Sex combs reduced

Targets of Activity

Dfd is repressed, both transcriptionally and at the phenotypic level, by Scr, Antp, Ubx and Abd-B, if their products are supplied in sufficient quantity (Gonzalez-Reyes, 1992).

Expression of Creb-A in salivary glands depends on Sex combs reduced, the master regulator of salivary gland fate, since Scr mutants do not express CrebA in salivary glands and embryos expressing Scr in new places also express CrebA in new places. Activation is blocked by the trunk gene, teashirt and the posterior homeotic gene Abdominal-B. As with two other salivary gland genes, forkhead and trachealess, activation of CrebA in the salivary gland by Scr is blocked by ectodermal dpp (Andrew, 1997).

Based on its pattern of expression, eyegone is thought to play a role in salivary gland organogenesis. Salivary gland primordium (SGP) development responds to positional information. On the anteroposterior axis, Sex combs reduced (Scr) specifies parasegment 2. In Scr minus embryos, no salivary glands are formed and eyg expression is lost, except for a small patch of cells present at the PS1/PS2 border. In a teashirt minus mutation, Scr is expanded to both PS2 and PS3 and results in enlarged SGPs. The SGP expression of eyg is duplicated in PS3, although its appearance and fading are delayed slightly. Along the dorsoventral axis, the SGP is restricted dorsally by decapentaplegic (dpp), and ventrally by the spitz group of genes. In dpp minus embryos, eyg expression expands dorsally to form a ring that is interrupted ventrally. In several spitz-group mutant embryos, such as single minded (sim), the SGPs from each side move ventrally, and eyg expression expands ventrally. Expression in the trunk is also disordered, which may be a secondary effect of the disruption of the mesoderm (Jun, 1998).

Salivary eyegone expression is regulated positively by Sex combs reduced and trachealess (trh) but is regulated negatively by forkhead. Scr, the homeotic gene responsible for patterning parasegment 2, is responsible for the activation of every salivary gene that has been tested. As expected, eyg is not expressed in the salivary primordium of Scr-mutant embryos (Jones, 1998).

Sulfation is a widespread modification for many biological active molecules. This modification has been implicated in an increasing number of biological processes such as the clotting of blood, the formation of connective tissue, the metabolism of drugs and toxins and the action of growth factors and hormones. Sulfation involves the transfer of a sulfuryl group from a sulfate donor, including protein, glycolipids, glycosaminoglycans and steroids. PAPS synthetase is a bifunctional enzyme containing both ATP sulfurylase and APS kinase activities required for the biosynthesis of PAPS, the sulfate donor in sulfation reactions. The PAPS synthetase is the first gene implicated in the sulfation pathway to be described in Drosophila; its specificity of expression in embryos is described. DDmPAPSS is a novel salivary gland marker. Expression in the salivary glands is regulated by Sex combs reduced. At the end of embryogenesis, expression of DmPAPSS is also observed at the entry and exit of the gut and the posterior spiracles. The pattern of expression of the DmPAPSS gene might reflect a major role for sulfation in mucus biosynthesis at the end of Drosophila embryogenesis (Jullien, 1997).

The gene proboscipedia (pb) is a member of the Antennapedia complex in Drosophila and is required for the proper specification of the adult mouthparts. In the embryo, pb expression serves no known function despite having an accumulation pattern in the mouthpart anlagen that is conserved across several insect orders. Several of the genes necessary to generate this embryonic pattern of expression have been identified. These genes can be roughly split into three categories based on their time of action during development. (1) Prior to the expression of pb, the gap genes are required to specify the domains where pb may be expressed. (2) The initial expression pattern of pb is controlled by the combined action of the genes Deformed (Dfd), Sex combs reduced (Scr), cap'n'collar (cnc), and teashirt (tsh). cnc and tsh act as as negative regulators of pb expression in the mandible and first thoracic segments, respectively. (3) Maintenance of this expression pattern later in development is dependent on the action of a subset of the Polycomb group genes. These interactions are mediated in part through a 500-bp regulatory element in the second intron of pb. Dfd protein binds in vitro to sequences found in this fragment. This is the first clear demonstration of autonomous positive cross-regulation of one Hox gene by another in Drosophila and the binding of Dfd to a cis-acting regulatory element indicates that this control might be direct (Rusch, 2000).

The early phase reflects a requirement for gap gene function for normal expression of pb to occur during later stages. Specifically, btd, gt, and hb have been identified as being required for proper gnathal expression of pb. The function of the head gap gene btd has been shown to be required only during early stages of embryogenesis. The expression patterns of gt and hb are such that they are no longer expressed in the labial segment at the time when pb expression begins. This is taken as a strong indication that the gap genes influence pb indirectly. Consistent with this hypothesis, no gt or hb binding sites could be detected in the regulatory elements of the pb reporter. In the case of hb, the role that various trans-acting factors might play in mediating loss of pb expression in the labial segment was investigated. Expression of Scr, the positive regulator of pb in the labial segment, is not eliminated. Further, repression of pb is not attributable to expansion of tsh expression. One possibility is that another negative regulator is being expressed such that Scr can no longer activate pb. Given the negative regulatory interactions that occur between the gap genes, it is possible that one of the other gap genes might be misexpressed and downregulate pb. However, it may be misexpression of cnc or some other gene that has yet to be identified. Alternatively, the 'hit-and-run' hypothesis, proposed to explain the long-term repression of Ultrabithorax (Ubx) by hb, may describe how transient expression of the gap genes is required very early in development to permit later expression of pb. In this hypothesis, heritable changes in chromatin structure, mediated by the PcG genes, are invoked to explain how hb regulates Ubx long after hb expression has ceased. In the case of pb regulation, one or more of these gap genes may be required to alter chromatin structure in and around the pb locus, thereby allowing the various trans-acting factors access to the pb cis-acting regulatory elements (Rusch, 2000).

During the middle phase, the initial expression pattern of pb is set by a variety of trans-acting factors. Focus was placed on the identification of those factors that determine the ectodermal pattern of pb expression along the A/P axis of the embryo. The Hox genes Dfd and Scr act as positive regulators of pb and Dfd can bind to pb regulatory elements in vitro. It is thought likely that Scr also regulates pb directly based on the similarity with which mutations in Dfd and Scr affect expression of pb and the pb reporter. In addition to the Hox genes, the region-specific homeotics cnc and tsh have been identified as negative regulators of pb and serve to restrict pb expression to the gnathos. It is not clear whether these genes regulate pb directly, though in the case of tsh the sequence TGGAAAAGT has been identified in the 500-bp regulatory fragment used in the pb reporter; this sequence is very similar to the identified tsh binding site. While this regulatory paradigm does not completely describe the regulation of the endogenous gene, based on the presence of pb residual expression, it is sufficient to explain the behavior of the 500-bp pb reporter. This mechanism of regulation places pb downstream of the Hox genes and is the first instance in Drosophila where one Hox gene is positively and directly regulated by another, a distinction previously accorded only to vertebrate Hox genes. Studies by others have suggested that wg may be mediating the nonautonomous residual expression of pb that is uncovered by mutations in Dfd and/or Scr. With the exception that wg has the strongest effect on pb expression of the segment polarity genes tested, the results shed little light on the mechanism that underlies this phenomenon. However, non-cell autonomous signaling has been implicated to explain regulation of ectodermal pb function by mesodermal expression of Scr; perhaps the residual expression in the embryo is an example of this pathway. Further experiments, including identification of an enhancer that mediates this residual expression, are needed (Rusch, 2000).

Cross-regulation of Homeotic Complex (Hox) genes by ectopic Hox proteins during the embryonic development of Drosophila was examined using Gal4 directed transcriptional regulation. The expression patterns of the endogenous Hox genes were analyzed to identify cross-regulation while ectopic expression patterns and timing were altered using different Gal4 drivers. Evidence is provided for tissue specific interactions between various Hox genes and the induction of endodermal labial (lab) by ectopically expressed Ultrabithorax outside the visceral mesoderm (VMS). Similarly, activation and repression of Hox genes in the VMS from outside tissues seems to be mediated by decapentaplegic gene activation. Additionally, it has been found that proboscipedia (pb) is activated in the epidermis by ectopically driven Sex combs reduced (Scr) and Deformed (Dfd); however, mesodermal pb expression is repressed by ectopic Scr in this tissue. Mutant analyses demonstrate that Scr and Dfd regulate pb in their normal domains of expression during embryogenesis. Ectopic Ultrabithorax and Abdominal-A repress only lab and Scr in the central nervous system (CNS) in a timing dependent manner; otherwise, overlapping expression in the CNS in tolerated. A summary of Hox gene cross-regulation by ectopically driven Hox proteins is tabulated for embryogenesis (Miller, 2001a).

The pb gene is normally expressed during embryogenesis but mutants have no apparent embryonic phenotype. However, ectopic Pb protein in the embryo does produce homeotic transformations in embryos. These observations suggest that the regulation of pb expression during embryogenesis may be important for proper development. Both Scr and Dfd are necessary for establishing the proper expression patterns of pb during embryogenesis. Although ectopic Scr and Dfd function equivalently to activate pb in the antennal segment epidermis, they have an opposite effect on native pb expression in the mandibular mesoderm (Mn). Ectopic Dfd accumulation has no significant effect on pb expression in the Mn, which is part of the Dfd expression domain. However, ectopic expression of Scr by the prd and 69B drivers represses pb expression in the Mn, demonstrating the opposite tissue specific regulation of pb by Scr (Miller, 2001a).

Genetic analyses demonstrate native regulatory interactions between pb, Dfd and Scr during embryogenesis. The reduction of pb expression in Dfd and Scr mutant backgrounds shows that normal pb expression is dependent on these genes. Dfd is required in the mandibular mesoderm (Mn) and anterior maxillary (Mx) segments (Miller, 2001a).

Similarly, Scr is necessary in the posterior Mx and Labial (Lb) segments. The functional significance of these regulatory interactions is debatable due to the lack of mutant embryonic phenotypes in pb nulls; however, the evolutionary implications are perhaps more interesting. Positive cross-regulation between Hox genes has not been previously demonstrated in Drosophila except through signal transduction. Nevertheless, vertebrate enhancers that are responsible for direct positive cross-regulation exhibit similar activity when tested in Drosophila suggesting that these differences are probably due to evolutionary changes at cis-regulatory elements. Since there is no clear mutant pb embryonic phenotype, these cis-regulatory elements apparently direct positive Hox regulatory interactions between Scr, Dfd and pb may be atavistic and non-functional in derived insects such as Drosophila. However, these cis-regulatory elements may also be necessary for proper Hox gene expression later in development (Miller, 2001a).

Homeosis and Homeotic Complex (Hox) regulatory hierarchies have been evaluated in the somatic and visceral mesoderm. Both Hox control of signal transduction and cell autonomous regulation are critical for establishing normal Hox expression patterns and the specification of segmental identity and morphology. Novel regulatory interactions have been identified associated with the segmental register shift in Hox expression domains between the epidermis/somatic mesoderm and visceral mesoderm. A proposed mechanism for the gap between the expression domains of Sex combs reduced (Scr) and Antennapedia (Antp) in the visceral mesoderm is provided. Previously, Hox gene interactions have been shown to occur on multiple levels: direct cross-regulation, competition for binding sites at downstream targets and through indirect feedback involving signal transduction. Extrinsic specification of cell fate by signaling can be overridden by Hox protein expression in mesodermal cells and the term autonomic dominance is proposed for this phenomenon. The endoderm was used to monitor target gene regulation by the Hox proteins (specifically wg, dpp and lab) through signal transduction (Miller, 2001b).

The redundancy of ectopic Scr and Dfd in transforming the mesothoracic muscle pattern to resemble the prothorax indicates that these proteins may regulate similar target genes in this developmental pathway. This may be a reflection of their strong homologies resulting in similar binding specificities. Transformations caused by ectopic Scr, Pb and Dfd in the thorax may also be due, in part, to their ability to compete with the Teashirt protein for targets. Additionally, the strong Dfd autoregulatory mechanism may be responsible for its more potent transformation of the thorax (Miller, 2001b).

The co-linear relationship to homeotic penetrance in the thoracic sm by ectopic Hox expression may be related to their relative homologies. For example, the frequency of transformation by these ectopic Hox proteins is representative of their chromosomal organization; namely (Lab<Pb<Dfd<Scr<Antp<Ubx). This ordering is similar to their respective homologies; this ordering is also a reflection of their dependence on Exd as a co-factor. Interestingly, this same order of penetrance is represented in reverse, when these Hox proteins are used to rescue lab mutant phenotypes in the embryonic CNS (i.e. Lab>Pb>Scr>Antp>Ubx>abdA>Abd-B). In summary, segmental identity was followed in ectopic Hox protein expression experiments by monitoring ventral muscle development. It was found that nearly all the homeotic proteins are capable of transforming the T2 segment in a pattern that is not predicted by previously described hierarchies such as posterior dominance and phenotypic suppression. Since the T2 segment musculature is at least in part inductively regulated by Hox gene expression in ectodermal tissue, 'autonomic dominance' is proposed as an additional component of the Hox regulatory hierarchy to explain this phenomena; namely, the ability of Hox encoded proteins to cell autonomously override an exogenous signal. A better understanding of signal transduction between germ layers is needed in order to determine the mechanism of autonomic dominance (Miller, 2001b).

During an investigation of Hox cross-regulation in the midgut visceral mesoderm it was demonstrated that both Antp and Ubx are responsible for the proper maintenance of the posterior boundary of Scr expression in ps4. It is proposed that Ubx represses Scr at this position extrinsically from nearby tissues. The segmental register shift in Hox expression domains found between the epidermis/somatic mesoderm/CNS and visceral mesoderm juxtaposes Ubx expression (ps5) to a position where it can influence Scr expression in the visceral mesoderm (ps4). Since Ubx activates dpp, which represses Scr in the visceral mesoderm, it seems reasonable to conclude that the interaction seen involves the action of dpp. Hox cross-regulation studies demonstrate that ectodermal Gal4 drivers producing ectopic Ubx repress Scr in the visceral mesoderm while stimulating dpp-LacZ expression. Ubx expression in the somatic mesoderm, which is between the epidermis and visceral mesoderm, may be the tissue that actually contributes the signaling influence demonstrated in this interaction. However, the responder only contains the visceral mesoderm regulatory elements and does not demonstrate that dpp gene activation is the signaling source in these outer tissues. Interestingly, ectopic expression of Abd-A outside the visceral mesoderm also demonstrates a posterior expansion of Scr expression in the visceral mesoderm, presumably since it represses Ubx there. Similarly, Antp repression of Scr in ps5 of the visceral mesoderm appears to be through signaling. Scr and Antp expression does not entirely fill the gap when Ubx expression is removed. Additionally, in Antp null mutants, Scr accumulation is seen in cells that normally express Antp in the presence of normal Ubx expression. By counting Scr expressing cells in the visceral mesoderm, it was found that Antp represses Scr in this tissue, contrary to previous reports. Interestingly, ectopic Antp in ectodermal tissues has no effect on ectodermal Scr expression. Thus, both Ubx and Antp contribute to define the Scr domain at its posterior visceral mesoderm boundary apparently through signal transduction (Miller, 2001b).

The segmental register shift in Hox gene expression domains between developing germ layers is well documented and appears to be a result of midgut morphogenesis. However, functional consequences of that shift have not been previously demonstrated. The regulation of Scr in the visceral mesoderm by Ubx suggests that the segmental register shift is functional and through signal transduction serves to specify the observed segmental register of Hox gene expression. In addition to Hox cross regulation and feedback loops, combined signal transduction pathways contribute to development and morphogenesis of the gastric caecae and midgut constrictions. However, a more thorough time dependent analysis of Hox expression and signaling domains in the embryo is necessary for a complete understanding of how these processes conspire to regulate these specific aspects of gut ontogeny (Miller, 2001b).

Scr inductively stimulates growth of the gastric caecae in the visceral mesoderm but seems to block it cell autonomously. Evidence was found for ectopic gastric caecae primordia in ps7 of the visceral mesoderm associated with ectopic Scr. Interestingly, the presumptive gastric caecae primordia at ps7 is generated in the same signaling environment as the native gastric caecae in ps3. During normal gastric caecae development at ps3, wg expression is observed at ps2 and dpp expression is observed at ps3. In ectopic Scr embryos polyps (ectopic gastric caecae?) are found at ps7. The signaling environment for this region of the midgut has dpp at ps7 and wg at ps8. Ectopic expression of Scr alters neither wg expression in ps8 nor dpp expression at ps7. Ectopic Hox expression (including Scr) suppresses complete gastric caecae development at ps3. This suggests that the signaling environment at ps3 may be responsible for development of the gastric caecae while Hox proteins block this morphogenesis cell autonomously (autonomic dominance). A complete map of signal transduction gene expression and Hox influences is critical for understanding this morphogenic process since other signaling agonists are likely also involved (Miller, 2001b).

Ectopic Hox protein expression in the mesoderm can induce lab, lab-LacZ and dpp-LacZ expression in the midgut. Typically, the anterior ectopic endodermal lab expression parallels the observed expression pattern in the visceral mesoderm. The lack of ectopic lab expression posterior to ps7 is probably due to the unaltered high levels of wg expression, that repress lab. Normal lab induction in the endoderm requires wg, dpp and vein; however, dFos dependent (wg independent) lab transcription can be accomplished with high Dpp levels. Typically, lab induction by dpp, wg and vein is coordinated by sgg (GSK3) which may be responsible for the ps4 gap in lab, lab-LacZ, and expression patterns seen in experiments involving ectopic Antp visceral mesoderm expression. Moreover, the lack of expanded lab-LacZ expression (unlike native lab) by ectopic Antp indicates the existence of presently undefined cis-regulatory elements at the lab locus that are not contained in genomic fragments of the identified enhancers. Antp protein may be regulating other influential signaling pathways while the corresponding cis-acting elements are not located in the genomic lab enhancers tested. Antp expression is functionally linked to another TGF-beta agonist 60A (glass bottom boat), as well as the Wnt pathway agonist DWnt4 (Miller, 2001b).

It is concluded that Hox gene interactions in the mesoderm are not always consistent with previous governing hierarchies: posterior dominance and phenotypic suppression. In the visceral mesoderm it is found that posterior dominance (Hox direct cross-regulation) seems legitimate but may be mediated by signal transduction. Phenotypic suppression is violated by morphological changes and target gene regulation. In the somatic mesoderm, more anterior Hox genes alter the identity of the ventral T2 segment, but this tissue is largely extrinsically regulated in the absence of direct Hox expression. In light of this result, the notion of autonomic dominance is proposed: Hox genes cell-autonomously dominate tissues regulated by signal transduction (Miller, 2001b).

The predominant paradigm depends on whether cells are extrinsically or autonomously specified by Hox gene expression. It is argued that non-typical homeosis caused by ectopically expressed Hox proteins (i.e. not following the dictates of posterior prevalence) can be taken to indicate inductively specified tissues and hence, confer autonomic dominance. Interestingly, ectopic expression of the Hox proteins also exhibit non-typical homeosis in the chordotonal organs of the PNS and the thoracic cuticle, suggesting that inductive specification and autonomic dominance may not be restricted to the mesoderm. However, Hox regulatory hierarchies seem to be of limited value in other tissues as well. The mechanism responsible for autonomic dominance has not been determined in this study; only the correlation between autonomous Hox dominance over inductively specified tissue. Signal transduction pathway cross-talk could be the predominant cause of autonomic dominance phenotypes (homeosis) due to Hox regulation of signaling agonists. These agonists could then contribute to the signaling environment to alter the tissue, since these morphogens are potent factors in differentiation. Meanwhile, Hox genes cross-regulate each other cell autonomously and in nearby tissues through signal transduction. This occurs in a tissue specific manner that likely depends on both the signaling environment, transcriptional co-factors, and perhaps any of an estimated 100 target genes for a given Hox protein. The signaling environment of any given tissue is dictated primarily by Hox genes, which is critical for maintenance of Hox expression domains and subsequent differentiation, determination and morphogenesis. This complex set of intrinsic and extrinsic Hox controls are likely responsible for the means by which Hox genes were genetically identified for their abilities to dominate segmental identities as homeotic selector genes (Miller, 2001b).

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 carried out 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).

In the 219-308 interval of a 1-kb promoter subfragment, bases 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).

Sex combs reduced and Ultrabithorax establish differential expression of the proneural gene achaete by modifying expression of the achaete prepattern regulator Delta in Drosophila legs

Many studies have shown that morphological diversity among homologous animal structures is generated by the homeotic (Hox) genes. However, the mechanisms through which Hox genes specify particular morphological features are not fully understood. This issue was addressed by investigating how diverse sensory organ patterns are formed among the legs of the Drosophila adult. The Drosophila adult has one pair of legs on each of its three thoracic segments (the T1-T3 segments). Although homologous, legs from different segments have distinct morphological features. Focus was placed is on the formation of diverse patterns of small mechanosensory bristles or microchaetae (mCs) among the legs. On T2 legs, the mCs are organized into a series of longitudinal rows (L-rows) precisely positioned along the leg circumference. The L-rows are observed on all three pairs of legs, but additional and novel pattern elements are found on T1 and T3 legs. For example, at specific positions on T1 and T3 legs, some mCs are organized into transverse rows (T-rows). The T-rows on T1 and T3 legs are established as a result of Hox gene modulation of the pathway for patterning the L-row mC bristles. The findings suggest that the Hox genes, Sex combs reduced (Scr) and Ultrabithorax (Ubx), establish differential expression of the proneural gene achaete (ac) by modifying expression of the ac prepattern regulator, Delta (Dl), in T1 and T3 legs, respectively. This study identifies Dl as a potential link between Hox genes and the sensory organ patterning hierarchy, providing insight into the connection between Hox gene function and the formation of specific morphological features (Shroff, 2007).

It is proposed that T-rows are formed on T1 and T3 legs in response to Scr or Ubx alteration of the L-row prepattern via repression of Dl expression. Dl is expressed in narrow longitudinal stripes that correspond to the L-row primordia. Dl-expressing cells in the L-row primordia signal to adjacent cells to activate N signaling and repress ac expression in the hairy-OFF interstripes and in one hairy-ON interstripe, between the L-row 1 and 8 proneural fields. It is suggested that in T1 and T3 legs, reduction of Dl expression in cells with high-levels of Scr or Ubx establishes a zone where there is no repressive influence on ac expression, resulting in expression of Ac in broad domains from which the T-row precursors will be selected. Cells in the center of T-row primordia are presumably out of range of the Dl signaling that takes place at the interface of Dl-expressing and Dl-non-expressing cells. The anterior and posterior boundaries of Ac expression in the T-row primordia of T1 prepupal legs are likely established by Dl/N signaling. In T3 legs, in contrast, it appears that Hairy rather than Dl/N signaling establishes the boundaries of ac on either side of the T-row primordia. Reduced Dl expression in the T-row primordia of T3 legs, however, is likely required to establish a broader domain of Ac expression than would be observed in the corresponding domain of T2 legs (Shroff, 2007).

A key feature of the model for mC patterning is that position-specific expression of ac expression in the mC proneural fields is established mainly by repression and that differential mC patterns are generated by altering expression or function of the repressive factors, Hairy and/or Dl. It is suggested that altered Dl expression is required in order to reduce N signaling, which allows proneural gene expression within the T-row primordia. An alternative hypothesis is that Dl function in during leg mC development is limited to selection of SOPs via lateral inhibition and that regulation of Dl by Scr/Ubx alters lateral inhibition within the T-row proneural fields. However, the hypothesis that Scr/Ubx regulation of Dl alters the proneural prepattern is supported by several observations. A prepattern function for Dl in mC patterning has been previously demonstrated in the notum. Similarly, it has been observed that in prepupal legs with reduced Dl function, proneural Ac expression expands along the leg circumference and is excluded only from Hairy-expressing cells. Furthermore, in prepupal legs, proneural Ac expression fills the center of large clones lacking Dl function. It is also observed that N signaling is activated only in narrow stripes on either side, but not within the T-row proneural fields. The genetic observations are substantiated by analysis of an enhancer that directs ac expression in both the L-row and T-row proneural fields. This enhancer consists of an activation element that directs uniform expression of ac along the leg circumference and two associated repression elements, one that is N-responsive and another that is Hairy-responsive. This is consistent with genetic studies suggesting that in the absence of repressive influences from Hairy and Dl, proneural ac expression would be uniformly along the leg circumference. Combined, these observations suggest that the mC patterning pathway is modified upstream of proneural gene expression by establishment of differential Dl expression in legs from different thoracic segments (Shroff, 2007).

The finding that Dl expression is down-regulated in the T-row primordia, does not necessarily imply that Dl expression is incompatible with mC formation. That this is not the case is suggested by the observation that Dl is expressed in the L-row primordia. Previous studies in a number of tissues have shown that high-level N-ligand expression renders cells non-responsive to N signaling. Hence, it appears that Dl/N signaling at the boundary of Dl-expressing and Dl-non-expressing cells, not Dl expression per se, is incompatible with mC development (Shroff, 2007).

Many studies have made clear the importance of establishing spatially defined proneural gene expression, largely via transcriptional regulation, for patterning of both the vertebrate and invertebrate nervous system. For example, it has been shown that ectopic proneural gene expression causes disruption of the sense organ pattern in adults. In the leg, compromised hairy function results in ectopic proneural ac expression and disorganization of the adult mC pattern, including formation of extranumerary mCs. However, other studies have implicated post-transcriptional regulation of proneural gene function in neural patterning. This was suggested by a study that showed that generalized and transient sc expression in a background devoid of ac and sc function results in an almost normal sense organ pattern in adult flies. Studies in the notum have provided an explanation for this observation by identification of the Extra macrochaetae (Emc) protein as a post-transcriptional regulator of proneural gene function. Emc, an HLH protein that lacks a basic DNA binding domain, binds proneural bHLH proteins, such as Ac, and inhibits their activity. In the notum, emc is expressed in a complex pattern that partially overlaps proneural gene expression, and it appears that SOPs are selected from cells with the lowest levels of Emc. This would suggest that on the notum, competence to acquire a neural fate depends on the balance of proneural protein to Emc levels. It is probable that similar mechanisms function in leg mC patterning as well, since largely normal sense organ patterns are found in legs ubiquitously expressing Sc. These observations indicate that sense organ patterning is a complex process that involves regulation of both proneural gene expression and function. Hence, it would be of interest to assess the relative contribution of post-transcriptional regulation of proneural gene function on leg mC patterning (Shroff, 2007).

It is proposed that T-row mCs are selected from domains of up-regulated Scr or Ubx expression and that one essential function for Scr and Ubx in T-row development is repression of Dl expression. This proposal is supported by several lines of evidence. The requirement of Scr and Ubx in T-row formation was suggested by prior reports that loss of Scr or Ubx function results in transformation of T1 or T3 legs, respectively, toward a T2 fate. This study shows that adult legs heterozygous for reduced function alleles of Scr (Scr^EdK6/Scr^EfW22) exhibit almost complete loss of T-rows in the adult. Moreover, ectopic expression of Scr or Ubx induces T-row formation on T2 legs, on which T-rows are never normally observed. The domains of elevated Scr and Ubx expression in T1 and T3 prepupal legs correspond to the respective positions of T-rows in adult T1 and T3 legs. Furthermore, comparison of Scr expression to that of an SOP marker, sca-Gal4, shows that T-row mCs are selected from groups of cells that express high-level Scr on T1 legs (Shroff, 2007).

Strong evidence is provided that Scr and Ubx repress Dl expression in the T-row primordia. First, a correlation is observed between up-regulated Scr and Ubx expression and domains of reduced Dl expression. Second, loss and gain of function studies indicate that Scr and Ubx negatively regulate Dl expression. In Scr^EdK6/Scr^EfW22, prepupal legs, Dl is expressed in two longitudinal stripes overlapping the region of high-level Scr expression, whereas in wild type legs Dl stripes flank but do not overlap this domain. In addition, Dl is ectopically expressed in either Scr or Ubx loss of function clones within the T-row primordia. Consistent with loss of function results, it was found that ectopic high-level expression of Scr or Ubx results in repression of Dl expression (Shroff, 2007).

Taken together, these observations suggest a function for Scr and Ubx in specification of a T-row fate. However, the finding that the formation of T-rows in response to ectopic Scr or Ubx is confined to ventro-lateral regions along the circumference implies that there may be other positional cues, in addition to elevated Scr or Ubx expression, that are required for T-row specification. Hence, it is plausible that these genes function combinatorially with other factors to induce T-row formation. Wg, for example, is a good candidate since it is expressed in ventro-lateral regions of the leg. In addition, ectopic Wg expression results in expansion of T-row bristles in T1 legs. It is also plausible that, in addition to or instead of T-row promoting factors in ventro-lateral leg regions, there are factors outside these domains that inhibit T-row formation (Shroff, 2007).

These studies have elucidated a general pathway for leg mC patterning in which an early event is establishment of position-specific expression of the prepattern genes hairy and Delta, presumably in response to the global regulators of limb patterning. The spatial regulation of hairy expression during leg development has been investigated ant it has been determined that hairy expression is controlled by modular enhancer elements that integrate patterning information provided by the signaling molecules known to pattern the leg along its circumference, Hedgehog (Hh), Decapentaplegic (Dpp) and Wingless (Wg). Periodic Dl expression is partially established by Hairy. However, it is likely that Dl expression in the mC proneural fields is also regulated, like hairy, by genes that pattern the leg along the circumference (circumferential patterning genes), such as hh, dpp and wg (Shroff, 2007).

Up-regulated expression of Scr and Ubx at specific positions along the circumference and P/D axis of T1 and T3 prepupal legs is key to generating the T-row pattern, raising the question of how Scr and Ubx expression in the T-row primordia is regulated. It is proposed that Scr and Ubx expression is controlled by the circumferential patterning genes and genes that pattern the leg along the P/D axis (P/D patterning genes). For example, Scr and/or Ubx expression might be regulated by Wg, which is known to specify ventral leg identity and patterns the ventral leg along the A/P axis in a concentration dependent manner. Along the P/D axis, a number of genes, such as Distal-less and dachshund, are expressed in defined and partially overlapping domains and might function to define the extent of up-regulated Scr and/or Ubx expression. This model would suggest that circumferential and P/D patterning information is integrated by Scr and Ubx, implying that these Hox genes link the global regulators of leg development to local acting genes, such as ac, that specify a neural fate (Shroff, 2007).

These studies indicate that Scr and Ubx function early in T-row development to repress Dl expression, which allows formation of the T-row proneural fields. It will be of interest to determine whether Dl is a direct target of these Hox genes, especially since few direct Hox-gene targets have been identified to date. However, it is probable that Scr and Ubx have other functions in T-row development. Establishment of ac expression in the T-row primordia is an early and essential step of T-row development, but, while ac specifies a neural fate, it does not specify sensory organ type. Hence, it is likely that Scr and Ubx function, in conjunction with other factors, to specify 'T-row-type' mCs. For example, since the T-row mCs are less pigmented than L-row mCs, a potential role of Scr and Ubx in T-row development is to regulate genes involved bristle pigmentation. A second potential function for these Hox genes is in controlling growth in the regions of the legs where T-rows are formed. In T1 legs, for example, the region between L-rows 7 and 8, in which the T-rows are found, is larger than the corresponding region on T2 legs, implying that there is additional growth in this domain. Inconsistent with this hypothesis, however, is the observation that posterior compartment clones lacking Ubx function in the T3 basitarsus did not have a significant effect on basitarsal width (Shroff, 2007).

Another potential role for Scr and Ubx in patterning T-row bristles is to implement a mechanism for selection and organization of T-row mCs into transverse rows. The mechanisms through which the L-row and T-row bristles are selected and organized within their respective proneural fields are likely to differ substantially. The regular spacing of L-row mCs suggests that the L-row SOPs send inhibitory signals in all directions to establish their proper spacing. This is also suggested by the observation that in hairy mutant legs, Ac is expressed in four broad domains, similar to the broad T-row proneural fields, and the supernumerary mCs that are formed on hairy mutant legs are well spaced along the leg circumference. This would suggest that the lateral inhibitory signals are sent along both the leg circumference and P/D axis. Unlike the L-row bristles, the T-rows mCs are positioned directly adjacent to one another in straight regularly spaced transverse rows. How the T-row precursors are selected from a broad field of ac-expressing cells and are arranged in tandem in straight rows is not understood. Previous studies have implicated N and EGFR signaling in formation of organized T-rows. Although the current studies indicate that N-signaling is down-regulated in the T-row primordia, it is conceivable that N functions at later stages of T-row development to pattern the T-row bristles. For example, N might function to set the register and spacing of the T-rows. Also of interest is how the T-rows are aligned in tandem within the rows. It has been suggested that homophilic adhesion between mC SOPs might be involved in organizing T-row bristles. Hence, it is plausible that Hox genes regulate expression of genes involved in adhesion, N signaling and/or EGFR signaling. Investigation of the mechanisms of T-row SOP selection and organization will provide an opportunity to uncover a potential connection between Hox gene function and morphogenesis (Shroff, 2007).

The proposed function for Scr and Ubx in T-row patterning bears some similarity to that described for Ubx in generation of diverse trichome patterns among the T2 legs of various Drosophila species. It has been shown that late pupal expression of Ubx in the T2 femur primordia correlates with lack of trichome formation in different Drosophila species, implying that Ubx inhibits formation of these structures. This role for Ubx, which has been termed a 'micromanaging role' is analogous to the function described here for Scr and Ubx in directing formation of T-rows in specific domains of T1 and T3 legs. Hence, micromanaging functions for Hox genes in generating complex and detailed morphologies may be a general phenomenon (Shroff, 2007).

Another common theme that has emerged from studies of the mechanisms through which Hox genes generate morphological diversity is that, in many cases, Hox genes function to suppress specific developmental pathways. For example, in legs, Antennapedia functions to repress expression of genes that promote antennal development, and Ubx functions to prevent development of specific macrochaete bristles on T3 legs. Furthermore, Ubx is known to act at several levels of the wing patterning hierarchy to suppress wing development in the haltere disc and as mentioned, Ubx functions late in leg development to suppress trichome formation. Less well understood, in contrast, is how and whether Hox genes act positively to direct the formation of morphological novelties among homologous structures, e.g., the T-row bristles on T1 and T3 legs. Further analysis of the mechanisms involved in T-row specification and morphogenesis is likely to provide insight into this question (Shroff, 2007).

Functional specificity of a Hox protein mediated by the recognition of minor groove structure

The recognition of specific DNA-binding sites by transcription factors is a critical yet poorly understood step in the control of gene expression. Members of the Hox family of transcription factors bind DNA by making nearly identical major groove contacts via the recognition helices of their homeodomains. In vivo specificity, however, often depends on extended and unstructured regions that link Hox homeodomains to a DNA-bound cofactor, Extradenticle (Exd). Using a combination of structure determination, computational analysis, and in vitro and in vivo assays, this study showed that Hox proteins recognize specific Hox-Exd binding sites via residues located in these extended regions that insert into the minor groove but only when presented with the correct DNA sequence. These results suggest that these residues, which are conserved in a paralog-specific manner, confer specificity by recognizing a sequence-dependent DNA structure instead of directly reading a specific DNA sequence (Joshi, 2007).

It is well established that homeodomain-DNA recognition utilizes hydrogen bonds formed between recognition helix side chains and base-specific moieties in the major groove. However, the residues making these contacts are identical in all Hox proteins. While some N-terminal arm residues have been seen in the minor groove, these interactions have not been sufficient to account for specificity differences among Hox proteins. In particular, although Arg5 is often observed in the minor groove, it is common to all homeodomains. Conversely, residues 1 to 4 are important for Hox specificity, but are often not observed in homeodomain-DNA structures. The structure reported in this study, of a complex formed between a Scr-Exd dimer and an in vivo paralog-specific binding site, fkh250 (see Overview of structures and sequences), reveals Hox-DNA contacts that provide new insights into the molecular basis of Hox specificity. Minor groove contacts from linker (His-12) and N-terminal arm (Arg3) residues are critical for Scr's specific in vitro and in vivo properties. Moreover, both residues insert into an unusually narrow region of the minor groove, which in turn creates a local dip in electrostatic potential through the phenomenon of electrostatic focusing (see Protein-DNA contacts). In contrast, in the fkh250^con* complex, the minor groove does not have these features, and, like many of the previous structures, there are no DNA contacts N-terminal to Arg5 (Joshi, 2007).

Based on these findings, it is suggested that there are two conceptually separable components to Hox-DNA binding. First, contacts between the DNA major groove and the recognition helix are sufficient to target Hox homeodomains to 'AT-rich' DNA sequences. Second, contacts made between the DNA minor groove and N-terminal arm/linker residues help to discriminate among AT-rich binding sites. Unlike recognition-helix residues in the major groove, the residues that insert into the minor groove recognize a specific DNA structure instead of forming base-specific hydrogen bonds. Below the implications are discussed of these findings for binding site recognition by Hox proteins as well as other DNA binding proteins (Joshi, 2007).

Consecutive ApA, TpT, or ApT base pair steps are known to result in a narrow minor groove due to negative propeller twisting that is stabilized by inter-base pair interactions in the major groove. In contrast, due to poor base stacking interactions, TpA steps tend to widen the minor groove and, for example, produce significant unwinding in the case of the TATA box. It is suggested that these sequence-dependent effects on DNA structure can account for the conformations of the two DNAs observed in this study. The fkh250^con* binding site is TGATTTATGG (TpA steps are underlined). ATTT is expected to have the observed narrow minor groove where Arg5 binds. The AT sequence 3' to the TpA step is too short to produce the pattern of inter-base pair contacts required for minor grove narrowing. Moreover, the minor groove that is widened by this TpA step remains wide, in part due to the 3' guanines which introduce amino groups into the minor groove. In contrast, the fkh250 binding site is AGATTAATCG. Here, the ATT and AAT sequences flanking the TpA step both have the pattern of inter-base pair contacts and propeller twisting required for minor groove narrowing. Consequently, two minor groove width minima are observed. The second minimum, where His-12/Arg3 insert, is reinforced by a positive roll introduced by a 3′-CpG step (Joshi, 2007).

The DNA conformations observed in the crystal structures were qualitatively reproduced by Monte Carlo simulations, and the importance of the TpA steps and 3' flanking G-C base pairs in affecting DNA structure were supported by the simulations of DNAs containing individual base pair differences. Interestingly, the standard deviations observed in these simulations are different for fkh250 and fkh250^con. This difference, which may reflect an inherent difference in flexibility, is also consistent with known sequence-dependent properties of DNA. The fkh250^con* sequence, which shows a smaller standard deviation, is expected to be rigid due to the presence of an 'A-tract', a sequence that consists of at least three consecutive ApA, ApT or TpT steps. In contrast, the larger deviations seen in the fkh250 simulations indicate greater conformational flexibility that can be attributed to the absence of an A-tract and the presence of a TpA step in the middle of the sequence (Joshi, 2007).

The N-terminal arm has been known for some time to play an important role in Hox specificity. Consistent with this idea, this study found Arg3 and Arg5 in the minor groove of fkh250. However, Arg5 is conserved in all homeodomains and Arg3 is present in many Hox proteins, raising the question of what makes Scr's N-terminal arm unique. One answer is that other N-terminal arm differences are important for Scr's properties. In agreement with this notion, it was found that changing RQR to RGR reduced the affinity for fkh250 by ∼six-fold, similar to the effect observed when Arg3 was mutated to Ala. These data suggest that, unlike RQR of Scr, it is energetically unfavorable for the RGR motifs of Antp, Ubx, and AbdA to assume the conformation of the RQR motif as seen in the fkh250 complex. This may be due in part to the increased entropic cost associated with fixing a Gly in any given conformation but also to the fact that its lack of a Cβ precludes the formation of the hydrophobic contact formed between Gln4 and Thr6 in the fkh250 complex (the distance between the Cδ of Gln4 and the Cγ of Thr6 is about 4.7 Å) (Joshi, 2007).

Taken together, these results suggest that the conformational preferences of Hox N-terminal arms are an important determinant of Hox specificity. However, there is clearly more to the story because, like Scr, Deformed (Dfd) also has an RQR motif in its N-terminal arm, but Dfd does not activate fkh250-lacZ in vivo. Thus, while the sequence of the N-terminal arm plays an important role, and allows Hox proteins to be categorized into RGR and RQR subgroups, other specificity-determining factors must also exist. Based on these results, and as discussed below, it is suggested that other important contributors are the paralog-specific residues neighboring the YPWM motif (Joshi, 2007).

His-12 is located in Scr's linker region, four residues away from its YPWM motif. Interestingly, not only is His-12 conserved in all Scr orthologs, residues on both sides of its YPWM motif are also well conserved (see Overview of structures and sequences). This pattern is not unique to Scr and its orthologs: residues in the vicinity of Hox YPWM motifs are generally conserved in a paralog-specific manner. In fact, the evolutionarily conserved sequences in the vicinity of YPWM are sufficient to distinguish between Hox paralogs, and can even discriminate between Scr and Deformed (Dfd), which, like Scr, also has a His in the same position relative to its YPWM motif. These observations suggest that paralog-specific residues near the YPWM motif, together with the N-terminal arm, may be considered as specificity-determining 'signature' residues. Analogous to the findings with Scr-fkh250, it is suggested that these paralog-defining residues in other Hox proteins are critical for the recognition of specific binding sites in vivo. These residues may, as shown here for His-12 and Arg3 of Scr, contact DNA. Alternatively, as shown here for Scr's Gln4, they may be important for specifying the correct conformation of the DNA-contacting residues. A general role for linker and N-terminal arm residues in Hox specificity is supported by the in vivo specificities of Hox protein chimeras (Joshi, 2007).

Although His-12 is conserved among all Scr orthologs, mutating it to an Ala had, for most readouts, only a partial effect on binding or in vivo activity. In contrast, the Arg3 to Ala mutation had a much larger effect, and the strongest effect was observed when both His-12 and Arg3 were mutated to Ala. Some simple considerations can in principle account for the data. First, it is suggested that the main contribution of His-12/Arg3 is to provide a positive charge and, consequently, a favorable electrostatic interaction between Scr and fkh250. Second, given the N-N distance of 2.9 Å in the His-Arg hydrogen bond, His-12 is likely neutral in the fkh250 complex, so that the net charge for both residues is +1. In the double mutant this charge is lost. The His-12 to Ala mutation leaves Arg3 intact and the net charge unchanged. The Arg3 to Ala mutation would likely result in the protonation of His-12 given the negative electrostatic environment in the minor groove, also leaving the net charge of the protein unchanged. While these considerations can explain why the effect of the double mutant is stronger than of either single mutant, they do not explain why Scr^Arg3A binds more weakly to fkh250 than ScrWT or Scr^His-12A. One possibility is that there is an unfavorable free energy cost of proton uptake to His-12 when it is bound to DNA since, as opposed to Arg3, the free His is only partially protonated (Joshi, 2007).

These results suggest that the interaction of Hox proteins with Exd/Pbx through the YPWM motif is important, not only because the presence of two homeodomains allows for a larger and more specific DNA sequence readout in the major groove, but also because it favors conformations of the linker and N-terminal arm residues such that they can recognize structural patterns in the minor groove. Indeed, it appears that these residues are unable to assume these conformations in the absence of Exd/Pbx. That these residues have not been observed in two other Hox-Exd/Pbx ternary complexes may suggest that their intrinsic flexibility is designed to inhibit binding to the wrong DNA site. That is, only when the protein sequence is compatible with the structure of the minor groove will the stabilizing interaction be strong enough to overcome the entropic loss associated with binding (Joshi, 2007).

Studies on homeodomain-DNA binary complexes also suggest that the N-terminal arm has a tendency to be disordered, unless presented with a DNA structure that provides sufficient stabilizing interactions to compete with conformational entropy. For example, residues 1 to 4 are not observed in the Antp and Engrailed X-ray complexes. In contrast, most of the N-terminal arm is structured in an Even-skipped-DNA complex where, notably, both Arg3 and Tyr4 insert into the minor groove. In that complex the minor groove is quite narrow where Arg3 inserts, consistent with the idea that a narrow groove is required to structure a region of the protein which is intrinsically disordered. In the HoxA9-Pbx-DNA ternary complex, the N terminal arm is also ordered but in that case, a very short linker severely limits the conformational freedom of the N-terminal arm (Joshi, 2007).

As seen in the crystal structure, binding of Scr-Exd to fkh250^con* involves residues that are present in all Hox proteins, thus providing an explanation for why this site is not specific for a particular paralog. As discussed above, the answer to the inverse question, of why fkh250 preferentially binds Scr-Exd, involves the insertion of His-12 and Arg3 into the minor groove, which is narrower than the equivalent region in fkh250^con*. That a narrow groove is an inherent feature of the fkh250 site suggests the more general idea that Hox proteins recognize their specific binding sites by reading a sequence-dependent DNA structure which, in turn, enhances the negative electrostatic potential and attracts the positively charged Arg/His pair. Thus, local differences in electrostatic potential provide an explanation for why sequence-dependent DNA conformations can attract basic amino acids. This shape-dependent DNA recognition mechanism is distinct from 'direct readout' mechanisms that involve specific hydrogen bond formation and hydrophobic contacts between amino acid side chains and bases. It is also distinct from 'indirect readout' where protein binding is influenced by the global shape of a DNA molecule or by sequence-dependent DNA bending and deformability (Joshi, 2007).

Scr's ability to recognize the shape of the minor groove via basic residues may provide an example of a more general class of protein-DNA recognition mechanisms. For example, an Arg of phage 434 repressor inserts into the minor groove of its operator and a His in the DNA binding domains of interferon regulatory factors (IRFs) inserts into a compressed minor groove. Moreover, the sequence (either FGR, RGR or RGGR) in the minor groove binding region of monomeric human estrogen related receptors, hERR, is an important specificity determinant for that family of transcription factors. The analogy between Hox and hERR2, a nuclear receptor, is particularly striking as the Zn finger domain of nuclear receptors makes major groove contacts while a normally extended peptide expands the binding site by making minor groove contacts. It will be interesting to determine if, as suggested in this study for Hox proteins, other families of DNA binding proteins use a common set of major groove contacts to recognize large sets of degenerate binding sites with individual family members distinguishing among these sites via more specific minor groove contacts. For Hox proteins, it is suggested that such a two-tiered recognition system gives them the flexibility to bind both shared and paralog-specific binding sites (Joshi, 2007).

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 Dfd^ScrSMδ23, 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).

Dfd^ScrSMδ23, 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 Dfd^ScrSM converted this protein from a repressor of fkh250-lacZ to an activator of fkh250-lacZ, while deletion of the same motifs from Dfd^WT did not change the regulatory output: The protein retained its ability to repress fkh250-lacZ. The only difference between Dfdγ23 (represses) and Dfd^ScrSMγ23 (activates) is the specificity module, and the only difference between Dfd^ScrSMγ23 (activates) and Dfd^ScrSM (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).

Homeotic effects

Ectopically expressed Teashirt protein induces an almost complete transformation of the labial to prothoracic segmental identity, when expressed during the first 8 hours of development. Positive autoregulation of the endogenous teashirt gene and the presence of SCR protein in the labium explain this homeosis. Trunk patterns partially replace those patterns in the maxillary and a more anterior head segment. teashirt function is overridden by homeotic gene function acting in the posterior trunk. Strong heat-shock regimes provoke novel defects: ectopic sense organs differentiate in posterior abdominal segments and trunk pattern elements differentiate in the ninth abdominal segment. It has been suggested that Teashirt and HOM-C proteins regulate common sets of downstream target genes (de Zulueta, 1994).

Zygotic expression of modifier of variegation Modulo depends on the activity of genes which pattern the embryo along dorsoventral and anteroposterior axes and specify diversified morphogenesis. dorsal and the mesoderm-specific genes twist and snail direct modulo expression in the presumptive mesoderm. The homeotic genes Sex combs reduced and Ultrabithorax positively regulate modulo in the ectoderm of parasegment 2 and abdominal mesoderm (Graba, 1994).

Protein Interactions

Antennapedia and Sex combs reduced determine cell fates in the epidermis and internal tissues of the posterior head and thorax. Genes encoding chimeric Antp/Scr proteins have been introduced into flies and their effects on morphology and target gene regulation observed. The N-terminus of the homeodomain is critical for determining the specific effects of these homeotic proteins in vivo, but other parts of the proteins have some influence as well. The N-terminal part of the homeodomain has been observed to contact the minor groove of the DNA. The different effects of Antennapedia and Sex combs reduced proteins in vivo may depend on differences in DNA binding, protein-protein interactions, or both (Zeng, 1993).

Sex combs reduced (SCR) is a Drosophila Hox protein that determines the identity of the labial and prothoracic segments. In search of factors that might associate with SCR to control its activity and/or specificity, a yeast two-hybrid screen was performed. A Drosophila homolog of the regulatory subunit (B'/PR61) of serine-threonine protein phosphatase 2A (dPP2A,B') specifically interacts with the SCR homeodomain. The N-terminal arm within the SCR homeodomain has been shown to be a target of phosphorylation/dephosphorylation by cAMP-dependent protein kinase A and protein phosphatase 2A, respectively. In vivo analyses reveal that mutant forms of SCR mimicking constitutively dephosphorylated or phosphorylated states of the homeodomain are active or inactive, respectively. Inactivity of the phosphorylated mimic form is attributable to impaired DNA binding. Specific ablation of dPP2A,B' gene activity by double-stranded RNA-mediated genetic interference results in embryos without salivary glands, an SCR null phenotype. These data demonstrate an essential role for Drosophila PP2A,B' in positively modulating SCR function (Berry, 2000).

PP2A exists as a multisubunit enzyme complex in a variety of organisms and cell types. The enzyme complex is composed of a catalytic and a scaffold subunit, which together form a core dimer that then associates with one of a number of regulatory subunits to constitute a trimeric enzyme complex. Regulatory subunits of PP2A are encoded by at least three unrelated gene families: B (PR55), B' (PR61) and B" (PR72). Each family consists of several members, which in addition can give rise to a number of splice variants, thereby greatly increasing the variety of distinct trimeric enzyme complexes. Several lines of evidence suggest that the regulatory subunits of PP2A may serve as specific adaptors that confer substrate specificity to the core domain of PP2A. Specific interaction of dPP2A,B' with the SCR homeodomain, as documented here, therefore reflects its potential of reversibly recruiting SCR into the PP2A complex (Berry, 2000).

The two phosphorylatable residues (T and S) within the N-terminal arm of the SCR homeodomain appear to be conserved, since at least one such site has been found in all SCR homologs from other species, except for PS12-B of Atlantic salmon. The homeodomain of PS12-B in fact seems more closely related to ANTP than to SCR. In vivo results suggest that in developing embryos, SCR is functionally inactive when the N-terminal arm of its homeodomain is phosphorylated and is active upon dephosphorylation. These results may have important implications for the functional specificity of homeotic proteins in general: since ANTP has a glutamine instead of threonine at position 6 (which is well conserved in all the SCR homologs), it is proposed that the differential modification of this residue plays an important role in determining the functional specificity of these two homeotic proteins (Berry, 2000).

The data from the functional knockout of dPP2A,B' by dsRNA interference prove unequivocally that expression of dPP2A,B' is essential for the functional activity of SCR. Genetic studies in Drosophila have shown that Ras-1 activity positively modulates the function of Hox proteins such as proboscipedia (PB) and SCR -- a finding that suggests that covalent modifications triggered by Ras-1-mediated signals might influence the activity of PB and SCR. The catalytic subunit of dPP2A has been identified as a component operating downstream of Ras-1. The observation that the functional activity of SCR is dependent upon the presence of dPP2A,B' seems to provide a missing link, suggesting that Ras-1 might influence the activity of SCR via dPP2A (Berry, 2000).

A model is proposed to describe the regulation of SCR activity: in a cell, where SCR function is not required continuously, the protein is locked in an inactive state by phosphorylation of residues 6 and/or 7 within the N-terminal arm of the homeodomain. The fact that, in older embryos, SCR is present but is no longer able to induce the expression of its target gene forkhead, may be a case in point. In response to positive signals, SCR-specific protein phosphatase (dPP2A) becomes activated, possibly through a signaling cascade involving Ras-1. In the absence of positive signals, or when negative signals, e.g. DPP and SP1 prevail, specific dPP2A activity is inhibited and, as a result, SCR can no longer be maintained in its dephosphorylated state. PKA or PKA-like enzymes will phosphorylate residues 6/7 of the SCR homeodomain, thus abrogating the ability of SCR to bind to its target genes. A delicate balance between the activities of SCR-specific PP2A and specific protein kinases would thus allow a cell to fine-tune SCR activity (Berry, 2000).

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, fkh250^CON (for 'consensus') is an artificial variant of fkh250 with two base pair substitutions that enable fkh250^CON-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, fkh250^CON 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 fkh250^CON 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 fkh250^CON-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 fkh250^CON-lacZ by AbdA. Note that both fkh250 and fkh250^CON-lacZ require direct binding by the Hox cofactor Exd. The primary distinction between these two readouts is that AbdA-Exd binds well to fkh250^CON 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 fkh250^CON, 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 fkh250^CON were measured in vitro. AbdA-Exd heterodimers bound more than twofold more tightly to fkh250^CON compared with Scr-Exd. Thus, at the same concentration, AbdA-Exd is more likely than Scr-Exd to be bound to fkh250^CON, consistent with the idea that competition depends on cofactor-dependent DNA binding (Noro, 2011).

Binding to fkh250^CON 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 (2W^Ala) was coexpressed with Scr in the phenotypic suppression assay, fkh250^CON-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 fkh250^CON-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 fkh250^CON 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 (Δ^200-220) exhibits a reduced ability to repress fkh250^CON-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 (2W^AlaΔ^200-220). Additional sequences in the C-tail of AbdA may account for the residual activity of variants lacking the UR motif (Δ^200-220 and 2W^AlaΔ^200-220). 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 fkh250^CON 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 fkh250^CON-lacZ (ΔC¹⁹⁷, Δ^200-220, and 2W^AlaΔ^200-220) were severely compromised in binding fkh250^CON 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 fkh250^CON 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 fkh250^CON-lacZ, AbdA mutants that are compromised in their ability to cooperatively bind DNA with Exd (e.g., ΔC¹⁹⁷, Δ^200-220, and 2W^AlaΔ^200-220) 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).

Principles of microRNA-target recognition: Post-transcriptional regulation

MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression in plants and animals. Although their biological importance has become clear, how they recognize and regulate target genes remains less well understood. This study systematically evaluates the minimal requirements for functional miRNA-target duplexes in vivo and classes of target sites with different functional properties are distinguished. Target sites can be grouped into two broad categories. 5' dominant sites have sufficient complementarity to the miRNA 5' end to function with little or no support from pairing to the miRNA 3' end. Indeed, sites with 3' pairing below the random noise level are functional given a strong 5' end. In contrast, 3' compensatory sites have insufficient 5' pairing and require strong 3' pairing for function. Examples and genome-wide statistical support is presented to show that both classes of sites are used in biologically relevant genes. Evidence is provided that an average miRNA has approximately 100 target sites, indicating that miRNAs regulate a large fraction of protein-coding genes and that miRNA 3' ends are key determinants of target specificity within miRNA families (Brennecke, 2005).

The 3' UTR of the HOX gene Sex combs reduced (Scr) provides a good example of a 3' compensatory site. Scr contains a single site for miR-10 with a 5mer seed and a continuous 11-base-pair complementarity to the miRNA 3' end. The miR-10 transcript is encoded within the same HOX cluster downstream of Scr, a situation that resembles the relationship between miR-iab-5p and Ultrabithorax in flies and miR-196/HoxB8 in mice. The predicted pairing between miR-10 and Scr is perfectly conserved in all six drosophilid genomes, with the only sequence differences occurring in the unpaired loop region. The site is also conserved in the 3' UTR of the Scr genes in the mosquito, Anopheles gambiae, the flour beetle, Tribolium castaneum, and the silk moth, Bombyx mori. Conservation of such a high degree of 3' complementarity over hundreds of millions of years of evolution suggests that this is likely to be a functional miR-10 target site. Extensive 5' and 3' sequence conservation is also seen for other 3' compensatory sites, e.g., the two let-7 sites in lin-41 or the miR-2 sites in grim and sickle (Brennecke, 2005).

Dimer formation via the homeodomain is required for function and specificity of Sex combs reduced in Drosophila

Hox transcription factors specify body segments along the anteroposterior axis of the embryo. Despite conservation of the homeodomain (HD), different Hox paralogs instruct remarkably different developmental fates. This study unexpectedly found that the Drosophila Sex combs reduced (Scr) protein dimerizes in vivo via the homeodomain, whereas its closest relative, Antennapedia (Antp), does not. Dimerization requires the conserved residue 19 in the ELEKEF motif of the HD and is facilitated by DNA binding. To study Scr dimerization in vivo, a giant transcriptional puff was created in live salivary gland cells, consisting of a controllable multiple Scr-binding site of the fork head enhancer, and Scr dimer formation was visualized upon specific DNA binding. Scr dimerization is required not only for transcriptional activation of the fork head gene but also for Scr homeotic function in the fly (formation of ectopic salivary glands, posterior transformations in the embryo and antenna-to-tarsus transformations). Finally, attempts were made to attribute the differential behavior in dimer formation observed between Antp and Scr to diverse amino acid regions between the two proteins that account for dimerization in Scr versus non-dimerization in Antp. By constructing hybrid Antp proteins, it was found that the C terminus and linker region between the YPWM motif and the HD of Scr are independently sufficient to confer dimer formation in Antp, whereas the long N terminus of the protein and the HD are largely dispensable. These results indicate that Scr functions as a homodimer to increase its transcriptional specificity and suggest that the formation of HD homo- or heterodimers might underlie the functional distinction between very similar HD proteins in vivo (Papadopoulos, 2012).

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