To determine if C15 lies downstream of Al or vice versa, their expression was examined in discs from the reciprocal mutant. Each was still expressed, but its expression domain was significantly reduced. In contrast, Lim1 expression is lost completely in both C15 and al mutant discs. In addition, although there is some variation, the expression domains of C15 and Al are only mildly reduced in lim1 mutants (Campbell, 2005).
If C15 is not downstream of the other homeobox genes expressed in the center of the disc, it must be activated by another mechanism. al expression is induced by EGFR signaling, raising the possibility that C15 may also be under EGFR control. This was confirmed by loss and gain of function experiments, as follows: (1) C15 expression was lost in discs from an Egfrts mutant grown at the restrictive temperature (29.1°C) at which al expression is lost; (2) misexpression of a constitutively active form of the EGFR (UAS-Egfr.lambdatop) results in ectopic expression of C15; similar to other EGFR targets, this ectopic expression is restricted to the ventral region (Campbell, 2005).
Requirements of Lim1 and Chip for maximal al and cll/C15 expression: Lim1 expression becomes discernible slightly later than expression al, cll and Bar, and maximal al expression in late third instar depends on Lim1 function (Tsuji, 2000). Cll expression is also significantly reduced in Lim17B2 (a null allele) clones in late third instar discs, indicating that not only al but also cll is positively regulated by Lim1 in the late third instar. Chi encodes a LIM domain binding protein and has been suggested to act as a co-factor for Lim1. Cll and Al signals are significantly reduced in clones of Chie5.5, a null allele of Chi. The concerted action of Lim1 and Chi is thus shown to be required for the maximal expression of cll and al in the late-third-instar pretarsus region (Kojima, 2005).
When Lim1 is misexpressed using blk-GAL4, the expression of al but not cll is induced (Kojima, 2005; Tsuji, 2000). Thus, unlike al, cll may require an additional component for its maximal expression. Alternatively, cll may be less sensitive to activation by Lim1 than al (Kojima, 2005).
Bar attenuates al and cll expression through Lim1 repression: Previous experiments have shown that al expression invades into Bar mutant clones generated in the distal tarsus and that Bar misexpression attenuates al expression in the pretarsus (Kojima, 2000), indicating that al expression is negatively regulated by Bar. As with al, cll is also under a negative regulation of Bar, since cll expression not only intrudes into Bar mutant clones generated in the distal tarsus but is also attenuated in the pretarsus cells misexpressing Bar (Kojima, 2005).
That Bar represses Lim1 expression (Tsuji, 2000) and that al and cll expression is positively regulated by Lim1 may indicate that Bar represses both al and cll expression through Bar-dependent repression of Lim1 expression (Tsuji, 2000) (Kojima, 2005).
To test whether the attenuation of al and cll expression due to Bar misexpression is caused only through Lim1 repression, Al and Cll expression was examined in Lim17B2 leg discs misexpressing Bar with blk-GAL4. Should Lim1 repression be the sole cause for al and cll repression, Al and Cll signal reduction in Bar-misexpressing cells would be no more than in surrounding cells under a Lim1 mutant background. This was the case for Al but not for Cll, indicating that Bar negatively regulates al expression mainly through Lim1 repression, while cll expression is repressed by Bar both through Lim1 repression and that independent of Lim1 (Kojima, 2005).
In eukaryotes, the ability of DNA-binding proteins to act as transcriptional repressors often requires that they recruit accessory proteins, known as corepressors, which provide the activity responsible for silencing transcription. Several of these factors have been identified, including the Groucho (Gro) and Atrophin (Atro) proteins in Drosophila. Strong genetic interactions are seen between gro and Atro and also with mutations in a third gene, scribbler (sbb), which encodes a nuclear protein of unknown function. Mutations in Atro and Sbb have similar phenotypes, including upregulation of the same genes in imaginal discs, which suggests that Sbb cooperates with Atro to provide repressive activity. Comparison of gro and Atro/sbb mutant phenotypes suggests that they do not function together, but instead that they may interact with the same transcription factors, including Engrailed and C15, to provide these proteins with maximal repressive activity (Wehn, 2006; full text of article).
Previous studies demonstrated that Atro acts as a corepressor in Drosophila, the most convincing of these being the demonstration that fusion of Atro to a heterologous DNA-binding domain confers repressive activity to the chimera. Atro has been shown to interact directly with two transcription factors, Even-Skipped (Eve) and Huckebein, and the repressive ability of Eve is compromised in Atro mutants, probably accounting for the loss of en expression in even-numbered parasegments in Atro mutant embryos (Wehn, 2006).
These studies here are consistent with Atro acting as a corepressor since it was shown that several genes, including run, tkv, al, and B, are completely or partially derepressed in Atro mutant clones in imaginal discs, suggesting that transcriptional repressors required to silence these genes recruit Atro. Atro-dependent repression of Bar (B) in the center of the leg disc is very likely due to interaction with the transcription factor C15, which is expressed in the center of the leg and is required for repression of B. Similarly, Atro-dependent repression of al in the posterior of the wing is very likely due to interaction with En, which is expressed in the posterior and required to exclude al from this compartment. At present it is unclear which transcription factors recruit Atro to repress run in the eye or tkv in the wing, although a strong candidate for run would be the Rough homeodomain protein, which is expressed in the same cells, R2 and R5, that exhibit ectopic run expression in Atro mutant clones. Whether Atro can, in fact, bind directly to C15, En, and possibly Rough, needs to be tested biochemically, since previous studies with Eve and Hkb did not identify a possible interaction motif for Atro nor do sequence comparisons among C15, En, Eve, and Hkb suggest a common motif (Wehn, 2006).
The sbb gene encodes a nuclear protein with unknown function. sbb mutations have many different phenotypes affecting multiple tissues. sbb and Atro interact very strongly genetically and that many of the phenotypes of sbb mutants are very similar to those of Atro mutants, including derepession of run, tkv, al, and B in imaginal discs. Thus, Atro is unable to silence these genes in the absence of Sbb, suggesting that it is required for Atro activity either to recruit Atro to transcription factors or possibly to assist binding of these factors to DNA. Since these transcription factors appear to function normally in some respects in the absence of Sbb (or Atro), it appears more likely that Sbb and Atro function together in a corepressor complex (Wehn, 2006).
One problem with the proposal that Atro activity is dependent upon Sbb is that the phenotypes of Atro and sbb mutants are not identical. For example, embryos lacking both maternal and zygotic Atro have a very severe, almost uncharacterizable phenotype, while embryos lacking both maternal and zygotic Sbb have a much less severe phenotype, characterized by a reduced number of abdominal segments, that is similar to that of embryos lacking only maternal Atro. This could be explained if Atro is partially active in the absence of Sbb, or if it is dependent upon Sbb for repression of some genes but not others. Alternatively, the difference between Atro and sbb mutant phenotypes could be related to Atro having functions other than that of a corepressor. It is has been implicated in positive regulation of Hox gene expression, and it also functions in the cytoplasm to control planar cell polarity. This analysis of sbb mutants does not reveal any potential involvement of Hox gene expression or planar cell polarity and, consequently, if Sbb is required only for Atro to act as a corepressor, then it is not surprising that Atro and sbb mutant phenotypes are not identical. Further experiments are required to determine the nature of the Atro dependence on Sbb for transcriptional repression and how direct any interactions might be (Wehn, 2006).
Mutations in sbb and Atro were originally uncovered in a genetic screen for enhancers of al. It is likely that they act as enhancers because they are utilized by the C15 transcription factor to repress genes such as Bar; C15 is expressed in the same cells as Al and it is thought that they bind together to regulate gene expression. Strong genetic interactions were uncovered among sbb, Atro, and en mutations, that could be explained if En also recruits Atro/Sbb (Wehn, 2006).
Curiously, genetic studies also revealed strong interactions among gro, sbb, and Atro. This could be explained if Gro was also required for Atro activity; i.e., all three proteins may form a corepressor complex. However, this appears to be unlikely because, in contrast to the similar phenotypes of sbb and Atro mutants, there are several distinct differences among the phenotypes of gro mutants and those of sbb and Atro mutants. For example, repression of tkv in the anterior of the wing is dependent on both Sbb and Atro but not on Gro, while repression of run in the antennal disc is dependent upon Gro but not upon Atro or Sbb. This suggests that a specific transcription factor recruits Atro/Sbb to repress tkv in the wing and another transcription factor recruits Gro to repress run in the antenna. The identity of these transcription factors remains to be uncovered (Wehn, 2006).
In some cases gro mutants do have a similar phenotype to those of Atro and sbb; this includes partial derepression of al expression in the posterior of the wing and Bar in the center of the leg. This can be explained if C15 (expressed in the center of the leg) and En (expressed in the posterior of the wing) recruit both Gro and Atro/Sbb and if each imparts some but not all the repressive activity to these transcription factors. Consistent with this, both C15 and En possess eh1-type Gro-interaction motifs and previous studies have revealed that En can repress in the absence of Gro. Further biochemical studies are required to determine if C15 and En can indeed recruit Atro (Wehn, 2006).
At present it is unclear whether Atro and Gro provide all the repressive activity to C15 and En; this will await the generation of Atro gro double-mutant clones. sbb gro double-mutant clones have been analyzed and these reveal that some targets of C15 and En are still at least partially repressed, although En activity appears to be somewhat compromised following the simultaneous loss of Sbb and Gro, in comparison to loss of one of these alone. Either Atro has some activity in the absence of Sbb or C15 and En can use mechanisms other than recruitment of Gro and Atro to repress transcription. Many transcription factors have been shown to have the ability to repress by several mechanisms; for example, although Brk recruits both CtBP and Gro, it can repress some genes in the absence of both, using additional repression domains (Wehn, 2006).
Why do C15 and En need to recruit both Gro and Atro? En can repress some genes completely in the absence of either Gro or Atro, for example, ci and dpp in the wing. However, for repression of al, the activity of En is clearly reduced in the absence of either, indicating that it needs to recruit both to completely repress this gene. This would suggest a quantitative explanation; i.e., En recruits both Gro and Atro to increase its activity, rather than to allow it to repress specific genes repressed more efficiently by one or the other. This is consistent with the suggestion that both corepressors function via a similar mechanism: both Gro and a vertebrate homolog of Atro have been shown to recruit a histone deacetylase. The recruitment of both may decrease histone acetylation to a level that cannot be achieved with either alone (Wehn, 2006).
Lim1 may be a direct target of C15: To investigate regulatory interactions between C15, Al, and Lim1, each was misexpressed in the leg and expression of the other two was examined. This was achieved initially with a UAS-C15 line and by generating Gal4 expressing clones using the FLPout technique and Tub-Gal4; the clones were monitored with UAS-GFP. This revealed that ectopic C15 could, in fact, induce ectopic expression of both Al and Lim1, although this was somewhat random with Al and Lim1 being expressed only in some cells ectopically expressing C15. Ectopic C15 can also repress Bar and loss of Bar results in expansion of the Al expression domains, but only in the cells immediately surrounding their normal domains. Repression of Bar does not appear to account for the ectopic Al and Lim1 expression induced by C15, because, Al and Lim1 can be induced some distance from their endogenous domains. In contrast, misexpression of al or lim1 in Tub-Gal4 clones has no effect on expression of the other genes. It has been shown that driving higher levels of lim1 can induce ectopic expression of al and this was confirmed using dpp-Gal4. However, there was no ectopic C15 in the UAS-lim1; dpp-Gal4 discs. Similarly, driving higher levels of al with dpp-Gal4 does not induce ectopic expression of C15 (Campbell, 2005).
Therefore, although Al is still expressed in C15 mutants, and vice versa, indicating that both are probably activated independently by EGFR signaling, C15 can induce expression of al and lim1. This may act as a feedback mechanism to ensure all three are expressed in the same cells. Since expression of Lim1 is completely lost in the center of discs from C15 and al mutants, it may simply be a direct target of either or both and may not be directly activated by EGFR signaling (Campbell, 2005).
Since al is still expressed, albeit in a much smaller domain, in C15 mutants and C15 is still expressed in al mutants, it appears possible that each may play an additional, redundant role, in patterning the leg. This was ruled out by examining alice, C152 double mutants (both alleles are either null or very close to being null), which have legs and antennae that are indistinguishable from either single mutant, indicating that, in the absence of the other, neither Al nor C15 provides any function during leg development (Campbell, 2005).
C15 acts directly to repress Bar in the center of the leg: In late third instar discs, Bar is expressed in the cells immediately surrounding C15, as has already been described for Al. In partially everted discs, this corresponded to C15 at the very tip and Bar in ta V and IV. The nubbin (nub) gene is expressed in ta V overlapping with Bar in ta V but not in IV. With antibody staining, Bar and C15 can first be detected in very early third instar and both appear to be expressed at the same time. Bar is already excluded from the center at this stage. However, using a lac-Z enhancer trap in Bar, which is more sensitive than antibody staining, β-gal expression was detected even earlier in late second instars. At this stage, when no C15 can be detected, β-gal expression is found throughout the center of the disc. Slightly later when C15 becomes detectable, β-gal is excluded from the center. Al is first detected at approximately the same time as C15 (Campbell, 2005).
The loss of Bar and Nub expression from the center of the disc can be explained by repression by Al and C15. Loss of al has been shown to result in expansion of Bar expression into the center of the disc, indicating Al is required to repress Bar in this position. Not surprisingly, C15 null mutant discs have the same phenotype. The diameter of the domain of Bar is slightly smaller than the diameter of the Bar ring in wild-type discs. Nub expression is also found in the center of C15 mutant discs, but in a smaller domain than Bar, indicating that there are still distinct differences between ta IV and V in C15 mutants (Campbell, 2005).
Repression of Bar by C15 is strictly autonomous, as shown in discs containing C15 mutant clones, where Bar expression expands into all the cells in the center that has lost C15. In addition, ectopic expression of C15 resulted in autonomous repression of Bar. Curiously, although studies that showed ectopic al cannot repress Bar, it was also found that it can actually induce ectopic expression of Bar in more proximal regions of the disc (Campbell, 2005).
C15 acts indirectly to repress ap in the center of the leg: Bar expression is absent from the center of the leg, specifically from the cells expressing Al and C15. However, other genes, including ap and bab, are absent from a more extensive region in the center, and there is a gap between the C15 expression domain and Ap and Bab. Consequently, Ap expression is restricted to presumptive tarsal segment IV, where it overlaps with Bar. It has been suggested that, as well as activating genes such as al and Bar, EGFR signaling may directly repress genes in the center of the disc, possibly accounting for the absence of ap and bab in this location. Surprisingly, ap and bab expression, as well as Bar, is regulated by C15/Al. In both C15 and al mutant discs, Ap and Bab expression expands into the center of the disc. Consequently, in regard to Ap expression, the distal region of the leg adopts a tarsal segment IV-like fate. However, Nub, which is normally only expressed in ta V, is now co-expressed with Ap in the very center, indicating that the distal-most segment in C15 legs has characteristics of both ta IV and V (Campbell, 2005).
In wild-type discs, Ap expression is first detected slightly later than Bar, Al, or C15, but even at this time there is a clear gap between Ap expression and C15, indicating that C15/Al acts non-autonomously to repress ap. This is supported by two further studies: (1) unless there is a complete loss of C15 in homozygous mutant discs, Ap expression is not derepressed in C15 mutant clones in the center if the clones are not too large, indicating surrounding wild-type C15-expressing cells can rescue the mutant tissue; (2) ectopic expression of C15 results in non-autonomous repression of Ap (Campbell, 2005).
These results suggest that EGFR signaling represses gene expression in the center of the disc only indirectly through activation of C15/Al. This is also supported by two other observations. (1) Al is still expressed in C15 mutant discs, indicating that EGFR signaling levels are still very high in the center of these discs, but ap is not repressed (if ap is repressed directly by EGFR, its threshold for this would be lower than the threshold for activation of al because ap is repressed further from the source in the center than al is activated). (2) Ectopic expression of C15 results in non-autonomous repression of ap, but, if this is due to increased EGFR signaling in surrounding cells, then it should result in activation of EGFR targets such as Bar immediately adjacent to the cells expressing C15 (outside of the normal Bar domain), but does not. Consequently, it seems very likely that C15 uses an alternative mechanism to repress ap, most likely by upregulation of a signaling pathway in surrounding cells (i.e., ta V) (Campbell, 2005).
Notch signaling can repress ap expression:
The ability of different signaling pathways to repress ap expression was tested and it was discovered that upregulation of the Notch pathway in ta IV (by misexpression of the Notch intracellular domain) results in loss of Ap expression. Curiously, however, Ap expression is not upregulated in Notch mutant cells, and is, in fact, lost or downregulated; the phenotype is somewhat variable), indicating low-level Notch signaling is required for Ap expression, possibly indirectly, because loss of Notch can also lead to downregulation or loss of Bar expression in ta IV and Bar is required for expression of ap (Campbell, 2005).
Bowl can repress ap and is activated non-autonomously by C15: Notch signaling usually represses gene expression indirectly by inducing expression of repressors, so known Notch targets in the distal leg were tested to determine if they are required for repression of ap. The best candidate appeared to be the bowl gene which encodes a zinc finger transcription factor that is expressed in a ring in the distal leg under the control of Notch signaling and can both activate and repress gene expression. To investigate if bowl is involved in repressing ap expression, mutant clones were generated in leg discs. In these clones, cells expressing high levels of Ap now directly abut those expressing C15, i.e., there is no gap between them. Low-level Ap expression is also detected in clones that extend into the C15 domain, indicating Bowl is also required here but that an additional factor, possibly C15/Al, can partially repress ap in this location (if so, C15/Al would be acting autonomously in a similar fashion to repression of Bar). Ectopic bowl expression can also repress Ap expression. The response to ectopic bowl is fairly weak, but it appears that ectopic expression of this gene does not result in high levels of protein expression (Campbell, 2005).
bowl represses ap and C15 regulates bowl expression: Examination of Bowl and Ap expression in leg discs reveals that there is a gap between their expression domains, even at a time when Ap expression is first detected in mid-third instars. This could indicate that Bowl acts non-autonomously to repress ap. However, the clonal analysis clearly shows that Bowl acts autonomously: any wild-type cells expressing Bowl has no influence on Ap expression in surrounding mutant tissue. It is possible that there is low-level Bowl expression in the 'gap' that cannot be detected with antibody staining. Another possible explanation is one of timing, and that Bowl is expressed in the cells in the 'gap' slightly earlier and that this is sufficient to silence the ap gene even before its expression can be detected more proximally. The possibility that bowl is expressed transiently in cells has been proposed to explain the observation that bowl mutant clones have effects in central regions of tarsus, i.e., in regions where its expression cannot be detected later (Campbell, 2005).
Thus, Bowl is required to repress ap expression in tarsal segment V and this predicts that C15 regulates bowl expression. This was confirmed by analysis of C15 mutant discs, in which Bowl expression in the center is lost, although other, more proximal, domains of expression are normal. The ring of Bowl in the distal tarsus is usually just two cells in width with the inner cell overlapping with C15, but the outer cell being outside the C15 domain, suggesting C15 can induce bowl non-autonomously. This is supported by the ability of cells ectopically expressing C15 to activate Bowl expression in surrounding cells. This ability is fairly limited, but would be expected because the endogenous C15-expressing cells only appear able to induce bowl in their immediate neighbor (resulting in a ring of bowl expression in a single row of cells surrounding the C15 domain (Campbell, 2005).
Delta activates bowl, but Delta expression is repressed by C15: If Notch signaling induces bowl expression and C15 is also required for bowl expression, it was predicted that C15 upregulates Notch signaling by regulating the expression of the Notch ligand responsible for activation of bowl. Although, both Notch ligands, Delta (Dl) and Serrate are expressed in leg discs, it was discovered that only Dl is required to induce expression of bowl. bowl expression is lost in homozygous Dl mutant clones, although, if positioned appropriately, wild-type cells can rescue bowl expression in adjacent cells laterally and distally. Curiously, nub, which was also thought to be a target of Notch signaling, is still expressed in Dl mutant cells (even far from wild-type cells), albeit in an irregular pattern (it is expressed at normal levels in some cells, but at lower levels or not at all in others. Misexpression of Dl can also induce ectopic bowl expression both in adjacent cells and in the cells misexpressing Dl. However, in large clones, cells in the center of the clone do not express bowl, which is only expressed in the cells at the edge of the clone and in the cells immediately adjacent to the clone. Nub is not ectopically expressed following misexpression of Dl (Campbell, 2005).
In wild-type mid-third instar discs, Dl expression is upregulated in ta V, overlapping with Nub, but not with C15. Distally, it overlaps partially with Bowl, although Bowl is also expressed even more distally. Proximally, however, Dl does not appear to induce expression of Bowl, suggesting there is a repressor of Bowl expressed in this location. This is supported by the inability of cells misexpressing Dl in this position (proximal ta V, ta IV) to activate Bowl. Although it might be predicted that C15 would induce expression of Dl, in fact the opposite was found, and C15 actually represses Dl in the center of the disc. In C15 mutants, Dl expression expands into the center of the disc and misexpression of C15 can repress expression of Dl. How C15-repression of Dl can result in upregulation of Notch signaling in cells in ta V surrounding the pretarsus is discussed below (Campbell, 2005).
clawless/C15 represses Bar expression cooperatively with al in the pretarsus: As with strong al mutants (Tsuji, 2000), Bar is de-repressed in the putative cll mutant pretarsus at late third instar. Cell-autonomous Bar de-repression is also observed in al and cll mutant clones generated in the pretarsus region. Since the sole misexpression of al could not induce Bar repression (Kojima, 2000), these findings may indicate that a cooperative action of al and cll is required for Bar repression in the future pretarsus.
blk-GAL4 (a dpp enhancer driven GAL4) is a GAL4 driver capable of inducing UAS-gene expression strongly on the dorsal side and weakly on the ventral side along the anterior/posterior boundary. cll was misexpressed using a moderate UAS-cll line and blk-GAL4. In contrast to al, cll misexpression causes endogenous Bar repression on the dorsal side of the future distal tarsus region. Furthermore, unexpectedly, ectopic al is found in the dorsal-side tarsal cells that have lost Bar expression, possibly suggesting that cll misexpression or Bar elimination induces the ectopic expression of al (Kojima, 2005).
To further clarify these points, whether cll is capable of inducing al expression was examined. Since pretarsus al expression intrudes into Bar mutant clones generated in the tarsus region in the late third instar (Kojima, 2000), cll-misexpressing clones were generated outside of the Bar domain using a flip-out technique, and the presence or absence of Al misexpression in the cll-misexpressing clones was examined. Al was induced in a considerable fraction of cll-misexpressing clones outside of the Bar domain, indicating that cll is capable of inducing al misexpression independent of Bar repression (Kojima, 2005).
Examined next was whether endogenous Bar expression in the future tarsus is repressed on the ventral side when al and cll are simultaneously misexpressed using blk-GAL4. Note that neither Bar reduction (Kojima, 2000) nor cll misexpression occurs upon blk-GAL4-driven al misexpression. A simultaneous misexpression of al and cll using blk-GAL4 causes Bar repression not only on the dorsal side but on the ventral side as well, strongly supporting the notion that endogenous Bar expression in the future tarsus is repressed by a concerted action of al and cll. It is concluded that cll is capable of inducing al and that Bar is repressed by a concerted function of al and cll (Kojima, 2005).
At early third instar, al, cll and Bar expression in the wild-type leg disc became discernible simultaneously. From the very beginning of the expression onward, Al and Cll signals localize in the disc center, while Bar signals are in a circular region immediately adjacent to the Al/Cll domain (Kojima, 2000), possibly suggesting that Bar is negatively regulated by Al and Cll from an early stage of expression. To test this possibility, Bar expression was examined in alex or clldl9-2 clones in early third instar discs. Bar is de-repressed cell-autonomously in both mutant clones, indicating that Bar has a potentiality to be expressed not only in the future distal tarsus but in the future pretarsus as well. It is concluded that, in the wild-type leg discs, pretarsus Bar expression is repressed by a concerted action of Al and Cll from a very early stage of expression (Kojima, 2005).
Positive regulation of Lim1 expression via concerted action of al and cll: That Lim1 expression becomes discernible in the future pretarsus shortly after the appearance of Al and Cll signals may imply that Lim1 is positively regulated by al and/or cll. However, it is difficult to directly determine whether al and cll are required for Lim1 expression, since Bar, serving as a repressor for Lim1 (Tsuji, 2000), is de-repressed in the pretarsus of al or cll mutants (Kojima, 2000; Tsuji, 2000). Thus, the effect was examined of cll or al misexpression on Lim1-lacZ, whose expression is essentially identical to that of Lim1. cll-misexpressing flip-out clones were generated in first-second instar, and Lim1-lacZ signals were detected in late third instar. Lim1-lacZ misexpression occurred in some cll-misexpressing cells, indicating that Cll can serve as a positive regulator of Lim1 (Kojima, 2005).
A previous experiment showed the sole misexpression of al is incapable of inducing Lim1 misexpression (Tsuji, 2000). However, this does not necessarily mean that al is not involved in Lim1 regulation. Rather the notion is preferred that Lim1 is positively regulated by a concerted action of al and cll, since Lim1-LacZ signals are detected in all cll-misexpressing cells, only in those cells in which al expression is simultaneously observable (Kojima, 2005).
The al/cll cooperation found in Bar repression in the pretarsus may possibly stem from the interactions between Al and Cll. GST pull-down assay was first conducted in vitro to confirm this possibility. Cll was tagged with GST, and a possible binding of Cll to Al was monitored by Western blotting of the eluents from a GST column with anti-Al antibody. GST-Cll was prepared using E. coli cells, and Al was synthesized using reticulocyte lysates. Al signals were detected only when a mixture of GST-Cll and Al was applied to and then eluted from the GST column, indicating that Al and Cll are capable of binding to each other in the absence of DNA (Kojima, 2005).
A polymerase chain reaction-based approach, the systematic evolution of ligands by exponential enrichment (SELEX), was undertaken to determine a possible consensus DNA sequence for the binding of the Al/Cll complex. The nucleotide sequence alignment of 48 fragments obtained after five rounds of enrichment revealed a consensus sequence of 5′-(T/C)TAATTAA(T/A)(T/A)G-3′, which differs from the consensus sequences for the vertebrate homologs of Al (TAATNNNATTA; Alx and Cart proteins; Qu, 1999) and those for Cll homologs (CGGTAA(T/G)(T/C)(G/C)G; Hox11/tlx proteins (Dear, 1993; Shimizu, 2000; Tang, 1995; Kojima, 2005).
Protein-DNA interactions were examined using the electrophoretic mobility shift assay (EMSA). A double-stranded oligonucleotide containing the SELEX consensus sequence was used as a probe. No or weak retardation bands were detected for Cll or All alone. In contrast, a very strong retardation signal was observed for a combination of Al and Cll. A few base substitutions in the consensus sequences results in a significant reduction in or the abolishment of retardation signals. Thus, the Al/Cll complex is significantly different in target-sequence specificity from Al and Cll, and only the Al/Cll complex can strongly bind to the SELEX-determined consensus sequence (Kojima, 2005).
It may thus be concluded that, in the pretarsus, Al and Cll form a complex capable of binding to specific sequences, which cannot be well recognized solely by Al or Cll, and that the resultant complex plays a central role in al/cll-dependent gene regulation in the future pretarsus. However, it should be noted that the possibility cannot be formally excluded that Al and Cll separately bind to their own consensus sequences and function cooperatively in the pretarsus (Kojima, 2005).
Thus al and cll seem to act cooperatively through the formation of the complex between their protein products. To determine whether vertebrate Al and Cll homologs possess similar properties, possible interactions between the Al/Cll consensus sequence and either one of vertebrate Al homolog, Cart1, or a Cll homolog, Hox11L1 (also called as Tlx2), were assessed. Cart1 is capable of binding to the Al/Cll consensus binding site to some extent, but Hox11L1 can not at all. A considerably strong signal is detected when a mixture of Cart1 and Hox11L1 is subjected to gel retardation. Moreover, strong retardation signals are detected for a mixture of Al and Hox11L1 and that of Cart1 and Cll. Thus, the formation of an Al/Cll-type complex may be an evolutionally conserved feature of Al-type and Hox11/tlx-type homeodomain protein family members (Kojima, 2005).
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