Interactive Fly, Drosophila

Mammalian Runt domain genes encode the alpha subunit of the heterometric DNA-binding factor PEBP2/CBF. The unrelated PEBP2/CBF beta protein interacts with the Runt domain to increase its affinity for DNA. The conserved ability of the Drosophila Runt protein to respond to the stimulating effect of mammalian PEBP2/CBF beta indicates that flies are likely to have a homologous beta protein. Using the yeast two-hybrid system to isolate cDNAs for Runt-interacting proteins, two Drosophila genes have been identifed: Brother and Big-brother. These genes have substantial sequence homology with PEBP2/CBF beta. Yeast two-hybrid experiments as well as in vitro DNA-binding studies confirm the functional homology of Brother, Big-brother, and PEBP2/CBF beta proteins, and demonstrates that the conserved regions of the Runt and Brother proteins are required for their heterodimeric interaction. The DNA-bending properties of Runt domain proteins, both in the presence and absence of their partners, has been examined. Runt domain proteins bend DNA: this bending is influenced by Brother protein family members, supporting the idea that heterodimerization is associated with a conformational change in the Runt domain. Analysis of expression patterns in Drosophila embryos reveals that Brother and Big-brother are likely to interact with Runt in vivo and further suggests that the activity of these proteins is not restricted to their interaction with Runt (Grolling, 1996).

The Drosophila gene runt plays multiple roles during embryogenesis, including one as a pair-rule class segmentation gene. The Runt protein contains an evolutionarily conserved domain (the Runt domain) that is found in several mammalian proteins including the human protein AML1, which is involved in many chromosome translocations associated with leukaemia. Specific DNA binding activity of a mammalian Runt domain is enhanced by a partner protein called PEBP2beta/CBFbeta. DNA binding activity of Drosophila Runt is also stimulated by this protein, suggesting the existence of a similar Runt partner protein in Drosophila. Two closely linked Drosophila genes have been cloned: runt domain partner (rp) beta1 and beta2. They encode homologs of mouse PEBP2beta/CBFbeta. They are highly homologous to each other and to their mammalian counterpart. Either of the rpb proteins is capable of forming a complex with Runt and stimulating its DNA binding activity, but their temporal and spatial distributions are quite dissimilar, suggesting that functional specificity of Runt may be conferred by the interacting partner. Runt represses transcription dominantly when coexpressed with either partner in cultured cells, a function consistent with a direct role for Runt in regulating expression of the even-skipped gene in Drosophila embryos. It is concluded that Drosophila Runt can interact with either of two Runt domain partners, and the resulting complex functions as an active repressor of transcription (Fujioka, 1996).

Runt domain family members are defined based on the presence of the 128-amino-acid Runt domain, which is necessary and sufficient for sequence-specific DNA binding. There exists an evolutionarily conserved protein-protein interaction between Runt domain proteins and the corepressor Groucho. However, the interaction is independent of the Runt domain and can be mapped to a 5-amino-acid sequence, VWRPY, present at the C terminus of all Runt domain proteins. Drosophila melanogaster Runt and Groucho interact genetically; the in vivo repression of a subset of Runt-regulated genes is dependent on the interaction with Groucho and is sensitive to Groucho dosage. Runt's repression of one gene, engrailed, is independent of VWRPY and Groucho, thus demonstrating alternative mechanisms for repression by Runt domain proteins (Aronson, 1997).

Unlike other transcriptional regulatory proteins that interact with Groucho, Runt domain proteins are known to activate transcription. The distinction between the Runt domain consensus (VWRPY) and the Hairy-related/HES consensus (WRPW) raises a question: are the C-termini of these families interchangeable? The ability of Runt domain proteins to activate transcription suggests that the interaction with Groucho is regulated: when Runt domain proteins assemble on a promoter that is to be activated, Groucho must either be absent or in a context where it cannot exert its repressive effects. The difference between the Groucho-recruiting C-termini of the Hairy-related/HES family and the Runt domain family may be the difference between a constitutive Groucho interaction and one that is regulated (Aronson, 1997).

Brother and Big brother were isolated as Runt-interacting proteins and are homologous to CBFb, which interacts with the mammalian CBFa Runt-domain proteins. In vitro experiments indicate that Brother family proteins regulate the DNA binding activity of Runt-domain proteins without contacting DNA. In both mouse and human there is genetic evidence that the CBFa and CBFb proteins function together in hematopoiesis and leukemogenesis. Functional interactions between Brother proteins and Runt domain proteins have been demonstrated in Drosophila. A specific point mutation in Runt has been shown to disrupt interaction with Brother proteins but does not affect DNA binding activity. The point mutation was introduced into Runt by a PCR based site-directed mutagenesis. The mutant is dysfunctional in several in vivo assays. The most sensitive targets of Runt are the odd-numbered stripes of engrailed (en) expression. Ectopic expression of the altered Runt[G163R] has no discernible effect on en expression, even at levels that are fivefold greater than required for repression of en by the wild-type Runt protein. To determine whether Runt[G163R] retains any residual activity a heat-shock driven ectopic expression assay was used. The high levels of Runt expression obtained by this method cause alterations in the expression of other pair-rule genes in addition to en. Even under these conditions, the pattern of en expression as well as that of even-skipped and fushi tarazu in embryos expressing Runt[G163R] is indistinguishable from that of wild-type embryos. These results indicate that Runt[G163R] is incapable of regulating expression of several of Runts targets in the pathway of segmentation (Li, 1999).

Interestingly, this mutant protein acts dominantly to interfere with the Runt-dependent activation of Sex-lethal transcription. To investigate further the requirements for Brother proteins in Drosophila development, an examination was carried out of the effects of expression of a Brother fusion protein homologous to the dominant negative CBFb::SMMHC fusion protein that is associated with leukemia in humans. This Bro::SMMHC fusion protein interferes with the activity of Runt and a second Runt domain protein, Lozenge. The effects of lozenge mutations on eye development are suppressed by expression of wild-type Brother proteins, suggesting that Brother/Big brother dosage is limiting in this developmental context. Results obtained when Runt is expressed in developing eye discs further support this hypothesis. These results firmly establish the importance of the Brother and Big brother proteins for the biological activities of Runt and Lozenge, and further suggest that Brother protein function is not restricted to enhancing DNA-binding (Li, 1999).

Repression of Sex lethal^Pe expression was observed in female embryos injected with Runt[G163R] mRNA. This dominant negative activity indicates that the Runt[G163R] protein interacts with some other factor(s) in the Drosophila embryos in a manner that interferes with the activity of the wild-type Runt protein. In contrast to this, no dominant negative interference is observed when runt[CK], a Runt derivative that is specifically impaired for DNA-binding, is used in this assay. If the Runt[G163R] protein is interfering by competing for interaction with some other limiting protein factors then Runt[CK] protein would also be expected to behave as a dominant negative. Taken together, these results suggest that DNA binding is required for the dominant negative activity of Runt[G163R]. This is somewhat surprising as the prevailing view, primarily from in vitro experiments, has been that the central function of the Bro/Bgb and CBFbeta proteins is to enhance DNA-binding by the Runt domain proteins. The data in this paper strongly suggest that the Bro proteins have other functions in addition to enhancement of DNA binding by Runt. What then might be the other functions of the Bro/Bgb proteins? One possibility is that Bro induces a conformational change in Runt that is required for transcriptional activation. Runt/Bro complexes induce a bend in DNA that is greater than that observed by the binding of Runt alone. Perhaps DNA-bending is critical for interactions between Runt and other transcription factors on the Sex lethal^Pe promoter. An alternative possibility is that Bro/Bgb may be a bridge between Runt and other proteins that are critical for transcription regulation. In this model Runt[G163R] would compete for binding to the early promoter region of Sex lethal^Pe-lacZ but when bound would fail to activate transcription because other Bro-interacting proteins are not recruited. In a two-hybrid screen for Bro-interacting proteins a number of proteins have been identified that appear to be members of the Trithorax group of transcriptional regulators (G. Golling, personal communication to Li, 1999) have been identified. Trithorax group proteins have been implicated as having widespread roles in transcription activation in Drosophila development and it is attractive to speculate that recruitment of such proteins by Runt and Bro contributes to the activation of Sxl transcription. It is clear from the results presented here that interactions between Runt domain proteins and Bro/Bgb/CBFbeta proteins are important for the functions of these conserved transcriptional regulators. Experiments that further address the functions of the Bro/Bgb and CBFbeta proteins will be essential for understanding the mechanisms that account for the pivotal regulatory roles of these proteins in diverse developmental contexts (Li, 1999).

Redundant function of runt domain binding partners, Big brother and Brother, during Drosophila development

The Core Binding Factor is a heterodimeric transcription factor complex in vertebrates that is composed of a DNA binding alpha-subunit and a non-DNA binding ß-subunit. The alpha-subunit is encoded by members of the Runt Domain family of proteins and the ß-subunit is encoded by the CBFß gene. In Drosophila, two genes encoding alpha-subunits, runt and lozenge, and two genes encoding ß-subunits, Big brother and Brother, have been identified. A sensitized genetic screen was used to isolate mutant alleles of the Big brother gene. Expression studies show that Big brother is a nuclear protein that co-localizes with both Lozenge and Runt in the eye imaginal disc. The nuclear localization and stability of Big brother protein is mediated through the formation of heterodimeric complexes between Big brother and either Lozenge or Runt. Big brother functions with Lozenge during cell fate specification in the eye, and is also required for the development of the embryonic PNS. ds-RNA-mediated genetic interference experiments show that Brother and Big brother are redundant and function together with Runt during segmentation of the embryo. These studies highlight a mechanism for transcriptional control by a Runt Domain protein and a redundant pair of partners in the specification of cell fate during development (Kaminker, 2001).

Sensitized genetic screens have proved to be powerful tools in identifying interacting proteins that participate in many different developmental pathways. A particularly impressive use of this technique in the Drosophila eye has led to the identification of the mutations in the components of the RTK pathway. Such a screening technique was used to generate mutations in genes that function with lz during eye development. The identification of mutations in a direct transcriptional target of Lz, D-Pax2, and the gene encoding a binding partner of Lz, Bgb, suggests that this screen is able to detect proteins whose function is directly related to that of Lz (Kaminker, 2001).

In this screen, two alleles of hsp83 were isolated as dominant enhancers of lz^ts1. Drosophila Hsp83 is a chaperone protein that has been shown to physically interact with Raf. Mutations in hsp83 were identified as downstream modifiers of the sevenless and EGFR RTK pathways. 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. 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. 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 (Kaminker, 2001).

The screen also uncovered two alleles of osa/eld, a member of the brahma (brm) complex, involved in chromatin remodeling. The identification of osa as a dominant enhancer suggests that Lz may have a function related to chromatin remodeling. This is not surprising since 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. Additionally, during myeloid differentiation, Runx1 has been shown to interact with p300/CBP, 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. 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 (Kaminker, 2001).

This paper focused on the function of the partner proteins since mutations in Bgb were identified as modifiers of lz. The similarity in the phenotype of lz^ts1; Bgb^D/Df(3L)Bgb^K4 mutants to the null allele of lz suggests an absolute functional requirement of the partner protein during eye development. Similarly, ds-RNA interference results suggest that both partner proteins are able to function with Run during embryonic pattern formation (Kaminker, 2001).

It remains to be proven whether the disorganization seen in the PNS of Bgb can be attributed to Bgb function with the known Runt domain 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 reveals two additional runt domain proteins (Kaminker, 2001).

S2 cell expression data show that Bgb is translocated to the nucleus only in the presence of Lz. Although Bgb has a nuclear localization signal (NLS), 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 as well as with Homothorax (Htx) and Extradenticle (Exd) heterodimers. In these examples, the localization to the nucleus of either Tgo or Exd, depends on the presence of Sim or Hth, respectively. Recent work has shown that Hth binding allows nuclear transport of Exd by simultaneously inhibiting its nuclear export signal (NES) while activating its NLS. Bgb does not have a leucine-rich sequence typically associated with an NES; 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 alpha- 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 (Kaminker, 2001).

The Lz/Bgb complex provides an interesting example of post-translational stabilization of proteins through the formation of heterodimeric complexes. While the possibility that low levels of Bgb protein remain in the cytoplasm of the cell in a lz mutant background cannot be ruled out, 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 alpha-partner. Similar mechanisms involving degradation 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. As an interesting contrast to these results, in mammalian systems it is the alpha-subunit, Runx1, that is stabilized by CBFß. In this case, the absence of the ß-partner causes a proteosome-mediated degradation of the alpha-subunit (Kaminker, 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, these proteins are 59% identical throughout the remainder of their sequence. Furthermore, Bro and Bgb have overlapping expression domains during embryogenesis. ds-RNA-mediated genetic interference experiments used in this study 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 revealed only in the absence of both Bro and Bgb (Kaminker, 2001).

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. 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. Yet, a compensating mechanism can be created in a background when one of the two genes is eliminated (Kaminker, 2001).

Detection of mutations in genes that function redundantly poses a difficult challenge to genetic analysis. The 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 (lz^ts1; 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 (Kaminker, 2001).

mRNA TRANSPORT

Molecular motors actively transport many types of cargo along the cytoskeleton in a wide range of organisms. One class of cargo is localized mRNAs, which are transported by myosin on actin filaments or by kinesin and dynein on microtubules. How the cargo is kept at its final intracellular destination and whether the motors are recycled after completion of transport are poorly understood. A new RNA anchoring assay in living Drosophila blastoderm embryos has been used to show that apical anchoring of mRNA after completion of dynein transport does not depend on actin or on continuous active transport by the motor. Instead, apical anchoring of RNA requires microtubules and involves dynein as a static anchor that remains with the cargo at its final destination. This study proposes a general principle that could also apply to other dynein cargo and to some other molecular motors, whereby cargo transport and anchoring reside in the same molecule (Delanoue, 2005).

This study has used a specific RNA anchoring assay to distinguish between the four main models that could explain how apical wg and pair-rule mRNA (runt, and fushi tarazu) are retained in the apical cytoplasm after their transport by dynein. The models that have been proposed could also apply to other molecular motors and their various cargos. (1) The dynein motor could release the RNA cargo at its final destination, allowing the RNA to bind to an actin-dependent static anchor and the motor to participate in further transport. (2) The anchor could be MT associated rather than actin based. (3) RNA could be retained in the apical cytoplasm by continuous active transport without anchoring. (4) The motor itself could retain the cargo and turn into a static anchor when it reaches the final destination (Delanoue, 2005).

At the outset of this study, it was anticipated that cargo anchoring via actin was the most likely possibility given that actin is thought to be involved in anchoring of many other RNAs. It was also thought that after a motor completes a transport cycle, it releases the cargo and is available for transport of new cargo. However, in general, there has not been very good direct evidence showing that such a model is correct because of the lack of an assay that could discriminate between the transport and anchoring steps. In this study, two specific assays were used: one for transport and another for anchoring. Both anchoring and transport were assayed at the same time in the same embryo using two distinct RNAs. These specific assays have allowed a test and refutation of the prevailing actin anchoring model at least in the case of runt, fushi tarazu and wg apical mRNA localization in the Drosophila blastoderm embryo. Against expectations, the results show that the fourth model is correct, namely that wg and pair-rule RNA are anchored by a dynein-dependent mechanism so that the motor molecules are maintained to the site of anchoring with the cargo. The data shows that the requirement for dynein to anchor the apical RNA is independent of the ATPase activity of the motor and its transport cofactors Egl and BicD, all of which are required for the active transport of the RNA. These observations are best explained by a model in which the dynein motor involved in apical transport of RNA does not release the cargo and acts as a static anchor at the final destination (Delanoue, 2005).

It is interesting to consider how a dynamic motor such as dynein could turn into a static anchor after completion of cargo transport. Dynein is a large multicomplex motor that is difficult to work with in vitro. Nevertheless, many of the subunits of dynein are defined and the force-generating protein, Dhc, is thought to contain physically distinct ATPase and MT binding domains. It is therefore easy to imagine how the motor could change to a static anchor by remaining attached to MTs via the MT binding domain and losing its ATPase force-generating capacity. Indeed, ATPase-independent MT binding has been observed with dynein under in vitro conditions. While it is difficult to compare in vitro studies with the current studies in vivo, the latter are likely to show much more complex and varied interactions with proteins in the cell. Indeed, anchoring may also involve interactions with additional components not present in vitro, such as MT-associated proteins (MAPs), which could stabilize the binding of dynein to the apical MTs or could physically obstruct the motor movement. Another possibility could be anchoring through association with ribosomes, but this can be ruled out in the case of wg and pair-rule RNA, since RNAs lacking a coding region can be transported and anchored correctly. Alternative hypotheses, which cannot be ruled out, include a change of conformation or modifications of the structure of the dynein-dynactin complex. While the data demonstrate conclusively a new RNA-anchoring function for dynein, they do not allow distinguishing between the various hypotheses of how this anchoring occurs at the molecular level, nor test definitively whether Dynactin is required for anchoring. p50/dynamitin is present with the anchored RNA, and overexpression of p50/dynamitin and a Glued/p150 allele cause a partial inhibition of RNA localization with no obvious effects on anchoring. These results suggest, but do not demonstrate conclusively, that Dynactin is not required for anchoring. Furthermore, while it is shown that the ATPase activity of the motor is not required for anchoring, this observation does not test whether dynactin is required in addition to dynein for anchoring (Delanoue, 2005).

Whatever the molecular basis for the dynein anchoring function that was uncovered, it seems likely that the described anchoring does not involve a single dynein molecule anchoring a single RNA molecule. Instead, the RNA cargo is likely to consist of particles containing many RNA molecules and probably many motor complexes. The cargo is thus likely to remain strongly attached to at least some motor molecules throughout transport and anchoring. However, it is not yet known what the linkers between the RNA and motors are (Delanoue, 2005).

Little is also known about the mechanism of anchoring of other dynein cargos, although the mechanism of transport of RNA by dynein could be very similar to other cargos such as lipid droplets. Dynein is also required for nuclear positioning and tethering in many systems, so its role as a static anchor may be widespread. Furthermore, some kinesin-like proteins are also thought to interact with static cell components, and recent in vitro studies show that myosin VI can switch from a motor to an anchor under tension. This process has been proposed to stabilize actin cytoskeletal structures and link protein complexes to actin structures. It is therefore proposed that myosins, kinesins, and dynein may all be able to switch under certain circumstances from dynamic motors to static anchors and that the observations of this study may represent a general principle for anchoring of some cargos following transport to their final cytoplasmic destination (Delanoue, 2005).

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