Beadex

Transcriptional Regulation

In wild-type discs dLMO protein is nuclear and is expressed at higher levels in the dorsal compartment of the mature third-instar disc than in the ventral compartment. This expression pattern mirrors that of the MS1096 GAL4 enhancer trap line. In early- to mid-third-instar discs both MS1096 and dLMO protein are restricted to the dorsal compartment. The observation that dLMO is initially expressed in dorsal cells and maintained at elevated levels in the dorsal compartment suggests that dLMO might be regulated by Apterous (Ap). To test this possibility ectopic Ap expression was forced using dpp-gal4 to direct UAS-Ap in a stripe of cells along the anterior-posterior boundary of the wing disc. dLMO is induced in Ap-expressing ventral cells to the same elevated level typical of cells in the dorsal compartment. Thus, Ap induces expression of dLMO in dorsal cells. The transition from exclusively dorsal expression to dorsal and ventral expression suggests that dLMO expression is initiated by Ap but comes under an additional control mechanism as the disc matures (Milan, 1998).

Compartment formation is a developmental process that requires the existence of barriers against intermixing between cell groups. In the Drosophila wing disc, the dorso-ventral (D/V) compartment boundary is defined by the expression of the apterous selector gene in the dorsal compartment. Ap activity is under control of dLMO (Beadex) which destabilizes the formation of the Ap-Chip complex. D/V boundary formation in the wing disc also depends on early expression of vestigial. These data suggest that vg is already required for wing cell proliferation before D/V compartmentalization. In addition, over-expression of vg can, to some extent, rescue the effect of the absence of ap on D/V boundary formation. Early Vg product regulates Ap activity by inducing dLMO and thus indirectly regulating ap target genes such as fringe and the PSalpha1 and PSalpha2 integrins. It is concluded that normal cell proliferation is necessary for ap expression at the level of the D/V boundary. This would be mediated by vg, which interacts in a dose-dependent way with ap (Delanoue, 2002).

Vnd/NK-2 homeodomain affinity column chromatography was used to purify Drosophila DNA fragments bound by the Vnd/NK-2 homeodomain. Sequencing the selected genomic DNA fragments led to the identification of 77 Drosophila DNA fragments that were grouped into 42 Vnd/NK-2 homeodomain-binding loci. Most loci were within upstream or intronic regions, especially first introns. Nineteen of the Drosophila DNA fragments cloned correspond to one locus, termed Clone A, which is 312 bp in length and contains five Vnd/NK-2 homeodomain core consensus binding sites, 5'-AAGTG, and is part of the first intron of the Beadex gene. The interactions between Clone A and Vnd/NK-2 homeodomain protein were further analyzed by mobility-shift assay, DNase I footprinting, methylation interference, and ethylation interference. The DNase I footprinting analysis of Clone A with Vnd/NK-2 homeodomain protein revealed three strong binding sites and one weak binding site between 15 and 130 bp of Clone A. Binding of the Vnd/NK-2 homeodomain to the 5'-flanking sequence of vnd/NK-2 genomic DNA was also analyzed. The DNase I footprinting result showed that there are two strong binding sites and five weak binding sites in the fragment between -385 and -675 bp from the transcription start site of the vnd/NK-2 gene (Wang, 2005).

Protein Interactions

Formation of the dorsal-ventral axis of the Drosophila wing depends on activity of the LIM-homeodomain protein Apterous (Ap). Ap activity levels are modulated by dLMO, the protein encoded by the Beadex (Bx) gene. Overexpression of dLMO in Bx mutants interferes with Apterous function. Conversely, Bx loss-of-function mutants fail to down-regulate Apterous activity at late stages of wing development. Biochemical analysis shows that dLMO protein binds to Chip, thus competing with Apterous binding to Chip. These data suggest that Apterous activity depends on formation of a functional complex with Chip and that the relative levels of dLMO, Apterous, and Chip determine the level of Apterous activity. The dominant interference mechanism of dLMO action may serve as a model for the mechanism by which LMO oncogenes cause cancer when misexpressed in T cells (Milan, 1998).

LIM domains are thought to mediate protein interactions and are found in a variety of different types of proteins, often in combination with other recognized protein domains, as in the LIM-homeodomain proteins. dLMO belongs to a class of LIM domain proteins that have two LIM domains and no other recognizable motifs (hence, the designation LMO, for LIM only). In view of the effects of dLMO on Ap function, experiments were designed to discover whether dLMO can interact with Ap, a LIM homeodomain protein and Chip, an LDB (LIM domain binding) protein. LDB proteins bind to the LIM domains of nuclear LMO proteins of the type encoded by Bx. Genetic interactions between chip and ap suggest that as with Ldb1 and XLim1, Chip binding might activate Ap function. When overexpressed, Bx appears to interfere with Ap function without affecting either Chip or Ap protein expression. This raises the possibility that dLMO might interfere with binding between Ap and Chip. This was tested using a coimmunoprecipitation assay in which the binding between constant amounts of Chip and Ap proteins was challenged by increasing concentrations of Bx protein. Chip protein can be immunoprecipitated with T7-epitope-tagged Ap protein and anti-T7 antibody, showing that Ap and Chip proteins bind in vitro. Binding between Chip and Ap was challenged by adding increasing amounts of in vitro-translated dLMO protein. A dose-dependent decrease in the amount of Chip immunoprecipitating with Ap is observed as the amount of dLMO protein is increased and a corresponding increase in the amount of Chip immunoprecipitating with dLMO is also observed. These observations indicate that dLMO can bind to Chip in vitro and can compete for binding between Chip and Ap in a concentration-dependent manner. As a further test, the LIM domains of Ap were expressed as a GST fusion protein and tested for binding to full-length dLMO, Chip, and Ap proteins. Ap binds to itself and to Chip but not to dLMO in the GST-pull-down assay. This suggests that dLMO interferes with formation of the active Ap-Chip complex by competing with Ap for binding to Chip (Milan, 1998).

A tetramer of dLBD (Chip) and Apterous confers activity and capacity for regulation by dLMO (Beadex). To test whether the active form of Apterous is a complex involving two molecules of Ap and two molecules of Chip, the effects of expressing dominant-negative forms of Chip that differ in their ability to bind Ap were compared. Overexpression of wild-type Chip has dominant-negative activity in vivo. It has been suggested that this could be due to formation of trimeric complexes (Ap:Chip:Chip) that lack a second Ap molecule because the dominant-negative activity of Chip can be suppressed by overexpression of Ap. It was reasoned that a truncated form of Chip lacking the LIM interaction domain (ChipdeltaLID) would also serve as a dominant negative but that its activity should not be suppressed by overexpression of Ap. Before testing the activity of the ChipdeltaLID construct in vivo, it was verified that the molecular interactions between Ap and Chip in vitro are consistent with the expectations based on analysis of the human LDB protein, NLI (Mil·n, 1999).

Complex formation between Ap, Chip, and ChipdeltaLID was assayed using in vitro translated proteins. Ap was expressed with a T7-epitope tag, incubated with 35S-labeled Chip or ChipdeltaLID, and immunoprecipitated with anti-T7. Full-length Chip coprecipitates with T7-Ap. ChipdeltaLID is not recovered above background levels when incubated with T7-Ap, confirming that Chip needs the LID to bind effectively to Ap. ChipdeltaLID coprecipitates when incubated with T7-Ap and full-length Chip, demonstrating formation of a three part Ap:Chip:ChipdeltaLID complex. To verify that a Chip dimer can bridge two Ap molecules, a tagged version of Ap (Ap-TAP) was used. The biological activity of Ap-TAP is comparable to that of wild-type Ap when ectopically expressed in vivo under GAL4 control. Ap-TAP was in vitro translated and bound to IgG beads. The beads were washed and incubated with labeled Ap with or without unlabeled Chip. Without Chip, only background levels of 35S-Ap are recovered. When Chip is present, Ap-TAP beads bind 35S-Ap, indicating formation of the tetrameric complex in vitro (Mil·n, 1999).

Overexpression of Chip represses the Ap targets fringe-lacZ and dLMO. Overexpression of ChipdeltaLID using patched GAL4 also represses fringe-lacZ and dLMO, indicating that both forms of Chip suppress Apterous activity when overexpressed. Overexpression of Chip under control of ap-GAL4 interferes with wing formation, producing a phenotype resembling the lack of ap function. This can be suppressed by coexpression of UAS-Chip and UAS-Ap. Overexpression of ChipdeltaLID using ap-GAL4 causes a mild apterous phenotype: distal notching of the wing margin and dorsal-to-ventral transformation of the alula. Although the phenotype suggests only partial reduction of Ap activity, the defects caused by overexpression of ChipdeltaLID cannot be suppressed by coexpression of Ap. These results suggest that ChipdeltaLID acts as a dominant negative for Ap activity in vivo by promoting the formation of a trimeric Ap:Chip:ChipdeltaLID complex that cannot bind another Ap molecule (Mil·n, 1999).

The LIM domain protein dLMO can compete with Ap for binding to its cofactor Chip. If the active form of Ap in vivo is a LIM-HD dimer bridged by a dimer of cofactor, dLMO might compete for Ap activity by displacing an Ap molecule from the Ap:Chip complex to form an Ap:dLMO heterodimer bridged by the cofactor. This model was tested by preparing a form of Ap that could dimerize but that could not be displaced by dLMO. To do so, a fusion protein consisting of the N-terminal dimerization domain of Chip linked to a C-terminal fragment of Ap containing the homeodomain (aa 270-469) was expressed. The new protein, called ChAp, lacks the LIM interaction domain of Chip and the LIM domains of Ap. Its structure should allow it to form an Ap dimer (Mil·n, 1999).

A test was performed to see whether ChAp has activity comparable to Ap in vivo. When expressed along the anteroposterior compartment boundary under control of dpp-GAL4, UAS-ap and UAS-ChAp produce essentially identical phenotypes. In both cases, ectopic wing margins are induced on both sides of the dpp-GAL4 stripe in the ventral compartment. The ectopic wing margin is due to the ectopic expression of Wingless in the ventral compartment. This correlates with ectopic induction of the dorsally expressed Ap targets fringe-lacZ and dLMO in ventral cells. These observations show that ChAp can mimic the effects of Ap in ectopic expression assays. A rescue assay was used to ask whether ChAp can functionally substitute for Ap in vivo. The wing defect in ap^GAL4/ap^rk568 flies is completely suppressed when wild-type Ap is expressed in dorsal cells using ap-GAL4. Dorsal expression of ChAp produces a comparable rescue. These results show that ChAp behaves like wild-type Ap when ectopically expressed and that ChAp can substitute for Ap in vivo (Mil·n, 1999).

According to the dimer model, ChAp should be sensitive to the dominant-negative activity of ChipdeltaLID but should not be subject to regulation by dLMO in vivo. dLMO is induced by Ap in the wing disc, and loss-of-function dLMO mutants produce defects that are thought to result from overactivation of Ap. ChAp overexpression in the dorsal compartment of an otherwise wild-type wing disc gives a phenotype that closely resembles the dLMO loss-of-function phenotype. The wings are smaller than wild type and are held in an abnormal position. The dorsal compartment is typically smaller than the ventral compartment, giving the wing a slightly curled appearance. The pattern of veins is also abnormal. Coexpression of the dominant-negative form of the cofactor, ChipdeltaLID, suppresses the ChAp overexpression phenotype. This indicates that dimerization is required for ChAp activity in vivo (Mil·n, 1999).

To ask whether ChAp is subject to regulation by dLMO, the ability of Ap and ChAp to suppress the effects of dLMO overexpression in vivo were compared. apGAL4/+; UAS-dLMO/+ wings show loss of the normal wing margin and sporadic patches of ectopic wing margin, thus promoting local overgrowth. Overexpression of wild-type Ap does not suppress the dLMO overexpression phenotype. Antibody staining shows that Wg is not expressed at the DV boundary in these discs. The wing pouch is very small, and the normally straight boundary between cells expressing Ap and adjacent nonexpressing cells is uneven. These observations suggest that Ap is nonfunctional in these discs in spite of being overexpressed. In contrast, coexpression of ChAp and dLMO restores Wg expression at the DV boundary even though dLMO is expressed at high levels. The resulting wings have a normal wing margin and resemble the dLMO mutant wing. These results suggest that ChAp overexpression phenocopies the dLMO loss-of-function mutant because ChAp is not sensitive to downregulation by dLMO. Consequently, ChAp remains active in the presence of excess dLMO (Mil·n, 1999).

The bridged dimer model suggests that removing dLMO activity would result in excess Ap activity. To test this, the properties of Chip and Ap interaction were exploited to regulate Ap activity in a dLMO mutant background. dLMO loss-of-function mutants were generated by excision of a GAL4-P element insertion in the second intron of the dLMO gene. Fortuitously, excision line hdp^R590 strongly reduces dLMO expression but leaves GAL4 and the cis-regulatory region unaffected, so that the mutant expresses GAL4 in the normal pattern of dLMO. hdp^R590 causes aberrant Serrate expression and a reduced dorsal wing pouch. The dLMO loss-of-function phenotype in this mutant can be suppressed by expression of ChipdeltaLID. The small wing size of hdp^R590 is fully rescued, and the abnormal venation is partially suppressed. Likewise, expression of a mutant form of Ap lacking only the homeodomain completely suppresses the hdp^R590 phenotype. Both of these constructs have mild dominant-negative effects that reduce Ap activity in vivo. These results confirm that the dLMO loss-of-function phenotype results principally from excess Ap activity at later stages of wing development. Further, they support the proposal that the normal function of dLMO is to regulate Ap activity levels by interfering with formation of an active complex consisting of two Ap molecules bridged by a dimer of Chip molecules (Mil·n, 1999).

The finding that ChAp can completely replace Apterous function in vivo suggests that the relevant feature of this tetrameric complex is the formation of a dimer of Ap. This molecule is not subject to negative regulation by dLMO and so remains constitutively active. The consequence is a failure to downregulate Ap activity as development proceeds. The phenotypic consequences of excess Apterous activity are comparable to those of the dLMO lack-of-function mutant. These findings support the view that the tetrameric complex between Ap and its cofactor Chip provides a means to generate the requisite bridged dimer of Ap, while allowing the activity of the complex to be regulated by the competitive inhibitor dLMO. It is suggested that this may provide a general model for regulation of LIM-HD activity. LMO family proteins may be generic antagonists of LIM homeodomain proteins through binding to their common LDB cofactors. The active complexes may be cofactor-bridged homodimers (as is the case for Ap in wing development) or heterodimers with other LIM-HD transcription factors or other types of LDB-binding transcription factors. Combinations of different transcription factors bridged by a cofactor dimer might broaden the range of possible target genes (Mil·n, 1999).

Apterous is a LIM-homeodomain protein that confers dorsal compartment identity in Drosophila wing development. Apterous activity requires formation of a complex with a co-factor, Chip/dLDB. Apterous activity is regulated during wing development by dLMO, which competes with Apterous for complex formation. Complex formation between Apterous, Chip and DNA stabilizes Apterous protein in vivo. A difference in the ability of Chip to bind the LIM domains of Apterous and dLMO contributes to regulation of activity levels in vivo (Weihe, 2001).

Apterous activity levels are spatially and temporally regulated in the wing disc by expression of dLMO. Comparing expression of Ap protein and mRNA in the wing imaginal disc suggested that Ap might be subject to post-transcriptional regulation. ap mRNA is expressed at similar levels in the presumptive wing hinge and wing pouch. By contrast, Ap protein levels are considerably lower in the wing pouch than in the hinge region. The region where Ap levels are low coincides with the region in which dLMO is expressed. This suggests that the difference in Ap protein levels reflects a post-transcriptional consequence of dLMO expression. To ask whether dLMO is responsible for reducing Ap levels where the two proteins are co-expressed, genetic mosaics were produced in which dLMO activity was removed from clones of cells. Ap protein was more abundant in cells homozygous mutant for dLMO^Delta39. The increased level of Ap protein in the clone was similar to the level detected in the hinge (Weihe, 2001).

These observations suggest that dLMO protein is responsible for the reduced level of Ap protein in the dorsal wing pouch. To further test this possibility, clones of cells overexpressing dLMO were created and Ap protein levels were assessed. As expected from the loss-of-function data, dLMO expression reduced Ap levels in the hinge region, where Ap levels are usually high. The lower level of Ap in the dorsal pouch was further reduced by elevated dLMO expression. It is therefore concluded that dLMO reduces Ap levels in third instar imaginal wing discs. To determine whether Ap protein might be degraded in dLMO-expressing cells by a proteasome-dependent mechanism, wing discs were incubated with the proteasome inhibitor MG-132. Ap protein levels were increased in the wing pouch relative to the levels in the hinge in drug treated. This suggests that Ap protein is more susceptible to proteasome-mediated degradation in cells expressing dLMO (Weihe, 2001).

Since dLMO competes with Ap for binding to Chip, the possibility that Ap protein may be protected when it is in a complex with Chip was examined. To test this, Chip^e5.5 mutant clones, which lack Chip protein and therefore lack Ap activity, were created. Ap protein levels were reduced in Chip mutant clones, and increased in the wild-type twin spots which contain a higher level of Chip protein. To verify that reduced Chip activity does not affect ap mRNA levels ap-lacZ reporter gene expression was examined in discs expressing the dominant negative form of Chip, ChipDeltaLID. Ap protein levels were reduced in cells expressing ChipDeltaLID but ap-lacZ levels were unaffected. Thus, loss of Chip leads to reduced levels of Ap protein. It was noted that Chip mutant clones also lack dLMO expression. Thus, loss of Ap protein in Chip mutant clones does not correlate with expression of dLMO, as in wild-type cells. Rather, reduction of Ap levels correlates with the availability of Chip as a binding partner. This suggested that binding to Chip contributes to stabilization of Ap (Weihe, 2001).

If Ap stability decreases when it is unable to bind DNA, it was reasoned that providing additional binding sites might stabilize the protein. To test this possibility a cell culture system was used in which the number of Ap-binding sites could be varied by transfection. It was first verified that co-expression of dLMO would decrease Ap stability in transfected cells. A constant amount of a plasmid directing expression of a Myc-tagged Ap protein was co-transfected with varying amounts of a plasmid directing expression of myc-tagged dLMO. As observed in the wing disc, overexpressed dLMO reduces Ap protein levels in S2 cells. It was noted that high levels of dLMO are required to reduce Ap levels. The relative levels of the two proteins can be directly compared in this assay by virtue of the myc-epitope tag. Comparison of relative levels of the endogenous proteins is not possible (Weihe, 2001).

dLMO has been proposed to act as a competitive inhibitor of Ap in vivo. This model suggests that overexpression of Ap should be able to produce phenotypes similar to those caused by reduced levels of dLMO; however, this has not been observed. Overexpression of Ap in its endogenous domain does not produce alterations in the wing comparable with those caused by loss of dLMO activity. By contrast, expression of fusion proteins between Chip and Ap, which are insensitive to repression by dLMO, produce the expected phenotypes. This suggests that Ap does not compete effectively with dLMO for interaction with Chip, even when overexpressed. These observations could be explained by an intrinsic difference in the affinities of the two LIM domain proteins for Chip. To test this possibility, a fusion protein was constructed that contains the LIM domains of dLMO (100 amino acids) but otherwise consists entirely of Ap sequences. This protein was called dLAp to indicate LIM-domains of dLMO in Ap. To distinguish dLAp from endogenous Ap and dLMO proteins, a C-terminal flag tag was included (Weihe, 2001).

To ask whether overexpression of dLAp in dorsal cells would compete effectively with dLMO to produce a net increase in Ap activity levels, wing development was compared in flies expressing dLAp or Ap under ap^Gal4 control. Ap overexpressing wings are normal. In ap^Gal4/+; uas-dLAp/+ flies defects were observed in wing veins, especially in the posterior crossvein and vein 5, and a held up wing phenotype. These defects resemble the dLMO mutant phenotype, which has been shown to be due to excess Ap activity. Another feature of dLMO mutant wings is overexpression of Serrate in the D compartment. Overexpression of wild-type Ap under ap^Gal4 control does not cause abnormal Serrate expression; however, expression of dLAp in ap^Gal4/+;uas-dLAp/+ wing discs induces ectopic Serrate in the dorsal compartment and causes mild reduction of the D compartment. Similar, though somewhat stronger effects were obtained by overexpression of the Chip/Ap fusion protein ChAp, which is insensitive to competition by dLMO. Thus, dLAp expression can increase Ap activity to levels above normal in the presence of dLMO (Weihe, 2001).

Ap activity can be abolished by overexpression of dLMO under ap^Gal4 control in the wing disc. Providing additional Ap protein by co-expression of Ap does not overcome the inhibitory effects of dLMO. Wingless is not expressed at the interface between D and V cells and the wing pouch is very small. By contrast, co-expression of dLAp restores Wingless expression along the DV boundary and wing pouch growth. This indicates that dLAp is able to restore Ap activity in the presence of dLMO. Taken together, these observations indicate that dLAp competes efficiently with dLMO for binding to Chip, whereas Ap does not. Since the only difference between Ap and dLAp is in the LIM domains, their different behavior is attributed to an intrinsic property of the LIM domains (Weihe, 2001).

This report addresses the problem of asymmetry in the competition between dLMO and Ap. The simplest model for competitive inhibition by dLMO would suggest that Ap should compete effectively with dLMO for binding to Chip when overexpressed. However, overexpression of Ap does not produce an excess of Ap activity. dLMO competes effectively for Ap activity, but the reverse is not true. Swapping the LIM domains of Ap for those of dLMO produces a functional Ap protein that is able to compete effectively with dLMO. This finding may provide an explanation for the non-reciprocal properties of Ap and dLMO. The effectiveness of dLMO as an inhibitor of Ap activity is attributed to an intrinsic difference in the ability of the LIM domains of these two proteins to bind to Chip. It is considered likely that the LIM domains of dLMO bind the LID of Chip with higher affinity than the LIM domains of Ap. However, these proteins have not been produced in soluble form at adequate concentrations and so the affinities of these binding interactions have not been determined directly (Weihe, 2001).

Osa modulates the expression of Apterous target genes in the Drosophila wing

The establishment of the dorsal-ventral axis of the Drosophila wing depends on the activity of the LIM-homeodomain protein Apterous. Apterous activity depends on the formation of a higher order complex with its cofactor Chip to induce the expression of its target genes. Apterous activity levels are modulated during development by dLMO (Beadex). Expression of dLMO in the Drosophila wing is regulated by two distinct Chip dependent mechanisms. Early in development, Chip bridges two molecules of Apterous to induce expression of dLMO in the dorsal compartment. Later in development, Chip, independently of Apterous, is required for expression of dLMO in the wing pouch. A modular P-element based EP (enhancer/promoter) misexpression screen was conducted to look for genes involved in Apterous activity. Osa, a member of the Brahma chromatin-remodeling complex, was found to be a positive modulator of Apterous activity in the Drosophila wing. Osa mediates activation of some Apterous target genes and repression of others, including dLMO. Osa has been shown to bind Chip. It is proposed that Chip recruits Osa to the Apterous target genes, thus mediating activation or repression of their expression (Milan, 2004).

This study presents evidence that Osa, a member of a subset of Brahma chromatin remodeling complexes, behaves overall as a general activator of Apterous activity in the Drosophila wing. Overexpression of Osa rescues and loss of Osa enhances the Beadex¹ phenotype. It does so by modulating the expression levels of Apterous target genes, some of them being activated (e.g. Serrate and probably other unknown target genes) and some repressed (e.g. Delta, fringe). Chip has been shown to bind Osa. The fact that Osa has different effects on the transcription of Apterous target genes suggests that Chip recruits Osa to the promoters and in combination with other unknown factors mediates either transcriptional repression or activation. Osa mediates repression of both Apterous dependent and independent expression of fringe, suggesting a direct and probably Chip independent effect of Osa on fringe transcription (Milan, 2004).

Apterous activity is regulated during development by dLMO. Osa is required to mediate repression of dLMO expression. Since both early and late expression of dLMO depend on Chip, it is postulated that Chip forms a transcriptional complex with Apterous in the D compartment and an unknown transcription factor expressed in the wing pouch. Osa may interact with Chip thus recruiting the Brahma complex to the dLMO locus and remodeling chromatin in a way that limits dLMO transcriptional activation. High levels of dLMO protein reduce Apterous activity and the Notch dependent organizer is not properly induced along the DV boundary. Osa mediated repression of dLMO expression may ensure moderate levels of expression of dLMO in the wing, thus allowing proper wing development. Gain of function mutations that cause misexpression of vertebrate LMO proteins have been implicated in cancers of the lymphoid system. Truncating mutations in the human SWI-SNF complex, the human homologues of the Brahma complex, cause various types of human cancers. The SWI-SNF complex may be required to mediate repression of LMO expression in lymphoid tissues. Thus, it would be very interesting to analyze if truncating mutations in members of the human SWI-SNF complex cause higher levels of LMO expression and are associated with lymphoid malignancies (Milan, 2004).

It has been shown that the Brahma complex plays a general role in transcription by RNA Polymerase II. Then, is Osa having a general effect on the expression levels of every gene involved in wing patterning? Several observations indicate this is not the case. (1) Osa is a component of a subset of Brahma (Brm) chromatin complexes. (2) Brahma and Polycomb were shown to have non-overlapping binding patterns in polytenic chromosomes. Those genes involved in wing patterning and regulated by Polycomb (i.e. Hedgehog) may not be affected by overexpression of Osa. (3) Overexpression of Osa has different effects on the expression levels of Serrate, Delta and fringe. (4) Osa has been shown to specifically regulate the expression of Wingless target genes and the Achaete-scute complex genes, interestingly by restricting their expression levels (Milan, 2004).

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