of the TIP60 complex, as determined by mass spectrometry. The TIP60 histone acetyltransferase complex has been shown to be involved in many processes, including both transcriptional activation and repression. The complex contains many components, including Bap55, Domino (Dom), and Enhancer of Polycomb (E(Pc)). Dom, homologous to human p400, is the catalytic DNA-dependent ATPase; its ATPase domain is highly similar to Drosophila Brahma and human BRG1 ATPase domains. E(Pc) is homologous to human EPC1 and EPC2 and is an unusual member of the Polycomb group; it does not exhibit homeotic transformations on its own, but rather enhances mutations in other Polycomb group genes (Tea, 2011).

This study provides evidence that Bap55 functions as a part of the TIP60 complex rather than the BRM complex in postmitotic PNs to control their dendrite wiring specificity (Tea, 2011).

To further understanding of dendrite wiring specificity in Drosophila olfactory PNs, a MARCM-based forward genetic screen was performed using piggyBac insertional mutants. MARCM allows visualization and genetic manipulation of single cell or neuroblast clones in an otherwise heterozygous background, permitting the study of essential genes in mosaic animals. In this screen, GH146-GAL4 was used to label a single PN born soon after larval hatching, which in wild-type (WT) animals always projects its dendrites to the dorsolateral glomerulus DL1 in the posterior of the antennal lobe. The DL1 PN also exhibits a stereotyped axon projection, forming an L-shaped pattern in the lateral horn, with additional branches in the mushroom body calyx. A mutant, called LL05955, was identified in which DL1 PNs mistargeted to the dorsolateral glomerulus DA4l in the anterior of the antennal lobe. This phenotype is strikingly specific, with 100% penetrance. Arborization of mutant axons, however, was not obviously altered. The piggyBac insertion site was identified using inverse PCR and Splinkerette PCR. LL05955 is inserted into the coding sequence of Bap55, encoding a homolog of human BAF53a and BAF53b. Precise excision of the piggyBac insertion reverted the dendrite mistargeting phenotype, confirming that disruption of the Bap55 gene causes the dendrite mistargeting (Tea, 2011).

In addition to causing DL1 mistargeting, Bap55 mutants also display neuroblast clone phenotypes. In WT, GH146-GAL4 can label three distinct types of PN neuroblast clones generated in newly hatched larvae. Two of these clones, the anterodorsal neuroblast clone and the lateral neuroblast clone, possess cell bodies that lie dorsal or lateral to the antennal lobe, respectively. PNs from these two lineages project their dendrites to stereotyped and nonoverlapping subsets of glomeruli in the antennal lobe. The third type of clone, the ventral neuroblast clone, has cell bodies that lie ventral to the antennal lobe and dendrites that target throughout the antennal lobe due to the inclusion of at least one PN that elaborates its dendrites to all glomeruli (Tea, 2011).

In Bap55^-/- PNs, anterodorsal neuroblast clones display a mild reduction in cell number, and their dendrites are abnormally clustered in the anterior dorsal region of the antennal lobe, including the DA4l glomerulus. Lateral neuroblast clones display a severe reduction in cell number, and the remaining dendrites are unable to target to many glomeruli throughout the antennal lobe. Ventral neuroblast clones display a mild reduction in cell number and a reduced dendrite mass throughout the antennal lobe. During development, the lateral neuroblast first gives rise to local interneurons before switching to produce PNs; in mutants affecting cell proliferation, this property of the lateral neuroblast displays as a severe reduction in GH146-labeled PNs. The severely reduced cell number in Bap55 mutants suggests that Bap55 is essential for neuroblast proliferation or neuronal survival. In the anterodorsal and ventral neuroblasts, PN numbers are not drastically changed; thus, the phenotypes indicate that Bap55 is important for dendrite targeting in multiple classes of PNs (Tea, 2011).

In WT, Mz19-GAL4 labels a subset of the GH146-GAL4 labeling pattern. It labels a small number of PNs derived from two neuroblasts, which can be clearly identified in WT clones generated in newly hatched larvae. Anterodorsal neuroblast clones target their dendrites to the VA1d glomerulus, as well as the DC3 glomerulus residing immediately posterior to VA1d (not easily visible in confocal stacks). Lateral neuroblast clones target all dendrites to the DA1 glomerulus. Unlike GH146-GAL4, WT Mz19-GAL4 never labels ventral neuroblast clones because it is not normally expressed in those cells (Tea, 2011).

In Bap55 mutant PN clones, however, Mz19-GAL4 labels additional PNs in anterodorsal, lateral, and ventral clones compared to their WT counterparts. This phenotype suggests that some Mz19-negative PNs now express Mz19-GAL4. In anterodorsal clones, Mz19-GAL4 labels additional cells, although not as many as are labeled by GH146-GAL4. The PNs also mistarget their dendrites to the anterior antennal lobe, similar to mutant GH146-GAL4 anterodorsal neuroblast clones. WT lateral neuroblast clones normally contain GH146-positive PNs and GH146-negative local interneurons. In Bap55^-/- lateral neuroblast clones, Mz19-GAL4 predominantly labels local interneurons that send their processes to many glomeruli throughout the antennal lobe and do not send axon projections to higher brain centers. Lateral clones also show ectopic PN labeling with a lower frequency. The Bap55 mutant appears to cause derepression of Mz19-GAL4, resulting in labeled local interneurons. Ventral neuroblast clones are never labeled in WT Mz19-GAL4, yet are labeled in Bap55 mutants. This further indicates a derepression of the Mz19-GAL4 labeling pattern (Tea, 2011).

Unlike GH146-GAL4, WT Mz19-GAL4 never labels single cell clones when clone induction is performed in newly hatched larvae. This is because Mz19-GAL4 is not expressed in the DL1 PN, the only GH146-positive cell generated during this heat shock time of clone generation. However, in Bap55 mutant PN clones, Mz19-GAL4 ectopically labels single cell anterodorsal PN clones targeting to the DA4l glomerulus, which show an L-shaped pattern in the lateral horn with branches in the mushroom body calyx, similar to GH146-GAL4 labeling. The simplest interpretation is that this compound phenotype reflects first a derepression of Mz19-GAL4 in the DL1 PN, and second a DL1 to DA4l mistargeting phenotype in Bap55 mutants (Tea, 2011).

To test whether Bap55 functions in the neuroblast or postmitotically in PNs, GH146-GAL4, which expresses only in postmitotic PNs, was used to express UAS-Bap55 in a Bap55^-/- single cell clone. The dendrite mistargeting phenotype was shown to be rescued to the WT DL1 glomerulus and it is concluded that Bap55 functions postmitotically to regulate PN dendrite targeting. The axon phenotype remains the stereotypical L-shaped pattern (Tea, 2011).

The Drosophila Bap55 protein is 70% similar and 54% identical to human BAF53a and 71% similar and 55% identical to human BAF53b. BAF53a and b are 91% similar and 84% identical to each other. Using GH146-GAL4 to express human BAF53a or b in a Bap55^-/- single cell clone, it was found that the human homologs can effectively rescue the Bap55^-/- dendrite mistargeting phenotype. Interestingly, both also cause the de novo DM6 dendrite and ventral axon mistargeting phenotypes in 6 out of 19 cases for BAF53a and 2 out of 32 cases for BAF53b. Thus, human BAF53a and b can largely replace the function of Drosophila Bap55 in PNs (Tea, 2011).

To address whether Bap55 functions as a part of the BRM complex in PN dendrite targeting, two additional BRM complex mutants were tested for their PN dendrite phenotypes. First, Brahma (brm), the catalytic ATPase subunit of the BRM complex, which is required for the activation of many homeotic genes in Drosophila, was tested. Null mutations have been shown to decrease cell viability and cause peripheral nervous system defects. RNA interference knockdown of brm in embryonic class I da neurons revealed dendrite misrouting phenotypes, although not identical to the Bap55 phenotype. The human homologs of brm, BRM and BRG1 (Brahma-related gene-1), both have DNA-dependent ATPase activity. Inactivation of BRM in mice does not yield obvious neural phenotypes, but inactivation of BRG1 in neural progenitors results in reduced proliferation. BRG1 is likely to be required for various aspects of neural development, including proper neural tube development (Tea, 2011).

In PNs, brm mutants displayed anterodorsal single cell clone mistargeting to non-stereotyped glomeruli throughout the antennal lobe, with each clone differing from the next. This is in contrast to the highly stereotyped DA4l mistargeting of Bap55 mutants. brm^-/- neuroblast clones also displayed phenotypes where dendrites make small, meandering projections throughout the antennal lobe, which does not resemble the Bap55^-/- phenotype. They additionally exhibit a perturbed cell morphology phenotype, which is markedly more severe than the Bap55 mutant phenotype (Tea, 2011).

Next, Snr1, a highly conserved subunit of the BRM complex, was tested. It is required to restrict BRM complex activity during the development of wing vein and intervein cells and functions as a growth regulator. Its human homolog, SNF5, is strongly correlated with many cancers, yet little is known about its specific role in neurons (Tea, 2011).

In PNs, Snr1 mutants displayed similar phenotypes to brm mutants. The single cell clones displayed mistargeting to non-stereotyped glomeruli throughout the antennal lobe, with each clone demonstrating a unique phenotype. The neuroblast clones exhibited small meandering dendrites throughout the antennal lobe, which also showed extremely perturbed cell morphology. These results, especially the non-sterotyped single cell clone phenotypes, indicate that the BRM complex functions differently from Bap55 in controlling PN dendrite targeting (Tea, 2011).

brm and Snr1 mutants were further analyzed with Mz19-GAL4 to determine whether their phenotypes resembled the Bap55 mutant phenotype of derepression. It was not possible to generate any labeled clones, indicating one of three possibilities: increased cell death, severe cell proliferation defects, or repression of the Mz19-GAL4 labeling pattern. In any of the three cases, the phenotype does not resemble the Bap55^-/- mutant phenotype of abnormal activation of Mz19-GAL4 in single cell or neuroblast clones, indicating that the BRM complex functions differently from Bap55 in PNs (Tea, 2011).

In the same screen in which the Bap55 mutation was identified, LL05537, a mutation in dom that resulted in a qualitatively similar phenotype to Bap55 mutants was identified. dom^-/- DL1 PNs split their dendrites between the posterior glomerulus DL1 and the anterior glomerulus DA4l. Their axons exhibit a WT L-shaped pattern in the lateral horn (Tea, 2011).

The LL05537 allele contains a piggyBac insertion in an intron just upstream of the translation start of dom. Because the piggyBac insertion contains splice acceptor sites and stop codons in all three coding frames, this allele likely disrupts all isoforms of dom. Similarly to Bap55, the piggyBac insertion site was identified using inverse PCR and Splinkerette PCR. Precise excision of the piggyBac insertion reverted the dendrite targeting phenotype, confirming that disruption of the dom gene causes the dendrite mistargeting. In addition, a BAC transgene that contains the entire dom transcriptional unit rescued the dom^-/- mutant phenotypes (Tea, 2011).

Dom is the catalytic DNA-dependent ATPase of the TIP60 complex and has been shown to contribute to a repressive chromatin structure and silencing of homeotic genes. Dom is a member of the SWI/SNF family and its ATPase domain is highly similar to the Drosophila Brahma and human BRG1 ATPase domains. The human homolog of Dom is p400, which is important for regulating nucleosome stability during repair of double-stranded DNA breaks and an important regulator of embryonic stem cell identity (Tea, 2011).

To determine whether Bap55 and Dom genetically interact, UAS-Bap55 was expressed in a dom^-/- PN. This manipulation did not significantly alter the dendrite phenotype. The axon branching pattern also was not altered (Tea, 2011).

Another component of the TIP60 complex, E(Pc), was also examined. In Drosophila, E(Pc) is a suppressor of position-effect variegation and heterozygous mutations in E(Pc) result in an increase in homologous recombination over nonhomologous end joining at double-stranded DNA breaks. Following ionizing radiation, heterozygous animals also exhibit higher genome stability and lower incidence of apoptosis. Yet little is known about its role in neurons (Tea, 2011).

In this study, it was found that E(Pc)^-/- DL1 PN dendrites also mistarget to the anterior glomerulus DA4l and exhibit the stereotyped L-shaped axon pattern in the lateral horn. A BAC transgene that contains the entire E(Pc) transcription unit rescued the E(Pc) mutant phenotypes. To determine whether Bap55 and E(Pc) genetically interact, UAS-Bap55 was expressed in an E(Pc)^-/- DL1 PN. This manipulation caused the dendrites to split between the DA4l and DM6 glomeruli, and resulted in axons targeting ventrally to the lateral horn (Tea, 2011).

Neuroblast clones mutant for dom also exhibit dendrite mistargeting phenotypes to inappropriate glomeruli throughout the antennal lobe. Anterodorsal and lateral neuroblast clones show a very mild reduction in cell number and their dendrites do not target to the full set of proper glomeruli. Ventral neuroblast clones, when compared to WT, exhibit incomplete targeting throughout the antennal lobe (Tea, 2011).

Further analysis of dom mutants by labeling with Mz19-GAL4 revealed the same derepression as in Bap55 mutants. dom mutant Mz19-GAL4 PN clones also label anterodorsal, lateral, and ventral neuroblast clones with phenotypes similar to GH146-GAL4 labeled neuroblast clones. In anterodorsal and lateral neuroblast clones, Mz19-GAL4 labels a large number of PNs that target to many glomeruli throughout the antennal lobe, although the cell number is smaller than GH146-GAL4 labeling. Ventral neuroblast clones are never labeled in WT Mz19-GAL4, yet are labeled in dom mutants. Mz19-GAL4 also labels single cell clones that split their dendrites between the DA4l and DL1 glomeruli and form the stereotypical L-shaped axon pattern in the lateral horn. As in Bap55 mutants, this compound phenotype likely results from ectopic labeling of a DL1 PN, which further mistargets to DA4l (Tea, 2011).

The E(Pc) phenotypes in GH146 and Mz19-GAL4 labeled neuroblast clones, as well as Mz19-GAL4 labeled single cell clones, displayed similar phenotypes to dom as described above. The phenotypic similarities in single cell clone dendrite mistargeting and derepression of a PN-GAL4 in mutations that disrupt Bap55, dom and E(Pc) strongly suggest that these three proteins act together in the TIP60 complex to regulate PN development (Tea, 2011).

This study has demonstrated a similar role for three members of the TIP60 complex in olfactory PN wiring. The TIP60 complex plays a very specific role in controlling dendrite wiring specificity, with a precise mistargeting of the dendrite mass in Bap55, dom, and E(Pc) mutants. This specific DL1 to DA4l mistargeting phenotype has only been seen in these three mutants, out of approximately 4,000 other insertional and EMS mutants screened, supporting the conclusion that the TIP60 complex has a specific function in controlling PN dendrite targeting. TIP60 complex mutants show discrete glomerular mistargeting, rather than randomly distributed dendrite spillover to different glomeruli. In contrast, perturbation of individual cell surface receptors often leads to variable mistargeted dendrites that do not necessarily obey glomerular borders, possibly reflecting the combinatorial use of many cell surface effector molecules. Even transcription factor mutants yield variable phenotypes. Interestingly, BRM complex mutants yield non-stereotyped phenotypes in PNs. No stereotyped glomerular targeting was seen for brm or Snr1 mutant dendrites; each PN spreads its dendrites across different glomeruli. These data suggest that different chromatin remodeling complexes play distinct roles in regulating neuronal differentiation. The uni- or bi-glomerular targeting to specific glomeruli implies that the TIP60 complex sits at the top of a regulatory hierarchy to orchestrate an entire transcriptional program of regulation (Tea, 2011).

This study suggests a function for Bap55 in Drosophila olfactory PN development as a part of the TIP60 complex rather than the BRM complex. Another possibility could be that Bap55 also serves as the interface between the BRM and TIP60 complexes. While loss of core BRM complex components results in a more general defect, loss of Bap55 could specifically disrupt interactions with the TIP60 complex but maintain other BRM complex functions, causing a more specific targeting phenotype mimicking loss of TIP60 complex components (Tea, 2011).

Interestingly, both human BAF53a and b can significantly rescue the Bap55^-/- phenotype. Though in mammals BAF53a is expressed in neural progenitors and BAF53b is expressed in postmitotic neurons, they can perform the same postmitotic function in Drosophila PNs. Further, both can function with the TIP60 complex in PNs to regulate wiring specificity. These data suggest that the functions for BAF53a and b (in neural precursors and postmitotic neurons, respectively) diverge after the evolutionary split between vertebrates and insects (Tea, 2011).

The discrete glomerular states of the mistargeting phenotypes may suggest a role for the TIP60 complex upstream of a regulatory hierarchy determining PN targeting decisions. It is possible that disrupting various components changes the composition of the complex. Additionally, overexpression of Bap55 in various mutant backgrounds might alter the sensitive stoichiometry of the TIP60 complex, resulting in targeting to different but still distinct glomeruli (Tea, 2011).

Several mutants have been identified that cause DL1 PNs to mistarget to areas near the DM6 glomerulus (Tea, 2010). Interestingly, WT DM6 PNs have the most similar lateral horn axon arborization pattern to DL1 PNs. It is hypothesized that the transcriptional code for DM6 is similar to that of DL1, which is at least partially regulated by the TIP60 complex. The genes described in this manuscript are the only mutants that have yielded specific DA4l mistargeting to date. It is possible that the targeting 'code' for DA4l, DL1, and DM6 may be most similar, such that perturbation of the TIP60 complex might result in reprogramming of dendrite targeting. PNs have previously been shown to be pre-specified by birth order. Yet DA4l is born in early embryogenesis, DL1 is born in early larva, and DM6 is born in late larva. This implies that the TIP60 transcriptional code does not correlate with PN birth order. The mechanisms by which the TIP60 complex specifies PN dendrite targeting remain to be determined (Tea, 2011).

This study has characterize PN phenotypes of mutants in the BRM and TIP60 complexes, with a focus on Bap55, which is shared by the two complexes. The TIP60 complex was found to play a very specific role in regulating PN dendrite targeting; mutants mistarget from the DL1 to the DA4l glomerulus. This specific mistargeting phenotype suggests that TIP60 controls a transcriptional program important for making dendrite targeting decisions (Tea, 2011).

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. PubMed ID: 11080158

Börner, K. and Becker, P.B. (2016). Splice variants of the SWR1-type nucleosome remodeling factor Domino have distinct functions during Drosophila melanogaster oogenesis. Development 143: 3154-3167. PubMed ID: 27578180

Boudreault, A. A., et al. (2003). Yeast Enhancer of Polycomb defines global Esa1-dependent acetylation of chromatin. Genes Dev. 17: 1415-1428. PubMed ID: 12782659

Ceol, C. J. and Horvitz, H. R. (2004). A new class of C. elegans synMuv genes implicates a Tip60/NuA4-like HAT complex as a negative regulator of Ras signaling Dev. Cell 6: 563-576. PubMed ID: 15068795

Chan, H. M., Narita, M., Lowe, S. W. and Livingston, D. M. (2005). The p400 E1A-associated protein is a novel component of the p53 --> p21 senescence pathway. Genes Dev. 19: 196-201. PubMed ID: 15655109

Doyon, Y. and Cote, J. (2004). The highly conserved and multifunctional NuA4 HAT complex. Curr. Opin Genet. Dev. 14(2): 147-54. PubMed ID: 15196461

Fuchs, M., et al. (2001). The p400 complex is an essential E1A transformation target. Cell 106: 297-307. PubMed ID: 11509179

Georlette, D., et al. (2007). Genomic profiling and expression studies reveal both positive and negative activities for the Drosophila Myb MuvB/dREAM complex in proliferating cells. Genes Dev. 21(22): 2880-96. PubMed ID: 17978103

Gévry, N., et al. (2007). p21 transcription is regulated by differential localization of histone H2A.Z. Genes Dev. 21(15): 1869-81. PubMed ID: 17671089

Guillemette, B., Bataille, A.R., Gévry, N., Adam, M., Blanchette, M., Robert, F. and Gaudreau, L. (2005). Variant histone H2A.Z is globally localized to the promoters of inactive yeast genes and regulates nucleosome positioning. PLoS Biol. 3: e384. PubMed ID: 16248679

Horikawa, I., et al. (1995). Forced expression of YL-1 protein suppresses the anchorage-independent growth of Kirsten sarcoma virus-transformed NIH3T3 cells. Exp. Cell Res. 220: 11-17. PubMed ID: 7664828

Hu, G., Cui, K., Northrup, D., Liu, C., Wang, C., Tang, Q., Ge, K., Levens, D., Crane-Robinson, C. and Zhao, K. (2013). H2A.Z facilitates access of active and repressive complexes to chromatin in embryonic stem cell self-renewal and differentiation. Cell Stem Cell 12: 180-192. PubMed ID: 23260488

Jin, J., Cai, Y., Florens, L., Swanson, S. K., Kusch, T., Li, B., Workman, J. L., Washburn, M. P., Conaway, R. C. and Conaway, J. W. (2005). The mammalian YL1 protein is a shared subunit of the TRRAP/TIP60 histone acetyltransferase and SRCAP complexes. J. Biol. Chem. 280: 13665-13670. PubMed ID: 16230350

Kobor, M. S., et al. (2004). A protein complex containing the conserved Swi2/Snf2-related ATPase Swr1p deposits histone variant H2A.Z into euchromatin. PLoS Biol. 2(5): E131. PubMed ID: 15045029

Korenjak, M., et al. (2004). Native E2F/RBF complexes contain Myb-interacting proteins and repress transcription of developmentally controlled E2F target genes. Cell 119(2): 181-93. PubMed ID: 15479636

Krogan, N. J., et al. (2003). A Snf2 family ATPase complex required for recruitment of the histone H2A variant Htz1. Mol. Cell 12(6): 1565-76. PubMed ID: 14690608

Kusch, T., et al. (2004). Acetylation by Tip60 is required for selective histone variant exchange at DNA lesions. Science 306(5704): 2084-7. PubMed ID: 15528408

Lewis, P. W., et al. (2004). Identification of a Drosophila Myb-E2F2/RBF transcriptional repressor complex. Genes Dev. 18: 2929-2940. PubMed ID: 15545624

Li, B., Pattenden, S. G., Lee, D., Gutierrez, J., Chen, J., Seidel, C., Gerton, J. and Workman, J. L. (2005). Preferential occupancy of histone variant H2AZ at inactive promoters influences local histone modifications and chromatin remodeling. Proc. Natl. Acad. Sci. 102: 18385-18390. PubMed ID: 16344463

Lu, J., Ruhf, M. L., Perrimon, N. and Leder, P. (2007). A genome-wide RNA interference screen identifies putative chromatin regulators essential for E2F repression. Proc. Natl. Acad. Sci. 104(22): 9381-6. PubMed ID: 17517653

Mizuguchi, G., Shen, X., Landry, J., Wu, W. H., Sen, S. and Wu, C. (2004). ATP-driven exchange of histone H2AZ variant catalyzed by SWR1 chromatin remodeling complex. Science 303(5656): 343-8. PubMed ID: 14645854

Morillo Prado, J. R., Srinivasan, S. and Fuller, M. T. (2013). The histone variant His2Av is required for adult stem cell maintenance in the Drosophila testis. PLoS Genet 9: e1003903. PubMed ID: 24244183

Ogawa, H., et al. (2002). A complex with chromatin modifiers that occupies E2F- and Myc-responsive genes in G0 cells. Science 296: 1132-1136. PubMed ID: 12004135

Qi, D., Jin, H., Lilja, T. and Mannervik, M. (2006). Drosophila Reptin and other TIP60 complex components promote generation of silent chromatin. Genetics 174(1): 241-51. PubMed ID: 16816423

Swaminathan J., Baxter, E. M. and Corces, V. G. (2005). The role of histone H2Av variant replacement and histone H4 acetylation in the establishment of Drosophila heterochromatin. Genes Dev. 19(1): 65-76. PubMed ID: 15630020

Tea, J. S., Chihara, T. and Luo, L. (2010). Histone deacetylase Rpd3 regulates olfactory projection neuron dendrite targeting via the transcription factor Prospero. J Neurosci 30: 9939-9946. PubMed ID: 20660276

Tea, J. S. and Luo, L. (2011). The chromatin remodeling factor Bap55 functions through the TIP60 complex to regulate olfactory projection neuron dendrite targeting. Neural Dev. 6: 5. PubMed ID: 21284845

Utley, R. T. and Cote, J. (2003). The MYST family of histone acetyltransferases. Curr. Top. Microbiol. Immunol. 274: 203-36. Review. PubMed ID: 12596909

Wismar, J., et al. (1995). The Drosophila melanogaster tumor suppressor gene lethal(3)malignant brain tumor encodes a proline-rich protein with a novel zinc finger. Mech. Dev. 53: 141-154. PubMed ID: 8555106

Raisner, R. M., Hartley, P. D., Meneghini, M. D., Bao, M. Z., Liu, C. L., Schreiber, S. L., Rando, O. J., and Madhani, H. D. (2005). Histone variant H2A.Z marks the 5' ends of both active and inactive genes in euchromatin. Cell 123: 233-248. PubMed ID: 16239142

Ruhl, D. D., Jin, J., Cai, Y., Swanson, S., Florens, L., Washburn, M. P., Conaway, R. C., Conaway, J. W., and Chrivia, J. C. (2006). Purification of a human SRCAP complex that remodels chromatin by incorporating the histone variant H2A.Z into nucleosomes. Biochemistry 45: 5671-5677. PubMed ID: 16634648

Yan, D., Neumuller, R. A., Buckner, M., Ayers, K., Li, H., Hu, Y., Yang-Zhou, D., Pan, L., Wang, X., Kelley, C., Vinayagam, A., Binari, R., Randklev, S., Perkins, L. A., Xie, T., Cooley, L. and Perrimon, N. (2014). A regulatory network of Drosophila germline stem cell self-renewal. Dev Cell 28: 459-473. PubMed ID: 24576427

Zhang, H., Roberts, D. N., and Cairns, B. R. (2005). Genome-wide dynamics of Htz1, a histone H2A variant that poises repressed/basal promoters for activation through histone loss. Cell 123: 219-231. PubMed ID: 16239141

date revised: 18 February 2024

Home page: The Interactive Fly © 2007 Thomas Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.