1 Department of Molecular, Cell and Developmental Biology,
2 Molecular Biology Institute,
3 Department of Biological Chemistry and Department of Human Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA
*Author for correspondence (e-mail: banerjee{at}mbi.ucla.edu)
Accepted April 26, 2001
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SUMMARY |
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Key words: Big brother, Brother, lozenge, runt, Runx, Eye development, Ommatidium, CBFß, RNAi
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INTRODUCTION |
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In humans, the RUNX1 and CBFß proteins have been extensively studied in the context of oncogenic forms that cause leukemia. The most frequent translocation [t(8;21)] associated with acute myeloid leukemia (AML) encodes a fusion product between RUNX1 and the protein ETO (reviewed by Downing, 1999). The fusion protein includes the RD, interacts with CBFß, and functions as a dominant negative transcription factor. The leukemic phenotype presumably results from the accumulation of secondary mutations. Additionally, a chromosomal inversion [Inv(16)] that generates a fusion protein, CBFß-SMMHC, between CBFß and the smooth muscle myosin heavy chain protein, has been associated with AML (Liu et al., 1993). This fusion protein complexes with RUNX1 but is retained in the cytoplasm, and therefore disrupts transcription (Adya et al., 1998; Kanno et al., 1998; Liu et al., 1993).
The Runt Domain was named after runt (run), best known for its role as a primary pair-rule gene in Drosophila embryonic patterning and segmentation (Gergen and Wieschaus, 1986). run also plays an important role in sex determination by directly controlling the expression of the sex lethal gene (Kramer et al., 1999). Additionally, run function is required for the development of the EL neurons in the embryonic CNS (Dormand and Brand, 1998; Duffy et al., 1991). A second protein containing the RD is Lozenge (Lz), which specifies both neuronal and non-neuronal cell types in the Drosophila eye (Daga et al., 1996; Flores et al., 1998). In the non-neuronal cone cells, Lz functions combinatorially with the transcription factors downstream of the Notch (N) and Epidermal growth factor receptor (EGFR) signaling pathways to activate the expression of D-Pax2 (also known as shaven; Flores et al., 2000). Similar mechanisms also operate in the control of the prospero (pros) gene in the eye (Xu et al., 2000). Additionally, lz functions during the development of olfactory sensory organ precursors (Gupta et al., 1998) where it regulates the expression of the proneural gene amos (Goulding et al., 2000). Finally, studies have defined a role for Lz in Drosophila hematopoiesis in establishing a sub-population of blood cells called the crystal cells (Lebestky et al., 2000; Rizki and Rizki, 1980). It is interesting to note that both vertebrate and Drosophila RD proteins are involved in the control of blood cell fate.
In Drosophila, two genes encoding ß-subunits, Brother (bro) and Big brother (Bgb), were first identified through homology searches (Fujioka et al., 1996; Golling et al., 1996). These are expressed in distinct but overlapping patterns during embryogenesis (Fujioka et al., 1996; Golling et al., 1996). Furthermore, Bro and Bgb have been shown to physically interact with Run and increase its affinity for DNA (Fujioka et al., 1996; Golling et al., 1996). Overexpression experiments using either Bro or Bgb suggest that such complexes can form in vivo (Li and Gergen, 1999). A more complete functional analysis of the Drosophila partner proteins was lacking because mutations in these genes were unavailable at the time.
Here we report a loss-of-function analysis of the partner proteins. A genetic screen using a sensitized background isolated a mutation in Bgb, and ds-RNA-mediated interference strategies illustrate a redundant role for Bro and Bgb during development. We further show that Bgb functions during embryonic and eye development in the context of Run and Lz and is stabilized only in the presence of these proteins.
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MATERIALS AND METHODS |
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Immunohistochemistry and ds-RNA experiments
A rabbit polyclonal antibody (-Bgb) was generated against a C-terminal peptide of the predicted Bgb sequence (RDNRQDEMEAVR) (Fujioka et al., 1996; Golling et al., 1996). The antibody was affinity purified using the peptide conjugated to a Pierce Immunopure column. The purified antibody was preadsorbed against fixed cuticle from wild-type larvae and used at a final dilution of 1:150. Additionally, the following antibodies were used:
-Run (1:250; Dormand and Brand, 1998),
Pros (1:10,000; Kauffmann et al., 1996), and DSHB antibodies
2B10 (1:20),
Elav (1:400),
Engrailed (1:200), and mAb22C10 (1:200). Secondary antibodies (goat
-mouse-HRP, goat
-rabbit-HRP) were obtained from Jackson ImmunoResearch and used at a final dilution of 1:200. Eye discs were stained as described previously (Rogge et al., 1995).
ds-RNA experiments were performed essentially as described previously (Kennerdell and Carthew, 1998). The results from the injections are summarized in Table 1. For each experiment, the observed segmentation phenotype is shown in three independent examples in Fig. 5. A certain fraction of the injected embryos either hatched as wild type or were grossly and non-specifically affected as a result of the injection. Their numbers relative to the total injected was independent of the nature of the ds-RNA and are included in Table 1.
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Df(3L)BgbK4 was generated by crossing y w / Y; P[1556 ry+] ry e / TM3 with y w / y w; 2-3 Ki /
2-3 Ki. Male progeny were mated to lzts1 virgins. The progeny from this cross were reared at 25°C and the males were scored for an enhanced eye phenotype. Mosaic clones were generated by crossing w / Y; BgbD FRT80B / TM6b with ey-flp w / ey-flp w; P[w+] FRT80B / P[w+] FRT80B. Mutant tissue was identified by the absence of pigment.
Mosaic clones in adult eyes were fixed and sectioned as described previously (Coyle-Thompson and Banerjee, 1993). For rescue experiments, full length Bgb cDNA (Fujioka et al., 1996) was cloned into the XbaI and NotI site of hsp70-pCaSpeR and germline transformants were generated.
High resolution sequence analysis of the BgbD and Bgb9 mutants was performed using the 33P-Thermo Sequenase Radiolabeled Terminator Cycle Sequencing Kit (Amersham Pharmacia Biotech).
Transient transfections were performed using the Drosophila Expression System Kit (Invitrogen) and antibody staining was performed as described by Fehon (Fehon et al., 1990).
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RESULTS |
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The en(lz)D/9 mutants were identified as alleles of Bgb based on several lines of evidence. First, we used a P-element excision strategy to generate a deletion belonging to the region because no chromosomal aberrations existed that eliminated this locus. A P-element located near Bgb was excised in a lzts1 sensitized background. About 20,000 lines were screened and one excision allele, en(lz)K4, was identified as a dominant enhancer of lzts1 at 25°C. This deletion completely eliminates the Bgb locus. The right breakpoint of this deletion maps within the transcribed region of an uncharacterized gene (Adams et al., 2000; Rubin et al., 2000) between Bro and Bgb. Thus, Df(3L)K4 does not eliminate any part of the Bro gene. The EMS-induced mutants, en(lz)D/9, fail to complement the lethality of Df(3L)K4 suggesting that they too carry a mutation in a gene uncovered by the deletion. Sequence analysis showed that en(lz)9 carries a mutation (A801C) in Bgb which results in a change from glutamic acid to aspartic acid in the Bgb sequence. Although non-conserved in the vertebrate protein, this amino acid is physically close to the segment that binds the -subunit (Goger et al., 1999; Tang et al., 2000; Warren et al., 2000). More strikingly, the en(lz)D chromosome carries a mutation (G808A) in Bgb which results in a change from a conserved glycine to an arginine. This glycine in Bgb is conserved in the vertebrate CBFß and Drosophila Bro proteins. This amino acid is also close to residues that physically interact with the Runt Domain (Goger et al., 1999; Tang et al., 2000; Warren et al., 2000). It is therefore likely that this mutation disrupts the stability of a Bgb/RD complex in vivo. Finally, flies carrying hsp70-Bgb constructs were generated using P-element mediated transformation (Rubin and Spradling, 1982) and assessed for rescue of the Bgb phenotype. Three independent hsp70-Bgb transformant lines were placed in homozygous en(lz)D and en(lz)9 genetic backgrounds. In each case, complete rescue to adult viability was obtained with the basal level of expression achieved without the application of a heat shock. Taken together, these data establish that en(lz)D and en(lz)9 carry mutations in Bgb and these lines were therefore renamed BgbD and Bgb9. The Df(3L)K4 was renamed Df(3L)BgbK4.
Expression studies
The Drosophila eye imaginal disc undergoes morphogenesis during the third larval instar. At this stage, a furrow sweeps across the disc from the posterior to the anterior. Cells entering the furrow undergo a synchronized round of cell division to give rise to a set of undifferentiated cells as well as pre-clusters of five neurons that differentiate in a stereotypical developmental order: R8, R2/R5, then R3/R4 (for a review of eye development see) (Wolff and Ready, 1993). The remaining undifferentiated cells subsequently give rise to R1/R6, R7, and the non-neuronal cone and pigment cells.
In order to determine the expression pattern of Bgb during morphogenesis of the eye disc, we generated a rabbit polyclonal antibody against a C-terminal peptide of Bgb that is not homologous with any peptide stretch of Bro. Bgb expression is seen in the basal nuclei of the undifferentiated cells immediately posterior to the furrow (Fig. 2A). Among the differentiated cells, Bgb expression is first obvious in the R8 cell (Fig. 2B). Posteriorly, Bgb is seen in the R1/R6 cells (Fig. 2B), and the R7 and cone cells (Fig. 2C). The level of Bgb is much higher in the R7 cell than in any of the other cells in the eye disc.
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The correlation between Lz and Bgb staining patterns is functionally relevant. In a lz mutant background, Bgb expression is eliminated from all cells in which Lz is normally expressed (Fig. 2F,G). Additionally, when Lz is misexpressed in R3 and R4, Bgb protein is seen in the nuclei of these cells (Fig. 2H). This misexpression is not accompanied by an increase in Bgb transcripts (not shown) which suggests that Lz does not transcriptionally regulate Bgb. Instead, as Lz and Bgb are binding partners, it is likely that Lz stabilizes the Bgb protein in cells in which they are co-expressed. We also examined the relationship between Run and Lz proteins. The expression of the Run protein is genetically downstream of Lz in the eye. In a lz mutant background, Run expression is limited to the R8 cell (Fig. 2I) and is eliminated from the presumptive R7 cell (Fig. 2J). Thus, in a lz mutant background, the only cell that expresses a RD protein is R8. This is significant since in this background, R8 is also the only cell that expresses Bgb (Fig. 2F). This suggests once again that the -subunit, in this case Run, is able to stabilize the ß-protein.
A correlation of the in vivo results described above was also seen in S2 cells. The nuclear localization of Lz is constitutive and does not require Bgb protein (Fig. 2M,N). In contrast, when Bgb alone was expressed in S2 cells, nearly all of the Bgb protein remained cytoplasmic (Fig. 2K). However, when a construct expressing wild-type lz was co-transfected, a majority of the Bgb protein was seen in the nucleus (Fig. 2L). Thus, Bgb cannot be translocated to the nucleus in the absence of Lz. The cytoplasmic protein can be detected in S2 cells because of the high level of expression. Presumably, in the in vivo situation, the cytoplasmic Bgb protein is rapidly degraded in the absence of an -subunit.
Phenotypic analysis
The identification of Bgb mutants as enhancers of lzts1 provides genetic evidence that Bgb functions during eye development. The enhanced roughness produced by the loss of one copy of Bgb in this sensitized background is rather mild (Fig. 3A,B). However, rare escaper flies of Df(3L)BgbK4/BgbD genotype can be generated in a lzts1 background. These flies are extremely weak and rarely seen. They can only be rescued if they are dissected from their pupal cases. Such flies have a very strong adult eye phenotype (Fig. 3D) that is identical to that of the lz null allele (Fig. 3C).
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Bgb and Bro function redundantly during development
The results described in Fig. 3A-D establish a role for Bgb in eye development. However, when BgbD/BgbD clones were generated in an otherwise wild-type (lz+) eye, the external phenotype of the facets (Fig. 4A) as well as the morphology of all retinal cells (Fig. 4B,C) appeared completely normal. Clones of BgbD were also examined in larval eye discs that were stained with antibodies directed against Elav, Cut and Pros. The expression pattern of these markers of neuronal and non-neuronal cells was wild type in all discs examined (not shown). Thus, all neuronal and non-neuronal cells in the eye are able to develop normally in the absence of the Bgb protein. The presence of a gene encoding the second partner protein, Bro, in the Drosophila genome, suggested to us the possibility that Bro and Bgb may function redundantly during development. This was investigated during embryonic development using the ds-RNA-mediated genetic interference method (Kennerdell and Carthew, 1998).
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Injection of ds-Bgb does not have any significant effect on the pattern of segmentation (Fig. 5G-I). In comparison, ds-Bro causes a segmentation phenotype (Fig. 5J-L) reminiscent of hypomorphic alleles of run (compare with Fig. 5B). Only a partial deletion of the naked cuticle between the second and third abdominal segments is apparent. Doubling the concentration of ds-Bro did not affect the severity of the phenotype (not shown). Strikingly, simultaneous injection of both ds-Bgb and ds-Bro resulted in embryos that have a much stronger segmentation phenotype (Fig. 5M-O), which is identical to that seen in a null allele of run (Fig. 5C), or in embryos injected with ds-run (Fig. 5D-F). These data show that although the disruption of Bgb does not generate a phenotype on its own, a role for Bgb in segmentation is revealed upon the disruption of both partner genes.
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DISCUSSION |
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In our screen, two alleles of hsp83 were isolated as dominant enhancers of lzts1. Drosophila Hsp83 is a chaperone protein that has been shown to physically interact with Raf (van der Straten et al., 1997). Mutations in hsp83 were identified as downstream modifiers of the sevenless and EGFR RTK pathways (Simon et al., 1991; van der Straten et al., 1997). Recent studies have indicated an extensive collaboration between RTK pathways and Lz in the regulation of direct target genes such as D-Pax2 and pros (Flores et al., 2000; Xu et al., 2000). It is therefore likely that hsp83 strengthens the RTK signal transduction cascade that functions with Lz in the regulation of target genes. In addition, HSP90, the mammalian homolog of hsp83, has been shown to associate with a variety of different transcription factors and has also been proposed to function in nuclear transport (reviewed by Helmbrecht et al., 2000). An analysis of the relationship between Hsp83 and Lz/Bgb might provide insight into the mechanism by which this transcription factor complex is translocated to the nucleus.
The screen also uncovered two alleles of osa/eld (Kennison and Tamkun, 1988; Treisman et al., 1997), a member of the brahma (brm) complex, involved in chromatin remodeling (Collins et al., 1999; Kennison and Tamkun, 1988; Vazquez et al., 1999). The identification of osa as a dominant enhancer suggests that Lz may have a function related to chromatin remodeling. This is not surprising as other Runx family members are thought to function in this manner. For example, Runx2 binding has been implicated in the remodeling of the rat osteocalcin promoter (Javed et al., 1999). Additionally, during myeloid differentiation, Runx1 has been shown to interact with p300/CBP (Kitabayashi et al., 1998), a protein involved in histone acetylation. Further, Drosophila Run has been shown to bend DNA and is likely involved in modifying the architecture of target enhancers (Golling et al., 1996). In the eye, Lz is essential for pre-patterning an undifferentiated population of cells and preparing them to activate different target genes in response to signal transduction cascades. It is possible that this process involves remodeling of the individual enhancers through the mediation of an Osa/Lz complex. The identification of osa as a genetic modifier of lz suggests the need for future biochemical experiments to establish if such protein complexes are indeed formed during development.
In this paper we have focused on the function of the partner proteins since mutations in Bgb were identified as modifiers of lz. The similarity in the phenotype of lzts1; BgbD/Df(3L)BgbK4 mutants to the null allele of lz suggests an absolute functional requirement of the partner protein during eye development. Similarly, the ds-RNA interference results suggest that both partner proteins are able to function with Run during embryonic pattern formation.
It remains to be proved if the disorganization seen in the PNS of Bgb is attributable to Bgb function with the known RD proteins. Similar PNS defects are seen in run mutants, but these phenotypes are difficult to interpret because of the additional segmentation phenotypes that could indirectly affect PNS development. It remains possible that Bgb functions with an as yet uncharacterized RD protein in the PNS. Consistent with this explanation, a survey of the sequence of the Drosophila genome (Adams et al., 2000) reveals two additional RD proteins.
Our S2 cell expression data show that Bgb is only translocated to the nucleus in the presence of Lz. Although Bgb has a nuclear localization signal (NLS; Fujioka et al., 1996), these data suggest an additional requirement of Lz binding for its transport to the nucleus. Similar regulation of nuclear transport has been reported with Single-minded (Sim) and Tango (Tgo) heterodimers (Ward et al., 1998) as well as with Homothorax (Htx) and Extradenticle (Exd) heterodimers (Pai et al., 1998). In these examples, the localization to the nucleus of either Tgo or Exd, depends on the presence of Sim or Hth, respectively (Pai et al., 1998; Ward et al., 1998). Recent work has shown that Hth binding allows nuclear transport of Exd by simultaneously inhibiting its nuclear export signal (NES) while activating its NLS (Abu-Shaar et al., 1999; Berthelsen et al., 1999). Bgb does not have a leucine-rich sequence typically associated with an NES (Fischer et al., 1995; Wen et al., 1995); co-localization into the nucleus in this case is likely to involve an unmasking of the NLS causing its exposure to the transport machinery. Obviously, nuclear localization of both the - and the ß-subunit is a prerequisite for activation of transcription. In fact, in human AML caused by Inv(16), the CBFß fusion protein is exclusively retained within the cytoplasm (Adya et al., 1998; Kanno et al., 1998; Liu et al., 1993).
The Lz/Bgb complex provides an interesting example of post-translational stabilization of proteins through the formation of heterodimeric complexes. While we cannot rule out the possibility that low levels of Bgb protein remain in the cytoplasm of the cell in a lz mutant background, the likely explanation for the Bgb protein not being detectable in the absence of Lz or Run is that the ß-subunit is degraded in the absence of the -partner. Similar mechanisms involving degredation of a subunit operate in creating stable Exd/Hth and Sim/Tgo complexes. Tissue lacking Hth or Sim will cause degradation of Exd and Tgo, respectively (Pai et al., 1998; Ward et al., 1998). As an interesting contrast to our results, in mammalian systems it is the
-subunit, Runx1, that is stabilized by CBFß. In this case, the absence of the ß-partner causes a proteosome-mediated degradation of the
-subunit (Huang et al., 2001).
The initial cloning of Bro and Bgb raised the possibility that these genes might function redundantly during development. Although there is a stretch of 156 amino acids at the N terminus of Bgb that is not present in Bro (Fujioka et al., 1996), these proteins are 59% identical throughout the remainder of their sequence. Furthermore, Bro and Bgb have overlapping expression domains during embryogenesis (Fujioka et al., 1996; Golling et al., 1996). ds-RNA-mediated genetic interference experiments used here clearly show that Bro and Bgb function redundantly during development as heterodimeric partners of Run. A loss-of-function phenotype equivalent to a complete run null allele is only revealed in the absence of both Bro and Bgb.
The two partner proteins do not function redundantly in all tissues. This is highlighted by the fact that Bgb mutants have a PNS defect on their own. Thus, at least in this tissue, Bgb function is not redundant with that of Bro. This is different from redundant gene pairs such as BarH1 and BarH2 which are co-regulated in all tissues and always function together (Higashijima et al., 1992). It is also interesting to note that injection of ds-Bro generates a fairly strong segmentation phenotype, while injection of ds-Bgb does not affect segmentation patterning at all. Therefore, it is possible that in the wild-type fly, when both partners are present, Run preferentially functions with Bro. However, only in the absence of Bro, can compensation of Run function be achieved through its binding Bgb. A comparable situation exists in mice. The paralogs Hoxa3 and Hoxd3 are expressed in the same tissue, but clearly have distinct functional requirements (Chisaka and Capecchi, 1991; Condie and Capecchi, 1993). Yet, a compensating mechanism can be created in a background when one of the two genes is eliminated (Greer et al., 2000).
Redundancy is a classical and important problem in genetics. As vertebrate genomes are analyzed in increasing detail, it is becoming evident that single mutant phenotypes may be masked due to the function of an alternate redundant gene (Müller, 1999). The completion of the sequence of the Drosophila genome (Adams et al., 2000) has revealed many gene families that function redundantly. Indeed, the identification of the Rhomboid family of genes led to the observation that Rho1 and Rho3 function redundantly during EGFR signaling (Wasserman et al., 2000). Another example is sloppy paired1 (slp1) and sloppy paired2 (slp2) which were identified as functionally redundant genes based on the fact that they are expressed in the same tissue but at slightly different levels (Bellen et al., 1989; Grossniklaus et al., 1992; Wilson et al., 1989). In spite of these advances, detection of mutations in genes that function redundantly poses a difficult challenge to genetic analysis. Our data show that at least for the case in study, dosage-sensitive screens involving sensitized genetic backgrounds can be used for the purpose of identifying redundant genes. Bro and Bgb together can be considered to contribute 4 copies of the partner gene. Loss of 1 out of these 4 copies in a sensitized background (lzts1; Bgb- Bro+/Bgb+ Bro+) gives rise to a detectable eye phenotype. Yet, loss of 2 copies in a wild-type background (lz+; Bgb- Bro+/Bgb- Bro+) does not generate a mutant phenotype. This remarkable sensitivity to dosage suggests that properly sensitized genetic screens could be used in the detection of redundant gene function.
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ACKNOWLEDGMENTS |
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