fork head


REGULATION

Promoter Structure

The 5' promoter region controls anterior midgut, hindgut, posterior midgut and salivary gland expression. A downstream enhancer responsible for fkh expression in the CNS lies between 2 and 6 kb 3' from the transcription unit. A regulatory element responsible for fkh expression in the foregut is located as far as 30 kb from the transcription unit (Weigel, 1990a).

The activity of the Drosophila gene trithorax is required to maintain the proper spatial pattern of expression of multiple homeotic genes of the bithorax and Antennapedia complexes. Trithorax protein binds at ectopic sites carrying fork head-specific enhancer sequences in transformed lines. Trithorax binding occurs within an 8.4-kb regulatory region that directs fork head expression in several embryonic tissues including salivary glands. Consistently, expression of endogenous Fork head RNA is greatly reduced in trithorax mutant embryos and in larval tissues (Kuzin, 1994).

In the early Drosophila embryo, a system of coordinates is laid down by segmentation genes and dorsoventral patterning genes. Subsequently, these coordinates must be interpreted to define particular tissues and organs. To begin understanding this process for a single organ, a study has been made of how one of the first salivary gland genes, fork head (fkh), is turned on in the primordium of this organ, the salivary placode. A placode-specific fkh enhancer was identified 10 kb from the coding sequence. Dissection of this enhancer shows that the apparently homogeneous placode is actually composed of at least four overlapping domains. These domains appear to be developmentally important because they predict the order of salivary invagination, are evolutionarily conserved, and are regulated by patterning genes that are important for salivary development. Three dorsoventral domains are defined by Egf receptor (Egfr) signaling, while stripes located at the anterior and posterior edges of the placode depend on wingless signaling. Further analysis has identified sites in the enhancer that respond either positively to the primary activator of salivary gland genes, Sex combs reduced (Scr), or negatively to Egfr signaling. These results show that fkh integrates spatial pattern directly, without reference to other early salivary gland genes. In addition, a binding site for Fkh protein was identified that appears to act in fkh autoregulation, keeping the gene active after Scr has disappeared from the placode. This autoregulation may explain how the salivary gland maintains its identity after the organ is established. Although the fkh enhancer integrates information needed to define the salivary placode, and although fkh mutants have the most extreme effects on salivary gland development thus far described, it is argued that fkh is not a selector gene for salivary gland development and that there is no master, salivary gland selector gene. Instead, several genes independently sense spatial information and cooperate to define the salivary placode (Zhou, 2001).

fkh regulatory sequences are spread over 25 kb of DNA that flank the gene. Within this large region, a 5-kb genomic DNA fragment was found that has both salivary gland and hind-gut enhancer activity. The salivary expression pattern driven by this 5-kb fragment is consistent and identical to the expression of fkh itself. Several subfragments of the 5-kb DNA were inserted upstream of lacZ and tested for enhancer activity by P element-mediated transformation. If the 5-kb DNA is denoted as 1-5000 (starting from the distal NotI site), only the first 1-kb subfragment (1-1008) directs detectable salivary lacZ expression. As measured by anti- ß-gal antibody staining, the enhancer activity of the 1-kb DNA is similar to that of the 5-kb fragment, indicating that most of the elements of the salivary enhancer are confined to this 1-kb region. The 3400-5000 fragment has at least partial hindgut enhancer activity but no salivary activity, indicating that these two enhancers are separable (Zhou, 2001).

Closer examination reveals that the salivary expression directed by the 1-kb fragment is not quite the same as that directed by the 5-kb fragment. With the 1-kb DNA, some transformed embryos show ectopic expression of variable strength in the cells located ventrally between the two placodes. This ventral expression, which is weaker than expression in the placode itself, is rarely observed for either the 1-5000 fragment or for Fkh protein itself. Within any particular transformed line, the amount of ventral expression is variable and is not dependent on the chromosomal location of the enhancer:lacZ transposon. These results suggest that there is at least one ventral repression element located outside the 1-kb enhancer. Without that element, the cells ventral to the placode can weakly express lacZ. In the normal chromosomal context, repression elements within and outside the 1-kb enhancer would interact for effective ventral repression. Consistent with these suggestions, in experiments described later, a region within the 1-kb enhancer DNA was identified to be necessary for ventral repression (Zhou, 2001).

Further dissection of the 1-kb enhancer (1-1008) showed that salivary expression of fkh is regulated in a spatially complex pattern. The first half of the 1-kb enhancer, 1-506, drives expression in most of the placode, but not in its most ventral two rows of cells. This lack of expression in the ventral cells of the placode can be clearly seen by the greater separation between the stained regions of the placodes in these embryos than in embryos expressing the 1-kb or 5-kb constructs or Fkh protein itself (six to eight cells apart across the ventral midline instead of about four cells apart). The overall expression level directed by 1-506 is easily detectable but significantly lower than that by the 1-kb enhancer. The other half of the enhancer does not direct the simple, complementary pattern. 507-1008 drives expression not only in the ventral part of the placode, but also in the cells ventral to the placodes. In addition, in more than half of the 507-1008:lacZ embryos, expression at the anterior and posterior edges of the placode extends dorsally, forming anterior and posterior stripes that overlap cells in which 1-506:lacZ would be expressed. The remaining embryos do not have these dorsal extensions. Again, the variability is not transformant line-dependent. When 507-1008:lacZ expression begins at late stage 10, only the two stripes are visible. They extend across the ventral midline, one at the front edge and one at the back edge of parasegment 2. It is imagined that the pattern detected at later times (stage 11) arises by adding to the stripes a ventral domain that includes the ventral cells of the placode and the cells ventral to the placode (Zhou, 2001).

In summary, both the 1-506 and 507-1008 halves of the enhancer direct reporter gene expression to salivary placode cells, but the patterns that they direct are very different. Within the placode, which had previously been seen as a homogeneous field of cells, fkh expression is regulated in at least four partially overlapping domains: the dorsal and ventral parts of the placode, and the anterior and posterior stripe domains (Zhou, 2001).

Expression of fkh is limited at the dorsal edge of the placode by dpp and at the ventral edge of the placode by Egfr and the spitz group genes. Expression driven by the two halves of the enhancer has identified a new dorsoventral limit, the boundary between the dorsal and ventral parts of the placode. What genes establish this boundary? Expression of 1-506:lacZ in sim or pnt embryos shows that the Egfr pathway is involved. In these embryos, staining is as strong in the dorsal placode as it is in wild-type embryos, but now there is also weak staining that extends all the way to the ventral midline. Thus, expression of 1-506:lacZ must normally be limited by Egfr signaling. In contrast to this result, dpp mutations have no effect on this boundary (Zhou, 2001).

In the anteroposterior dimension, establishment of parasegmental borders depends on interaction between wingless expressing cells at the posterior edge of one parasegment and hedgehog or engrailed expressing cells at the anterior edge of the next, more posterior parasegment. Since 507-1008:lacZ is expressed in anterior and posterior stripes adjacent to the borders of parasegment 2, its reliance on wg was tested. At the placode stage in wg-mutant embryos, the posterior stripe is not seen, and the anterior stripe may also be missing. At slightly earlier times, when only the stripes are seen in wild-type embryos, no expression of 507-1008:lacZ is seen in wg-mutant embryos. These results suggest that fkh responds to wg signaling by expression in both posterior and anterior stripes (Zhou, 2001).

Drosophila virilis and D. melanogaster diverged about 60 million years ago. During this separation, it is expected that unimportant sequences diverged more rapidly than functionally important ones. To clone the virilis fkh homolog, a virilis lambda genomic library was screened and clones that hybridized to both the melanogaster fkh structural gene and the 1-kb enhancer were isolated. Sequence comparisons between melanogaster and virlilis reveal that the fkh salivary enhancer is a highly conserved regulatory region, both in its overall organization and in its sequence. The fact that multiple regions are conserved and that several of these are 80-100 bp long suggests that many elements are involved in fkh transcriptional control (Zhou, 2001).

To test whether the virlilis and melanogaster DNAs are functionally equivalent, pieces of the virlilis DNA were used to drive ß-gal expression in melanogaster embryos. The results for two different constructs showed that the virlilis DNA does work as a salivary enhancer in melanogaster. For virlilis DNA corresponding to 550-1008 of the melanogaster enhancer, the ß-gal expression was found mostly in the anterior part of the glands and in the ducts, just like the expression pattern driven by the 507-1008 fragment of the melanogaster enhancer. In addition, when a piece of virlilis DNA corresponding to 300-1008 of the melanogaster sequence was used, the expression was very similar to that of the complete 1-kb melanogaster enhancer or its 350-1008 fragment. The glands were well stained and there was little expression in the ducts. These results suggest that fkh transcriptional control and salivary patterning have been conserved during evolution (Zhou, 2001).

This conserved organization along with additional dissection of the enhancer has allowed the identification of several important regulatory regions that have distinct functions. First, because 219-1008 activity is stronger than 350-1008, there is likely to be an activating element between 219 and 350. In addition, there is little difference between the enhancer activities of 1-308 and 1-356, suggesting that this activator does not lie between 308 and 356. From this analysis, it is concluded that there is probably an activating element within the 219-308 region. By similar reasoning, there is likely to be an activating element within the 357-506 region and multiple activating elements within the 507-1008 region (Zhou, 2001).

A negative element was identified in the 350-506 region. In addition to its placode expression, 507-1008 gives strong ectopic expression in the cells ventral to the placodes, expression that is not seen for fkh itself and is only occasionally and weakly seen with the 1-kb enhancer. The strong ventral derepression indicates that 507-1008 lacks a ventral repression element that is present in the 1-kb enhancer. Further dissection located this element between 350 and 506, because 350-1008 is ventrally repressed. Further analysis has concentrated on the positive 219-308 region and on the 357-506 region that contains both positive and negative elements (Zhou, 2001).

In the 219-308 interval, 242-277 is strongly conserved in the D. virlilis enhancer, and it has been designated as the 250 region for further analysis. To test the activity of this 36-bp region, three copies of 250 were placed upstream of the minimal heat shock promoter and lacZ. Multiple transformed lines containing this construct were obtained, and all showed ß-galactosidase expression throughout the ectoderm of PS2, as well as in a few cells in each trunk segment and patches in the head. These results suggest that, in its normal context, 250 activates fkh expression in the salivary placode, perhaps by binding the homeotic regulator Scr. Within the 250 element, there is one region with two overlapping sequences of ATTAAT that are similar to homeodomain core binding sites. To test whether this consensus site is important for PS2 expression, the two contiguous As were changed to Gs. These changes should destroy homeodomain protein binding, but have minimal effect on the surrounding regions. Staining of transformants carrying this 250-GG construct is consistent with the direct involvement of Scr in the enhancer activity of the 250 element (Zhou, 2001).

Comparison of the 507-1008 and 350-1008 constructs has identified an element between 350 and 506 that mediates ventral repression. If this location is correct, deletion of this region from the otherwise intact 1-kb enhancer should lead to ventral derepression. D361-506, an internally deleted construct that removes most of this region, does indeed direct strong expression in both the placode and the ventral cells. The ectopic expression in the ventral cells is as strong as in the placodes, indicating that few if any, functional ventral repression elements exist in the remaining 0.85 kb of the enhancer. This is the same pattern of expression that was seen for fkh itself or for the 1-kb fkh enhancer in embryos that lack Egfr signaling. Therefore, it is proposed that an Egfr-dependent repressor normally acts on the 361-506 region to prevent fkh expression in the cells ventral to the placode. Comparison to the virlilis enhancer sequence does not help to localize the negative element more precisely because almost the entire 350-506 region is conserved. However, preliminary footprinting experiments with crude embryonic extracts have shown protection of the 415-424 region which overlaps two adjacent E-box sequences separated by a single nucleotide. To test whether this region mediates the ventral repression signal, three internally deleted versions of the 1-kb enhancer were constructed that lack either 361-424, 413-424, or 413-506. All three lack 413-424. When tested in embryos after transformation, all three constructs gave stronger duct staining than the 1-kb enhancer. The 413-506 deletion, which extends furthest to the right has the most dramatic effect, suggesting that there is more than one repression element within the 361-506 region. The smallest deletion, 413-424, must disrupt at least one of these elements (Zhou, 2001).

These experiments showed that this region (hereafter called the 420 region) is necessary for ventral repression. To test whether it is sufficient, three copies of a 21-bp fragment (411-431) were added to the D361-506 construct, which by itself is expressed throughout the salivary gland and duct primordia. As a result, ventral repression was restored. Similarly, when this 21-bp fragment was inserted between the 507-1008 fragment and the basal promoter, lacZ expression was limited to parts of the placode, or at later times, to the salivary glands. It is concluded that the 420 region contains an element that mediates the ventral repression signal. This repression normally overrides activation signals for the reporter or fkh expression in the preduct region and limits fkh expression to the salivary placode (Zhou, 2001).

There is not only a ventral repression element, but also an activating element in 361-506. To locate this element, this 146-bp region was divided into small elements and a concatemer of each region was put in front of 507-1008 to test for enhancement of its salivary gland activity. This strategy should allow detection of even minor enhancements, because 507-1008 by itself is not expressed in all cells of the placode or salivary gland. The 460 element, which includes nucleotides from 457 to 495, gave a strong response in this assay, indicating that 460 contains an activating element. To determine whether this element itself is sufficient to be a salivary enhancer, a concatemer including four copies of just the 460 element was tested. Staining shows that this element can by itself activate lacZ expression that is limited to the salivary glands. However, expression of (460)4:lacZ begins during salivary invagination, later than either fkh or the Scr-dependent construct 250:lacZ. This temporal difference suggests that the enhancing activities of 250 and 460 are different (Zhou, 2001).

Sequence comparison has shown that the 460 element includes a consensus Fkh/HNF3 binding site sequence that is very similar to an established Fkh binding site from the Kruppel promoter. This similarity suggests that endogenous Fkh might be activating the 460 construct. To test this idea, (460)4:lacZ was transferred to fkh-mutant embryos. Its activity is absolutely dependent on Fkh, suggesting that fkh expression in salivary glands is autoregulatory. Binding experiments indicate that Fkh can bind to the 460 region through the 11 nucleotides of the consensus Fkh binding site and suggest that this binding accounts for (460)4:lacZ expression in vivo (Zhou, 2001).

If embryos expressing one of the partial fkh enhancer constructs are allowed to finish invagination, only a subset of the salivary cells will be marked by lacZ expression. Since the salivary cells do not divide during this time, a fate map can be constructed by correlating the position of marked cells at the placode and gland stages. The 1-506 construct gives rise to expression in the dorsal part of placodes. Older embryos show staining in the distal 3/4 of the glands. This result suggests not only that the dorsal part of the placode becomes the distal part of the salivary gland, but also that the ventral part of the placode gives rise to the proximal salivary gland. The 507-1008 enhancer construct confirms this suggestion by showing its strongest placode expression in the ventral cells and later showing strong expression in the proximal cells of the invaginated glands. With this construct, variable expression is also seen in the posterior part of invaginated glands, and it is proposed that this staining comes from the variable anterior and posterior stripes seen at the placode stage. The fact that some embryos have no stripes is consistent with the observation that some invaginated glands have little distal staining. This construct consistently shows little staining in mid-dorsal placode cells and in cells on the medial side of the invaginated gland about halfway along its length. It is thought that these are the same cells at different stages. These results, along with the fact that invagination in uniformly stained placodes can be seen to initiate at the dorsal, posterior edge of the placode, can be combined to produce a consistent hypothesis for salivary morphogenesis. The first cells to invaginate are those at the dorsal end of the posterior stripe. They are immediately followed by the remaining cells of the posterior stripe and by the cells of the mid-dorsal placode. All of the cells of the dorsal placode invaginate before any cells of the ventral placode. The ventral cells are the last placode cells to invaginate, and they form the proximal end of the gland. Finally, the duct forms after the gland has invaginated (Zhou, 2001).

At stage 11, both 507-1008 and D361-506 give ectopic expression in cells ventral to the placodes. Examination of these embryos at later stages shows that the salivary ducts are stained in addition to part or all of the glands. By examining embryos at intermediate stages during the invagination of the glands and ducts, a continuous progression can be seen from the cells ventral to the placode to the invaginated ducts. Therefore, it is concluded that the cells ventral to the placodes are the duct precursors (Zhou, 2001).

Dissecting the functional specificities of two Hox proteins

Hox proteins frequently select and regulate their specific target genes with the help of cofactors like Extradenticle (Exd) and Homothorax (Hth). For the Drosophila Hox protein Sex combs reduced (Scr), Exd has been shown to position a normally unstructured portion of Scr so that two basic amino acid side chains can insert into the minor groove of an Scr-specific DNA-binding site. This study provides evidence that another Drosophila Hox protein, Deformed (Dfd), uses a very similar mechanism to achieve specificity in vivo, thus generalizing this mechanism. Furthermore, it was shown that subtle differences in the way Dfd and Scr recognize their specific binding sites, in conjunction with non-DNA-binding domains, influence whether the target gene is transcriptionally activated or repressed. These results suggest that the interaction between these DNA-binding proteins and the DNA-binding site determines the architecture of the Hox-cofactor-DNA ternary complex, which in turn determines whether the complex recruits coactivators or corepressors (Joshi, 2010).

Previous work on Scr's ability to specifically regulate its target gene, fkh, revealed that the N-terminal arm of its homeodomain and preceding linker region are positioned in such a manner as to allow the insertion of two basic side chains into the minor groove of the target DNA, fkh250 (Joshi, 2007). Importantly, the correct positioning of these residues depends on an interaction between Scr's YPWM motif and the cofactor Exd. This study shows that an analogous mechanism is required for Dfd to bind productively to a Hox-Exd-binding site in the EAE element and to activate EAE-lacZ in vivo. Specifically, it was found that Dfd's YPWM motif is required for cooperative binding to EAE's site I in vitro, and for executing Dfd-specific functions in vivo. Like Scr, Dfd has the same two basic residues -- a histidine (likely to be protonated when bound to DNA) and an arginine -- at the equivalent positions relative to its YPWM motif and homeodomain. Moreover, these residues are also required for Dfd to execute its specific functions in vivo. Thus, the activation of fkh by Scr and the activation of Dfd by Dfd appear to use analogous mechanisms, whereby linker and N-terminal arm residues are used to bind paralog-specific binding sites in an Exd-dependent manner (Joshi, 2010).

The YPWM-to-YPAA mutation severely impaired Dfd's ability to carry out its specific in vivo functions, such as activation of EAE-lacZ and production of cirri. Thus, the YPWM motif of Dfd is critical for Dfd function in vivo. This situation contrasts with other apparently more complex scenarios. For example, mutation of the YPWM motif of the Hox protein Ultrabithorax (Ubx) did not significantly impair some of its in vivo functions. In this case, it appears that other sequence motifs, in particular a domain C-terminal to the Ubx homeodomain, are important for Ubx to carry out its specific functions in vivo. These Ubx sequences also appear to help recruit Exd to DNA, and therefore may be used for binding site selection in conjunction with YPWM at a subset of Ubx target-binding sites. Interestingly, a sequence motif immediately C-terminal to Dfd's homeodomain also plays a role in in vivo specificity, although its impact on DNA binding has not been examined. As these sequences are still present in DfdScrSMδ23, it may explain why this chimera retains some Dfd-specific functions, such as the formation of cirri and ability to activate EAE-lacZ. The picture that emerges from all of these data is that Hox proteins may use different motifs to interact with cofactors such as Exd, depending on the specific in vivo function and target gene being regulated (Joshi, 2010).

In general, the sequences surrounding Hox YPWM motifs and the N-terminal arms of their homeodomains are highly conserved, from invertebrates to vertebrates, in a paralog-specific manner (Joshi, 2007). Thus, based on the results presented in this study, it is hypothesized that these sequences, which are referred to as Hox specificity modules, may in general be used for the recognition of specific DNA-binding sites in a cofactor-dependent manner. In the case of Scr binding to fkh250, an X-ray crystal structure revealed that the histidine and arginine side chains recognize an unusually narrow minor groove that is an intrinsic feature of the fkh250-binding site. Without the benefit of a Dfd-Exd-site I crystal structure, it cannot be know with certainty if Dfd's His-15 and Arg3 also read the shape of a narrow minor groove. However, the fact that the same two basic residues are required for both Scr and Dfd suggests the possibility that this is the case for Dfd binding to EAE site I as well (Joshi, 2010).

DfdScrSMδ23, which has the specificity module of Scr in place of Dfd's, exhibited clear Scr-like functions in vivo, as assayed by fkh250-lacZ activation and larval cuticle transformation. Other attempts to swap Hox specificities by generating chimeric Hox proteins have had variable success. For example, when the linker and N-terminal arm of Scr is used to replace the equivalent region of Antennapedia (Antp), the chimera behaved like Scr. This finding supports the importance of specificity modules in conferring Hox specificity. When the homeodomain and C-terminal region of Ubx were replaced by the equivalent domains from Antp, the chimera behaved like Antp, suggesting that the identity of the linker region may not be critical in all cases. Other Hox chimeras have generated less clear changes of specificity. For example, chimeras between Ubx and Dfd generated a cuticle phenotype that was dissimilar to that produced by either parent protein. Similarly, a chimera between Ubx and Abd-B had novel properties that were unlike those produced by either parent protein. It is noteworthy that the cleanest changes in specificity occurred when the chimera was generated between Hox genes that are adjacent to each other in the Hox complex. This correlation may be due to the fact that adjacent Hox genes are more similar to each other, both in sequence and in function, than nonadjacent Hox genes. This higher degree of similarity is likely a consequence of how these genes are thought to have duplicated during evolution (Joshi, 2010).

Previous work on the regulation of fkh by Scr, the reporter gene used to study the activity of the Exd-Scr-binding site had a multimerized version of the minimal 37-bp fkh250 element. In contrast, in the work described in this study, an intact regulatory element from the Dfd gene was characterized, revealing significantly more complexity. In particular, the 570-bp modC element contains a single 'classical' Exd-Hox composite site, but also four additional Dfd sites and several additional Exd-Hth-binding sites. Mutagenesis studies suggest that all of these inputs are important for the full activity of this enhancer. Also noteworthy is that there are additional Dfd-Exd-binding sites in the larger 2.7-kb EAE element that, in principle, could also be used in vivo. Thus, the picture that emerges from this analysis is that native enhancer elements may use a combination of classical Exd-Hox-binding sites together with additional arrangements that may not always conform to the classical spacing of the Exd and Hox half-sites. This picture raises the question of how the linker and N-terminal arm residues are positioned correctly in these nonclassical arrangements. The answer may lie in the fact that, in vivo, the assembly of the complete multiprotein complex -- which is likely to include factors in addition to Dfd, Exd, and Hth -- promotes the recognition of Dfd-binding sites in ways that are not fully revealed by experiments that examine binding to individual or small groups of binding sites in isolation (Joshi, 2010).

Depending on the context, most transcription factors have the capacity to activate and repress transcription. In most cases, it is not understood how this choice is made. One established scenario is that other proteins that get recruited to an enhancer element determine the sign of the regulation. However, this type of model is not sufficient to explain the results presented in this study. The results suggest that the DNA-binding properties of the Exd-Hox complex influence the regulatory output of the bound protein-DNA complex. Deletion of two motifs (γ23) from the N-terminal region of DfdScrSM converted this protein from a repressor of fkh250-lacZ to an activator of fkh250-lacZ, while deletion of the same motifs from DfdWT did not change the regulatory output: The protein retained its ability to repress fkh250-lacZ. The only difference between Dfdγ23 (represses) and DfdScrSMγ23 (activates) is the specificity module, and the only difference between DfdScrSMγ23 (activates) and DfdScrSM (represses) is the presence or absence of motifs 2 and 3. These results imply that the relevance of motifs 2 and 3, which are far from the DNA-binding domain, depends on the identity of the specificity module. These findings lead to a suggestion that the DNA-binding site, together with how it is read by the specificity module, plays an important role in determining the overall conformation of the Hox-Exd complex, which eventually determines whether there will be recruitment of a coactivator or corepressor. This idea fits well with a DNA allostery model that was supported recently by cell culture experiments with the glucocorticoid receptor. In these experiments, it was discovered that small differences in the DNA-binding site lead to differences in conformation and the degree of transcriptional activation. This study extends this idea by showing that Hox proteins with different specificity modules, and therefore with slightly different DNA recognition properties, result in unique regulatory outputs in an in vivo context. Furthermore, in these experiments, a complete change was observed in the sign of the regulation from repression to activation, instead of a more subtle change of activation amplitude. Thus, the transcriptional output of a Hox-cofactor complex depends both on the ability of these complexes to bind to their binding sites with high specificity, in part by reading structural features of the DNA, and on the three-dimensional architecture of the bound complex, which is a consequence of both protein-DNA and protein-protein interactions. An important goal for the future will be to use structural biology methods to see how different Hox specificity modules result in distinct conformations of Exd-Hox complexes (Joshi, 2010).

Competition for cofactor-dependent DNA binding underlies Hox phenotypic suppression

Hox transcription factors exhibit an evolutionarily conserved functional hierarchy, termed phenotypic suppression, in which the activity of posterior Hox proteins dominates over more anterior Hox proteins. Using directly regulated Hox targeted reporter genes in Drosophila, this study shows that posterior Hox proteins suppress the activities of anterior ones by competing for cofactor-dependent DNA binding. Furthermore, a motif in the posterior Hox protein Abdominal-A (AbdA) was identified that is required for phenotypic suppression and facilitates cooperative DNA binding with the Hox cofactor Extradenticle (Exd). Together, these results suggest that Hox-specific motifs endow posterior Hox proteins with the ability to dominate over more anterior ones via a cofactor-dependent DNA-binding mechanism (Noro, 2011).

fkh250 is a 37-base-pair (bp) element from the forkhead (fkh) gene, which is directly regulated by Scr; it contains a single Hox-Exd-binding site that, compared with other Hox-Exd heterodimers, is preferentially bound by Scr-Exd in vitro (Ryoo, 1999). When lacZ is placed under the control of fkh250, fkh250-lacZ is specifically expressed in PS2 in an exd- and Scr-dependent manner. Indeed, misexpression of Scr throughout the Drosophila embryo can ectopically activate fkh250-lacZ. Notably, ectopic activation of fkh250-lacZ occurs even in the abdomen, in the presence of endogenous, more posterior Hox (Noro, 2011).

In contrast to fkh250, fkh250CON (for 'consensus') is an artificial variant of fkh250 with two base pair substitutions that enable fkh250CON-lacZ to be directly regulated by four Hox genes in an exd-dependent manner: Scr, Antp, and Ubx activate this reporter in PS2-PS6, while AbdA represses it in abdominal segments (Ryoo, 1999). Consistent with its relaxed specificity in vivo, fkh250CON binds well to Scr-Exd, Antp-Exd, Ubx-Exd, and AbdA-Exd heterodimers in vitro. The promiscuous binding and regulation by multiple Hox proteins classifies fkh250CON as a shared Hox target gene, while the Scr-specific regulation of and binding to fkh250 suggests that it is a specific Hox target gene (Mann, 2009; Noro, 2011).

Because of their distinct specificities, fkh250-lacZ and fkh250CON-lacZ provide an ideal system to examine the molecular mechanism of phenotypic suppression. In accordance with the premise of posterior dominance, coexpression of Scr and AbdA throughout the fly embryo leads to repression of fkh250CON-lacZ by AbdA. Note that both fkh250 and fkh250CON-lacZ require direct binding by the Hox cofactor Exd. The primary distinction between these two readouts is that AbdA-Exd binds well to fkh250CON but not to fkh250. Accordingly, it is concluded that AbdA cannot suppress the activities of Scr if it cannot bind to the target element. Furthermore, in this system, posterior dominance cannot be mediated by miR activity or competition for factors, such as Exd, off DNA. Rather, these data support a model in which competition for cofactor-dependent DNA binding underlies phenotypic suppression for shared Hox target genes (Noro, 2011).

If AbdA is outcompeting Scr for binding to fkh250CON, AbdA would be expected to have a higher affinity for this sequence compared with Scr. To test this prediction, the affinities of AbdA-Exd and Scr-Exd heterodimers for fkh250CON were measured in vitro. AbdA-Exd heterodimers bound more than twofold more tightly to fkh250CON compared with Scr-Exd. Thus, at the same concentration, AbdA-Exd is more likely than Scr-Exd to be bound to fkh250CON, consistent with the idea that competition depends on cofactor-dependent DNA binding (Noro, 2011).

Binding to fkh250CON is Exd-dependent for both AbdA and Scr, implying that AbdA has domains that allow higher binding affinity with Exd to this target site. In general, Hox interactions with Exd are mediated by the highly conserved, four-amino-acid motif YPWM, which directly binds to a hydrophobic pocket established by the three-amino-acid loop extension (TALE) in the Exd homeodomain (Mann, 2009). For some Hox proteins, the YPWM-TALE interaction is necessary and sufficient for cooperative DNA binding with Exd and target gene regulation in vivo (Joshi, 2010). In addition to the YPWM motif, AbdA, but not Scr, has a second well-conserved tryptophan-containing motif, TDWM, which could play a role in mediating AbdA-Exd interactions. However, when a mutant form of AbdA in which both the YPWM and TDWM motifs are mutated to alanines (2WAla) was coexpressed with Scr in the phenotypic suppression assay, fkh250CON-lacZ was repressed to the same extent as by wild-type AbdA. Thus, although the YPWM and TDWM may contribute to interactions with Exd, these motifs are not necessary for AbdA to dominate over Scr (Noro, 2011).

Immediately C-terminal to its homeodomain, AbdA contains a so-called UbdA motif, a nine-amino-acid sequence also present in Ubx, which has been suggested to mediate cooperative binding with Exd to some DNA sequences (Merabet, 2007). In fact, UbdA is part of a larger 23-residue conserved region adjacent to the AbdA homeodomain, which is referred to as the UR motif (for UbdA-RRDR). To determine whether this or other regions in the C-tail of AbdA are involved in mediating phenotypic suppression, a series of C-terminal truncations were tested for their ability to compete with Scr for the repression of fkh250CON-lacZ in vivo. All AbdA variants were epitope-tagged, allowing use of transgenes that express at similar levels (Noro, 2011).

AbdA's ability to compete with Scr for fkh250CON regulation is eliminated when the entire C terminus is removed (ΔC197). Adding back only the UR motif partially restores AbdA's ability to dominate over Scr (ΔC220). Consistently, an internal deletion that removes most of the UR motif (Δ200-220) exhibits a reduced ability to repress fkh250CON-lacZ. No additional loss of repressive activity is displayed by an AbdA variant in which both the YPWM and TDWM motifs are mutated in combination with this internal deletion (2WAlaΔ200-220). Additional sequences in the C-tail of AbdA may account for the residual activity of variants lacking the UR motif (Δ200-220 and 2WAlaΔ200-220). All AbdA variants used in this study are capable of repressing the exd-independent target gene spalt in the wing imaginal disc, confirming that these mutants are still functional transcription factors. Furthermore, these mutants retain the ability to repress gene expression in vivo, arguing that AbdA's repressive activity is not sufficient to account for its ability to dominate Scr. Together, these data highlight the importance of the UR motif for phenotypic suppression (Noro, 2011).

The above data show that the UR motif is required for AbdA to compete with Scr in vivo. To test the hypothesis that UR carries out this function by facilitating cooperative DNA binding with Exd, the ability of the truncated AbdA variants to bind fkh250CON in complex with Exd was analyzed. In general, the results correlate with the in vivo phenotypic suppression assay: Those mutants that failed to suppress Scr's ability to activate fkh250CON-lacZ (ΔC197, Δ200-220, and 2WAlaΔ200-220) were severely compromised in binding fkh250CON with Exd in vitro. Together, these data strongly suggest that cooperative DNA binding with Exd is required for phenotypic suppression and that domains unique to AbdA are critical for its ability to dominate over Scr. More specifically, they argue that AbdA's UR motif is necessary for cooperative binding of AbdA and Exd to fkh250CON and that the YPWM and TDWM motifs are not sufficient to mediate this interaction on this binding site. The insufficiency of the YPWM motifs to mediate cooperative binding with Exd has been observed for other Hox proteins, suggesting that the use of paralog-specific motifs such as UR may be a general phenomenon (Noro, 2011).

To test the generality of AbdA's dependency on its UR motif for posterior dominance, the same AbdA variants were analyzed for their ability to suppress the activity of the thoracic Hox protein Antp in the patterning of the larval epidermis. When ectopically expressed, Antp transforms the head and first thoracic segment (T1) toward the identity of the second thoracic segment (T2), where Antp is normally expressed). In contrast, when AbdA is ectopically expressed, the head and thorax acquire abdominal segmental identities. Consistent with the rules of phenotypic suppression, wild-type AbdA is able to produce this transformation even in the presence of exogenous Antp. However, similar to the results with fkh250CON-lacZ, AbdA mutants that are compromised in their ability to cooperatively bind DNA with Exd (e.g., ΔC197, Δ200-220, and 2WAlaΔ200-220) fail to suppress the activity of Antp (Noro, 2011).

Taken together, these data support a model in which phenotypic suppression depends on a competition for cofactor-dependent DNA binding. It follows that this mechanism would only apply to readouts that depend on regulatory elements that are targeted by multiple Hox proteins. For example, ectopic Scr can activate fkh and other target genes required for salivary gland development in more posterior segments, illustrating that this Hox-specific function does not obey phenotypic suppression. Furthermore, it is particularly noteworthy that, compared with the anterior Hox protein Scr, AbdA has additional motifs that facilitate complex formation with Exd on DNA. These data suggest that when phenotypic suppression is observed, the more posterior Hox proteins may have a higher affinity for shared binding sites; this higher affinity is a consequence of the quantity and quality of motifs that mediate cooperative DNA binding with Exd. It is speculated that these motifs may be used differently at different target genes and binding sites. It is suggested that the YPWM motif provides a common, basal level of interaction between Hox proteins and Exd. In the context of Hox-specific regulatory elements, this motif may be sufficient to enable Hox-Exd regulation of some target genes. In contrast, in the context of shared enhancers and when multiple Hox proteins are coexpressed, additional, paralog-specific motifs present in the more posterior Hox proteins enable tighter binding of Hox-Exd dimers to DNA, leading to more posterior phenotypes. This was shown to be the case for a single shared Hox-Exd enhancer and suggest that the generality of this mechanism for phenotypic suppression will become apparent as more shared and specific targets for Hox proteins are characterized at high resolution (Noro, 2011).

Transcriptional Regulation

Mutants in the torso group of maternal genes delete all structures where fkh is expressed in posterior segments. Mutants of the zygotic terminal gene tailless affect only ectodermal fkh expression. fkh expression in the anterior depends strictly on the activity of the gap gene huckebein. Expression in salivary glands is under control of Sex combs reduced (Weigel 1990a), maintained by fkh itself and repressed by spitz group genes (Zhou 1995).

During early embryogenesis in Drosophila, Caudal mRNA is distributed as a gradient with its highest level at the posterior of the embryo. This suggests that the Caudal homeodomain transcription factor might play a role in establishing the posterior domains of the embryo, which undergo gastrulation and give rise to the posterior gut. By generating embryos lacking both the maternal and zygotic mRNA contribution, caudal has been shown to be essential for invagination of the hindgut primordium and for further specification and development of the hindgut. Mature embryos lacking cad activity (maternal and/or zygotic contributions) were examined to assess the requirement for cad in establishing the structures that arise from the posterior ~15% of the blastoderm embryo, namely the posterior midgut, Malpighian tubules and hindgut (Wu, 1998b).

The stages of gastrulation can be observationally followed by using expression of brachyenteron byn as a marker for the hindgut primordium. In the wild-type embryo, byn is expressed in a ring at the circumference of the amnioproctodeal plate. The edges of this ring come together as the posterior midgut primordium invaginates during stages 6 and 7; the ring of the hindgut primordium then sinks inward during stage 8 and is completely internalized by the end of stage 9. The zygotically expressed cad stripe and the posterior wg stripe are also expressed in the bordering ring (i.e., the hindgut primordium) of the invaginating amnioproctodeal plate. Strikingly, in cad-deficient embryos, the byn-expressing ring of hindgut primordium draws together, but fails to invaginate, remaining on the outside of the embryo. Thus, although internalization of the Malpighian tubule and posterior midgut primordia is normal in cad-deficient embryos, the gastrulation movements necessary for internalization of the hindgut primordium do not occur in embryos lacking cad activity (Wu, 1998b).

The absence of the hindgut primordium from cad-deficient embryos suggests that Caudal regulates genes required for establishing and/or maintaining the hindgut primordium. tailless, fork head, byn, bowl and wingless are likely targets for cad regulation, since all are required for some aspect of hindgut development: the hindgut is missing from both tll and fkh embryos, and severely reduced in wg, byn and bowl embryos. bowl, also called bowel, codes for a zinc finger transcription factor related to odd-skipped. Since maternally provided Caudal, which persists only through the blastoderm stage, is sufficient for essentially normal hindgut formation, the fact that all of these genes are expressed at the posterior of the embryo during the blastoderm stage means that they are potential targets for regulation by Caudal. The effect of absence of maternal and/or zygotic cad activity on the expression of these genes was assessed by in situ hybridization with appropriate probes. For tll, byn and bowl, absence of cad activity does not result in a detectable effect on expression. As described below, however, cad activity is essential for expression of fkh and wg. Both maternal and zygotic cad contributions are necessary for posterior wg expression. During early stage 5, just prior to its expression in 14 stripes that are required to establish the segmental pattern, wg is expressed in two domains at the anterior, and in a broad posterior stripe. This terminal wg stripe is located at approximately 8-12% EL, overlapping with the posterior of the zygotic cad stripe and with the position of the hindgut and Malpighian tubule primordia in the blastoderm fate map. Expression of the wg terminal stripe has been shown to be independent of other segmentation genes, but has not been otherwise characterized. All embryos from mutant cad germline mothers (even those expressing zygotic cad) fail to express the terminal stripe of wg. These results demonstrate that maternal cad activity is essential for the transcription of wg in the terminal stripe. Among embryos from wg heterozygous parents, approximately one-quarter (presumably those lacking only the zygotic component of cad expression) lack the terminal wg stripe. Thus both maternal and zygotic cad activities are required for expression of the terminal wg stripe (Wu, 1998b).

The expression of the early cap of fkh also requires cad activity; approximately half of the embryos from mutant cad germline females mated to cad heterozygous males (i. e., cad m-z - embryos) show a dramatic reduction in both the size and intensity of the posterior cap of fkh expression. However, if cad is supplied either maternally or zygotically, fkh expression is normal. Thus expression of the posterior cap of fkh requires cad activity, which can be provided either maternally or zygotically. Later, by stage 10, fkh expression is as strong in cad-deficient as in wild-type embryos, indicating that this later expression is independent of cad activity. Since tll and hkb are also required to activate early fkh expression but are not themselves regulated by cad, cad must act combinatorially with these two genes to promote early fkh expression (Wu, 1998b).

cad also regulates wg in combination with other genes. In addition to the demonstrated requirement for cad, expression of the posterior wg stripe requires positive input from fkh and tll, since the stripe is absent from the respective mutant embryos. Since embryos lacking either maternal or zygotic cad fail to express the posterior wg stripe, but still express fkh and tll, cad must act combinatorially with fkh and tll to promote formation of the posterior wg stripe. Expression of the terminal stripe thus requires the combinatorial action of cad, tll and fkh; the posterior limit of the stripe is known to be defined by repression by hkb (Wu, 1998b).

The failure of the hindgut to become internalized in caudal-deficient embryos raises the question of whether cad might regulate a zygotically expressed gene required for the invagination of the amnioproctodeal plate. One gene known to be required for gastrulation is fog; fog mutant embryos lack not only the posterior midgut, but, as revealed by anti-Crb staining, the Malpighian tubules and hindgut as well. In the blastoderm stage embryo, fog expression is first activated in the region that will become the ventral furrow; shortly thereafter, expression is initiated in a posterior cap, in the region that will become the amnioproctodeal invagination. In cad-deficient embryos, fog expression in the prospective ventral furrow is normal, but is significantly reduced in the posterior cap. Thus, cad is required for the normal level of expression of fog in the prospective amnioproctodeal plate; decreased fog expression in cad-deficient embryos is likely responsible for the failure of the hindgut primordium to be internalized during gastrulation. Since fkh or wg mutant embryos do not display detectable defects in gastrulation, fog is the only gene presently known to mediate the effects of cad on gastrulation. In fog mutant embryos, none of the posterior gut primordia invaginate, while in cad-deficient embryos the posterior midgut and Malpighian tubule primordium do invaginate; thus, consistent with the in situ hybridization results, a low level of fog activity is present at the posterior of embryos lacking cad (Wu, 1998b).

In addition to cad, three other genes (fkh, byn and wg,), which are required at the posterior of the Drosophila embryo for formation of the hindgut, are related to genes found throughout the metazoa, known as HNF-3 (alpha, beta, and gamma), Brachyury (also known as T) and Wnt, respectively. In many cases, these homologs are expressed in portions of the 'blastopore equivalent' at the posterior of the embryo, that overlap with domains of expression of cad (Cdx). In C. elegans, a Wnt homolog is expressed, and required for proper posterior development, in the same posterior blastomere where the cad homolog pal-1 functions. In sea urchin, HNF-3 and Brachyury homologs are expressed in the vegetal plate just prior to gastrulation. In fish and frog, Caudal, Brachyury and Wnt (Wnt8 and Wnt11) are initially expressed around most or all of the blastopore lip while HNF-3 expression is dorsally localized. As gastrulation proceeds, the expression of these genes becomes more restricted and non-overlapping, with HNF-3 and Brachyury expression becoming localized to the notochord and Wnt8 expression retreating from the dorsal position and becoming exclusively ventral. Patterns of expression of HNF-3 and Brachyury consistent with this general description have been found in ascidians, amphioxus, chick and mouse. Required roles for some of these genes have been demonstrated by analysis of mutants: mouse HNF-3beta knockouts reveal requirements in the formation of the node, notochord and head process; fish no tail and mouse T mutants reveal a requirement for Brachyury in migration of mesoderm through the primitive streak and in formation of the notochord. There is thus a constellation of conserved genes -- cad (Cdx), fkh (HNF-3), wg (Wnt8 and Wnt11) and byn (Brachyury) -- whose overlapping expression patterns in the blastopore equivalent suggests function in a related process. The phenotypes of the available mutations in these genes suggest that the common function is to specify cell fate at the blastopore; in most cases, essential parts of this fate are internalization and forward migration, two of the cellular movements that occur during gastrulation (Wu, 1998b and references).

The striking conservation in expression (and likely in function) of cad suggests that the regulation of posterior terminal development in Drosophila by Caudal may represent a more ancient regulatory mechanism than the tor receptor and the two genes that it activates: tll and hkb. Of these three genes, a vertebrate homolog is known only for tll; the function of this vertebrate gene, Tlx, is related to that of Drosophila tll not in the posterior, but rather in the anterior, in the establishment of the brain. Thus the Torso receptor pathway and its activation of tll and hkb has probably been superimposed relatively recently (in evolutionary terms) upon a more ancient, Caudal-regulated network of gene activity controlling gastrulation and gut formation. The fact that the same four genes are expressed at both the blastopore equivalent of chordates and at the amnioproctodeal invagination of Drosophila suggests that these two highly dynamic domains are homologous. Given the regulatory hierarchy that is present in Drosophila, it is proposed that in embryos of the proximate ancestor to arthropods and chordates, the posterior was defined by a posterior-to-anterior gradient of Cad activity. Cad is thought to have then activated expression of downstream network of genes in control of invagination (gastrulation) and gut specification. Cad expression in the archenteron probably continued during evolution and played an essential developmental role, since this structure differentiated into the gut. Going beyond the bilaterian ancestor to chordates and arthropods, it is worth considering that this nexus of gene expression may have evolved even more basally in the metazoa. The foregoing, by homologizing the insect amnioproctodeal invagination with the echinoderm and vertebrate blastopore, does not fit with the classical definition of protostomes and deuterostomes. This view categorizes arthropods as protostomes, in which the mouth is derived from the primary invagination of gastrulation; chordates are categorized as deuterostomes, where the mouth arises from a secondary invagination. More recently, comparisons of gastrulation patterns in many different species, as well as construction of molecularly based cladograms, have called into question the utility of these classically defined groups. While there continues to be uncertainty in understanding of 'protostome' and 'deuterostome' phyla, the significant conclusion of the information presented here is that there may be a homology between the blastopore of vertebrates and the amnioproctodeal (posterior) invagination of insects (Wu, 1998b and references).

Drosophila development is coordinated by pulses of the steroid hormone 20-hydroxyecdysone (20E). During metamorphosis, the 20E-inducible Broad-Complex (BR-C) gene plays a key role in the genetic hierarchies that transduce the hormone signal, being required for the destruction of larval tissues and numerous aspects of adult development. Most of the known BR-C target genes, including the salivary gland secretion protein (Sgs) genes, are terminal differentiation genes that are thought to be directly regulated by BR-C-encoded transcription factors. Repression of Sgs expression is indirectly controlled by the BR-C through transcriptional down-regulation of fork head, a tissue-specific gene that plays a central role in salivary gland development and is required for Sgs expression. Integration of a tissue-specific regulatory gene into a 20E-controlled genetic hierarchy provides a mechanism for hormonal repression. Furthermore, these results suggest that the BR-C is placed at a different position within the 20E-controlled hierarchies than previously assumed, and that at least part of its pleiotropic functions are mediated by tissue-specific regulators (Renault, 2001).

DNaseI footprinting experiments with bacterially produced recombinant protein show that BR-C proteins can bind to the control regions of the BR-C-dependent genes Ddc, L71-6 and Sgs4. Consensus DNA-binding sequences have been defined in these studies for each of the four isoform-specific zinc-finger domains. However, the high variability of these A/T-rich sequences suggests that additional determinants are important for specific DNA recognition. One such determinant could be the BTB/POZ domain that is present in all BR-C isoforms. Nuclear proteins with this domain, like the GAGA or Mod(mdg4) proteins, are thought to act by remodeling chromatin structure. The BR-C dependence of a DNase I-hypersensitive site in the hsp23 gene suggests that BR-C proteins might act in a similar manner. The BTB/POZ domain of the GAGA factor mediates strong cooperative DNA binding to multiple sites but, like other BTB/POZ domains, inhibits binding to single sites. BTB/POZ domains are thus likely to play a critical role in targeting proteins to specific chromosomal loci. The data support this concept, as they show that DNA binding sites that are bound by BR-C proteins in vitro are not sufficient to bind BR-C in a chromosomal context. Such sites may, therefore, turn out to be irrelevant for the control of the gene with which they are connected. Mutation of in vitro BR-C binding sites in the promoter of the death gene reaper (rpr), for instance, has no effect on the expression of a reporter gene. It was therefore concluded that the BR-C might only indirectly regulate rpr transcription in the larval salivary glands, which foreshadows the BR-C-dependent destruction of this tissue during metamorphosis (Renault, 2001).

Mutations that eliminate BR-C binding to Sgs4 in vitro do lead to reduced reporter gene expression in vivo. However, these mutations also affect Fkh binding sites that are required for Sgs4 expression, and this effect may therefore be unrelated to the ability of these sites to be bound by BR-C. This interpretation is favored, not only because BR-C proteins cannot be detected at Sgs4 in situ, but also because BR-C proteins extracted from salivary gland nuclei or produced by in vitro translation are not able to recognize these sites in mobility shift DNA-binding assay. It is important to note that BR-C protein could not be detected at the Sgs4 locus 3C11, but also could not be detected at loci of other Sgs genes like 68C, which harbors Sgs3, Sgs7 and Sgs8. Interestingly, although expression of these three genes depends on BR-C function to a much larger extent than that of Sgs4, Fkh binding sites, but no BR-C binding sites, could be detected in the transcriptional control region of Sgs3. Fbp1 is another example of a gene that is clearly under BR-C control, although it does not seem to be bound directly by BR-C proteins. Fbp1 is activated in response to 20E in the larval fat body at about the time when the Sgs genes are activated in the salivary glands. Expression of an Fbp1 transgene depends on different BR-C isoforms, but attempts to demonstrate direct binding of these isoforms to elements within the transgene were not successful (Renault, 2001).

In this study, evidence has been presented not only for an indirect control of Sgs genes by the BR-C, but also for a possible mechanism that explains how this indirect control is achieved. Based on these results, a new model is proposed for the regulatory interactions between BR-C, Fkh and the 20E receptor EcR/Usp in Sgs gene regulation. The 2Bc function of the BR-C is required at puparium formation for downregulation of the Fkh transcription factor, which is involved in tissue-specific activation of the Sgs genes. Ectopic expression of fkh, which overrides repression of the endogenous gene, mimics the effects of 2Bc mutations on Sgs4 expression. The high level of Sgs4 mRNA that is maintained in the presence of ectopic Fkh clearly shows that the downregulation of fkh is necessary for Sgs repression. Together with downregulation of the Sgs activator SEBP3, it may even be sufficient for repression, leading to the surprising conclusion that direct binding of a repressor may not be required for this process. This study not only provides strong evidence for an indirect mode of action of the BR-C in Sgs gene repression but also suggests that activation might be indirect. BR-C proteins cannot be detected at Sgs loci when the genes are actively transcribed, however, they are present at 98D, the cytogenetic region that includes fkh and other genes. The presence of BR-C protein at 98D in mid- and late-third instar larvae is consistent with a model of direct repression of fkh by the BR-C. However, since fkh seems to be normally expressed in mid-third instar larvae of BR-C null mutants, BR-C activation of Sgs genes is likely to be mediated by a factor other than Fkh. Alternatively, BR-C protein might transiently bind to the Sgs genes at an earlier time when the polytene chromosomes are not yet accessible for immunolocalization studies (Renault, 2001).

A central player in this model is the Fkh protein, which plays an important role in development of the salivary glands as well as, later on, in tissue-specific gene control in this organ. Since fkh mutants show homeotic transformations, it has been suggested that Fkh is required for the establishment of tissue identity. Downregulation of fkh by the BR-C 2Bc function at puparium formation may therefore have a more global effect on the salivary glands than just repression of glue genes, altering the determined state of the glands. Downregulation of fkh could be the first step towards destruction of the larval salivary glands by programmed cell death that occurs about 14 hours later, at pupation. Interestingly, fkh mutations result in removal of the embryonic salivary gland placode by apoptosis, suggesting that Fkh might function as a survival factor that prevents the salivary glands from being eliminated by programmed cell death. Unfortunately, little is known about the mechanisms through which Fkh and other winged helix proteins exert their functions. Studies on the mouse serum albumin enhancer have implicated HNF3/Fkh proteins in chromatin organization. However, efforts to identify interacting proteins, including attempts to demonstrate a direct interaction between Fkh and BR-C proteins, have failed so far (Renault, 2001).

The study presented here integrates fkh in the 20E-controlled regulatory hierarchies that are active at the onset of metamorphosis and assigns BR-C to a new position within these hierarchies. Future studies will show if similar regulatory connections between the BR-C and tissue-specific factors exist in other responses to 20E signaling during development (Renault, 2001).

CrebA, a target of Forkhead, regulates secretory activity in the Drosophila salivary gland and epidermis

Understanding how organs acquire the capacity to perform their respective functions is important for both cell and developmental biology. This study examines the role of early-expressed transcription factors in activating genes crucial for secretory function in the Drosophila salivary gland. Expression of genes encoding proteins required for ER targeting and translocation, and proteins that mediate transport between the ER and Golgi is very high in the early salivary gland. This high level expression requires two early salivary gland transcription factors; CrebA is required throughout embryogenesis and Fkh is required only during late embryonic stages. Because Fkh is required to maintain late CrebA expression in the salivary gland, Fkh probably works through CrebA to affect secretory pathway gene expression. In support of these regulatory interactions, CrebA is shown to be important for elevated secretion in the salivary gland. Additionally, CrebA is required for the expression of the secretory pathway genes in the embryonic epidermis, where CrebA has been shown to be essential for cuticle development. Zygotic mutations in several individual secretory pathway genes result in larval cuticle phenotypes nearly identical to those of CrebA mutants. Thus, CrebA activity is linked to secretory function in multiple tissues (Abrams, 2005).

To test whether regulation of CrebA by Fkh is direct, a 2.8 kb fragment upstream of the CrebA transcription unit was identified that could drive salivary gland expression of a lacZ reporter gene (K. D. Henderson, PhD Thesis, Johns Hopkins University School of Medicine, 2000). Two smaller fragments from this enhancer resulted in salivary gland expression of the lacZ reporter gene either only after invagination had begun and later (CrebA-1100) or prior to invagination and later (CrebA-770). Since the later expression pattern fit the timeframe for Fkh-dependent salivary gland expression of CrebA, the CrebA-1100 construct, which contains six consensus Fkh-binding sites was further characterized. ß-Gal expression in the salivary glands with the CrebA-1100 construct was significantly reduced in fkh homozygotes although expression in the amnioserosa was unaffected, indicating that a Fkh-dependent salivary gland enhancer of CrebA had been identified. Flies were transformed with a CrebA-1100 reporter construct in which all six consensus Fkh-binding sites were mutated (CrebA-1100 fkh1-6 lacZ). Both lines carrying the mutated construct had significantly diminished salivary gland expression of ß-Gal, although ßGal expression in other tissues, including the amnioserosa and hemocytes was unaffected. It is conclude that Fkh functions directly to maintain late high-level expression of CrebA in the salivary gland (Abrams, 2005).

It was predicted that secretory pathway component encoding gene (SPCG) expression is controlled directly by CrebA. As a first step toward testing this possibility, lacZ reporter constructs for six of the 34 SPCGs were analyzed in this study. Each SPCG enhancer fragment spanned the 5' end of the most 5' cDNA for each gene and included ~1-2 kb of DNA further upstream. Transformant lines generated from five of the six constructs resulted in embryonic salivary gland expression. The srp68 lacZ enhancer construct did not express in the embryonic salivary gland. Salivary gland lacZ expression from the spase25 and sec61ß enhancer constructs wasdetected from early stage 12 and throughout embryogenesis. Salivary gland lacZ expression from the p24-1, zCop and SrpRa enhancer constructs was first detected during stage 13 and later. Expression of three of the constructs was examined in CrebA mutants: expression of ß-Gal from both the zCop-lacZ and sec61ß-lacZ constructs was completely absent in the salivary glands, whereas salivary gland ß-Gal expression from the spase25-lacZ construct was significantly reduced. Thus, CrebA-dependent salivary gland enhancers have been identified for at least three of the SPCGs (Abrams, 2005).

A search of the regions immediately upstream of the translation start sites of the SPCGs using MEME revealed a motif that is an excellent match for a mammalian Creb-binding site and that is present within 2 kb upstream of 32 of the 34 SPCGs. (The translation start site is used as a reference point since transcription start sites have not been mapped for any of the SPCGs.) Interestingly, of the two SPCGs that do not contain this consensus, one (sec62) is among the least affected by mutations in CrebA and the other, srp19, is one of only two genes examined that had ubiquitously high levels of expression in all tissues, including the salivary gland. Even more compelling is the finding that 13/32 have the site within 100 bp, another 7/32 have the site within 200 bp and another 5/32 have the site within 500 bp of the translation start site. All of the SPCG reporter gene constructs built contain this consensus site. Thus, not only is it predicted that the site is important for salivary gland expression of the SPCGs, but this could be the site through which CrebA acts to elevate transcription. The proximal location of these putative binding sites with respect to the start site of translation is consistent with the finding that mammalian Creb proteins bind close to the start of transcription. Also of relevance to these studies was the failure to discover consensus Fkh-binding sites conserved among the SPCGs through MEME analysis, further supporting an indirect role for Fkh in SPCG regulation (Abrams, 2005).

This study demonstrates that the Drosophila salivary gland prepares soon after specification to generate the machinery for its high-level secretory activity. The machinery includes components of the early secretory pathway crucial for targeting and translocating proteins into the ER and for vesicle transport between the ER and Golgi. Thus, one way the gland distinguishes itself from surrounding tissues is to greatly increase the relative transcriptional levels of the secretory pathway component genes (SPCGs). The leucine zipper transcription factor CrebA has a crucial and probably direct role in activating increased levels of SPCG expression not only in the salivary gland, but also in the epidermal cells, which secrete the larval cuticle. Fkh, the Drosophila FoxA/PHA-4 homolog, is required to maintain SPCG expression in the salivary gland, but acts indirectly, by maintaining CrebA expression. Hkb, the other early transcription factor examined in this study, is not required for elevated SPCG expression (Abrams, 2005).

Fkh has several roles in salivary gland development and function, including mediating the cell shape changes of invagination, maintaining secretory cell viability and transcriptional activation of the sgs genes in late larval life. In addition to these positive roles, FKH also represses the expression of salivary duct-specific genes in the secretory cells. In this paper, yet another role for fkh in the salivary gland was discovered: the maintenance of SPCG expression (Abrams, 2005).

fkh is a direct transcriptional target of Scr and Exd and the temporal expression of CrebA and the presence of consensus Scr/Exd-binding sites upstream of the gene suggest that CrebA may also be directly controlled by Scr and its co-factors. Late expression of CrebA, however, requires fkh, as does late expression of fkh itself. This study shows that Fkh functions directly to maintain CrebA expression in the salivary gland. Based on the requirement for CrebA for expression of the SPCGs at all embryonic stages and the requirement for fkh only at late stages, these data support a model in which CrebA controls the expression of the SPCGs and Fkh is required only because of its role in maintaining CrebA expression. A direct test of this model would be to express CrebA in the salivary glands of embryos missing fkh function; this experiment, unfortunately, could not be carried out because Fkh-independent drivers capable of providing high-level salivary gland-specific expression of CrebA are not yet available (Abrams, 2005).

A subset of the SPCGs that encode proteins required for retrograde vesicle transport from the Golgi to the ER are still expressed at low levels in CrebA mutants. However, in late but not early fkh mutants, expression of these genes is not above levels in surrounding tissues. It is proposed that the residual expression of the genes observed in the CrebA mutants would be controlled through other early transcription factor genes that, like CrebA, would require Scr and its co-factors for their initial expression and would require Fkh for maintaining late expression. Taken together, these studies suggest that regulation of salivary gland genes does not fit the simple paradigm suggested by studies of the C. elegans pharynx. The genes that specify the salivary gland (Scr/Exd/Hth) are distinct from the genes that activate and maintain gene expression in the organ. Moreover, no single gene takes over for the organ-specifying genes as even Fkh, the homolog of C. elegans PHA-4, is not required for expression of every salivary gland gene. In cases where Fkh is required, it is often indirect, such as with the SPCGs. Fkh does appear to have direct roles, however, much later in development, as demonstrated by regulation studies involving the sgs glue genes. Thus, the involvement of Fkh in salivary gland development and function is complicated and more consistent with the complexity of gene regulation seen in the liver than that suggested for the C. elegans pharynx. The existence of a single 'organ-specifying gene' may be more the exception than the rule (Abrams, 2005).


fork head: Biological Overview | Evolutionary Homologs | Targets of Activity | Developmental Biology | Effects of Mutation | References

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