InteractiveFly: GeneBrief

Brahma associated protein 55kD: Biological Overview | References


Gene name - Brahma associated protein 55kD

Synonyms -

Cytological map position- 54B7-54B7

Function - chromatin protein

Keywords - TIP60 complex, dendrite wiring specificity in olfactory projection neurons, wing veins, regulation of EGFR pathway

Symbol - Bap55

FlyBase ID: FBgn0025716

Genetic map position - 2R:13,316,854..13,318,440 [-]

Classification - actin

Cellular location - nuclear



NCBI links: Precomputed BLAST | EntrezGene
BIOLOGICAL OVERVIEW

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

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

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

Bap55 is an actin-related protein, the majority of which physically associates with the Brahma (BRM) chromatin remodeling complex in Drosophila embryo extracts (Armstrong, 2002). There are two distinct BRM complexes: BAP (Brahma associated proteins; homologous to yeast SWI/SNF) and PBAP (Polybromo-associated BAP; homologous to yeast RSC), both of which contain Brahma, Bap55, and Snf5-Related 1 (Snr1). The human homologs of the BAP and PBAP complexes are called the BAF (Brg1 associated factors) and PBAF (Polybromo-associated BAF) complexes, respectively. The BRM/BAF complexes are members of the SWI/SNF family of ATP-dependent chromatin-remodeling complexes, and have been shown to both activate and repress gene transcription, in some cases, of the same gene (Tea, 2011).

In experiments purifying proteins in complex with tagged Drosophila Pontin in S2 cells, Bap55 was also co-purified as a part of the TIP60 complex, as determined by mass spectrometry. The TIP60 histone acetyltransferase complex has been shown to be involved in many processes, including both transcriptional activation and repression (Sapountzi, 2006). The complex contains many components, including Bap55, Domino (Dom), and Enhancer of Polycomb (E(Pc)) (Kusch, 2004). Dom, homologous to human p400, is the catalytic DNA-dependent ATPase; its ATPase domain is highly similar to Drosophila Brahma and human BRG1 ATPase domains. E(Pc) is homologous to human EPC1 and EPC2 and is an unusual member of the Polycomb group; it does not exhibit homeotic transformations on its own, but rather enhances mutations in other Polycomb group genes (Tea, 2011).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Interestingly, both human BAF53a and b can significantly rescue the Bap55-/- phenotype. Though in mammals BAF53a is expressed in neural progenitors and BAF53b is expressed in postmitotic neurons (Wu, 2007), they can perform the same postmitotic function in Drosophila PNs. Further, both can function with the TIP60 complex in PNs to regulate wiring specificity. These data suggest that the functions for BAF53a and b (in neural precursors and postmitotic neurons, respectively) diverge after the evolutionary split between vertebrates and insects (Tea, 2011).

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

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

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

Osa, a subunit of the BAP chromatin-remodelling complex, participates in the regulation of gene expression in response to EGFR signalling in the Drosophila wing

Gene expression is regulated in part by protein complexes containing ATP-dependent chromatin-remodelling factors of the SWI/SNF family. In Drosophila there is only one SWI/SNF protein, named Brahma, which forms the catalytic subunit of two complexes composed of different proteins. The protein Osa defines the Bramha associated protein (BAP) complex, and the proteins Polybromo and Bap170 are only present in the complex named PBAP. This work analysed the functional requirements of Osa during Drosophila wing development, and found that osa is needed for cell growth and survival in the wing imaginal disc, and for the correct patterning of sensory organs, veins and the wing margin. Other members of the BAP complex, such as Snr1, Bap55, Mor and Brahma, also share these functions of Osa. Focus was placed on the requirement of Osa during the formation of the wing veins. Genetic interactions between osa alleles and mutations affecting the activity of the EGFR pathway suggest that one aspect of Osa is intimately related to the response to EGFR activity. Thus, loss of osa and EGFR signalling results in similar wing vein phenotypes, and osa alleles enhance the loss of veins caused by reduced EGFR activity. In addition, Osa is required for the expression of several targets of EGFR signalling, such as Delta, rhomboid and argos. It is suggested that one role of Osa and Brm in the wing is to establish a chromatin environment in the regulatory regions of EGFR target genes, making them available for both activators and repressors and facilitating transcription in response to EGFR signalling (Terriente-Félix, 2009).

Chromatin structure is critical to modulate gene expression during development, and is affected by a variety of alterations such as histone modification, DNA methylation and changes in conformation. Proteins related to Drosophila Brm, such as yeast SNF2 modify chromatin in an ATP-dependent manner, causing repositioning of nucleosomes along the DNA and re-distribution of histone proteins between nucleosomes. The SWI/SNF complexes are conserved in all eukaryotes, and display specific interactions with distinct transcription factors to regulate different subsets of genes. There are several examples where sequence-specific transcription factors interact specifically with SWI/SNF complexes. For example, the ATPase BRG1 binds Zn-finger proteins and hBRM interacts specifically with CBF-1/Su(H), which recruits hBRM to Notch target promoters such as those of HES1 and HES5 (Terriente-Félix, 2009).

A key aspect in the analysis of Brm function is the identification of targets accounting for the functions of the complex. A necessary step in this analysis is the description of its functional requirements using genetic approaches; which helps to identify the specific processes affected by loss of BAP function. The current data indicate that Osa is required during wing disc development for cell viability, cell proliferation, and for the formation of wing veins and the wing margin. Interestingly, increased expression of Osa in the wing also causes phenotypes related to wing growth and patterning, such as reduced wing size, ectopic sensory organs and hairs and the formation of extra vein tissue in most interveins. This analysis focused mostly on Osa, and this raises the question of whether its requirement reflects the function of the BAP complex. This is the most likely scenario, because the preliminary analysis of other BAP members, such as Snr1, Bap55, Mor and Brm uncovers similar phenotypes in the wing. Thus, lowering Snr1, Bap55 or Mor levels reduces wing size, disrupts the wing epithelium and causes the differentiation of ectopic sensory organs and hairs. These wings also display loss of veins, and in general the overall phenotypes are similar to those of loss of Osa. The phenotype of iRNA expression directed against brm is much milder, perhaps due to a lower efficiency of this construct, but still these wings show a loss of veins phenotype. The reduction of Bap170, a member of the PBAP complex, causes the formation of ectopic veins, which is the opposite phenotype to loss of function in osa and in other members that are present in both the BAP and PBAP complexes. Thus, although Brm is the catalytic subunit in both BAP and PBAP, these complexes could act in opposite manners on the same target genes at least during wing vein formation (Terriente-Félix, 2009).

Some Osa requirements can be explained by modifications in the transcriptional response to the activity of the Wg signalling pathway and by effects on wg expression. The function of Wg is required for the formation of the wing margin, including the development of sensory organs and veins along the anterior wing margin. In the absence of Wg signalling the wing margin does not form, and when Wg signalling is inappropriately activated ectopic sensory organs and hairs differentiate throughout the wing blade. In addition to affecting the response to Wg signalling, Osa is also required for the expression of wg along the dorso-ventral boundary. This requirement might be related to Notch signalling in these cells, and explains why the remnants of wing tissue formed in osa mutant wings do not form the wing margin or ectopic sensory organs (Terriente-Félix, 2009).

This study focused on the characterisation of Osa during the formation of the longitudinal wing veins. This process is independent of Wg signalling, and requires the activities of the Notch, Dpp and EGFR signalling pathways. Osa is needed for the expression of bs in the interveins, because bs is not expressed in cells mutant for osa. The regulation of bs expression involves the activity of Ash2 and the function of the Hh and Dpp pathways. It is suggested that Osa participates in the activation of bs facilitating the availability of its regulatory regions to these activators. This aspect of Osa function does not explain the phenotype of loss of veins characteristic of osa mutant cells, because the loss of Bs expression is normally associated with the differentiation of ectopic veins. The only context where bs mutant cells differentiate as interveins is when the activity of the EGFR pathway is reduced. Therefore, it is suggested that loss of bs expression is accompanied in osa mutant cells by a failure in the response to EGFR activity, leading to the differentiation of intervein tissue. Interestingly, the expression of bs is also severely reduced when Osa is present at higher than normal levels, and in this case loss of Bs is accompanied, as expected, by the formation of ectopic veins. The effects of increased Osa on bs expression can also be explained if Osa facilitates EGFR activity, because this pathway mediates the repression of bs in the proveins. In both cases, the common aspect mediated by Osa might be to regulate bs expression in collaboration with its transcriptional activators and repressors (Terriente-Félix, 2009).

Because the failure of osa mutant cells to differentiate the veins is not due to changes in bs expression, nor to changes in the expression of provein genes such as kni and caup, the search for Osa candidate targets was narrowed to the EGFR pathway. Several results suggest a close relationship between Osa and EGFR signalling in the wing. First, the phenotypes of changing osa expression in the veins are very similar to those resulting from the same manipulation in EGFR activity. Thus, a reduction in any core component of the EGFR pathway eliminates the veins, whereas the increase in EGFR signalling activity causes the formation of extra veins in intervein territories. Second, genetic interactions were observed between osa and several components of the EGFR pathway compatible with a function of Osa promoting EGFR activity in the veins. Finally, the extra veins caused by excess of Osa are suppressed when the activity of EGFR is reduced, indicating that Osa cannot substitute for EGFR activity. The changes in vein and intervein expression patterns are already detected in the wing disc, before other signalling pathways, such as Dpp, act to promote vein formation. Taken together, these observations suggest that Osa facilitates the response to EGFR activity in the wing disc, but cannot promote the transcription of EGFR targets in the absence of EGFR signalling (Terriente-Félix, 2009).

The changes in the expression of EGFR target genes observed in osa mutant cells or in osa gain-of-function experiments are compatible with a direct function of Osa/BAP is the transcriptional regulation of EGFR targets such as Dl, rho and aos. How Osa and the BAP complex are targeted to specific genomic regions is not entirely clear, although it is likely that sequence-specific transcription factors are involved in this process. Transcription in response to EGFR signalling is mediated by proteins belonging to the ETS family, such as Pointed-P2, Pointed-P1 and Yan in Drosophila. However, these genes are not required during wing vein formation, suggesting that other ETS proteins or uncharacterised transcription factors bring about interactions between the regulatory regions of EGFR target genes and the BAP complex (Terriente-Félix, 2009).

It is unlikely that Osa participates in any step of the EGFR pathway previous to the transcription of its target genes. It was noticed, however, that the expression of dP-ERK, a direct read-out of the pathway activity, is also affected in osa mutant cells. Thus, these cells frequently fail to express normal levels of dP-ERK, a result indicating that EGFR activity is reduced. The most likely explanation for this observation is that, in the wing, the EGFR pathway is engaged in a positive feedback loop mediated by the activation of rho expression, which maintains EGFR activity in cells where it has already been activated. Thus, loss of osa leads to a failure to express rho and subsequently to a reduction in the activity of the pathway detected as a loss of dP-ERK expression. There is one experimental situation in which Osa function appears to be dispensable for the expression of EGFR target genes. Thus, when a constitutive active form of Ras, RasV12, is driven in the wing, the augmented expression of Dl and aos, and the accumulation of dP-ERK are not affected by a reduction in Osa levels. It is possible that in this situation of strong and constitutive activity of the pathway, the possible modifications to chromatin structure brought about by Osa/BAP on EGFR target genes are not necessary, perhaps because at this level of EGFR activation the transcriptional repressors antagonising EGFR target gene transcription, such as Cic and Gro, are inactivated by the pathway, and this might make dispensable the function of Osa (Terriente-Félix, 2009).

It is not entirely clear to what extent the link observed between BAP function and EGFR signalling during wing disc development is conserved in other developmental systems and in other organisms. Some phenotypes of osa and brm alleles described in the eye disc, such as the loss of photoreceptor cells, are also observed upon a reduction in EGFR activity. Similarly, the loss of distal growth in the legs is also characteristic of reduced EGFR activity. These data are indicative of a general requirement for Osa in the expression of EGFR target genes at least in imaginal discs. The genetic approach that was used identifies transcription downstream of EGFR signalling as a relevant in vivo function of BAP complexes. Subsequent biochemical analysis should determine whether the functional interactions observed are mediated by direct binding of BAP to the regulatory regions of bs and other EGFR target genes (Terriente-Félix, 2009).


REFERENCES

Search PubMed for articles about Drosophila Bap55

Armstrong, J. A., et al. (2002). The Drosophila BRM complex facilitates global transcription by RNA polymerase II. EMBO J. 21:5245-5254. PubMed ID: 12356740

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

Sapountzi, V., Logan, I. R. and Robson, C. N. (2006). Cellular functions of TIP60. Int. J. Biochem. Cell. Biol. 38: 1496-1509. PubMed ID: 16698308

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

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Biological Overview

date revised: 20 April 2011

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