nejire

Targets of Activity

The dorsal ectoderm of the Drosophila embryo is subdivided into different cell types by an activity gradient of two TGFbeta signaling molecules, Decapentaplegic and Screw. Patterning responses to this gradient depend on a secreted inhibitor, Short gastrulation and a newly identified transcriptional repressor, Brinker, which are expressed in neurogenic regions that abut the dorsal ectoderm. The expression of a number of Dpp target genes has been examined in transgenic embryos that contain ectopic stripes of Dpp, Sog and Brk expression. These studies suggest that the Dpp/Scw activity gradient directly specifies at least three distinct thresholds of gene expression in the dorsal ectoderm of gastrulating embryos. Brk was found to repress two target genes, tailup/islet (tup) and pannier, that exhibit different limits of expression within the dorsal ectoderm. These results suggest that the Sog inhibitor and Brk repressor work in concert to establish sharp dorsolateral limits of gene expression. Evidence is provided that the activation of Dpp/Scw target genes depends on the Drosophila homolog of the CBP histone acetyltransferase (Ashe, 2000).

Previous studies have identified mutations in the Drosophila homolog of the mammalian CBP histone acetyltransferase gene, nejire. nej is maternally expressed so that the detection of early patterning defects depends on the analysis of embryos derived from females containing nej germline clones. The complete loss of nej+ activity results in a failure to make mature eggs. However, it is possible to obtain embryos from a strong hypomorphic allele, nej¹. These embryos exhibit dorsoventral patterning defects. Recent studies have shown that CBP interacts with Smad proteins including the Drosophila protein Mad, a transcription factor downstream of Dpp signaling. In nej mutant embryos, there is a loss of the amnioserosa and other derivatives of the dorsal ectoderm. The expression of target genes requiring peak levels of Dpp signaling is essentially abolished. For example, hnt expression is lost in the presumptive amnioserosa, but persists at the posterior pole where it might be separately regulated by the torso signaling pathway (Ashe, 2000).

There is a similar loss of the dorsal rho pattern in mutant embryos. In contrast, the lateral, neurogenic stripes are unaffected, indicating that the nej mutant does not cause defects in the patterning of the neurogenic ectoderm. Moreover, the fact that the rho stripes are excluded from ventral regions, as seen in wild-type embryos, suggests that the patterning of the mesoderm is also normal. Thus, the nej mutation does not appear to cause a general loss of transcriptional activation, but instead results in specific patterning defects in the dorsal ectoderm. Target genes that are activated by lower levels of Dpp signaling such as ush and pnr are also affected by the nej mutation. In the case of ush, there is a loss of staining in central regions of the dorsal ectoderm. Moreover, the residual staining pattern is narrower than the wild-type pattern. This is reminiscent of the ush pattern seen in dpp/+ heterozygotes. However, the nej mutation also causes a narrowing of the pnr pattern, whereas expression is normal in dpp/+ embryos (Ashe, 2000).

dpp expression has been examined in two groups of dorsal ectoderm cells at the posterior end of the embryo, in abdominal segment 8 and the telson. These dpp-expressing cells become tracheal cells in the posterior-most branches of the tracheal system (Dorsal Branch10, Spiracular Branch10, and the Posterior Spiracle). These branches are not identified by reagents typically used in analyses of tracheal development, suggesting that dpp expression confers a distinct identity upon posterior tracheal cells. dpp posterior ectoderm expression begins during germ band extension and continues throughout development. The sequences responsible for these aspects of dpp expression have been isolated in a reporter gene. An unconventional form of Wingless (Wg) signaling, Dpp signaling, and the transcriptional coactivator Nejire (CBP/p300) are required for the initiation and maintenance of dpp expression in the posterior-most branches of the tracheal system. These data suggest a model for the integration of Wg and Dpp signals that may be applicable to branching morphogenesis in other developmental systems (Takaesu, 2002).

During early stages of embryogenesis, wg and dpp are expressed in undifferentiated dorsal ectoderm. wg mRNA expression, in 15 stripes along the entire dorsal-ventral axis of the embryo (including the dorsal ectoderm), begins at stage 8. wg expression persists in this striped pattern through stage 17. dpp mRNA is expressed on the dorsal side of the embryo along the entire anterior-posterior axis, beginning at stage 4. dpp mRNA expression persists in a large portion of the dorsal ectoderm through stage 8 and resolves into leading edge cell-specific expression in stage 12 embryos. At this time, the embryonic expression pattern of nej has not been reported. However, some information can be obtained from nej mutant phenotypes. nej zygotic mutant embryos show visible defects in the tracheal system at stage 12. The tracheal system is derived from the dorsal ectoderm, suggesting that nej is expressed in this tissue prior to stage 12 (Takaesu, 2002).

dpp expression in posterior tracheal branch anlagen appears to be initiated by prior episodes of wg and dpp expression in the undifferentiated dorsal ectoderm. The maintenance of dpp expression in posterior tracheal branches appears to require continuous input from wg and from a dpp feedback loop. The initiation and maintenance of dpp expression in posterior tracheal branches also requires continuous nej activity. Overall, the data are consistent with the following combinatorial signaling model. The transcriptional activator Med (signaling for the Dpp pathway) interacts with the transcriptional activator Arm (signaling for the Wg pathway) via the transcriptional coactivator Nej. This multimeric complex initiates and, with continuous signaling, maintains dpp expression in posterior tracheal branches with the help of Zw3. These data extend previous studies of dpp expression and Dpp signaling in several ways. nej has been reported to participate in Dpp signaling. Expression from Dpp responsive enhancers is reduced in nej zygotic mutant embryos. While they show that nej3 enhances dpp wing phenotypes, this study shows that Med¹ enhances nej³ embryonic phenotypes. The Dorsal Trunk Branch forms normally in Mad¹² zygotic mutant embryos, and the Dorsal Trunk Branch appears normal in Med¹ mutants. nej is involved in mediating combinatorial signaling by the Wg and Dpp pathways and the involvement of nej in morphogenesis of Dorsal Branch, Spiracular Branch, and the Posterior Spiracle is demonstrated. A region of the histone acetyltransferase domain of Nej binds to Mad. Further study is needed to reveal the mechanisms used by Nej to interact with Wg and Dpp signaling. Several questions remain about the regulation of dpp expression by Wg, Dpp, and Nej. Two questions arise about the mechanism of signal integration: how is zw3 involved and how is Nej recruited to bridge the two pathways? It is tempting to speculate that, in response to a Wg or a Dpp signal, Zw3 (a serine-threonine kinase) is involved in Nej recruitment. Numerous studies have shown that p300/CBP transcriptional coactivation functions are stimulated by its phosphorylation, but the site of phosphorylation has never been mapped. Other questions concern the molecular nature of the enhancers that direct dpp expression in the posterior tracheal branches. A 54-nucleotide region has been identified that contains two sets of conserved, overlapping consensus binding sites for dTCF and Mad/Med. Analyses of DNA-protein interactions predicted by the data involving this candidate combinatorial enhancer have begun (Takaesu, 2002).

From a broader perspective, mammalian homologs of Dpp and orthologs of Wg are important in branching morphogenesis in a variety of developing tissues. For example, BMP2 is involved in renal branching and Wnt4 plays a role in mammary gland branching. The widespread use of TGF-ß and Wnt signals in branching suggests that a greater understanding of the regulation of dpp tracheal expression and dpps role in specifying the unique identities of posterior tracheal branches will have wide relevance (Takaesu, 2002).

Rapid induction of the Drosophila melanogaster heat shock gene hsp70 is achieved through the binding of heat shock factor (HSF) to heat shock elements (HSEs) located upstream of the transcription start site. The subsequent recruitment of several other factors, including Spt5, Spt6 and FACT, is believed to facilitate Pol II elongation through nucleosomes downstream of the start site. This study reports a novel mechanism of heat shock gene regulation that involves modifications of nucleosomes by the TAC1 histone modification complex. After heat stress, TAC1 is recruited to several heat shock gene loci, where its components are required for high levels of gene expression. Recruitment of TAC1 to the 5'-coding region of hsp70 seems to involve the elongating Pol II complex. TAC1 has both histone H3 Lys 4-specific (H3-K4) methyltransferase (HMTase) activity and histone acetyltransferase activity through Trithorax (Trx) and CREB-binding protein (CBP), respectively. Consistently, TAC1 is required for methylation and acetylation of nucleosomal histones in the 5'-coding region of hsp70 after induction, suggesting an unexpected role for TAC1 during transcriptional elongation (Smith, 2004).

Protein Interactions

T-cell factor (TCF), a high-mobility-group domain protein, is the transcription factor activated by Wnt/Wingless signaling. When signaling occurs, TCF binds to its coactivator, beta-catenin/Armadillo, and stimulates the transcription of the target genes of Wnt/Wingless by binding to TCF-responsive enhancers. Inappropriate activation of TCF in the colon epithelium and other cells leads to cancer. It is therefore desirable for unstimulated cells to have a negative control mechanism to keep TCF inactive. Drosophila CREB-binding protein (dCBP) binds to Drosophila TCF (Pangolin). dCBP mutants show mild Wingless overactivation phenotypes in various tissues. Consistent with this, dCBP loss-of-function suppresses the effects of armadillo mutation. Moreover, dCBP is shown to acetylate a conserved lysine in the Armadillo-binding domain of dTCF, and this acetylation lowers the affinity of Armadillo binding to dTCF. Although CBP is a coactivator of other transcription factors, these data show that CBP represses TCF (Waltzer, 1998).

Attempts to demonstrate trans-activation activity by the Drosophila Myb gene product have been unsuccessful so far. Co-transfection of Schneider cells with a plasmid expressing the Drosophila homolog of transcriptional co-activator CBP (dCBP) results in transactivation by Myb. Using this assay system, the functional domains of Myb have been analyzed. Two domains located in the N-proximal region, one of which is required for DNA binding and the other for dCBP binding, are both necessary and sufficient for trans-activation. In this respect, D-Myb is similar to c-Myb and A-Myb, but different from mammalian B-Myb. These results shed light on how the myb gene diverged during the course of evolution (Hou, 1997).

In the visceral mesoderm, dpp is expressed in parasegment (ps) 7 under the control of the homeotic gene Ultrabithorax (Ubx). In this cell layer, dpp stimulates its own expression and the expression of Ubx. dpp also stimulates the expression of wingless (wg), an extracellular signaling molecule of the Wnt family, in the neighbouring ps8. wg in turn feeds back to stimulate Ubx and dpp expression in ps7. Thus, dpp is part of a parautocrine feedback loop by which Ubx maintains its own expression indirectly through controlling dpp and wg. Dpp also diffuses from its mesodermal source through the endodermal cell layer of the embyonic midgut, where it stimulates the expression of D-Fos and of the homeotic gene labial. These inductive steps ultimately specify the differentiation of distinct cell types in the larval midgut epithelium. In order to understand the mechanism by which dpp stimulates transcription, a short enhancer fragment of Ubx, called Ubx B, has been characterized that contains response sequences for dpp and wg signaling in the embryonic midgut. The dpp response sequence of this enhancer is bipartite, consisting of a tandem repeat of Mad binding sites and a cAMP response element (CRE). The presence of the latter raised the question whether the co-activator CBP (CREB-binding protein, binding to CREs) might participate in Dpp-induced transcriptional activation (Waltzer, 1999).

Drosophila CBP loss-of-function mutants show specific defects that mimic those seen in mutants that lack the extracellular signal Dpp or its effector Mad. CBP loss severely compromises the ability of Dpp target enhancers to respond to endogenous or exogenous Dpp. CBP binds to the C-terminal domain of Mad. These results provide evidence that CBP functions as a co-activator during Dpp signaling, and they suggest that Mad may recruit CBP to effect the transcriptional activation of Dpp-responsive genes during development (Waltzer, 1999).

The embryonic midgut of nejire (nej) mutants (whose CBP function is reduced) show phenotypes related to wg gain-of-function phenotypes: increased labial expression in the endoderm, and derepression of the Ubx B enhancer in the visceral mesoderm. These phenotypes do not resemble those seen in dpp or Mad mutants: in Mad mutants, labial expression is strongly reduced, and so is the beta-galactosidase (lacZ) staining mediated by the Ubx B enhancer in the middle midgut. However, the narrow band of lacZ staining normally visible in the visceral mesoderm of the gastric caeca (in ps3) is absent in nej mutant embryos. Indeed, closer inspection reveals that the gastric caeca frequently fail to elongate in these mutants. A similar phenotype is observed in Mad and in dpp mutants. Thus nej, like dpp, is required for the formation of the gastric caeca, and also for the activity of the Ubx B enhancer in the caecal primordia. The activity of this enhancer in these primordia coincides with Dpp expression and depends on dpp function. The formation of the first midgut constriction is often impeded. While this could reflect overactive Wg signaling, it also mimics loss of glass bottom boat (gbb) signaling: Gbb is a Dpp homolog expressed in the visceral mesoderm and whose function is required for the formation of the first midgut constriction (Waltzer, 1999).

The hypothesis that CBP is a co-activator of dpp-induced transcription was tested by examining the Dpp response of the Ubx enhancer in nej mutants. Because it was expected that the repressive effect of CBP on this enhancer would mask a possible activating effect of CBP in cells in which the enhancer is stimulated by Wg signaling, a mutant version of Ubx B, called B4, was used whose positive response to Wg is abolished. B4 activity in the midgut is reduced compared with the wild-type enhancer; however, B4 still contains a fully functional dpp-response sequence and can be efficiently stimulated by ectopic Dpp. B4 can thus be used to selectively monitor the stimulation of Ubx by Dpp in the visceral mesoderm. The activity of Ubx B4 is significantly reduced in nej mutants. LacZ staining is particularly weak in ps6/7 (near the Dpp source), but also in ps10, and is barely detectable in the gastric caeca. Furthermore, in nej mutant embryos derived from nej mutant germlines (nej), lacZ staining mediated by B4 is even weaker than in the zygotic nej mutants: although these nej GLC embryos are somewhat variable in terms of their phenotypes the most severely mutant embryos show lacZ staining in only a few cells in the ps8 region. Similarly, in Mad12 mutant embryos, lacZ staining is much reduced, with some staining remaining in ps6 and ps8. This implies that CBP, like Mad, is required for the Dpp response of the Ubx B4 enhancer (Waltzer, 1999).

The response of B4 to GAL4-mediated ectopic Dpp was examined in nej mutant embryos. If Dpp is expressed throughout the mesoderm, B4-mediated lacZ staining is increased and detectable throughout the midgut mesoderm. In nej mutants, this response of B4 to ectopic Dpp is strikingly disabled: there is barely any lacZ staining in the anterior midgut, and only a moderate increase of lacZ staining in the ps8/9 region, indicating a residual Dpp response in this region. These results strongly support the conclusion that CBP is required for the transcriptional response of the Ubx enhancer to Dpp signalling. They argue that CBP functions downstream of the Dpp signal (Waltzer, 1999).

In the early blastoderm embryo, dpp mediates the subdivision of the dorsal ectoderm into two embryonic tissues: the amnioserosa and the dorsal epidermis. High Dpp levels in the dorsal-most cells specify amnioserosa while lower Dpp levels in dorsolateral regions specify epidermis. Expression of the gene Race (related to angiotensin converting enzyme; the earliest known marker for the amnioserosa) in the dorsal blastoderm embryo depends on dpp signaling. Thus it was asked whether the activity of the Race enhancer depends on CBP function. This enhancer mediates lacZ staining in the presumptive amnioserosa and in the anterior midgut primodium: the former, but not the latter, staining requires dpp. In nej GLC embryos, there is no detectable lacZ staining in the presumptive amnioserosa, although staining remains, and is even slightly enhanced, in the head and in the anterior midgut primordium. This demonstrates that the Race enhancer depends on an activating function of CBP exclusively in a subset of the blastoderm cells, namely in the dorsal-most cells of the embryonic trunk. It suggests that CBP is required for the response of this enhancer to dpp (Waltzer, 1999).

To see whether CBP may be required in other developmental contexts in which dpp functions, the developing tracheae were examined in nej mutant embryos. The tracheal system develops from segmentally repeated clusters of ectodermal cells, the tracheal placodes. These cells undergo a complex process of migration and fusion to generate the final branched structure of the tracheal system. dpp signaling plays a crucial role in this process, and has been implicated in the dorsoventral migration of certain tracheal branches. For example, in punt or thick veins mutants, the branches that normally migrate dorsally or ventrally (the dorsal and ganglionic branches, respectively) fail to develop, whereas the branches that grow out anteriorly (the dorsal trunk and the visceral branches) are essentially not affected. The tracheae in nej mutant embryos were examined using an antibody that stains the lumina of the tracheal trees (2A12). The dorsal trunk and the visceral branches are essentially normal in these mutants. However, in most nej mutant embryos, branching defects are seen: usually, one or two dorsal branches fail to form at each side, and ganglionic branches fail to fuse. Essentially the same defects are also seen in in nej GLC embryos. These defects resemble those found in punt hypomorphs and in Mad12 mutant embryos, although the most apparent defects in the latter mutants are the fusion defects in their ganglionic branches. Once again, the similarity of the tracheal phenotypes of nej mutants when compared to dpp, punt and Mad mutants suggests that CBP may be required during Dpp signaling (Waltzer, 1999).

dpp promotes vein development during pupal stages, and a subclass of dpp mutant alleles cause loss of veins. In particular, in dpp^S1 homozygous flies, vein 4 fails to reach the margin. This weak dpp allele was exploited to see whether there would be a genetic interaction between dpp and nej. Indeed, while nej heterozygosity on its own shows no abnormality whatsoever in the wing, this condition clearly enhances the vein phenotype of dpp^S1 homozygotes: in many of the wings from flies of this genetic constitution, neither vein 4 nor vein 2 reaches the margin. This synergy in the wing between nej and dpp loss-of-function alleles is consistent with the notion that CBP functions during Dpp signaling. To clarify the position of CBP in this Dpp response in the wing, it was asked whether the mild dpp overactivation phenotype due to overexpression of a constitutively active form of Sax (Sax*), a Dpp type I receptor, depends on nej gene dosage. Expression of Sax* under the control of engrailed.GAL4 induces ectopic venation and overgrowth of the posterior part of the wing. Moreover, removal of one copy of genes required for Sax signaling, such as Mad or Medea suppresses this phenotype. Likewise, nej heterozygosity suppresses to a considerable extent the wing phenotypes caused by Sax*. This result is consistent with CBP being required for Sax signaling, and it indicates that CBP functions downstream of this Dpp receptor (Waltzer, 1999).

Since Mad mediates transcriptional activation by dpp and appears to be a transcription factor required for every aspect of dpp signaling, it was asked whether CBP might be recruited by Mad as a transcriptional co-activator. To test whether CBP might bind to Mad, the yeast two-hybrid system was used. When these binding studies were begun, Drosophila CBP had not yet been discovered. So fragments of mouse CBP were used to test whether these might bind to Mad, assuming that a putative interaction between the two proteins would be conserved. Indeed, there is a strong degree of homology between mouse and Drosophila CBP. A set of fragments of mouse CBP were used that cover the whole protein and these were fused to a transcriptional activation domain (the 'prey'). This series of prey was tested in two-hybrid assays in yeast with full-length Mad protein fused to the LexA DNA-binding domain (the 'bait'). The C-terminal domain of mouse CBP (CBP1678 to 2441) interacts specifically with Mad in this assay. This interaction was confirmed using a similar set of prey with fragments from the Drosophila CBP protein, which was tested against a series of baits containing different Mad domains. This reveals that a fragment of Drosophila CBP that spans amino acids 2240-2608 (which overlaps the above mentioned C-terminal domain of mouse CBP) interacts specifically with the MH2 domain of Mad (Mad219-455). These specific interactions in the yeast two-hybrid assay between CBP fragments and Mad almost certainly reflect direct binding since yeast does not encode any proteins homologous to either of these. Interactions between CBP fragments and Mad are significantly stronger if the N-terminal domain of Mad is removed, suggesting that MH1 inhibits the binding of CBP to MH2. Inhibitory interactions between MH1 and MH2 have been described previously (Waltzer, 1999).

To confirm these results, direct binding between Mad and CBP in vitro were tested with pull-down assays. In these assays, [35S]methionine-labelled Mad domains and various fragments from Drosophila CBP expressed as GST fusion proteins and immobilized on GST-Sepharose beads were used. Either full-length Mad or its MH2 domain binds to the same Drosophila CBP fragment that interacts with Mad in the yeast assay while Mad's MH1 domain does not bind CBP. Interestingly, deletion of the C-terminal of Mad's MH2 domain (amino acids 372-455) abolishes CBP interaction, demonstrating that the C-terminal 84 amino acids of Mad are required for binding to CBP. The linker domain (L) between MH1 and MH2 seems to be dispensable for Mad interaction with CBP. Weak binding of full-length Mad to CBP2 (CBP2240-2507), the highly conserved domain of Drosophila CBP that overlaps the Mad-binding fragment of CBP, was observed. However, the significance of this binding is uncertain, as this binding activity could not be detected in the reciprocal assay nor in yeast. Finally, to characterize more precisely the mutual binding domains within Mad and Drosophila CBP, the reciprocal experiment was performed using [35S]methionine-labelled C-terminal fragments of CBP and various GST-Mad fusion proteins. CBP binds to GST-MH2, but not to GST alone, nor to GST-MH1+L fusion proteins, which include extended MH1 fragments (Mad1-241), nor to GST-MH2C fusion protein in which the last 84 amino acids of Mad's MH2 domain are deleted. Deletion mapping of the C-terminal region of CBP reveals a minimal fragment of CBP (CBP2413-2608) that is sufficient for binding to Mad. This domain partially overlaps the highly conserved CBP2 domain but in most binding assays, CBP2 by itself is not sufficient for binding to Mad. Altogether, these experiments demonstrate that CBP and Mad bind to one another, and that the stretch between amino acids 2507 and 2640 within Drosophila CBP is critical for CBP's binding to the MH2 domain of Mad (Waltzer, 1999).

tinman encodes an NK-2 class homeodomain transcription factor that is required for development of the Drosophila dorsal mesoderm, including heart. Genetic evidence suggests its important role in mesoderm subdivision, yet the properties of Tinman as a transcriptional regulator and the mechanism of gene transcription by Tinman are not completely understood. Tinman can activate or repress target genes in cultured cells, based on evaluation of functional domains that are conserved between the tinman genes of Drosophila melanogaster and Drosophila virilis. Using GAL4-tinman fusion constructs, a transcriptional activation domain (amino acids 1-110) and repression domains (amino acids 111-188 and the homeodomain) have been mapped and an inhibitory function for the homeodomain has been found upon transactivation by Tinman (Choi, 1999).

Tinman is regulated by Twist and autoregulates its own promoter. The properties of Tin as a transcription factor were assessed using tinman P1 and P1E2m promoters and truncated forms of the Tin expression vectors (d8 and d6). The P1 reporter contains Tin-responsive elements (the E2 cluster) and is activated by Tin. The P1E2m reporter contains mutated Tin binding sites but otherwise is exactly the same as the wild-type P1 reporter, which also contains several weak Tin binding sites. Tin can activate the P1 reporter (6-fold activation). In contrast, Tin down-regulates the P1E2m reporter gene (3-fold repression). In this case, Tin binds to weak binding sites and represses the P1E2m reporter gene. These results indicate that Tin can act either as a transcriptional activator or repressor, depending on the context of the reporters (P1 or P1E2m). These phenomena are dependent on the functional domains of Tin. For example, deletion of the amino-terminal region of Tin abrogates activation of the P1 reporter, indicating that the amino terminus (aa 1-110) of Tin is required for transcriptional activation. Indeed, this Tin mutant (d8) represses gene expression of both the P1 and the P1E2m reporter. Further deletion of Tin (construct d6) relieves this repression, irrespective of the reporter gene used. These results suggest that the region following the amino terminus of Tin (aa 111-188) is required for the repressor activity of Tin. Taken together, these results indicate that, depending on the context of the target genes (for example P1 or P1E2m), Tin can act as either a transcriptional activator or repressor and that these different transcriptional activities are dependent on functional domains of Tin (Choi, 1999).

Tinman-dependent transactivation is augmented by the p300 coactivator; Tinman physically interacts with p300 via the activation domain. In addition, cotransfection experiments indicate that the repressor activity of Tinman is strongly enhanced by the Groucho corepressor. Using immunoprecipitation and in vitro pull-down assays, Tinman is shown to directly interact with the Groucho corepressor, for which the homeodomain is required. Together, these results indicate that Tinman can act as either a transcriptional activator or repressor. The first evidence of Tinman interactions with the p300 coactivator and the Groucho corepressor is provided (Choi, 1999).

CREB-binding protein (CBP) is a coactivator for multiple transcription factors that transduce a variety of signaling pathways. Current models propose that CBP enhances gene expression by bridging the signal-responsive transcription factors with components of the basal transcriptional machinery and by augmenting the access of transcription factors to DNA through the acetylation of histones. To define the pathways and proteins that require CBP function in a living organism, a genetic analysis of CBP has been carried out in flies. Drosophila CBP (dCBP) was overproduced in a variety of cell types and distinct adult phenotypes were obtained. An uninflated-wing phenotype, caused by the overexpression of dCBP in specific central nervous system cells, was used to screen for suppressors of dCBP overactivity. Two genes with mutant versions that act as dominant suppressors of the wing phenotype were identified: the PKA-C1/DCO gene, encoding the catalytic subunit of cyclic AMP protein kinase, and ash1, a member of the trithorax group (trxG) of chromatin modifiers. Using immunocolocalization, it has been shown that the Ash1 protein is specifically expressed in the majority of the dCBP-overexpressing cells, suggesting that these proteins have the potential to interact biochemically. This model was confirmed by the findings that the proteins interact strongly in vitro and colocalize at specific sites on polytene chromosomes. The trxG proteins are thought to maintain gene expression during development by creating domains of open chromatin structure. One model for the function of CBP and p300 is bridging DNA binding transcription factors to components of the basal transcriptional machinery. These results thus suggest a second model for dCBP function, namely interaction with trxG proteins, and imply that dCBP might be involved in the regulation of higher-order chromatin structure (Bantignies, 2000).

Screens for enhancers and suppressors of overexpression phenotypes have been useful in identifying components of regulatory pathways. Nevertheless, overexpression systems have drawbacks and can potentially identify secondary effectors of a nonspecific phenotype. However, it is thought that this screen has identified genes that affect dCBP function for several reasons. (1) The number of deficiencies that suppress the uninflated-wing phenotype is small. A large number of suppressors might suggest that the overexpression of dCBP is not eliciting a specific cell phenotype. (2) Two of the deletions suppress both the wing and the eye overexpression phenotypes, suggesting that the overexpression of dCBP in the two tissues has some common effects. One of the deletions demonstrates that the dosage of PKA can affect the dCBP overexpression phenotype. CBP and dCBP are known to play a role in PKA signaling, so the fact that PKA was identified in this screen is consistent with the idea that dCBP overexpression reflects an overactivation of the PKA pathway. Trivial explanations for the suppression of dCBP overexpression by ASH1 have been ruled out; dCBP overexpression does not cause the death of ASH1-expressing cells, nor do ash1 mutations affect the overexpression of dCBP. A characterization of dCBP loss of function in these cells both in wild-type and ash1 mutant backgrounds is necessary to complete this analysis. A clonal analysis of dCBP mutant cells is not feasible because dCBP is required for cell viability and only small clones can be generated. This analysis will have to await reagents that allow dCBP function to be knocked out in the GAL4-386 cells in the ash1 mutant background. In addition, it will be important to identify the targets of dCBP and ASH1 in these cells as well as the pathways that activate them. Although the genetic analysis is not complete, it is likely that the genetic suppression of dCBP overexpression by ash1 mutations reflects a functional association between ASH1 and dCBP because these two proteins have specific interactions in vitro (Bantignies, 2000).

Overexpression of dCBP in specific CNS cells causes wing inflation defects. In many tissues, overexpression of dCBP causes lethality, suggesting that the dose of this effector is important for its function. The overproduction of dCBP in specific cells of the CNS with two different GAL4 lines produces defects in wing inflation with various degrees of penetrance. However, overexpression of dCBP in wing tissues throughout development does not interfere with wing inflation (Bantignies, 2000).

Previous studies have implicated specific CNS cells in the regulation of wing inflation. In Drosophila, the death of specific cells is triggered after eclosion and is strongly correlated with wing inflation behavior. In addition, two specific neurons in the fly brain are responsible for the production of the neuropeptide eclosion hormone (EH). The specific knockout of EH-producing cells (EH cells) during early development results in eclosion delays and a disruption of eclosion behaviors, such as wing inflation. In the moth Manduca sexta, EH triggers a neuroendocrine cascade that regulates both ecdysis and postecdysis processes such as wing inflation. It was suggested that the frequent failure of EH cell knockout flies to inflate their wings successfully is due to a lack of excitability of neuroendocrine-responsive EH cells that release important signals for proper eclosion behaviors. In Manduca, different neuropeptides, such as bursicon and the cardioacceleratory peptides, are usually released after eclosion to aid in wing expansion. It may be that the neurons that overexpress dCBP are the neurosecretory cells that are targeted by the EH cascade and that produce the peptides that signal the wing inflation process. In this case, the overexpression of dCBP interferes with normal cell function. Of course the wing inflation defect could be due to the death of the neurons caused by the overexpression of dCBP. However, the pattern of cells that overexpress LacZ and dCBP in the GAL4-386 background remains the same throughout development, and cells that overexpress dCBP and express ASH1 are viable at least 24 h posteclosion, so the overexpression of dCBP does not appear to affect the viability of these cells. Two additional GAL4 lines, GAL4-c929 and GAL4-c191, also drive specific expression in the CNS, specifically in most of the peptidergic neurons of the brain and ventral ganglion. At 25°C, escapers were obtained only with the GAL4-c191 line. Approximately 30% of these flies have uninflated or partially inflated wings (Bantignies, 2000).

It is proposed that the overexpression of dCBP in specific CNS cells affects the regulation of signaling pathways that involve dCBP and that are important for proper eclosion behaviors. Preliminary data suggest that at least some of the cells that overexpress dCBP are neuropeptidergic neurons and colocalize with the neuropeptides FMRFamide and PHM. However, antibody incompatibility does not allow for a determination of whether these cells also express ASH1. Clearly, more characterization will be required to determine the exact pathways affected by dCBP. The dominant wing phenotype obtained by overexpressing dCBP with GAL4-386 is a good model to elucidate some of the cells and signaling pathways involved in wing inflation (Bantignies, 2000).

Biochemical experiments show that coactivator dCBP binds strongly to trxG protein ASH1. This observation supports the idea that ASH1 and dCBP interact in vivo and implicates a novel class of chromatin binding proteins in mediating dCBP function. The ASH1 protein contains three motifs that are characteristic of some proteins that regulate transcription and/or are bound to chromosomes: there are two AT hook motifs in the N-terminal region, a SET domain, and a PHD finger in the C-terminal domain. The AT hook motif is important for the binding of some proteins to DNA. PHD fingers are Cys-rich Zn finger-like motifs implicated in protein-protein interactions and are found in other trxG proteins. The SET domain is an approximately 130-aa region found in a number of other chromatin-associated proteins, including the TRX factor, PcG protein Enhancer of Zeste [E(Z)], and the modifier of position effect variegation Suppressor of variegation 3-9. The TRX SET domains have been proposed to mediate association with components of chromatin-remodeling complexes, and ASH1 and TRX interact directly through their SET domains. Binding assays indicate that two N-terminal regions and the SET domain of ASH1 interact strongly with dCBP. However, no interaction with the PHD domain was observed. Thus, the SET and the PHD domains of ASH1 might function for the recruitment of other chromatin-associated proteins, such as TRX, and the N-terminal region could serve to interact with the DNA, possibly through the AT motifs, to direct the targeting of HATs to the promoter. Further biochemical characterization will be necessary to confirm this model, but the interaction between dCBP and ASH1 provides new insights on the possible function of ASH1 in gene regulation (Bantignies, 2000).

The binding of ASH1 to dCBP requires the C-terminal C/H3 domain. In mammalian CBP and p300, this region mediates interactions with numerous sequence-specific transcription factors, the adenovirus E1A protein, TFIIB, RNA helicase A, and P/CAF, a GCN5-like histone acetylase. In dCBP, the C/H3 domain mediates the interaction with transcription factor dTCF and Mad, demonstrating an important role for this domain in dCBP function. This domain contributes to the interaction with chromatin-associated protein ASH1, suggesting that dCBP may function in epigenetic regulatory complexes. The C/H3 domain is adjacent to HAT and might contribute to the regulation of the histone acetylation activity of CBP and p300 or might recruit targets of acetylation close to the enzymatic domain. Thus, it will be interesting to determine whether ASH1 has any effect on dCBP HAT functions or if it is a target of dCBP acetyltransferase activity (Bantignies, 2000).

The bromodomain of P/CAF has been shown to bind histone peptides in an acetylation-dependent manner. The bromodomain of GCN5, a member of the SAGA complex, is required for SWI/SNF remodeling of the nucleosome and stabilizing the SWI/SNF complex on the promoter. Thus, it appears that the bromodomain interacts with acetylated proteins and may form a link between different regulatory complexes. Although the full-length ASH1 does not interact with the bromodomain of dCBP, both the ASH1-458-853 polypeptide and the SET domain do interact with this domain. It may be that full-length ASH1 undergoes a modification, upon binding with the dCBP C/H3 domain, that allows other regions of ASH1 to interact with the dCBP bromodomain. In this case, it would appear that the interaction is not dependent on acetylation (Bantignies, 2000).

These results also show that dCBP and ASH1 colocalized to a number of specific sites on polytene chromosomes, suggesting that they might serve as coregulators of a specific set of genes including the homeotic selector genes. The mapping of the specific sites where dCBP and ASH1 colocalize will help identify target genes that are regulated by ASH1 and dCBP. An analysis of these genes, their promoters, and their regulation by dCBP and ASH1 will further define the functional role of the dCBP-ASH1 interaction (Bantignies, 2000).

The development of Drosophila requires the function of the CREB-binding protein, dCBP. In flies, dCBP serves as a coactivator for the transcription factors Cubitus interruptus, Dorsal, and Mad, and as a cosuppressor of Drosophila T cell factor. Current models propose that CBP, through its intrinsic and associated histone acetyltransferase activities, affects transient chromatin changes that allow the preinitiation complex to access the promoter. Evidence is provided that dCBP may regulate the formation of chromatin states through interactions with the modulo (mod) gene product, a protein that is thought to be involved in chromatin packaging. dCBP and Modulo bind in vitro and in vivo, mutations in mod enhance the embryonic phenotype of a dCBP mutation, and dCBP mutations enhance the melanotic tumor phenotype characteristic of mod homozygous mutants. These results imply that, in addition to its histone acetyltransferase activity, dCBP may affect higher-order chromatin structure (Bantignies, 2002).

Aberrant histone acetylation, altered transcription, and retinal degeneration in a Drosophila model of polyglutamine disease are rescued by CREB-binding protein

Sequestration of the transcriptional coactivator CREB-binding protein (CBP), a histone acetyltransferase, has been implicated in the pathogenesis of polyglutamine expansion neurodegenerative disease. A Drosophila model was used to demonstrate that polyglutamine-induced neurodegeneration is accompanied by a defect in histone acetylation and a substantial alteration in the transcription profile. Furthermore, complete functional and morphological rescue by up-regulation of endogenous Drosophila CBP (dCBP) is demonstrated. Rescue of the degenerative phenotype is associated with eradication of polyglutamine aggregates, recovery of histone acetylation, and normalization of the transcription profile. These findings suggest that histone acetylation is an early target of polyglutamine toxicity and indicate that transcriptional dysregulation is an important part of the pathogenesis of polyglutamine-induced neurodegeneration (Taylor, 2003).

Huntington's disease, spinobulbar muscular atrophy, dentatorubro-pallidoluysian atrophy, and six forms of spinocerebellar ataxia are caused by expansion of CAG trinucleotide repeats, resulting in pathological polyglutamine expansion in the disease proteins. These diseases likely share a common mechanism involving a toxic gain of function by the expanded polyglutamine tract. Evidence indicates that the nucleus is an important site of polyglutamine toxicity and that transcriptional dysregulation may be a primary pathogenic process in polyglutamine disease. Mutant proteins with expanded polyglutamine have been shown to bind and sequester the transcriptional coactivator CREB-binding protein (CBP) in cell culture, animal models, and tissue derived from patients with these diseases. Physical interaction between CBP and mutant protein has been found to depend on the acetyltransferase domain of the former and the polyglutamine tract of the latter. Sequestration of CBP results in reduced acetyltransferase activity and loss of CBP-mediated coactivation in cell culture models of polyglutamine disease. Live-cell dynamic imaging shows that coexpression of GFP-CBP with various proteins with polyglutamine expansions in cell cultures results in not merely colocalization, but functional sequestration of CBP in polyglutamine inclusions. CBP is an important cofactor in transcriptional activation mediated by CREB. A recent report demonstrates that disruption of CREB function leads to progressive neurodegeneration in a pattern similar to that observed in transgenic mouse models of polyglutamine disease. CBP also serves as a cofactor for other transcription factors in addition to CREB, but this finding indicates that disruption of the CREB pathway alone is sufficient to lead to neurodegeneration and further implicates CBP as a target of polyglutamine toxicity (Taylor, 2003 and references therein).

The results presented in this study demonstrate that CBP is a potent modifier of polyglutamine-induced neurodegeneration in vivo and support the hypothesis that histone acetylation may be a target of polyglutamine toxicity. Alterations in CBP activity by sequestration, increased degradation, or decreased expression as seen in this study may contribute to altered acetylation. The loss of polyglutamine aggregation seen with rescue by CBP suggests that CBP itself, as a polyglutamine protein, may directly block accumulation of polyglutamine monomers into aggregates, similar to a blocking peptide, making it more susceptible to degradation. Alternatively, genes regulated by CBP may influence aggregation or accelerate degradation of polyglutamine. The findings presented in this study add to the accumulating evidence that pharmacologics capable of influencing histone acetylation may be of benefit in polyglutamine disease (Taylor, 2003).

DEVELOPMENTAL BIOLOGY

A genetic screen identifies putative targets and binding partners of CREB Binding Protein (CBP) in the developing Drosophila eye

Drosophila CREB Binding Protein (dCBP) is a very large multi-domain protein, which belongs to the CBP/p300 family of proteins, which were first identified by their ability to bind the CREB transcription factor and the adenoviral protein E1. Since then CBP has been shown to bind to over 100 additional proteins and functions in a multitude of different developmental contexts. Among other activities, CBP is known to influence development by remodeling chromatin, by serving as a transcriptional co-activator and by interacting with terminal members of signaling transduction pathways. Reductions in CBP activity are the underlying cause of Rubinstein-Taybi syndrome, which is, in part, characterized by several eye defects including strabismus, cataracts, juvenile glaucoma and coloboma of the eyelid, iris, and lens. Development of the Drosophila compound eye is also inhibited in flies that are mutant for CBP. However, the vast array of putative protein interactions and the wide-ranging roles played by CBP within a single tissue such as the retina can often complicate the analysis of CBP loss-of-function mutants. Through a series of genetic screens several genes have been identified that could either serve as downstream transcriptional targets or encode for potential CBP binding partners and whose association with eye development has hitherto been unknown. The identification of these new components may provide new insight into the roles that CBP plays in retinal development. Of particular interest is the identification that the CREB transcription factor appears to function with CBP at multiple stages of retinal development (Anderson, 2005).

Through the use of several genetic screens attempts have been made to identify genes that function cooperatively with CBP to influence eye development. Either wild type or one of three variants of CBP was expressed within the developing eye of Drosophila. The expression of each CBP protein resulted in a disruption of eye development that is easily visualized by an examination of the external surface of adult eyes. The degree of structural alteration and the underlying cause of said disruptions are unique to the expression of each individual CBP protein. The retinal phenotypes that are generated by the expression of CBP variant proteins reveals considerable information on the role that CBP plays in normal eye development. Each variant protein retains a unique combination of protein domains and is expected to act as a protein sink by binding and soaking up signaling pathway components and transcription factors. Thus the changes in eye specification and ommatidial cell fate are due to a 'loss' of factors that normally function in these processes. Since CBP appears to be normally expressed in every cell within the developing retina it is likely that the putative transcriptional targets and binding partners of CBP that were identified in these screens are biologically relevant for eye development (Anderson, 2005).

Advantage was taken of the Bloomington Stock Center Deficiency Kit to rapidly identify regions of the genome that potentially harbor interacting genes. Through this method, 71 such regions were initially identified. Extant single gene mutants were then screened and 35 complementation groups were identified that potentially interact with CBP. Interacting genes for the remaining 36 genomic intervals could not be identified. This is likely due to the lack of single gene mutations for all predicted genes. Alternatively it is possible that these regions contain two or more genes that must be removed simultaneously in order to modify the rough eye phenotypes associated with CBP expression (Anderson, 2005).

Among the collection of potential interacting genes, the screens identified members of the eye specification cascade, two Hox genes (Ultrabithorax and Sex combs reduced), the CREB transcription factor and several members of the Epidermal Growth Factor Receptor (Egfr) and TGFbeta signaling cascades. Recovery of these factors in the course of an 'eye screen' is supported by known interactions between CBP and many of these factors in other developmental contexts. For instance, CBP is known to interact with the TGFbeta signaling pathway during several stages of embryonic development. Likewise, CBP is known to participate in the regulation of several Hox genes. As another example, CBP is known to interact with the retinal determination genes in both the fly retina and mouse muscle. Furthermore, interactions between CBP and CREB have been well documented in a number of contexts including learning and memory and synapse formation. And finally, the CREB-CBP complex is known to be phosphorylated by RSK, a known component of receptor tyrosine kinase signaling cascades. Thus, it is now possible to link CBP to the Egfr pathway during eye development. These results are further supported by the fact that loss-of-function mutations in either CBP or members of the Egfr cascade lead to near identical phenotypes in the eye, wing, leg and embryo of Drosophila. The screens connect CBP directly to this signaling system (Anderson, 2005).

It should be noted that the approach used in this report to find genes that potentially interact with CBP revealed interactions that would not have been found if the study were limited to a single screen for modifiers of the GMR-CBP FL (GMR driven full length CBP) rough eye phenotype. In that screen only two deficiency stocks were uncovered that could modify the rough eye. Since CBP interacts with well over 100 different proteins the reduction in any one is unlikely to alter significantly the GMR-CBP FL rough eye phenotype. The empirical results bear this out. By expressing CBP protein variants, each containing a unique combination of functional domains, it was possible to induce retinal phenotypes that could be modified by reductions in single gene dosages. Another novel aspect of these genetic screens is that have not only been able to find factors that interact with or are regulated by CBP, but some information is now available about how they modulate the activity of this co-activator. For instance, CBP deltaBHQ (lacking the Histone acetyltransferase domain and the Poly glutamine stretch) retains the N-terminal half of protein, which includes a zinc finger domain, a nuclear hormone receptor binding domain and the CREB binding domain. It is predicted that the genes identified as suppressors of the GMR-CBP deltaBHQ rough eye phenotype may be bound by the zinc finger domain of CBP or the CREB transcription factor. Alternatively, these genes may encode proteins that interact with the N-terminal portion of CBP. For example, the CBP deltaBHQ protein can still interact with the CREB transcription factor through the KIX domain. Previous reports have demonstrated that CREB is phosphorylated by RSK, a member of receptor tyrosine kinase signaling. The screen for modifiers of the GMR-CBP deltaBHQ phenotype yielded several members of the EGFR signaling cascade. The results from these screens will provide insight into how the modular activity of CBP can be integrated with the remaining interacting genes and proteins (Anderson, 2005).

In a significant fraction of cases it remains to be determined which of the newly identified factors will turn out to be binding partners, transcriptional targets or upstream regulators of CBP. This is especially true of many genes that have had no previously ascribed role in eye development. In addition to standard genetic practices of examining phenotypes of loss-of-function mutants, several high-throughput methods including yeast two hybrid assays and genomic microarrays will certainly be useful in sorting out the regulatory relationship between CBP and the interacting genes that are described in this report. Since CBP is thought to function as a biochemical scaffold, the results support a role for CBP in integrating instructions from multiple signaling pathways and regulatory networks. One of the most interesting results from this screen is the identification of CREB as a potential regulator of eye development. The interactions between CREB and CBP are well known and have been described for several developmental contexts. However, this is the first report of a potential role for CREB in Drosophila eye development. It has been further demonstrated that CREB-A is expressed in cells ahead of the morphogenetic furrow and in all developing photoreceptor cells. Loss-of-function retinal mosaic clones indicate that the CREB-A transcription factor is required in all photoreceptor cells with the exception of R8. This phenotype is consistent with that seen in CBP loss-of-function retinal mosaic clones. Interestingly, ectopic expression of CrebA is sufficient to induce ectopic eye formation on the ventral surface of the fly head, a phenotype seen when the Pax protein Eyg is itself ectopically expressed. This might place the CBP-CREB complex within the retinal determination network. While it has been reported that activation of CREB in the vertebrate retina blocks degeneration of retinal ganglion cells (RGCs), it is exciting to speculate on potential roles for CREB in the determination of the vertebrate eye and for the specification of vertebrate cell types. It would also be interesting to determine if mutations within human CREB might also be associated with the same retinal phenotypes observed in patients harboring lesions with CBP (Anderson, 2005).

Of the 36 genomic regions for which the genetic loci responsible for the modification of the rough eye phenotypes has not been identified, three regions are of particular interest because these results suggest the interacting gene or the encoded protein interacts specifically with the N-terminal half of CBP. Deletion of the 76B4-77B1 interval suppresses the rough eye phenotypes that are associated with expression of CBP FL, CBP deltaBHQ and CBP deltaQ proteins while enhancing the CBP deltaNZK (lacking the Nuclear hormone receptor binding domain, the Zinc finger domains and the KIX or CREB binding domain) retinal phenotype. These results suggest that at least one interacting gene resides within this interval and that it is either a target of or the encoded protein is a binding partner of the N-terminal region of CBP. Deletions of the 96F1-97B1 interval enhance the rough eye phenotypes that result from expression of CBP deltaBHQ and CBP deltaQ proteins. Likewise, deletions of the 5C3-6C12 interval suppress the retinal phenotypes of CBP deltaBHQ and CBP deltaQ expression. In both situations it is likely that the interacting gene or its encoded protein functions specifically with the N-terminal domain of CBP. The remaining 33 regions modified the expression of specific constructs. It will be interesting to determine if they encode for members of a common pathway or do they represent a set of factors within differing activities (Anderson, 2005).

Effects of Mutation or Deletion

At germband elongation, a mutation in Drosophila CBP results in a clockwise or counter-clockwise twisting of the embryo, just behind the cephalic furrow, often with the posterior side down. The ventral and cephalic furrows appear normal, but the mesodermally derived internal tissues and a block of ectodermal cells are often missing. On the basis of this phenotype, the Drosophila CBP mutant was named nejire, which means 'twist' in Japanese. Among the four Drosophila CBP mutants, the nej³ mutant exhibits the most severe phenotypes. In addition to the twisting of the embryo, cuticles of the late-lethal embryos exhibit several types of defects: disruption and twisting of the embryonic head skeleton; atrophy of the cephalo-pharyngeal skeleton and the thoracic region; sporadic deletion of naked cuticle between adjacent denticle belts; a decrease in the number of setae found in the denticle belts of the abdominal segments, and occasional reduction of the Filzkorper (Akimaru, 1997b)

The Drosophila X chromosome has been screened for genes whose dosage affects the function of the homeotic gene Deformed. One of these genes, extradenticle, encodes a homeodomain transcription factor that heterodimerizes with Deformed and other homeotic Hox proteins. Mutations in the nejire gene, which encodes a transcriptional adaptor protein belonging to the CBP/p300 family, also interact with Deformed. The other previously characterized gene identified as a Deformed interactor is Notch, which encodes a transmembrane receptor. These three genes underscore the importance of transcriptional regulation and cell-cell signaling in Hox function. Four novel genes were also identified in the screen. One of these, rancor, is required for appropriate embryonic expression of Deformed and another homeotic gene, labial. Both Notch and nejire affect the function of another Hox gene, Ultrabithorax, indicating they may be required for homeotic activity in general (Florence, 1998).

The extant nejire³ allele has been molecularly characterized as a null. However, in the Dfd interaction test, the viability of Dfd hypomorphs is not affected by heterozygous nej³. The failure of a nej null to interact with Dfd suggests that the Dfd-interacting nej alleles are not amorphic. Consistent with this interpretation, the lethal phases of the nej alleles in the current study differ from that of the nej³. Only ~15% of nej³ males die as embryos, demonstrating that the nej maternal component is sufficient for embryogenesis. However, 100% of both nej^Q7 males and nej^TA57j/nej^Q7 females, as well as ~35% of nej^S342/nej^Q7 females, die as embryos. The premature lethality of the Dfd-interacting alleles indicates that they provide a less functional maternal component, perhaps because of an antimorphic Nej protein. In nej^TA57/nej^Q7 or nej^Q7/Y cuticles, the maxillary and antennal sense organs often show a slight disruption in patterning; the mouth hooks and median tooth are reduced, and the proventriculus is sclerotized. No other phenotypes are consistently observed in cuticles of any nej genotype (Florence, 1998).

At the larval NMJ, an experimental decrease in postsynaptic excitation causes a compensatory enhancement of presynaptic release (see Glutamate receptor IIA and Glutamate receptor IIB). Thus there is a homeostatic regulatory system that maintains postsynaptic excitation through a retrograde signal(s) from muscle to nerve. A homeostatic regulatory system will likely include mechanisms that can monitor postsynaptic excitation and transduce this information through a retrograde signal to modulate presynaptic transmitter release. In principle, homeostatic regulation will require both positive and negative regulation of synaptic function (Marek, 2000).

Nuclear dCBP (Drosophila homolog of the CREB binding protein) located in the postsynaptic cell is required for presynaptic functional development. Viable, hypomorphic dCBP mutations have a ~50% reduction in presynaptic transmitter release without altering the Ca2+ cooperativity of release or synaptic ultrastructure (total bouton number is increased by 25%-30%). Exogenous expression of dCBP postsynaptically, in muscle, rescues impaired presynaptic release in the dCBP mutant background, while presynaptic dCBP expression does not. In addition, overexpression experiments indicate that elevated dCBP can also inhibit presynaptic functional development in a manner distinct from the effects of dCBP loss of function. Pre- or post-synaptic overexpression of dCBP (in wild type) reduces presynaptic release. However, no increase in bouton number is observed, and presynaptic overexpression impairs short-term facilitation. These data suggest that dCBP participates in a postsynaptic regulatory system that controls functional synaptic development (Marek, 2000).

This study used novel insertions in the dCBP sequence to regulate the expression of the gene. Four P(EP) elements have been identified that are located in a region ~3 kb upstream of the dCBP open reading frame. Two of these elements -- —P(EP)¹¹⁷⁹ and P(EP)¹¹⁴⁹— -- are oriented to overexpress dCBP, and two are oriented in the opposite orientation. P(EP) elements are mobile genetic elements (P elements) containing upstream transcription activation sequences (UAS sequence) that exploit the tendency for P elements to insert in 5' regulatory regions of a gene. Insertion of such an element in the proper 5'-3' orientation places random genes under the control of the UAS element, allowing them to be expressed in specific tissues under the control of an appropriate GAL4 driver. P(EP)¹¹⁷⁹ and P(EP)¹¹⁴⁹ can initiate overexpression of dCBP. Crossing P(EP)¹¹⁷⁹ or P(EP)¹¹⁴⁹ to GAL4 drivers that promote expression in nerve or muscle causes tissue-specific overexpression of dCBP, as detected by in situ hybridization using probes specific to the dCBP gene. Overexpression of dCBP has also been demonstrated by RT-PCR, using primers from the P(EP) elements and primers within the dCBP open reading frame. Each PCR product isolated by RT-PCR was sequenced to ensure that the correct open reading frame was driven by the P(EP) element. To ensure that these elements do not regulate overexpression of an additional message located between the P(EP) elements and the start of the dCBP open reading frame, the entire 3 kb genomic region between P(EP)¹¹⁷⁹ (the furthest insertion upstream of dCBP) and the start of dCBP was subcloned and sequenced. This region does not contain an additional transcript. Furthermore, the overexpression phenotype is phenocopied by overexpression of the dCBP cDNA under UAS control. Based on this sequence data and data obtained from RT-PCR from the P(EP)¹¹⁷⁹ and P(EP)¹¹⁴⁹ elements, a more complete characterization of the 5' untranslated region of dCBP is presented that extends to, and most likely beyond, these P(EP) elements. These results also predict that these P(EP) elements will generate a hypomorphic loss of dCBP function. Genetic, histological, and electrophysiological evidence demonstrates that the P(EP) elements P(EP)¹¹⁴⁹ and P(EP)¹¹⁷⁹ are hypomorphic mutations in dCBP. dCBP expression in muscle nuclei, as detected by an anti-dCBP antibody, is significantly reduced in the P(EP)¹¹⁴⁹ hypomorphic mutant (expression being decreased by ~75%–80%, based on reduced fluorescence intensity. Genetic experiments demonstrate that null or strong hypomorphic mutations in dCBP die as late embryos or first instar larvae. P(EP)¹¹⁷⁹ and P(EP)¹¹⁴⁹ fail to complement previously characterized hypomorphic alleles of dCBP (including dCBP^TA57 and dCBP^S342) for synaptic transmission defects. P(EP)¹¹⁷⁹ or P(EP)¹¹⁴⁹ trans-heterozygous with dCBP^TA57 or dCBP^S342 are semiviable as third instar larvae. These trans-heterozygous larvae are developmentally delayed by ~1 day and emerge as sluggish third instar larvae. It was therefore possible to proceed with anatomical and electrophysiological characterization of these dCBP loss-of-function mutations at the third instar larval synapse (Marek, 2000).

It is hypothesized that dCBP is centrally involved in the mechanisms that monitor postsynaptic activity. dCBP P(EP) element mutations are described, detected in a screen for mutations in genes that participate in the homeostatic regulation of presynaptic transmitter release. Perturbations in postsynaptic dCBP can affect presynaptic transmitter release. Furthermore, postsynaptic dCBP can act as both a positive and negative regulator of synaptic function. Finally, CBP in Drosophila and other systems is well suited to participate in a system that monitors postsynaptic activity. CBP function can be regulated by Ca2+ influx through voltage-gated channels and ionotropic receptors (Hardingham, 1999; Hu, 1999). Furthermore, CBP can act as a transcriptional coactivator with CREB and other transcription factors, as well as function as an intracellular signaling molecule via acetylase activity. In conclusion, it is proposed that dCBP is an essential component of the postsynaptic homeostatic mechanism that monitors activity and regulates presynaptic transmitter release (Marek, 2000).

It has been proposed that the homeostatic retrograde increase in presynaptic release is due to a signal that can enhance presynaptic transmission, similar to that proposed for the presynaptic expression of long-term potentiation. The current data suggest an additional model, one whereby a homeostatic increase in presynaptic transmission could also be achieved by relieving an inhibitory signal derived from postsynaptic dCBP function. Homeostatic control of presynaptic function at the Drosophila NMJ ensures that presynaptic release is precisely coupled to the growth of the postsynaptic muscle throughout development. To achieve constant muscle depolarization during development, homeostatic signaling must achieve a progressive and gradual increase in synaptic function. A progressive and gradual increase in synaptic function could be achieved through dCBP-dependent mechanisms since it can both promote and inhibit synaptic development. Activation of dCBP could promote synaptic strengthening, while sustained dCBP activation could inhibit further synaptic development, preventing runaway excitation. This is consistent with the demonstration that postsynaptic dCBP is necessary for normal synaptic development but that sustained overexpression of dCBP can inhibit presynaptic functional development (Marek, 2000).

Newly eclosed flies have wings that are highly folded and compact. Within an hour, each wing has expanded, the dorsal and ventral cuticular surfaces bonding to one another to form the mature wing. To initiate a dissection of this process, two mutant phenotypes were examined. (1) The batone (bae) mutant blocks wing expansion, a behavior that is shown to have a mutant focus anterior to the wing in the embryonic fate map. (2) Ectopic expression of protein kinase A catalytic subunit (PKAc) using certain GAL4 enhancer detector strains mimics the batone wing phenotype and also induces melanotic 'tumors'. Surprisingly, these GAL4 strains express GAL4 in cells, which seem to be hemocytes, found between the dorsal and ventral surfaces of newly opened wings. Ectopic expression of Ricin A in these cells reduces their number and prevents bonding of the wing surfaces without preventing wing expansion. It is proposed that hemocytes are present in the wing to phagocytose apoptotic epithelial cells and to synthesize an extracellular matrix that bonds the two wing surfaces together. Hemocytes are known to form melanotic tumors either as part of an innate immune response or under other abnormal conditions, including evidently ectopic PKAc expression. Ectopic expression of PKAc in the presence of the batone mutant causes dominant lethality, suggesting a functional relationship. It is proposed that batone is required for the release of a hormone necessary for wing expansion and tissue remodeling by hemocytes in the wing (Kiger, 2001).

Comparison of the effects of Ricin A and of PKAc on wing maturation indicates that ectopic PKAc does not simply inactivate hemocytes. Instead, it appears to substitute one normal function of hemocytes for another. Rather than carry out phagocytosis and ECM synthesis, hemocytes enter into an innate immune response in which lamellocytes are differentiated and crystal cells melanize target cells. Evidently, aggregation of lamellocytes within the wing blade interferes with wing expansion, and loss of normal hemocyte function interferes with bonding of dorsal and ventral surfaces. The observation that the effect of ectopic PKAc on the wing is suppressed by overexpression of Pan, the Drosophila homolog of mammalian blood cell transcription factors (lymphocyte enhancer-binding factor 1 and T cell factor), suggests that ectopic PKAc inhibits (or represses synthesis of) Pan, which in turn inhibits Wingless target gene expression. This conclusion is strengthened by the observation that ectopic expression of UAS-dCBP(nej+) using GAL4-30A produces phenotypes similar to those caused by ectopic PKAc. Pan is bound and its transcriptional activity inhibited by dCBP. Expression of PanDeltaN, a dominant-negative inhibitor of Wingless target gene expression, elicits what seems to be a massive induction of the cellular innate immune response. Thus, the Wingless signal transduction pathway may be involved in regulating a choice between the innate immune response and the apoptotic/ECM response (Kiger, 2001).

The dominant-lethal interaction between ectopic PKAc and bae is intriguing. When and how death occurs needs closer examination, as does the cellular focus of bae activity. What role PKAc normally plays in regulating hemocyte behavior remains to be investigated. The association of a wing phenotype with altered hemocyte behavior should provide a means of identifying additional genes involved in hemocyte function during wing maturation (Kiger, 2001).

A genetic system has been developed based upon the hobo transposable element in Drosophila melanogaster. hobo, like the better-known P element, is capable of local transposition. A hobo enhancer trap vector has been mobilized and two unique alleles of decapentaplegic (dpp) have been generated . A detailed study of one of those alleles (dpp^F11) is reported. This is the first application of the hobo genetic system to understanding developmental processes. LacZ expression from the dpp^F11 enhancer trap accurately reflects dpp mRNA accumulation in leading edge cells of the dorsal ectoderm. Combinatorial signaling by the Wingless (Wg) pathway, the Dpp pathway, and the transcriptional coactivator Nejire (CBP/p300) regulates dpp^F11 expression in these cells. This analysis of dpp^F11 suggests a model for the integration of Wg and Dpp signals that may be applicable to other developmental systems. This analysis also illustrates several new features of the hobo genetic system and highlights the value of hobo, as an alternative to P, in addressing developmental questions (Newfeld, 2002).

During early stages of embryogenesis, wg and dpp are expressed in undifferentiated dorsal ectoderm. wg mRNA expression, in 15 stripes along the entire dorsal-ventral axis of the embryo (including the dorsal ectoderm), begins at stage 8. wg expression persists in this striped pattern through stage 17. dpp mRNA is expressed on the dorsal side of the embryo along the entire anterior-posterior axis, beginning at stage 4. dpp mRNA expression persists in a large portion of the dorsal ectoderm through stage 8 and resolves into leading edge cell-specific expression in stage 12 embryos. The embryonic expression pattern of nej has not been reported. However, some information can be inferred from nej mutant phenotypes. nej zygotic mutant embryos show visible defects in the tracheal system at stage 12. The tracheal system is derived from the dorsal ectoderm, suggesting that nej is expressed in this tissue prior to stage 12 (Newfeld, 2002).

Analysis of dpp^F11 suggests that dpp expression in leading edge cells is initiated by prior episodes of wg and dpp expression in the undifferentiated dorsal ectoderm. The maintenance of dpp expression in leading edge cells appears to require continuous input from wg and from a dpp feedback loop. The initiation and maintenance of dpp expression in leading edge cells also require continuous nej activity. Overall, these data are consistent with the following combinatorial signaling model: Med (signaling for the Dpp pathway) interacts with Arm (signaling for the Wg pathway) via the transcriptional coactivator Nej. This multimeric complex initiates and, with continuous signaling, maintains dpp expression in leading edge cells (Newfeld, 2002).

These data extend previous studies of dpp expression in leading edge cells and Dpp signaling in several ways. A role for Wg signaling in the regulation of dpp expression in the leading edge has been suggested. dpp leading edge expression is not maintained in arm² zygotic mutants and does not initiate in arm² germline clones. nej and Med are involved in the regulation of dpp expression in leading edge cells. While nej³ enhances dpp wing phenotypes, Med¹ enhances nej³ embryonic phenotypes. This study suggests a role for nej in mediating combinatorial signaling by the Wg and Dpp pathways (Newfeld, 2002).

Several questions remain about the combinatorial regulation of dpp expression by Wg, Dpp, and Nej. One question is, how is Nej recruited to bridge the two pathways? Numerous studies have shown that p300/CBP transcriptional coactivation functions are stimulated by its phosphorylation but the site of phosphorylation has never been mapped. Perhaps Zeste white3 (a serine-threonine kinase in the Wg pathway) or Thickveins (a serine-threonine kinase in the Dpp pathway) are involved in recruiting Nej to participate in combinatorial signaling (Newfeld, 2002).

Control of chromosome structure is important in the regulation of gene expression, recombination, DNA repair, and chromosome stability. In a two-hybrid screen for proteins that interact with the Drosophila CREB-binding protein (dCBP), a known histone acetyltransferase and transcriptional coactivator, the Drosophila homolog of a yeast chromatin regulator, Sir2, was identified. In yeast, Sir2 silences genes via an intrinsic NAD+-dependent histone deacetylase activity. In addition, Sir2 promotes longevity in yeast and in Caenorhabditis elegans. In this report, the Drosophila Sir2 gene and its product were characterized and the generation of Sir2 amorphic alleles is described. It was found that Sir2 expression is developmentally regulated and that Sir2 has an intrinsic NAD+-dependent histone deacetylase activity. The Sir2 mutants are viable, fertile, and recessive suppressors of position-effect variegation (PEV), indicating that, as in yeast, Sir2 is not an essential function for viability and is a regulator of heterochromatin formation and/or function. However, mutations in Sir2 do not shorten life span as predicted from studies in yeast and worms (Newman, 2002).

Sir2 mutations are recessive suppressors of PEV, consistent with the model that Sir2 is involved in heterochromatin regulation across phylogenetic lines. Furthermore, CBP mutations dominantly suppress PEV, suggesting that Sir2 and CBP may act together to control the pattern of heterochromatin histone acetylation. For example, CBP may be inactivated by acetylation and Sir2 may be required to deacetylate CBP for proper heterochromatin function and/or formation. Alternatively, because CBP is known to acetylate proteins other than histones, Drosophila CBP may facilitate heterochromatin formation through modification of other proteins in the complex. The fact that Sir2 mutations are recessive suppressors of PEV while CBP mutations are dominant modifiers of PEV suggests that CBP's role in maintaining heterochromatin formation or function is more dosage sensitive. It is possible that Sir2 helps to stabilize heterochromatin but is not absolutely required for its formation. Sir2 antibodies can co-immunoprecipitate CBP from Drosophila Kc cells, demonstrating that Sir2 and CBP interact in vivo. Dosage studies of Sir2 and CBP on PEV will clarify the nature of the Sir2-CBP interaction. It will also be important to determine whether the deacetylase activity of Sir2 and the acetyltransferase activity of CBP are important for their functions in heterochromatin formation and/or activity (Newman, 2002).

Myb and CBP/Nejire work together to promote mitosis

Drosophila melanogaster possesses a single gene, Myb, that is closely related to the vertebrate family of Myb genes, which encodes transcription factors involved in regulatory decisions affecting cell proliferation, differentiation and apoptosis. In proliferating cells, Myb promotes both S-phase and M-phase, and acts to preserve diploidy by suppressing endoreduplication. The CBP and p300 proteins are transcriptional co-activators that interact with a multitude of transcription factors, including Myb. In transient transfection assays, transcriptional activation by Myb is enhanced by co-expression of the Drosophila CBP protein, dCBP/Nejire. Genetic interaction analysis reveals that these genes work together to promote mitosis, thereby demonstrating the physiological relevance of the biochemical interaction between the Myb and CBP proteins within a developing organism (Fung, 2003).

The cellular basis of the cuticular defects is observed in the wings and abdomens of myb mutants. In the wings, the lower hair density reflects a reduction in cell number, a consequence of the majority of mutant myb cells being arrested in the G₂ phase of their final cell cycle. A fraction of the arrested cells also lose the ability to suppress endoreduplication, resulting in DNA contents of higher than 4C. In the abdomens, epidermal cells that are mutant for Myb proliferate much more slowly than wild type cells and display a variety of mitotic defects, including abnormal numbers of centrosomes, resulting in aneuploidy and polyploidy. Therefore, Myb function appears to be required for multiple aspects of the cell cycle (Fung, 2003).

Do the enhanced cuticular phenotypes observed in myb mutants with reduced CBP levels reflect qualitative or quantitative defects at the cellular level? To address this question, pupal wing and abdominal tissue samples were prepared from females that were: wild type for Myb but heterozygous for a mutation in CBP (w,nej³/w,+); homozygous for a mutation in Myb but wild type for CBP (w,myb¹/w,myb¹), and simultaneously homozygous for a mutation in Myb and heterozygous for a mutation in CBP (w,nej³,myb¹/w,+,myb¹). During embryonic and larval development, the animals were incubated at 18°C, but to maximize differences in cellular morphology between the various genotypes, they were shifted to 24°C at puparium formation, thereby reducing Myb function during pupal development (Fung, 2003).

Postmitotic wing tissues were double stained with DAPI to visualize nuclei and rhodamine-labeled phalloidin, an F-actin specific stain, to visualize either cell boundaries (28-30 h after puparium formation, APF) or 'prehairs' (developing hairs at 34-36 h APF). In control samples (heterozygous for a mutation in CBP), each cell boundary encircled a single nucleus of relatively uniform size. After initiation of hair formation, the pattern and morphology of nuclei and prehairs was highly ordered and uniform, with each cell producing a single distally protruding hair. In myb¹ mutants, cell and nuclear sizes and shapes were more variable and generally larger than in controls. A few cells with bi-lobed nuclei (appearing like two fused nuclei) were also observed, an abnormality not detected in previous experiments, presumably because of its rarity. The size, shape and orientation of prehairs were also less uniform, and two prehairs extending from a single cell were occasionally observed. The variability in nuclear and cellular sizes and shapes became more pronounced when CBP levels were reduced within the context of a myb¹ mutant, with enlarged, misshapen or multi-lobed nuclei and/or or multiple separated nuclei within a single cell being commonly observed. The enhanced cellular defects associated with decreased CBP levels generated more extreme variability in the number, size and orientation of prehairs. Most notably, single cells producing two or more prehairs were common in these samples, correlating with the adult phenotype (Fung, 2003).

To quantitate the visual observations the areas of pupal wing cells and nuclei from the relevant genotypes were measured at their largest photographic cross-section. These results confirm that on average, myb¹ nuclei and cells are larger than controls, but also more variable. Both properties are substantially enhanced by decreased levels of CBP as shown by increased averages, ranges, and standard deviations. Six microscopic fields of wing cells (measuring 0.055 mm×0.07 mm) for each genotype were also examined for the presence of cells with either multi-lobed or more than one nucleus, and for cells with more than one protruding hair. No examples of these abnormalities were detected in control samples. For myb¹, a total of six cells with bi-lobed nuclei and 16 cells with multiple wing hairs were observed. These defects were greatly enhanced in nej³,myb¹/myb¹ samples, where a total of 43 cells with multi-lobed or 2 or more separated nuclei and 83 cells with multiple wing hairs were observed (Fung, 2003).

The presence of multi-lobed or multiple separated nuclei in cells that were homozygous for myb¹ and heterozygous for nej³, indicates that the cells enter, but do not complete mitosis or cytokinesis, a phenotype that is qualitatively different from most of the myb¹ mutant wing cells, which appear to have been arrested before entering into their final mitosis. On the surface, this result is counter-intuitive since it indicates that the myb¹ mutant cells with reduced CBP levels appear to be progressing further in the cell cycle than the myb¹ mutant cells with normal levels of CBP. However, the presence of fewer, larger wing cells when CBP levels are reduced, indicates that at least a portion of these cells are failing to complete cell division in the previous (second to last) cell cycle, thereby accounting for the enhanced phenotype (Fung, 2003).

The cells that form the wings and other adult thoracic and head structures proliferate throughout larval development, completing their final one or two cell divisions during early pupation. In contrast, the cells that form the adult abdominal epidermis do not divide during larval development, but undergo rapid proliferation after puparium formation. The first detectable defect in abdominal cells that are mutant for Myb is that they proliferate considerably more slowly than wild type cells. Therefore, the rate of abdominal epidermal development was compared between the same three genotypes used for the wing analysis. By 27 h APF, replacement of the larval abdominal epidermal cells by adult cells is already well underway in controls. This process is clearly retarded in myb¹ mutants, but the delay is much more dramatic when CBP levels are reduced within the context of a myb¹ mutant. In controls, the majority of polyploid larval cells have already been replaced with adult cells by 32 h APF, even though cell proliferation continues until about 40 h APF. In contrast, small regions of larval cells at the segment boundaries (which are the last to be replaced) were still present in myb¹ mutants at 42 h APF in, and much larger regions of larval cells remain when CBP levels are reduced, even at 45 h APF. Since the animals were shifted to a non-permissive temperature at puparium formation (24°C), neither the myb¹/myb¹ nor the nej³,myb¹/+,myb¹ females survived to adulthood. Therefore, the cellular defects observed in these experiments are expected to be more extreme than those represented by the cuticular defects in adults that were raised entirely at permissive temperature. However, the dramatic delay in replacement of larval cells by adult epidermal cells is likely to account for the undifferentiated cuticle observed between segments and along the dorsal midline in nej³,myb¹/+, myb¹ adults (Fung, 2003).

Although mutant myb cells proliferate slowly, the mitotic index is actually higher in the mutant cells than in controls throughout pupal development, indicating a specific delay in progression through mitosis. Using an antibody for a mitotic-specific phospho-epitope on histone H3 (PH3) to identify mitotic abdominal histoblasts, it was found that at 32 h APF, the average mitotic index was 4.5±1.1% for w,nej³/w,+; 10.3±0.9% for w,myb¹/w,myb¹; and 17.4±1.1% for w,nej³,myb¹/w,+,myb¹. Similar results were also observed at other timepoints. This data demonstrate that delayed progression through mitosis is dramatically enhanced when CBP levels are reduced within the context of a myb¹ mutant. The sluggish mitotic progression could account for most, if not all, of the associated reduction in the rate of cell proliferation (Fung, 2003).

In the later cell cycles of abdominal epidermal cells, abnormal mitoses associated with multiple functional centrosomes, unequal chromosome segregation, formation of micronuclei, and/or failure to complete cell division are common in cells that are mutant for Myb. It seemed likely that the mitotic abnormalities and slowed rates of cellular proliferation in myb mutants are directly related to each other, and it was therefore anticipated that the occurrence of mitotic abnormalities would also be enhanced by reduced levels of CBP. However, the data do not support this expectation. No changes were detected in the timing or rate of centrosomal and chromosomal abnormalities between w,myb¹/w,myb¹ and w,nej³,myb¹/w,+,myb¹ samples, suggesting that these defects may be at least partially independent of the reduced rate of proliferation. The size and morphology of the cells and nuclei from the two genotypes were also very similar, an observation that is consistent with the rate of mitotic defects not being increased in these samples. These findings are also consistent with the observation in adults that in regions where differentiated cuticle has formed, the phenotype is not appreciably different between myb¹ mutants that are wild type or heterozygous for nej³ (Fung, 2003).

Although there are some discrepancies, these results confirm the conclusions of (Hou, 1997) that co-expression of CBP with Myb enhances the ability of Myb to activate transcription of a reporter construct in transient transfection assays. Taken together with their demonstration of direct binding between CBP and Myb in vitro, it is concluded that like their vertebrate counterparts, the Drosophila CBP and Myb proteins physically interact and that CBP acts as a transcriptional co-activator of Myb (Fung, 2003).

CBP-related protein, p300, can acetylate lysines within a highly conserved region of the human c-Myb protein, and the acetylation enhances the DNA-binding and transactivation capabilities of c-Myb. Of the three lysine residues within the conserved region (region III) that may be acetylated (K471, K480 and K485), the first two are conserved at equivalent positions in the Drosophila Myb protein (K450 and K459). The evolutionary conservation of these lysine residues suggests that they may be targets for acetylation by Drosophila CBP and that the mechanism of activating the Drosophila Myb protein via acetylation may also be conserved (Fung, 2003).

Reducing the levels of CBP in animals mutated for Drosophila Myb enhances virtually all aspects of the mutant phenotype: viability is reduced and cuticular and cellular defects are increased. The genetic interaction between Myb and CBP provides direct evidence that the biochemical interaction between CBP and Myb proteins (demonstrated in mammalian and Drosophila systems) is physiologically relevant within the context of a developing animal. Previous studies have shown that Drosophila CBP functions during multiple stages of development and that mutations in CBP/nej produce pleitropic phenotypes, indicating that CBP may be required for multiple developmental processes. Indeed, CBP/nej has been implicated in several signal transduction pathways that regulate developmental patterning, including the Decapentaplegic, Hedgehog, and Wingless pathways. However, the analysis presented here provides the first explicit evidence that Drosophila CBP is directly involved in regulating cell proliferation (Fung, 2003).

A paradox of CBP/p300 function is that these proteins appear to be capable of having opposing effects on cell proliferation. Mice or humans with mutations that led to reduced levels or activity of CBP display markedly increased susceptibility to tumorigenesis, indicating that they function as tumor suppressors. However, a plethora of biochemical and cell culture studies have shown that CBP/p300 physically interacts with, and activates a number of transcription factors known to promote cellular proliferation, including E2F1 and oncoproteins such as JUN, FOS and MYB. Still, direct evidence for CBP/p300 being able to cooperate with any of these transcription factors to drive proliferation within an animal has been lacking. Therefore, the finding that Drosophila CBP is required in concert with Myb for positive regulation of the cell cycle during Drosophila development validates a physiological role for CBP/p300 in promoting cell proliferation in vivo, and supports the proposal that the pro-or anti-proliferative effects of CBP/p300 are dependent on cellular context (Fung, 2003).

The CBP coactivator functions both upstream and downstream of Dpp/Screw signaling in the early Drosophila embryo

The CBP histone acetyltransferase plays important roles in development and disease by acting as a transcriptional coregulator. A small reduction in the amount of Drosophila CBP (dCBP) leads to a specific loss of signaling by the TGF-ß molecules Dpp and Screw in the early embryo. The expression of Screw itself, and that of two regulators of Dpp/Screw activity, Twisted-gastrulation and the Tolloid protease, is compromised in dCBP mutant embryos. This prevents Dpp/Screw from initiating a signal transduction event in the receiving cell. Smad proteins, the intracellular transducers of the signal, fail to become activated by phosphorylation in dCBP mutants, leading to diminished Dpp/Screw-target gene expression. At a slightly later stage of development, Dpp/Screw-signaling recovers in dCBP mutants, but without a restoration of Dpp/Screw-target gene expression. In this situation, dCBP acts downstream of Smad protein phosphorylation, presumably via direct interactions with the Drosophila Smad protein Mad. It appears that a major function of dCBP in the embryo is to regulate upstream components of the Dpp/Screw pathway by Smad-independent mechanisms, as well as acting as a Smad coactivator on downstream target genes. These results highlight the exceptional sensitivity of components in the TGF-ß signaling pathway to a decline in CBP concentration (Lilja, 2003).

These results suggest that several transcription factors that regulate expression of Dpp/Screw signaling components require the dCBP coactivator for their function in Drosophila embryos, and implicate dCBP in regulation of the Dpp/Screw pathway independently of an interaction with Smad proteins. An additional role of dCBP is to regulate Dpp-target genes, acting at a step downstream of Smad protein phosphorylation. It is likely that direct interactions between dCBP and Mad/Medea contribute to regulation of Dpp target genes). Such interactions have been observed in vitro, both in mammalian systems and using Drosophila proteins. However, a major cause of impaired Dpp/Screw signaling in dCBP mutant embryos is due to reduced tolloid expression. This prevents Dpp/Screw from initiating a signaling event in cells that would normally receive the Dpp/Screw signal, presumably by a failure to cleave the Dpp-Sog and/or Screw-Sog complexes. In fact, a majority of embryos that do not express the Dpp/Screw-target gene rhomboid in dorsal cells, also do not contain phosphorylated Smad proteins. Furthermore, the pattern of phosphorylated Smad proteins correlates closely with that of tolloid expression. For example, in many early, cellularizing dCBP mutant embryos, an anterior patch of both tolloid expression and phosphorylated Smad staining remains. At later stages, tolloid expression recovers in dCBP mutant embryos, as does Dpp/Screw signaling as revealed by Smad protein phosphorylation. This recovery of tolloid expression at later stages of development might explain the recovery of phosphorylated Smad proteins in dCBP mutant embryos, by allowing Dpp/Screw to signal. For these reasons, regulation of tolloid expression appears to be a major means of controlling Dpp/Screw signaling by dCBP (Lilja, 2003).

It is likely that reduced screw expression also contributes to the reduction of phosphorylated Smad proteins observed in dCBP mutant embryos. In both screw and tolloid mutants, phosphorylation of Mad is eliminated. Furthermore, progressive reduction in Screw activity leads to a corresponding progressive deletion of dorsal-most cell fate, the amnioserosa. Tsg is required together with Sog to generate peak Dpp activity in dorsal midline cells. Reduced tsg expression in dCBP mutants may therefore contribute to the lack of Dpp/Screw-target gene expression. However, it is not believe that this lack can explain the defects in dCBP mutants, because in tsg mutants, low levels of Dpp signaling persist in a broad dorsal domain, leading to expanded rhomboid expression in dorsal cells. By contrast, in dCBP mutant embryos, expression of genes in response to a low threshold of Dpp activity, such as U-shaped and the dorsal rhomboid pattern, is eliminated (Lilja, 2003).

These experiments do not address whether dCBP regulation of tolloid, screw, and tsg expression is direct or indirect. However, since expression of these genes begins at about the time when zygotic transcription initiates in the embryo, and the effect of dCBP is evident from the onset of expression of tolloid and screw, the notion is favored that dCBP is acting directly on the enhancers of these genes. It is not yet understood whether HATs such as CBP primarily act to acetylate large chromosomal domains, or are directed to specific genes. In the case of the tolloid gene, the results indicate that dCBP is being recruited to the enhancer by a DNA-binding protein, since the isolated enhancer removed from its normal chromosomal location requires dCBP for its activity (Lilja, 2003).

Given its central position in gene regulation and the great number of mammalian transcription factors shown to interact with CBP, relatively few genes are affected by the dCBP mutation. For example, activation and repression mediated by the Dorsal protein are unaffected in the dCBP mutant embryos, as demonstrated by the expression patterns of Dorsal target genes. Also, no defects in early segmentation gene expression could be observed in germline clone mutants. However, the nej¹ mutation used in this study to create dCBP mutant germline clone embryos is a weak mutation that results in a very modest reduction in dCBP levels. Since other means of reducing the dCBP amount by approximately two-fold results in similar gene expression defects, Smad proteins and the unidentified activators of tolloid, tsg, and screw expression are particularly sensitive to a decline in dCBP concentration. It may not be a coincidence that screw, tsg, tolloid, and Dpp-target gene expression are all specifically affected by a small dCBP reduction. Perhaps components of the Dpp/Screw signal transduction pathway have evolved to be coordinately regulated by a common coactivator. Given the phylogenetic conservation of the CBP protein and the TGF-alpha signal transduction pathway, as well as the ability of CBP and Smad proteins to interact in vitro, CBP is likely to play an equally important role in TGF-ß signaling in other metazoans (Lilja, 2003).

CREB binding protein functions during successive stages of eye development in Drosophila

During the development of the compound eye of Drosophila several signaling pathways exert both positive and inhibitory influences upon an array of nuclear transcription factors to produce a near-perfect lattice of unit eyes or ommatidia. Individual cells within the eye are exposed to many extracellular signals, express multiple surface receptors, and make use of a large complement of cell-subtype-specific DNA-binding transcription factors. Despite this enormous complexity, each cell will make the correct developmental choice and adopt the appropriate cell fate. How this process is managed remains a poorly understood paradigm. Members of the CREB binding protein (CBP)/p300 family have been shown to influence development by (1) acting as bridging molecules between the basal transcriptional machinery and specific DNA-binding transcription factors, (2) physically interacting with terminal members of signaling cascades, (3) acting as transcriptional coactivators of downstream target genes, and (4) playing a key role in chromatin remodeling. In a screen for new genes involved in eye development the Drosophila homolog of CBP has been identified as a key player in both eye specification and cell fate determination. A variety of approaches was used to define the role of CBP in eye development on a cell-by-cell basis (Kumar, 2004).

The early development of the compound eye is regulated in part by a regulatory network of genes that include the Pax genes twin of eyeless (toy), eyeless (ey), twin of eyegone (toe), and eyegone (eyg); the founding members of the Dach and Eya gene families dachshund (dac) and eyes absent (eya), and the Six class genes optix and sine oculis. Extracellular instructions from the Hh, Dpp, Egfr, Notch, and Wg signaling cascades are integrated into this network at several levels creating additional layers of complexity. A dominant allele of sine oculis, so^D, was used as the starting material for a genetic screen to isolate new genes involved in eye specification. The so^D allele appears to function as a dominant negative mutant: (1) so^D heterozygotes lack compound eyes while heterozygotes of the so³ null allele have wild-type eyes; thus so^D is a dominant mutant; (2) embryonic lethality results if the so^D mutation is placed in trans to the so³ allele (so^D/so³); (3) compound eye development is restored in so^D mutants by the addition of wild-type SO protein via UAS-so transgenes -- thus so^D has an inhibitory function. The open reading frame of the so^D mutant was sequenced and a single valine-to-aspartic acid substitution was found at amino acid 200 (V200D). This mutation occurs within the Six domain, which is implicated in both DNA-binding and protein-protein interactions with EYA. Mutations within this domain of So could negatively affect eye development by either altering its interactions with potential binding partners or causing inappropriate transcriptional regulation of downstream target genes (Kumar, 2004).

The retinal phenotypes of the eye-specific so¹ loss-of-function mutant and the so^D dominant negative allele differ slightly from one another. SO protein levels are below detection in so¹ mutant eye discs while remaining at wild-type levels in so^D discs. Similarly, the expression of several other genes that are required for eye development, such as dpp and dac, are not reduced in so^D mutants while being disrupted in so¹ mutants. Furthermore, in so¹ adults the region normally occupied by the compound eyes is replaced by surrounding head tissue. In contrast, so^D flies have a large nonpigmented and nondifferentiated field. The lack of retinal tissue in so^D adults can be traced back to a complete lack of photoreceptor differentiation during larval eye imaginal disc development as assayed by the absence of ELAV, a pan-neural protein. The presence of this nondifferentiated field in so^D adults allows for the isolation of both suppressor and enhancer mutations. Six complementation groups were discovered that suppress, and one complementation group that enhances the so^D no-eye phenotype. The enhancing locus is nej, the gene that encodes CBP in Drosophila (Kumar, 2004).

Removal of one copy of nej in a so^D background results in an eye phenotype that is now indistinguishable from so¹ loss-of-function mutants. Similar to so¹ mutants, eye imaginal discs from nej³/+; so^D/+ heterozygotes (nej³ is a null allele) are small and undergo increased levels of cell death, while adults lack the nondifferentiated field and instead contain only head tissue. Conversely, expression of CBP throughout the so^D retinal field suppresses the no-eye phenotype. Eye imaginal discs are near normal in size and contain large numbers of photoreceptor cell clusters, and adult eyes are fully pigmented although not normally patterned (Kumar, 2004).

CBP is expressed in all cells within the developing eye imaginal disc. Loss-of-function CBP mutations affect the expression of several eye specification genes within the embryonic visual system, protocerebrum, mesoderm, and the developing eye imaginal disc. Using viable loss-of-function allelic combinations, loss-of-function retinal clones, and RNAi interference, this study has demonstrated that each cell type in the developing eye, with the exception of the founder R8 photoreceptor, requires CBP for its specification. Using a 'pathway interference' approach it has been shown that CBP likely functions in the R3/R4 cell fate choice and in the specification of the R7 photoreceptor (Kumar, 2004).

The results presented here indicate a role for CBP in a myriad of developmental decisions within the developing fly retina. It remains to be determined if these effects are through repeated interactions with a small set of master regulatory proteins or with a larger set of signaling molecules and cell-subtype-specific transcription factors. It is more likely that the latter scenario will be correct. This is based on the large body of biochemical data that suggest CBP interacts with more than 100 proteins that are members of many diverse signaling cascades. Furthermore, no single gene has been shown to affect all of the processes that require the activity of CBP. Thus it is hypothesized that CBP functions as a connecting point for signaling, transcription, and chromatin remodeling during all phases of fly eye development (Kumar, 2004).

The sheer number of potential interactions mediated by CBP makes an analysis of this protein inherently difficult. To circumvent this potential problem, a pathway interference approach was used to dissect CBP function by expressing a series of truncated CBPs within the developing eye. The underlying idea behind this approach is that each protein variant will act as a protein sink and soak up a unique set of endogenous factors, thus providing insight into the processes that are affected by CBP. It also provides a first step toward understanding the role that each domain of CBP plays in the developmental process and lays the groundwork for identifying critical components using more biochemical methods. The target proteins are likely to interact with CBP at stoichiometric levels during normal development. However, by increasing the dosage of CBP, the amount of these proteins within a cell becomes limiting and loss-of-function phenotypes can be observed. This approach successfully revealed roles for CBP in the R3/R4 cell fate choice and in R7 fate specification (Kumar, 2004).

How CBP functions in any of these processes is still an unanswered question. Attempts to identify additional components of the regulatory network disrupted by expression of variant CBPs through the restoration of putative interacting and downstream factors were unsuccessful. The addition of any one single factor was insufficient to rescue the effects of any of the CBP variants. Although it is possible that none of the correct factors were tested, it is more likely that the observed phenotypes result from the loss of several proteins and adding just one is insufficient to restore normal eye development (Kumar, 2004).

How are so many developmental decisions in the developing eye regulated by CBP? On the basis of reported roles for CBP/p300 in mammalian development, CBP would appear to be the perfect candidate to act as a 'network manager' during eye development. A scenario can be envisioned in which every cell within the eye disc expresses CBP and a specific combination of transcription factors; some are present in restricted expression patterns while other are more promiscuously expressed. As signals are interpreted at the cell surface and transmitted into the nucleus, the CBP-transcription factor scaffold would interact with terminal members of signaling cascades and execute these instructions by modulating transcription of downstream target genes. Late in development this would translate into the differentiation of specific cell types -- photoreceptors, cone cells, pigment cells, and mechanosensory bristles. This is an attractive model for several reasons. (1) The uncommonly high number of described biochemical interactions suggests that CBP may act as a link between signaling pathways, specific DNA-binding proteins, and the basal transcriptional machinery. These qualities have been shown to be true in vitro. (2) It allows for individual cells to receive several common-use signals but then personalize the output. (3) The ability to interact with members of signaling pathways as well as remodel chromatin allows for very efficient transduction of extracellular instructions. This may be important for the recruitment of photoreceptors into the ommatidial cluster, a process that occurs over a relatively short period of time. This model can be extended to early events in eye specification. CBP is expressed in all cells of the eye and antennal tissues during early development, while expression of selector genes is restricted to the individual tissues. Signaling pathways that include Notch, Egfr, Hh, Dpp, and Wg are known to influence both eye and antennal development. CBP may mediate the interactions between signaling pathways and these selector genes, thereby participating in the process of subdividing the eye-antennal disc into the eye and antenna proper (Kumar, 2004).

Previous reports of CBP in the eye have focused on the role of CBP in the modulation of polyglutamine diseases and retinal degeneration. The work presented here extends these results and points to a role for CBP both in early eye determination and later in cell fate specification. The results that pertain to early eye determination are supported by the synergistic interactions between CBP and SIX, EYA, and DACH proteins observed in mammals. Furthermore, this study has demonstrated a role for CBP in the development of several photoreceptor cell subtypes including the R7 neuron. In recent years it has become increasingly clear that the molecules and mechanisms that control eye development have been preserved in both mammalian and invertebrate retinal systems. It will be interesting to elucidate the molecular and biochemical mechanisms by which CBP influences early eye specification and later photoreceptor cell fate decisions in both invertebrate and mammalian retinal systems (Kumar, 2004).

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