runt

Targets of Activity

Expression of hairy stripes can be generated in a two-step process involving regulatory interactions between the primary pair rule genes hairy and runt. Expression of h stripes 3 and 4 is directed by a common cis-acting element that results in an initial broad band of gene expression covering three stripe equivalents. Subsequently, this expression domain is split by repression in the forthcoming interstripe region, a process mediated by a separate cis-acting element that responds to Runt activity (Hartmann, 1994).

The so called primary pair-rule genes are involved in refinement rather than establishment of the fushi tarazu stripes. The order of appearance of ftz stripes has no relationship with the order of appearance of hairy stripes as would be expected if ftz stripes were generated by h repression. Furthermore, the seven ftz stripes are correctly established in embryos carrying mutations in h, even-skipped or runt, with normal expression patterns decaying in the absence of primary pair-rule genes only after cellularization (Yu, 1995).

Transient over-expression of runt under the control of a Drosophila heat-shock promoter caused stripe-specific defects in the expression patterns of the pair-rule genes hairy and even-skipped but had a more uniform effect on the secondary pair-rule gene fushi tarazu. The expression of the gap segmentation genes, upstream of runt in the segmentation hierarchy is also altered in heat shock/runt embryos. A subset of these effects are interpreted as due to an antagonistic effect of Runt on transcriptional activation by the maternal morphogen bicoid. Regulation of gap gene expression by runt is a normal component of the regulatory program that generates the segmented body pattern of the Drosophila embryo (Tsai, 1994).

Runt and Hairy act on ftz through fDE1, a common 32 base-pair element. The pair-rule expression of reporter gene constructs containing multimerized fDE1 elements depends on activation by Runt and repression by Hairy. Examination of reporter genes with mutated fDE1 elements provides further evidence that this element mediates both transcriptional activation and repression. Genetic experiments indicate that the opposing effects of runt and hairy are not due solely to cross-regulatory interactions between these two genes and that fDE1-dependent expression is regulated by factors in addition to runt and hairy (Tsai, 1995).

The runt gene is required to generate asymmetries within parasegmental domains. Ectopic runt expression leads to rapid repression of EVE stripes and a somewhat delayed expansion of FTZ stripes. Ectopic Runt is a rapid and potent repressor of odd-numbered EN stripes. Two remarkably different segmental phenotypes are generated as a consequence of these effects. The positioning of EN stripes is largely determined by the actions of negative regulators. runt is required to limit the domains of en expression in the odd-numbered parasegments, while the odd-skipped gene is required to limit the domains of en expression in the even-numbered parasegments. Activation of en at the anterior margins of both sets of parasegments requires the repression of runt and odd by the product of the eve gene (Manoukian, 1993).

Do Hairy and Runt repress target gene transcription independently of DNA binding, or as promoter bound regulators? Hairy-related transcriptional repressors show similar basic and HLH domains, and all terminate with an identical C-terminal tetrapeptide (WRPW), mutations of which largely or completely abolish repressor activity. It has proved difficult to define the precise molecular mechanism of Hairy action during segmentation. Although Hairy's embryonic patterning activity requires an intact basic (DNA binding) domain, none of the sequences in fushi tarazu promoter implicated in ftz repression by Hairy contain Hairy consensus binding sites. It is uncertain whether Runt acts primarily as a gene repressor or activator, as it behaves as a repressor of even-skipped and as an activator of fushi tarazu. In order to explore the ability of Hairy and Runt to act as promoter-bound transcriptional regulators, heterologous transcriptional activation domains (Act) were substituted for the WRPW repression domain (of Hairy) and the activation domain of Runt and the effects of such substitution were examined on presumed targets of Hairy and Runt. Expression of Hairy-Act during the blastoderm stage disrupts embryonic segmentation by driving ectopic expression of ftz, runt and odd-skipped. Activation depends on an intact basic domain, indicating that direct regulation occurs via sequence-specific binding to DNA. Expression of Runt-Act during the blastoderm stage likewise drives ectopic even-skipped, and shows that the normal apparent activation of fushi-tarazu by Runt is indirect, suggesting that Runt acts predominantly as a repressor. Hairy-Act has also been used to study sex determination. Ectopic Hairy mimics the activity of Deadpan in repressing early Sex-lethal transcription. Expression of Hairy-Act activates Sxl and causes male lethality, implying that Deadpan recognizes the Sxl promoter directly, and excludes models for Sxl regulation in which DPN functions as a passive repressor (Jiménez, 1996).

Ectopic expression of the pair-rule gene runt in the anterior end of the Drosophila embryo antagonizes transcriptional activation of the head gap gene orthodenticle (otd) by the anterior morphogen bicoid. The relevance of runt's activity as a repressor of otd in normal Drosophila embryogenesis has been investigated. otd expression is activated in the posterior region of embryos that are mutant for runt. This posterior expression domain of otd depends on the activity of the orphan nuclear receptor protein Tailless. Repression of otd by runt does not require the conserved VVVRPY motif, which mediates interaction between Runt and the co-repressor protein Groucho. It is speculated that the genetic interactions between runt and tll involve physical interactions between the two proteins. It is interesting to note that interactions between Runt and another orphan nuclear receptor protein, Ftz-F1 have been invoked to explain runt's regulation of the pair-rule gene fushi tarazu. However, in this case runt functions to activate, rather than repress Ftz-F1 dependent transcription. It will be interesting to determine if there are binding sites for Tll that are essential for the activation of otd in the posterior region and whether these sites respond to the repressive activity of runt. It is noted that the activity of tll is necessary, but not sufficient for otd expression in the posterior region of the embryo. The observed functional interactions between runt and tailless on otd expression may indicate there are other contexts where members of these two families of transcriptional regulators interact to regulate gene expression during development (Tsai, 1998).

The X-linked gene runt plays a role in the regulation of Sex lethal. Reduced function of runt results in female-specific lethality and sexual transformation of XX animals that are heterozygous for Sxl. The presence of a loss-of-function runt mutation masculinizes triploid intersexes. However, runt duplications cause a reduction in male viability by ectopic activation of Sex-lethal/runt is needed for the initial step of Sex-lethal activation, but does not have a major role as an X-counting element (Torres, 1992).

Three X-linked genes have been identified (scute, sisterless-a and runt) that determine the initial functional state of Sex lethal in the soma. These three X-linked genes do not seem to be required to activate Sex-lethal in the germ line (Granadino, 1993).

Runt functions as a transcriptional regulator in multiple developmental pathways in Drosophila melanogaster. Recent evidence indicates that Runt represses the transcription of several downstream target genes in the segmentation pathway. runt also functions to activate transcription. This paper documents the direct activation of Sex-lethal transcription by the Drosophila Runt protein. The initial expression of the female-specific sex-determining gene Sex-lethal in the blastoderm embryo requires runt activity. Male embryos mutant for deadpan show ectopic activation of Sxl expression, preferentially within the central, pre-segmented region of the embryo. Thus, it is possible that a major role for runt in the regulation of Sxl transcription is to counteract repression by dpn. Groucho is required to repress Sxl in male embryos. Thus it is possible that Runt bound to Sxl interacts with Groucho in a manner that blocks Groucho-mediated repression (Kramer, 1999 and references).

In situ hybridization was used to define the earliest effects of runt on transcription from the Sxl early embryonic promoter (Sxl_Pe). Wild-type female embryos containing a SxlPe:lacZ reporter gene begin to express lacZ transcripts during the syncitial nuclear division cycles preceding formation of the cellular blastoderm. Expression at nuclear division cycle 12 is observed in punctate dots distributed throughout the embryo except in pole cells. Later, this expression is seen as uniform staining throughout the embryo except in pole cells. Females homozygous for the amorphic runt^LB5 mutation fail to express the SxlPe:lacZ reporter gene within a broad central region of the embryo. This defect is observed concomitant with the earliest detectable expression of this reporter gene, demonstrating an early requirement for runt in Sxl_Pe activation. The alterations in Sxl expression observed in runt mutants correspond well to the initial expression of runt in a broad central domain of syncitial blastoderm stage embryos. This expression precedes the formation of the seven-stripe pair-rule pattern during cellularization, suggesting that runtís function in Sxl activation can be temporally separated from its role in segmentation. To test this idea, a temperature-sensitive runt mutation, runt^YP17, was used. Female embryos homozygous for runt^YP17 display normal Sxl_Pe expression when reared and collected at the permissive temperature. At the restrictive temperature of 29ƒC, these embryos show non-uniform Sxl_Pe expression identical to that observed in embryos deleted for runt. To examine runtís effects on segmentation, the expression pattern of the segment polarity gene engrailed (en) was examined in these embryos. In runt^YP17 embryos maintained at 18ƒC, En is expressed in a regular, well-spaced 14-stripe pattern, whereas at 29ƒC this pattern is disrupted. In collections of embryos aged at the non-permissive temperature for two hours and then shifted to the permissive temperature, female embryos with the abnormal Sxl_Pe expression pattern typical of runt mutants show normal En expression. In reciprocal temperature-shift experiments, female embryos, aged at the permissive temperature to the cellular blastoderm stage and then shifted to the non-permissive temperature, show normal Sxl_Pe expression and abnormal En expression. These results demonstrate that runtís role in the activation of Sxl_Pe is temporally distinct from and precedes the requirement for runt in segmentation, and provide strong evidence that runtís role as an activator of Sxl transcription occurs prior to cellularization, during the earlier syncitial blastoderm stages of Drosophila embryogenesis (Kramer, 1999).

Consistent with a role as a direct activator, Runt shows sequence-specific binding to multiple sites in the Sex-lethal early promoter. The early regulation of Sxl transcription by runt is readily explained if Runt interacts directly with the Sxl early promoter to activate transcription. Previous work has identified a 1.1 kb fragment of the Sxl_Pe promoter that contains sequences essential for sex-specific transcriptional activation. A test was performed for direct interactions between Runt and these DNA sequences. Probes that span this DNA fragment were generated and tested in electrophoretic mobility-shift assays (EMSAs). Runt binds only weakly to each of these DNA fragments. However, upon addition of the Brother partner protein (Bro, a homolog of mammalian PEBP2/CBF beta, a protein unrelated to Runt), multiple complexes are obtained with each of these probes. These complexes are Runt-dependent as they are not detected when only Bro protein is added. Competition with a bona fide CBF-binding site from the Polyoma enhancer prevents detection of these complexes. Competition is not observed when a mutant CBF-binding site is used, indicating that the binding is sequence specific. Recombinant mammalian CBF also recognizes multiple sites within these fragments from the Sxl_Pe promoter. Inspection of the sequence for matches to the consensus CBF-binding sequence TG(T/C)GGT(T/C) has identified ten sites that match this consensus at positions two through five that also match at least one of the three other, less critical positions. Interestingly, no perfect matches to the consensus are found. The presence of multiple binding sites is consistent with the hypothesis that activation of Sxl transcription involves direct interactions between Runt and the Sxl promoter. One prediction of this hypothesis is that Runtís DNA-binding activity should be required for Sxl activation: an in vitro assay shows this to be true (Kramer, 1999).

The 128 amino acid Runt domain confers sequence-specific DNA binding as well as heterodimerization with Brother, Runt's cofactor. As an initial test of the importance of Runtís DNA-binding domain, a form of runt that is deleted for its Runt domain, runtdeltaRD was injected into the central region of female homozygous runt^LB5 embryos. No evidence of rescue is seen in runtdeltaRD-injected embryos, indicating that the DNA-binding domain is important for runtís function as an activator of Sxl_Pe. However, since this is a large deletion, the effects could be attributed to improper folding and/or protein stability. Random- and site-directed mutagenesis experiments have identified several amino acids within the Runt domain that specifically affect DNA binding without disrupting association with the partner protein CBFbeta. Two conserved amino acids in Runt that are important for DNA binding correspond to a cysteine at position 127 and a lysine at position 199. In order to obtain a DNA-binding-defective form of Runt, a protein containing mutations at both of these sites (C127S, K199A) was generated. The DNA-binding activity of this mutant was compared with that of wild-type Runt in EMSAs with the high-affinity CBF-binding site from the Polyoma virus enhancer. The mutant protein, Runt[CK] shows only very low levels of complex formation on this DNA, and this is only in the presence of Brother. Similar experiments with a DNA probe from the Sxl promoter confirm the reduced DNA-binding activity of Runt[CK]. It is estimated that these mutations reduce DNA-binding affinity at least 50-fold. The observation that Brother stimulates DNA binding by Runt[CK] suggests that the two mutations do not disrupt interaction between the Runt and Brother proteins. Thus, these two mutations specifically impair DNA binding without affecting the overall structure of the Runt domain. The mRNA injection assay was used to examine the in vivo activity of this DNA-binding- defective form of Runt, and no evidence for rescue of Sxl_Pe expression was found in runt mutant female embryos. These results are consistent with the hypothesis that Runt activates Sxl transcription by binding to sequences in the Sxl_Pe promoter. Additional experiments further reveal that increasing the dosage of runt alone is sufficient for triggering the transcriptional activation of Sex-lethal in males. In addition, a Runt fusion protein, containing a heterologous transcriptional activation domain activates Sex-lethal expression, indicating that this regulation is direct and not via repression of other repressors. A small segment of the Sex-lethal early promoter that contains Runt-binding sites mediates Runt-dependent transcriptional activation in vivo (Kramer, 1999).

Although the truncated reporter gene (Sxl_Pe0.4kb:lacZ), isolated from the proximal 400 basepair fragment of Sxl_Pe, exhibits an abnormal pattern of expression in wild-type females, with higher levels found in the anterior and posterior, the expression is sex-specific. There are several putative Runt-binding sites found within this 400 bp fragment. Deletion of a small 70 bp segment within this fragment, which contains at least two putative binding sites for Runt, results in a loss of SxlPe expression. Conversely, a reporter gene that contains multiple copies of this segment, Sxl_PeGOF:lacZ, is expressed at high levels in WT female embryos. Interestingly, the Sxl_PeGOF:lacZ reporter gene is also expressed in males, however, at much lower levels and not in the anterior regions of the embryo. EMSA with Runt and Brother proteins demonstrates that Runt binds to sequences within this small segment. This interaction is sequence specific as it is competed by a DNA fragment from the Polyoma enhancer containing a wild-type CBF-binding site, but not by a similar DNA fragment with a mutant CBF-binding site. The differential expression in female and male embryos indicates that this reporter gene retains the ability to respond to numerator gene dosage. The observation that this transgene is expressed in males suggests that the activation mediated by multimerization of this small segment of DNA is sufficient to overcome the repression that is normally established in males for the parental Sxl_Pe0.4kb:lacZ reporter gene. Furthermore, the preferential expression within the segmented region of the embryo strongly suggests that this reporter gene is responding to runt. To test this, Sxl_PeGOF:lacZ expression was examined in embryos mutant for runt. Expression is reduced in most, but not all, regions of runt mutant male embryos. Thus, the region that is multimerized in the Sxl_PeGOF:lacZ reporter gene mediates runt-dependent transcriptional activation (Kramer, 1999).

Low-level ectopic expression of the Runt transcription factor blocks activation of the Drosophila melanogaster segmentation gene engrailed (en) in odd-numbered parasegments and is associated with a lethal phenotype. By using a genetic screen for maternal factors that contribute in a dose-dependent fashion to Runt-mediated repression, it is shown that there are two distinct steps in the repression of en by Runt. The initial establishment of repression is sensitive to the dosage of the zinc-finger transcription factor Tramtrack. By contrast, the co-repressor proteins Groucho and dCtBP, and the histone deacetylase Rpd3, do not affect establishment but instead maintain repression after the blastoderm stage. The distinction between establishment and maintenance is confirmed by experiments with Runt derivatives that are impaired specifically for either co-repressor interaction or DNA binding. Other transcription factors can also establish repression in Rpd3-deficient embryos: this indicates that the distinction between establishment and maintenance may be a general feature of eukaryotic transcriptional repression (Wheeler, 2002).

Ftz modulates Runt-dependent activation and repression of segment-polarity gene transcription

A crucial step in generating the segmented body plan in Drosophila is establishing stripes of expression of several key segment-polarity genes, one stripe for each parasegment, in the blastoderm stage embryo. It is well established that these patterns are generated in response to regulation by the transcription factors encoded by the pair-rule segmentation genes. However, the full set of positional cues that drive expression in either the odd- or even-numbered parasegments has not been defined for any of the segment-polarity genes. Among the complications for dissecting the pair-rule to segment-polarity transition are the regulatory interactions between the different pair-rule genes. An ectopic expression system that allows for quantitative manipulation of expression levels was used to probe the role of the primary pair-rule transcription factor Runt in segment-polarity gene regulation. These experiments identify sloppy paired 1 (slp1), most appropriately classified as segment polarity genes, as a gene that is activated and repressed by Runt in a simple combinatorial parasegment-dependent manner. The combination of Runt and Odd-paired (Opa) is both necessary and sufficient for slp1 activation in all somatic blastoderm nuclei that do not express the Fushi tarazu (Ftz) transcription factor. By contrast, the specific combination of Runt + Ftz is sufficient for slp1 repression in all blastoderm nuclei. Furthermore Ftz is found to modulate the Runt-dependent regulation of the segment-polarity genes wingless (wg) and engrailed (en). However, in the case of en the combination of Runt + Ftz gives activation. The contrasting responses of different downstream targets to Runt in the presence or absence of Ftz is thus central to the combinatorial logic of the pair-rule to segment-polarity transition. The unique and simple rules for slp1 regulation make this an attractive target for dissecting the molecular mechanisms of Runt-dependent regulation (Swantek, 2004).

The role of Runt as a primary pair-rule gene complicates interpreting the alterations in segment-polarity gene expression that are observed in run mutants. Recent experiments utilizing a GAL4-based NGT-expression system [the transgene construct used to express GAL4 maternally contains the nanos promoter and the 3' untranslated region of an alpha-tubulin mRNA and is thus referred to as an NGT transgene (nanos-GAL4-tubulin)] to manipulate expression in the blastoderm embryo have demonstrated that low levels of Runt repress en in odd-numbered parasegments without altering expression of the pair-rule genes eve and ftz. This observation suggested that this approach might provide a useful tool for defining the role of Runt in regulating other segment-polarity genes. A systematic survey was undertaken of the response of other segmentation genes to increasing levels of NGT-driven Runt expression. These experiments revealed significant differences in sensitivity as well as interesting differences in the nature of the response of different genes to ectopic Runt. The odd-numbered en stripes are repressed at both intermediate and high levels of ectopic runt. After en, the second most sensitive target is slp1. This gene shows a partially penetrant and subtle defect in the spacing of the segmentally repeated stripes in embryos with low levels of NGT-driven Runt. A more pronounced alteration is obtained in embryos with intermediate levels of Runt. In these embryos the slp1 pattern is converted from a segment-polarity-like, 14-stripe pattern to a pair-rule-like, seven-stripe pattern. At this level, expression of other segmentation genes is normal although there are subtle changes in the spacing of the wg stripes and a partial loss of the odd-numbered hh stripes. All three of these genes show clearer alterations at higher levels of NGT-driven Runt, with wg responding in a manner similar to slp1 and hh responding in a manner similar to en. High Runt levels also produce spacing defects in the expression of odd and gsb, as well as a more subtle effect on prd. Several of the changes observed at high levels of ectopic Runt are likely to be indirect and due to alterations in the expression of eve, ftz and hairy. The response of slp1 to ectopic Runt is notable both because of its sensitivity and apparent simplicity, thus suggesting that Runt plays a pivotal role in regulating slp1 transcription (Swantek, 2004).

The differential combinatorial effects of Runt and Ftz on segment-polarity gene regulation emerged as a result of analyzing the sensitive and relatively simple response of slp1 to ectopic Runt. The slp1 transcription unit is one of two redundant genes that comprise the slp locus. This locus was initially characterized as having a pair-rule function in the segmentation gene hierarchy based on a weak pair-rule phenotype associated with loss of slp1 function. The slp1 and slp2 genes are expressed in similar patterns during early embryogenesis. Embryos deficient for both slp1 and slp2 have an unsegmented lawn cuticle phenotype similar to that produced by wg mutations. This raises the question of whether it is most appropriate to consider slp as a pair-rule or segment-polarity locus. In the most straightforward interpretation of the segmentation hierarchy, the role of the pair-rule genes is to establish the initial metameric expression patterns of the segment-polarity genes. The initial expression of the key segment polarity genes en and wg is normal in gastrula stage embryos that are deleted for both slp1 and slp2. The expression of wg begins to become abnormal and is lost during early germband extension. These observations are consistent with the proposal that slp expression identifies cells that are competent to maintain wg expression subsequent to the blastoderm stage. Based on these observations, it is concluded that slp1 and slp2 are most appropriately classified as segment polarity genes, not pair-rule genes (Swantek, 2004).

The expression of slp1 (and slp2) differs from several other segment-polarity genes in that the metameric pattern is comprised of two-cell wide, rather than single-cell wide stripes. These two cell-wide stripes comprise the posterior half of each parasegment. slp1 activation in odd-numbered parasegments requires the cooperative action of Runt and Opa, whereas in even-numbered parasegments Runt works together with Ftz to repress slp1 expression. The simple rules involving these three factors fully account for slp1 regulation in all of the Runt-expressing cells in the blastoderm embryo but also raise a question regarding the positional cues that regulate slp1 expression in cells that do not express Runt (Swantek, 2004).

There are four other pair-rule transcription factors that could be involved in slp1 regulation: Eve, Hairy, Odd and Prd. Expression of both Odd and Prd overlaps the slp1 stripes in a manner that suggests that neither of these factors provides positional information crucial for slp1 regulation. Consistent with this, there are no substantial changes in the early 14-striped slp1 pattern in embryos mutant for either odd or prd. By contrast, elimination of either Eve or Hairy leads to changes in both the number and spacing of the slp1 stripes. However, as these are both primary pair-rule genes some of these changes are certainly indirect and due to alterations in Runt and Ftz expression (Swantek, 2004).

Several lines of evidence indicate that Eve has a direct role in slp1 repression. Experiments with the temperature-sensitive eve[ID19] mutation indicate that transient elimination of Eve at the cellular blastoderm stage leads to expanded six cell-wide slp1 stripes because of de-repression in the anterior two cells of each odd-numbered parasegment. These two are the cells with the highest level of Eve, indicating that the primary role of Eve at this stage is to repress slp1 expression. Complementary experiments with an inducible hs-Eve transgene reveal that ectopic Eve blocks slp1 activation in both odd- and even-numbered parasegments. This result not only confirms Eve's role as a repressor, but also reveals a crucial difference between Eve and Ftz-dependent repression. Ftz-dependent repression is restricted to odd-numbered parasegments unless Runt is also ectopically expressed. This same restriction is observed in experiments with hs-Ftz transgenes, indicating that the difference between Eve and Ftz is not due to the mode of ectopic expression. Taken altogether these results indicate that Eve and Ftz normally have comparable roles in repressing slp1 transcription in the anterior half of the odd- and even-numbered parasegments, respectively, in late blastoderm stage embryos. The key distinction in the regulation of slp1 by these two homeodomain transcription factors is the critical role that Runt plays in Ftz-dependent repression (Swantek, 2004).

One aspect of slp1 expression not accounted for by the above rules is the factor (or combination of factors), referred to here as factor X, that is responsible for slp1 activation in the posterior half of the even-numbered parasegments. Activation in these cells is blocked either by the combination of Runt+Ftz or by ectopic Eve. Runt and Ftz are co-expressed anterior to these even-numbered stripes and presumably both play a role in defining the anterior margin of these stripes. Conversely, Eve is expressed posterior to these cells and probably has a role in defining the posterior margins of these stripes. The sole pair-rule transcription factor that remains as a candidate for Factor X is Hairy, which is expressed in the posterior half of even-numbered parasegments. However, it is not thought that factor X is Hairy for several reasons. All of the evidence to date indicates that Hairy functions as a repressor. Furthermore, NGT-driven expression of Hairy does not lead to slp1 activation in anterior blastoderm cells similar to that produced by the co-expression of Runt and Opa. Identification of factor X is clearly important for a complete understanding of slp1 regulation (Swantek, 2004).

Previous studies have indicated that Runt has roles in both activating and repressing transcription of different target genes in the Drosophila. The current results provide additional compelling evidence for this dual activity and also provide insight on factors that contribute to this context-dependent regulation. The dramatic effects of Ftz on Runt-dependent slp1 regulation clearly demonstrate that one important component of context is the specific combination of other transcription factors that are present in a cell. Indeed, the unique and relatively simple rules for slp1 regulation make this an especially attractive target for dissecting the molecular mechanisms whereby Ftz converts Runt from an activator to a repressor of transcription. It seems likely that the rules governing the Runt-dependent regulation of slp1 will provide a foundation for understanding the regulation of wg and gsb, two segment-polarity genes that are expressed in a subset of slp-expressing cells and that respond to Runt in a manner similar, but not identical to slp1 (Swantek, 2004).

The results also point to a second important component of context-dependent regulation by Runt. The specific combination of Runt + Ftz, which represses slp1, does not always give repression, since these same two factors work together to activate en in some of these same cells at the same stage of development. Thus, cellular context alone cannot fully account for the regulatory differences and there must be a target-gene specific component of context-dependent regulation. A similar gene-specific example of context-dependent regulation has recently been described for the Runx protein Lozenge. In this case, the presence of binding sites for the Cut homeodomain protein helps to stabilize a complex that leads to repression of deadpan transcription in the same cells in which Lozenge is responsible for activation of Drosophila Pax2. In a strict parallel of this model, it would be speculated that the slp1 regulatory region contains binding sites for some factor that helps to stabilize a repressor complex that includes the Runt and Ftz proteins. In a reciprocal, and not mutually exclusive model, perhaps there are binding sites for a factor in the en regulatory region that helps to stabilize a Runt- and Ftz-dependent transcriptional activation complex. Further studies on the en and slp1 cis-regulatory regions are needed in order to address these questions at the molecular level. This future work is crucial for understanding the context-dependent activity of Runt and thus the molecular logic of the control system that underlies the pair-rule to segment-polarity transition in Drosophila segmentation (Swantek, 2004).

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