Targets of Activity

A major challenge in interpreting genome sequences is understanding how the genome encodes the information that specifies when and where a gene will be expressed. The first step in this process is the identification of regions of the genome that contain regulatory information. In higher eukaryotes, this cis-regulatory information is organized into modular units [cis-regulatory modules (CRMs)] of a few hundred base pairs. A common feature of these cis-regulatory modules is the presence of multiple binding sites for multiple transcription factors. Transcription factor binding sites have a tendency to cluster; the extent to which they do can be used as the basis for the computational identification of cis-regulatory modules. By using published DNA binding specificity data for five transcription factors active in the early Drosophila embryo, genomic regions containing unusually high concentrations of predicted binding sites were identified for these factors. A significant fraction of these binding site clusters overlap known CRMs that are regulated by these factors. In addition, many of the remaining clusters are adjacent to genes expressed in a pattern characteristic of genes regulated by these factors. One of the newly identified clusters, mapping upstream of the gap gene giant (gt) was tested; it acts as an enhancer that recapitulates the posterior expression pattern of gt (Berman, 2002).

The transcription factors Bicoid (Bcd), Caudal (Cad), Hunchback (Hb), Krüppel (Kr), and Knirps (Kni) act at very early stages of Drosophila development to define the anterior-posterior axis of the embryo. Bcd and Cad are maternal activators broadly distributed in the anterior and posterior portions of the embryo, respectively. Hb, Kr, and Kni are zinc-finger gap proteins that act primarily as repressors in specific embryonic domains. Sequences of previously described binding sites were collected for these five factors present in the cis-regulatory regions of known target genes. The binding sequences for each factor were aligned by using the motif-assembly program, and the binding specificities of each factor were modeled with position weight matrices (PWMs). PWMs are a useful way to represent binding specificities and provide a statistical framework for searching for novel instances of the motif in genome sequences (Berman, 2002).

A freely available program PATSER was used to search the genome for sequences that match these PWMs, and a web-based visualization tool, CIS-ANALYST ( was devised to display the location of predicted binding sites along with genome annotations in selected genomic regions. PATSER assigns a score to each potential site that reflects the agreement between the site and the corresponding PWM. These scores approximate the free energy of binding between the factor and site, and CIS-ANALYST uses a user-defined cutoff parameter to eliminate predicted low-affinity sites (Berman, 2002).

Using CIS-ANALYST, the distribution of Bcd, Cad, Hb, Kr, and Kni binding sites were examined in a 1-Mb genomic region surrounding the well-characterized eve locus at a site_p value of 0.0003. At this relatively high-stringency value, most experimentally verified binding sites are retained; at more restrictive values, many of these sites would be lost (Berman, 2002).

To investigate whether binding site clustering could help to explain the specificity of these factors for eve, a simple notion of binding site clustering was incorporated into CIS-ANALYST, allowing searches for segments of a specified length containing a minimum number of predicted binding sites. When the 1-Mb region surrounding eve was searched for dense clusters of predicted high-affinity sites (at least 13 Bcd, Cad, Hb, Kr, or Kni sites in a 700-bp window), three discrete regions were identified. Strikingly, these three clusters are all adjacent to eve, and overlap the previously characterized stripe 2, stripe 3 + 7, and stripe 4 + 6 enhancers (Berman, 2002).

To generalize and quantify these promising results, a broader collection of 19 well-defined CRMs from 9 Drosophila genes known to be required for proper embryonic development was compiled. Each of these CRMs is sufficient to direct the expression of a distinct anterior-posterior pattern in early embryos; genetic evidence suggests that each CRM is regulated by at least one of the following: Bcd, Cad, Hb, Kr, and Kni. Mutation and in vitro DNA binding studies completed on a subset of the CRMs provide evidence for a direct regulatory relationship. The same clustering criteria that were successful for identifying CRMs in eve (700-bp regions with at least 13 predicted binding sites) identified clusters overlapping 14 of these 19 known CRMs (Berman, 2002).

A search of the entire genome for 700-bp windows containing at least 13 predicted binding sites identified 133 clusters in addition to the 19 described above, or ~1 per 700 kb of noncoding sequence. As expected, when more stringent clustering criteria are used, both the number of known CRMs recovered and the number of novel clusters identified decrease. The novel clusters identified with a density of at least 15 binding sites per 700 bp, a level at which half of the known CRMs are still recovered, were further examined. Binding site plots for the 22 novel clusters identified at this high stringency condition, and 6 additional novel clusters identified with an equally stringent search by using only Bcd, Hb, Kr, and Kni have been published as supporting information on the PNAS web site). Twenty-three of these 28 clusters fall in regions between genes, whereas the remaining 5 fall in introns. There are therefore 49 genes that either contain a novel cluster of binding sites or flank an intergenic region that does. The expression patterns of these 49 genes in early embryos were examined by whole-mount RNA in situ hybridization and DNA microarray hybridization. At least 10 of the 28 clusters were adjacent to a gene that showed localized anterior-posterior expression in the syncitial or cellular blastoderm stages, consistent with early regulation by maternal effect or gap transcription factors. Although the numbers are small, this is significantly more than the 1 or 2 expected if the positions of clusters had been chosen at random (Berman, 2002).

One of these clusters is located ~2 kb upstream of the gap gene giant (gt). During cellularization, gt is expressed in two broad domains, one in the anterior and one in the posterior portion of the embryo. The pattern of expression of the posterior expression domain is determined by the activities of Cad, Hb, and Kr. However, the cis-regulatory sequence controlling this posterior expression pattern has not been precisely identified. Whether this cluster of binding sites might be the gt posterior enhancer was evaluated. A 1.1-kb fragment containing this cluster was placed in a reporter construct containing the eve minimal promoter fused to a lacZ reporter gene. The expression pattern of this construct largely recapitulates the early expression pattern of the gt posterior expression domain. In the absence of Kr function, the anterior border of the gt posterior domain shifts anteriorly, indicating repression by Kr. The construct containing the gt posterior enhancer exhibits a similar shift in the absence of Kr (Berman, 2002).

How are morphogenetic gradients interpreted in terms of embryonic gene transcription patterns within a syncytium such as the Drosophila blastoderm? A hypothetical model (Kerszberg, 1994) postulates a morphogen which is itself a spatially distributed transcription factor M or which generates a distribution of such a morphogenic factor. This model also postulates an additional, zygotically transcribed "vernier" factor V. M and V form all possible dimers: MM, MV, and VV. These are differentially translocated to the nuclei and bind with various affinities to responsive elements in the V promoter, thereby contributing to activation/inactivation of V transcription. A hypothetical model is presented in which the different dimers serve to activate the vernier factor in various distributions through the embryo. Interpretations in terms of Drosophila genes bicoid and hunchback are proposed (Kerszberg, 1994).

Organization of developmental enhancers in the Drosophila embryo

Most cell-specific enhancers are thought to lack an inherent organization, with critical binding sites distributed in a more or less random fashion. However, there are examples of fixed arrangements of binding sites, such as helical phasing, that promote the formation of higher-order protein complexes on the enhancer DNA template. This study investigated the regulatory 'grammar' of nearly 100 characterized enhancers for developmental control genes active in the early Drosophila embryo. The conservation of grammar is examined in seven divergent Drosophila genomes. Linked binding sites are observed for particular combinations of binding motifs, including Bicoid-Bicoid, Hunchback-Hunchback, Bicoid-Dorsal, Bicoid-Caudal and Dorsal-Twist. Direct evidence is presented for the importance of Bicoid-Dorsal linkage in the integration of the anterior-posterior and dorsal-ventral patterning systems. Hunchback-Hunchback interactions help explain unresolved aspects of segmentation, including the differential regulation of the eve stripe 3 + 7 and stripe 4 + 6 enhancers. Evidence is presented that there is an under-representation of nucleosome positioning sequences in many enhancers, raising the possibility for a subtle higher-order structure extending across certain enhancers. It is concluded that grammar of gene control regions is pervasively used in the patterning of the Drosophila embryo (Papatsenko, 2009).

Nearly 100 characterized enhancers and ~30 associated binding motifs control the patterning of the early Drosophila embryo, probably the best understood developmental process. These enhancers and sequence-specific TFs regulate the expression of ~50 genes controlling AP and DV patterning, including segmentation and gastrulation. The known TFs controlling embryogenesis represent less than ~10% of all TFs in the Drosophila genome. Thus, this analysis of regulatory grammar was restricted to the ~100 AP and DV enhancers and their ~30 TF inputs (31) (Papatsenko, 2009).

The recent completion of whole-genome sequence assemblies for 12 divergent Drosophila species has created an unprecedented opportunity for analyzing enhancer evolution. In this study 96 selected enhancer sequences from D. melanogaster were mapped to all 12 Drosophila genomes, using the UCSC Browser. The resulting collection combined 1420 kb of genomic sequence data in 1127 sequences, representing 60 enhancers in 23 AP genes and 36 enhancers in 31 DV genes. The entire collection of sequences and binding motifs is available at the Berkeley on-line resource (Papatsenko, 2009).

Inspection of aligned enhancer sequences among all 12 Drosophila species revealed strong conservation within the D. melanogaster subgroup (D. melanogaster, D. simulans, D. seichellia, D. yakuba and D. erecta) and also within the D. obscura group (D. pseudoobscura and D. persimilis). In order to focus on evolutionary changes in these enhancers the seven most divergent Drosophilids were analyzed: D. melanogaster, D. ananassae, D. pseudoobscura, D. willistoni, D. mojavensis, D. virilis and D. grimshawi. The remaining five species contain conservation patterns that are similar to those present in D. melanogaster or D. pseudoobscura (Papatsenko, 2009).

Short-range TF-binding linkages (0-80 bp) were examined in the collection of 96 enhancers from seven species for homo- and heterotypic pairs of binding motifs. Binding sites for the 30 most reliable TF motifs (see the Berkeley online resource) were mapped in enhancers using position weight matrices with match probability cutoff values set to ~2E-04. Distance histograms were generated for distances smaller than 80 bp, measured between the putative centers of each pair of neighboring site matches. Periodic signals were identified in the distance histograms using Fourier analysis, and statistical significance was estimated by bootstrapping positions of site matches in each enhancer sequence (Papatsenko, 2009).

Fourier analysis has identified helical phasing (~11 bp spacing) for several different homotypic activator-activator motif pairs. Such periodic signals were found in the distributions of Bcd-binding sites. Weaker helical-phasing signals were also identified for Caudal (Cad) and Dl-binding sites. Periodic signals close to two DNA turns (~20-22 bp) were found for Twi, Hb and Kruppel. Such helical phasing raises the possibility of direct protein-protein interactions (Papatsenko, 2009).

A weaker, ~11.4-bp periodic signal was detected in the distribution of heterotypic activator-activator site pairs, including Dl-Twi and Bcd-Cad. In contrast, there is a significant reduction in helical phasing signatures for activator-repressor motif pairs, and in fact, an over-representation of site pairs with 'anti-helical' spacing (15.2 bp). A similar 15.2 bp anti-helical signal was detected in distributions of all possible pair-wise combinations of the 30 binding motifs examined in this study. Thus, it would appear that any two randomly chosen binding sites are more likely to occupy the opposite sides of the DNA duplex as compared with helical phasing. This observation raises the possibility that most TFs function either additively or antagonistically to one another and just a special subset of TFs function in a synergistic fashion as reflected by helical phasing of the associated binding sites (Papatsenko, 2009).

The preceding analysis considered 'short-range' organizational constraints, involving linked binding sites separated by <25-30 bp. The possibility of 'long-range' constraints were also considered. The 96 enhancers under study possess characteristic 'unit lengths' of ~500 bp to 1.5 kb (300 bp minimum). The minimal/maximal sizes of the functional enhancers and the 'optimal' site densities can be determined by the amount of encoded information (pattern complexity), mechanisms of TF-DNA recognition such as lateral diffusion, or structural chromatin features like nucleosome positioning (Papatsenko, 2009).

Differential distance histograms reveal an over-representation of short-range linkages (<50 bp), but a depletion in mid-range distances (100-500 bp). These observations raise the possibility that TFs are distributed in a non-uniform manner across the length of the enhancer. That is, there may be sub-clusters, or 'hotspots', of binding sites within a typical enhancer. Such hotspots are observed in the prototypic eve stripe 2 enhancer, whereby 8 of the 12 critical binding sites are observed within two ~50-bp fragments located at either end of the minimal 480 bp enhancer. Homotypic motifs display the greatest propensity for such sub-clustering. Homotypic clusters (38) usually contain 3-5-binding sites distributed over 50-100 bp. Heterotypic activator-activator motif pairs also demonstrate sub-clustering, but these clusters are smaller (<25-30 bp) and usually contain just a pair of heterotypic sites. Heterotypic activator-repressor pairs show moderate enrichment over a distance of 50-70 bp, which is in agreement with the well-documented phenomenon of 'short-range repression'. Depletion of mid-range spacing constraints (around ~200 bp) is especially striking in the case of heterotypic motif pairs. Thus, activator synergy is like short-range repression: it appears to depend on closely linked binding sites (Papatsenko, 2009).

A possible explanation for this depletion of mid-range spacing is the occurrence of positioned nucleosomes, which might separate functionally distinct regions within an enhancer, and also separate neighboring enhancers. To test this hypothesis, nucleosome formation potential was compared with the distributions of TF-binding motifs in enhancers using the 'Recon' program. Three of the four eve enhancers that were examined (eve 1+5, eve 2 and eve 4+6) display a clear negative correlation between potential nucleosome formation and the distribution of TF-binding sites. This observation is consistent with the depletion of nucleosomes near TF-binding sites in vertebrates. This anti-correlation is especially striking in the case of the bipartite eve stripe 1+5 enhancer, where two enhancer regions (stripe 1 and stripe 5) are separated by a 400 bp 'spacer' DNA (in positions 600-1000), which might promote positioning of two nucleosomes and associated linker sequences (Papatsenko, 2009).

To investigate nucleosome positioning further, nucleosome-forming potential was measured in two sets of sequences, previously identified based on clustering of Dl sites and tested in vivo for enhancer activity. One set of sequences functioned as bona fide enhancers and produced localized patterns of gene expression across the DV axis of early embryos. The other set produced no expression in transgenic embryos, despite the presence of the same quality Dl-binding site clusters. The nucleosome-forming potential of the enhancers (true positives) was lower than that of the non-functional sequences (false-positives). These observations raise the possibility that the false Dl-binding clusters fail to function due to the formation of inactive nucleosomal structures (Papatsenko, 2009).

All 465 possible pairwise motif combinations for the 30 relevant binding motifs were tested for conservation in divergent drosophilids. Only linked binding sites, separated by a distance with small variations (max. distance bin = five bases) were considered. In the case of motif pairs, statistical significance was evaluated by bootstrapping columns in the binding motif alignments, thus preserving patterns of conservation. Pairs of homotypic motifs strongly prevailed in this type of analysis (28% of total pairs versus 6.5% expected), suggesting that homotypic interactions are important and pervasive in embryonic patterning. The strongest linkages were found for Bcd, Cad and Hb homotypic pairs. Each of these pairs was shared by five to six different enhancers and conserved in four to seven species. Among the identified heterotypic motif pairs, the most interesting were Bcd-Dl, Bcd-Cad and Dl-Twi (Papatsenko, 2009).

To identify cases of binding site pairs organized in a more flexible fashion, significant motif combinations were extracted using large distance bins or large distance variations. Along with the previously identified motif pairs, this analysis revealed several additional combinations, mainly involving the 'TAG-team' sequence motif, which is recognized by Zelda, a ubiquitous zinc finger TF. Zelda participates in the activation of the early zygotic genome and regulates a wide range of critical patterning genes. Indeed, significant combinations were identified for the TAG motif and Bcd, Dl and Hb. However, all of these TAG-X combinations exhibit spacing variability in different Drosophilids (Papatsenko, 2009).

It is conceivable that these results represent an underestimate of significantly linked motif combinations since very conservative cutoff values were used for statistical evaluation. A database of shared and/or conserved motif pairs, including those below the selected significance cutoff P = 0.03 is available from the Berkeley online resource (Papatsenko, 2009).

Conserved Bcd-Dl-binding site pairs were identified in the enhancers of several AP- and DV-patterning genes, including sal (AP), brk and sog (DV). The sites were found at similar distances, in the same orientation and were conserved in all seven species. It was suggested that the Bcd sites in the brk enhancer might augment gene expression in anterior regions, but this possibility was not directly tested. In wild-type embryos, both brk and sog exhibit significantly broader patterns of gene expression in anterior regions. This expanded pattern is lost in bcd mutants (Papatsenko, 2009).

Highly conserved Hb tandem repeats were detected in the regulatory regions of pair-rule genes, in the gap gene Kruppel, and in the Notch-signaling gene nubbin. Most of the homotypic Hb-Hb site pairs fall into two major groups, separated by either 6-8 or 13-15 bases. Some of the pair-rule enhancers selectively conserve either the 'short' or 'long' arrangement. For example, the eve stripe 4 + 6 enhancer contains two short Hb elements, while the stripe 3 + 7 enhancer contains a single long element. The odd 3 + 6 enhancer contains both short and long elements with various degrees of conservation. The hairy stripe 2,6,7 enhancer contains a single short element. Among the known gap genes, the long and short Hb elements were widely present in the enhancers of Kruppel, and in the blastoderm enhancer of nubbin, but not in any of the known knirps enhancers. It is conceivable that the distinct Hb site arrangements are important for the differential regulation of pair-rule genes by the Hb gradient (Papatsenko, 2009).

In conclusion, the systematic analysis of TF-binding sites in AP and DV patterning enhancers suggests a much higher degree of grammar, or fixed arrangements of binding sites, than is commonly believed. Developmental enhancers are thought to be highly flexible, with randomly distributed binding sites sufficing for the integration of multiple TFs. The results suggest that a large number of enhancers contain conserved short-range arrangements of pairs of binding sites. For instance, virtually all of the enhancers that respond to intermediate and low levels of the Dl gradient contain conserved arrangements of Dl-binding sites along with recognition sequences for other critical DV determinants, such as Twist and Zelda. Cooperating pairs of Bcd sites are found in enhancers responding to low Bcd concentrations, such as Knirps. Finally, distinctive arrangements of Hb-binding sites might influence whether the associated target genes are activated or repressed by high or low levels of the Hb gradient (Papatsenko, 2009).

Hunchback regulation of gap genes

Cis-acting elements for the expression of buttonhead head stripe expression are contained in a 1 kb DNA fragment, located about 3 kb upstream of the promoter, The four maternal coordinate systems are necessary for correct btd head stripe expression, most likely by acting through the 1 kb cis-acting control region. Expression of the btd head stripe depends on bicoid. bcd-dependent activation also involves the activity of the morphogens of the posterior and dorsoventral systems, hunchback and dorsal, respectively, which act together to control the spatial limits of the expression domain. Finally, tailless, a torso dependent repressor of btd, takes part in the regulation of btd head stripe expression by enhancing activation at low levels of activity and repression at high levels of activity (Wimmer, 1995).

Drosophila pair-rule gene expression, in an array of seven evenly spaced stripes along the anterior-posterior axis of the blastoderm embryo, is controlled by distinct cis-acting stripe elements. In the anterior region, such elements mediate transcriptional activation in response to (1) the maternal concentration gradient of the anterior determinant Bicoid and (2) repression by spatially distinct activities of zygotic gap genes. In the posterior region, activation of hairy stripe 6 has been shown to depend on the activity of the gap gene knirps, suggesting that posterior stripe expression is exclusively controlled by zygotic regulators. The zygotic activation of hairy stripe 6 expression is preceded by activation in response to maternal caudal activity. Thus, transcriptional activation of posterior stripe expression is likely to be controlled by maternal and zygotic factors as has been observed for anterior stripes. To establish the potential of Cad and Kni to interact with the cis-acting DNA that mediates hairy stripe 6-like expression in the embryo, in vitro footprinting experiments were performed with the 532 bp hairy stripe 6-element DNA. Cad and Kni bind to thirty six in vitro binding sites, some of which overlap, throughout the element. The sequence of the Cad and Kni binding sites matches the consensus described for each of the two proteins. Most of the potential Cad and Kni binding sites are close to or overlapped by binding sites for Kruppel (eight sites), Hunchback (eight sites), and Tailless (five sites). Tests using fragments of the 532 bp enhancer and of another element, 284-HT, show that sequences mediating activation of reporter expression are not maintained within a minimal activation element but instead are dispersed throughout the enhancer (Hader, 1998).

The initial expression of Krüppel occurs in a precisely bounded central region of the Drosophila blastoderm embryo. The spatial limits of the Kr expression domain are controlled by the morphogenetic activities of bicoid and the anterior gap gene hunchback. A 730 bp Kr control element, (CD1) drives gene expression in the endogenous KR central domain. This cis-acting element, Kr730, is composed of BCD and HB responsive sequences. They map into regions of multiple HB and BCD binding sites. A 142 bp core fragment, containing one low affinity HB and five medium to strong BCD binding sites, drives gene expression in a KR-like location in the center of the embryo. Thus, this fragment represents a target for the redundant activator/repressor system provided by the anterior morphogens BCD and HB (Hoch, 1991).

High hunchback activity represses Krüppel in the anterior (Schulz, 1994).

A promoter region of 287 bp, 2 kb upstream of the knirps structural gene is sufficient to drive kni expression in both anterior and posterior stripes under the positive regulation of Bicoid and Caudal, and the negative regulation by Hunchback. Bicoid and Caudal binding elements overlap (Rivera-Pomar, 1995a).

Most of the thoracic and abdominal segments of Drosophila are specified early in embryogenesis by the overlapping activities of gap genes hb, Krüppel, knirps, and giant. The orderly expression of these genes depends on two maternal proteins: Bicoid, which activates hb transcription anteriorly, and Nanos, which blocks translation of hb transcripts posteriorly. The resulting gradient of HB protein dictates where the Krüppel, knirps, and giant genes are expressed by providing a series of concentration thresholds that regulate each gene independently. Thus, HB protein functions as a classical morphogen, triggering several distinct responses as a function of its graded distribution (Struhl, 1992).

The caudal gene is both repressed and activated by Hunchback. caudal is a maternally and zygotically expressed gene in Drosophila. The two phases of expression can functionally replace each other. The zygotic expression forms an abdominal and a posterior domain, although only the posterior domain has so far been studied with respect to its regulation and function. The abdominal cad domain is regulated by the Hunchback gradient through repression at high concentrations and activation at low concentrations of HB protein (Schulz, 1995)

The interaction of hunchback and caudal has ramifications for insect evolution. Primitive insects develop segmentation by heterchronic gene activation. Each posterior segment forms independently in a time dependent manner. nanos activity eliminates Hunchback from the posterior precellular blastoderm region of primitive insects, and caudal, otherwise repressed by Hunchback, functions there at a later stage. Caudal proceeds to drive the addition of new segments over time. Thus, a gradient of Caudal regulates the asynchronous segment development of primitive insects (Rivera-Pomar, 1995a).

Anterior patterning of the Drosophila embryo is specified by the localized expression of the gap genes, controlled by the gradient of the maternal morphogen Bicoid. hunchback can substitute for bcd in the thorax and abdomen. hb is required for bcd to execute all of its functions, including activation of head gap genes. Removal of both maternal and zygotic hb produces embryos with disrupted polarity that fail to express all known bcd target genes correctly. The Torso system (including huckebein and tailless) is also required for head gap genes. Proper expression of hb and the head gap genes requires synergistic activation by hb and bcd. It is the combined activity of bcd and hb, not just bcd alone, that forms the morphogenetic gradient specifying polarity along the embryonic axis and correctly patterning the embryo. bcd may be a new gene to Drosophila (in evolutionary terms), one that is gradually replacing some of the functions performed by maternal hb in other species (Simpson-Brose, 1994).

The closely linked POU domain genes pdm-1 and pdm-2 are first expressed early during cellularization in the presumptive abdomen in a broad domain that soon resolves into two stripes. This expression pattern is regulated by the same mechanisms that define gap gene expression domains. The borders of pdm-1 expression are set by the terminal system genes torso and tailless, and the gradient morphogen encoded by hunchback. The resolution into two stripes is controlled by the gap gene knirps. (Cockerill, 1993).

Given the apparent transient overlap between Hb and Pdm-1 expression in the CNS, and Hb's established role as a repressor of pdm expression in the cellular blastoderm, it is likely that Hb also silences pdm expression during early NB sublineage development. Pdm-1 immunolocalizations performed on hb- embryos confirm the hypothesis that Hb functions as a repressor during CNS development. Comparisons between hb- and wild-type Pdm-1 immunostaining patterns shows that in the absence of Hb, Pdm-1 is ectopically expressed in all CNS ganglia. Transverse sections of Pdm-1 immunostained hb- embryos also revealed that early NBs that normally do not maintain Pdm-1 expression after their ectoderm delaminations fail to terminate Pdm-1 expression. The presence of ectopic Pdm-1 is also found in many early GMCs and by stage 12, Pdm-1 positive cells occupy both dorsal and inner regions of the developing ventral cord neuromeres. The expanded dorsal-ventral zone of Pdm-1 expression in hb- ventral cords indicates that many early GMCs and their progeny, normally marked by Hb expression, now ectopically maintain Pdm-1. However, ectopic Pdm-1 expression is not detected throughout the developing ganglia. After stage 11 in hb- embryos there is no Pdm-1 expression in the ventral most regions of the ventral cord ganglia. Similarly, no ectopic Pdm-1 is detected along the outer/superficial surfaces of hb- cephalic lobes after stage 11. This suggests that other mechanism(s) are regulating pdm expression in late developing sublineages (Kambadur, 1998).

Localized gene expression patterns are critical for establishing body plans in all multicellular animals. In Drosophila, the gap gene hunchback is expressed in a dynamic pattern in anterior regions of the embryo. Hb protein is first detected as a shallow maternal gradient that prevents expression of posterior gap genes in anterior regions. HB mRNA is also expressed zygotically, first as a broad anterior domain controlled by the Bicoid morphogen, and then in a stripe at the position of parasegment 4 (PS4). The PS4-hb stripe changes the profile of the anterior Hb gradient by generating a localized peak of protein that persists until after the broad domain has started to decline. This peak is required specifically for the formation of the mesothoracic (T2) segment. At the molecular level, the PS4-hb stripe is critical for activation of the homeotic gene Antennapedia, but does not affect a gradient of Hb repressive activity formed by the combination of maternal and Bcd-dependent Hb. The repressive gradient is critical for establishing the positions of several target genes, including the gap genes Kruppel, knirps, and giant, and the homeotic gene Ultrabithorax. Different Hb concentrations are sufficient for repression of gt, kni, and Ubx, but a very high level of Hb, or a combinatorial mechanism, is required for repression of Kr. These results suggest that the individual phases of hb transcription, which overlap temporally and spatially, contribute specific patterning functions in early embryogenesis (Wu, 2001).

Primary zygotic expression of HB mRNA (P2-hb) covers much of the anterior half of wild-type embryos early in nuclear cleavage cycle 14. This pattern is soon transformed into the secondary pattern, which includes the PS4-hb stripe. This stripe appears just before midcycle 14, and persists until the onset of gastrulation. The distribution of Hb protein is similar to the mRNA profile, but the protein seems to degrade more slowly. Thus, in midcycle-14 embryos, the Hb pattern consists of a broad anterior domain, with a peak at the position of PS4. Later, when cellularization is complete, Hb in anterior regions degrades further, leaving a clear stripe at PS4 (Wu, 2001).

To specifically remove this stripe, a misexpression transgene (st2-kni) was used that directs barely detectable levels of the gap gene kni at the position of PS3. Ectopic kni expression completely abolishes the PS4-hb stripe, and the peak of Hb protein observed in wild-type embryos. By the end of cellularization, no protein is detectable at the PS4 position of st2-kni embryos. The relative levels of Hb were further quantified at midcycle 14 in wild-type and st2-kni embryos. The PS4 position was determined by simultaneous fluorescence in situ hybridization with an RNA probe directed against fushi tarazu, a pair-rule gene expressed in stripes that correspond to the even-numbered parasegments (ftz stripe 2 corresponds to PS4). In st2-kni embryos, nuclei at the center of ftz stripe 2 contain only about 50% of the Hb normally present at this position. Thus, in wild-type embryos, the PS4-hb stripe alters the profile of the anterior Hb gradient by creating a peak of protein concentration at midcycle 14, and also ensures the perdurance of high Hb levels at this position until the end of cellularization (Wu, 2001).

In contrast to previous hb rescue experiments, the st2-kni transgene removes the PS4-hb stripe without changing the normal maternal and P2-hb gradients. Embryos carrying this transgene were examined for morphological defects later in development. More than 75% of these embryos die by the end of embryogenesis and show deletions of the T2 denticle band, which is derived from cells in PS4. While approximately 50% show a complete T2 deletion in dorsal and ventral regions, the phenotype is more severe on the ventral side, with ~90% completely lacking the T2 ventral denticle band. The cause of the phenotypic difference between dorsal and ventral regions is not clear. These results indicate that high levels of Hb at the position of PS4 are critical for T2 development (Wu, 2001).

To investigate the role of PS4-hb in T2 development, the expression patterns of segmentation and homeotic genes was examined in st2-kni embryos, and those fully rescued by the st2DeltaK-hb-1 transgene (a transgenic line that directs high levels of hb in a wide stripe that overlaps the PS4 position). No changes in the expression patterns of the gap genes Kr, kni, or gt were detected in st2-kni embryos. This suggests that Hb-mediated repression of these genes is not dependent on the PS4-hb stripe. However, several genes normally expressed in PS4 were significantly altered. One such gene is ftz. In st2-kni embryos, activation of ftz stripe 2 is delayed, and reduced in intensity. To test whether this reduction affects ftz function, expression of the ftz target gene engrailed was examined. en stripe 4, which is normally activated by ftz stripe 2, is also significantly reduced in st2-kni embryos (Wu, 2001).

The observed reductions in ftz stripe 2 and en stripe 4 could lead to the T2 deletion caused by the removal of the PS4-hb stripe. Surprisingly, however, addition of the st2DeltaK-hb-1 transgene, which mediates complete rescue of morphological defects in st2-kni embryos, does not detectably alter the level of ftz stripe 2 or en stripe 4. The failure to rescue these stripes may be due to the ectopic Kni in this region. However, since these embryos are rescued to adulthood, the reduced ftz and en stripes must be capable of supporting the establishment of the segment (Wu, 2001).

Previous experiments have implicated Hb as a repressor of the gap genes Kr, kni, and gt, but the expression domains of these genes are not affected by the removal of the PS4-hb stripe. Another gene controlled by Hb-mediated repression is the homeotic gene Ultrabithorax, which is strongly expressed as a stripe at the position of PS6 in late-blastoderm embryos. Since the PS4-hb stripe is also expressed at this stage, whether it is required for setting the anterior Ubx expression border was tested. Like the gap gene targets (Kr, kni, and gt), Ubx expression is undisturbed in embryos specifically lacking the PS4-hb stripe. Thus, the peak of protein provided by this stripe is not required for repression of any of these four genes (Wu, 2001).

In wild-type embryos, the anterior borders of Kr, kni, gt, and Ubx are located at different positions along the anterior-posterior axis, suggesting that they respond to different thresholds of Hb concentration. The position of the gt border, which lies most posteriorly, is established by the maternal Hb gradient. There is very little change in the position of this border in zygotic hb mutants, suggesting that the maternal gradient is sufficient for gt repression. The anterior kni border, which lies six to eight nuclear diameters from nuclei that produce zygotic HB mRNA, is also initially established by the maternal gradient. In mutants lacking zygotic hb, this border is correctly positioned early in cycle 14, but there is an anterior expansion at midcycle 14, possibly due to degradation of the maternal gradient. Whether this expansion is sensitive to ectopic Hb driven by the st2DeltaK-hb construct was tested. These experiments show a dose-dependent repression of kni back to its original position. Since only one copy of the transgene causes a strong repression, kni is sensitive to very low levels of Hb. The homeotic gene Ubx, which is normally expressed about four nuclear diameters from the posterior edge of the hb domain, also expands anteriorly in zygotic hb mutants. Addition of the st2DeltaK-hb transgene causes a dose-dependent repression of this gene as well. In this case, two copies of the transgene are significantly more effective than one, suggesting that Ubx repression requires a higher level of Hb than kni (Wu, 2001).

Finally, the gap gene Kr, whose anterior border overlaps the hb expression domain in wild-type embryos, was tested. This gene expands anteriorly in zygotic hb mutants, but cannot be repressed even by two copies of the st2DeltaK-hb transgene. This suggests that a very high level of Hb, or a combinatorial mechanism, is required for repression of Kr (Wu, 2001).

To further dissect the functions of subregions of anterior zygotic hb expression, larval cuticles were tested from zygotic hb mutants that also contain the st2DeltaK-hb-1 transgene. Externally, hb mutant cuticles lack all three thoracic segments (T1-T3), and exhibit an enlarged first abdominal segment A1. Addition of the st2DeltaK-hb-1 transgene results in specific rescue of the prothorax (T1), and returns A1 to its normal size. Rescue with two copies of the transgene further increases the size of the thorax, and rescues the dorsal part of T3. T2, however, is never present in these embryos, suggesting that the Hb levels generated by the transgene alone are not sufficient to rescue this segment (Wu, 2001).

In presumptive cephalic regions, zygotic hb mutants lack elements of the labial segment, including the H-piece and labial sense organs, and other head structures are severely disorganized, probably due to a disruption of head involution. Introduction of the st2DeltaK-hb-1 transgene causes a very weak rescue of these defects, compared to the mostly wild-type head structures found in hb mutants rescued by P2-hb transgenes. Since the st2DeltaK-hb transgene is activated at a later stage than P2-hb, perhaps high levels of Hb present earlier in anterior regions are required for the correct formation of these structures. However, the expression patterns of the head gap genes orthodenticle, button-head, and empty-spiracles are not affected in zygotic hb mutants, raising the possibility that Hb may function indirectly by preventing the expression of repressors of head development. A candidate for such a repressor is Kr, which cannot be repressed by even two copies of the st2DeltaK-hb-1 transgene. Perhaps the expanded Kr domain causes the remaining head defects in hb mutants that are partially rescued by the st2DeltaK-hb transgene (Wu, 2001).

To test this, one copy of the Kr gene was genetically removed from these embryos. This manipulation causes a dramatic rescue of most head structures, and a few embryos (~10%) display anterior phenotypes that are indistinguishable from wild type. This suggests that the expansion of Kr into presumptive head regions prevents normal head development, and that an important function of P2-hb is to restrict Kr to more posterior regions. However, the larval heads of Kr/+;hb/hb embryos still exhibit many morphological defects, suggesting that Hb also patterns the head by mechanisms that are independent of its role in Kr repression (Wu, 2001).

Dynamical analysis of regulatory interactions in the gap gene system of Drosophila

Genetic studies have revealed that segment determination in Drosophila melanogaster is based on hierarchical regulatory interactions among maternal coordinate and zygotic segmentation genes. The gap gene system constitutes the most upstream zygotic layer of this regulatory hierarchy, responsible for the initial interpretation of positional information encoded by maternal gradients. A detailed analysis of regulatory interactions involved in gap gene regulation is presented based on gap gene circuits, which are mathematical gene network models used to infer regulatory interactions from quantitative gene expression data. The models reproduce gap gene expression at high accuracy and temporal resolution. Regulatory interactions found in gap gene circuits provide consistent and sufficient mechanisms for gap gene expression, which largely agree with mechanisms previously inferred from qualitative studies of mutant gene expression patterns. These models predict activation of Kr by Cad and clarify several other regulatory interactions. This analysis suggests a central role for repressive feedback loops between complementary gap genes. Repressive interactions among overlapping gap genes show anteroposterior asymmetry with posterior dominance. Finally, these models suggest a correlation between timing of gap domain boundary formation and regulatory contributions from the terminal maternal system (Jaeger, 2004b).

Although activating contributions from Bcd and Cad show some degree of localization, positioning of gap gene boundaries during cycle 14A is largely under the control of repressive gap-gap cross-regulatory interactions. Thereby, activation is a prerequisite for repressive boundary control, which counteracts broad activation of gap genes in a spatially specific manner. In addition, gap genes show a tendency toward autoactivation, which increasingly potentiates activation by Bcd and Cad during cycle 14A. Autoactivation is involved in maintenance of gap gene expression within given domains and sharpening of gap domain boundaries during cycle 14A (Jaeger, 2004b).

Regulatory loops of mutual repression create positive regulatory feedback between complementary gap genes, providing a straightforward mechanism for their mutually exclusive expression patterns. Such a mechanism of 'alternating cushions' of gap domains has been proposed previously. The results suggest that this mechanism is complemented by repression among overlapping gap genes. Overlap in expression patterns of two repressors imposes a limit on the strength of repressive interactions between them. Accordingly, repression between neighboring gap genes is generally weaker than that between complementary ones. Moreover, repression among overlapping gap genes is asymmetric, centered on the Kr domain. Posterior to this domain, only posterior neighbors contribute functional repressive inputs to gap gene expression, while anterior neighbors do not. This asymmetry is responsible for anterior shifts of posterior gap gene domains during cycle 14A (Jaeger, 2004b).

Repression by Tll mediates regulatory input to gap gene expression by the terminal maternal system. Tll provides the main repressive input to early regulation of the posterior boundary of posterior gt, and activation by Tll is required for posterior hb expression. Note that these two features form only during cycle 13 and early cycle 14A, while other gap domain boundaries are already present at the transcript level during cycles 10-12 and largely depend on the anterior and posterior maternal systems for their initial establishment. The delayed formation of posterior patterning features and their distinct mode of regulation are reminiscent of segment determination in primitive dipterans and intermediate germ-band insects, supporting a conserved dynamical mechanism across different insect taxa (Jaeger, 2004b).

The set of regulatory interactions presented here provides a consistent and sufficient dynamical mechanism for gap gene expression. In summary, this set of interactions consists of the following five basic regulatory mechanisms: (1) broad activation by Bcd and/or Cad, (2) autoactivation, (3) strong repressive feedback between mutually exclusive gap genes, (4) asymmetric repression between overlapping gap genes, and (5) feed-forward repression of posterior domain boundaries by the terminal gap gene tll. In the following subsections, evidence is discussed concerning specific regulatory interactions involved in each of these basic mechanisms in some detail (Jaeger, 2004b).

Activation by Bcd and Cad: Activation of gap gene expression by Bcd and Cad is supported by the following. Bcd binds to the regulatory regions of hb, Kr, and kni. The kni regulatory region also contains binding sites for Cad. The anterior domains of gt and hb are absent in embryos from bcd mothers. The posterior domain of gt is missing in embryos mutant for both maternal and zygotic cad, while the posterior domain of kni is absent in embryos mutant for maternal bcd plus maternal and zygotic cad. These results suggest partial redundancy of activation of kni by Bcd, consistent with evidence from zygotic cad embryos from bcd mothers, where maternally provided Cad is sufficient to activate kni (Jaeger, 2004b).

Kr expression expands anteriorly in embryos from bcd mothers, which is due to the absence of the anterior gt and hb domains. Bcd has been shown to activate expression of Kr reporter constructs. The fact that Kr is still expressed in embryos from bcd mutant mothers has been attributed to activation by general transcription factors or low levels of Hb. In contrast, the models predict that this activation is provided by Cad. Although Kr expression is normal in embryos overexpressing cad, repressive control of Kr boundaries could account for the lack of expansion of the Kr domain in such embryos (Jaeger, 2004b).

The activating effect of Cad on hb found in gap gene circuits is likely to be spurious. The anterior hb domain is absent in embryos from bcd mutant mothers, which show uniformly high levels of Cad. Moreover, the complete absence of the posterior hb domain in tll mutants suggests activation of posterior hb by Tll rather than by Cad. It is believed that this spurious activation of hb by Cad is due to the absence of hkb in gap gene circuits. The posterior hb domain fails to retract from the posterior pole in hkb mutants, suggesting a repressive role of Hkb in regulation of the posterior hb border. Consistent with this, the posterior boundary of the posterior hb domain never fully forms in any of the circuits. Moreover, Tll is constrained to a very small or no interaction with hb due to the absence of the posterior repressor Hkb, since activation of hb by Tll would lead to increasing hb expression extending to the posterior pole (Jaeger, 2004b).

Autoactivation:: A role for autoactivation in the late phase of hb regulation is supported by the fact that the posterior border of anterior hb is shifted anteriorly in a concentration-dependent manner in embryos with decreasing doses of zygotic Hb. Weakened and narrowed expression of Kr in mutants encoding a functionally defective Kr protein suggests Kr autoactivation. Similarly, a delay in the expression of gt in mutants encoding a defective Gt protein indicates gt autoactivation. However, the results suggest that gt autoactivation is not essential. It is generally weaker than autoactivation of other gap genes, and circuits lacking gt autoactivation show no specific defects in gt expression. Finally, in the case of kni, there is no experimental evidence for autoactivation, while some authors have even suggested kni autorepression. No such autorepression has been detected in any gap gene circuit (Jaeger, 2004b).

Repression between complementary gap genes: Mutual repression of gt and Kr is supported by the following. gt expression expands into the region of the central Kr domain in Kr embryos. In contrast, Kr expression is not altered in gt mutants before germ-band extension. However, Gt binds to the Kr regulatory region, and the central domain of Kr is absent in embryos overexpressing gt. Moreover, Kr expression extends further anterior in hb gt double mutants than in hb mutants alone. The above is consistent with this analysis, which shows no significant derepression of Kr in the absence of Gt even though repression of Kr by Gt is quite strong (Jaeger, 2004b).

Hb binds to the kni regulatory region, and the posterior kni domain expands anteriorly in hb mutants. Embryos overexpressing hb show no kni expression at all, and embryos misexpressing hb show spatially specific repression of kni expression.There is no clear posterior expansion of kni in hb mutants. This could be due to the relatively weak and late repressive contribution of Hb on the posterior kni boundary or due to partial redundancy with repression by Gt and Tll. The posterior hb domain expands anteriorly in kni mutants, but anterior hb expression is not altered in these embryos. Nevertheless, a role of Kni in positioning the anterior hb domain is suggested by the fact that misexpression of kni leads to spatially specific repression of both anterior and posterior hb domains. Moreover, only slight posterior expansion of anterior hb is observed in Kr mutants, while hb is completely derepressed between its anterior and posterior domains in Kr kni double mutants (Jaeger, 2004b).

Repression between overlapping gap genes: gt, kni, and Kr show repression by their immediate posterior neighbors hb, gt, and kni, respectively. Retraction of posterior Gt from the posterior pole during midcycle 14A fails to occur in hb mutants, and no gt expression is observed in embryos overexpressing hb. The posterior kni boundary is shifted posteriorly in gt mutant embryos, and kni expression is reduced in embryos overexpressing gt. Note that these effects are very subtle and were not reported in similar studies by different authors. A weak but functional interaction of Gt with kni is consistent with these results. This interaction was found to be essential even in a circuit where it was deemed below significance level. Finally, Kni has been shown to bind to the Kr regulatory region, and the central Kr domain expands posteriorly in kni mutants (Jaeger, 2004b).

In contrast, no effect of Kr on hb was detected. However, hb expression expands posteriorly in Kr mutants. This effect is likely to involve repression of hb by Kni. Kni levels are reduced in Kr embryos. hb is completely derepressed between its anterior and posterior domains in Kr kni double mutants, whereas anterior hb does not expand at all in kni mutants alone. Taken together these results suggests that there is direct repression of hb by Kr in the embryo, but it is at least partially redundant with repression of hb by Kni (Jaeger, 2004b).

Unlike repression by posterior neighbors, no or only weak repression was found of posterior kni, gt, and hb by their anterior neighbors Kr, kni, and gt, respectively. Most gap gene circuits show weak activation of hb by Gt. Graphical analysis failed to reveal any functional role for such activation. Moreover, no functional interaction was found between gt and Kni. Although relatively weak repression of kni by Kr was found in 6 out of 10 circuits, no specific patterning defects could be detected in the other 4. Consistent with the above, expression of posterior hb is normal in gt mutants, and both the anterior boundaries of posterior gt and kni are positioned correctly in kni and Kr mutant embryos, respectively (Jaeger, 2004b).

Note that activation of kni by Kr, which has been proposed to explain decreased expression levels of kni in Kr mutants, was never found. The results strongly support the view that this interaction is indirect through Gt, which is further corroborated by the fact that kni expression is completely restored in Kr gt double mutants compared to that in Kr mutants alone (Jaeger, 2004b).

A significant repressive effect of Hb on Kr was found. Consistent with this, Hb has been shown to bind to the Kr regulatory region, and the central Kr domain expands anteriorly in hb mutants. However, partial redundancy of this interaction is suggested by correct positioning and shape of the anterior Kr domain in a circuit that does not show repression of Kr by Hb (Jaeger, 2004b).

It has been proposed that Hb plays a dual role as both activator and repressor of Kr. In the framework of the gene circuit model, concentration-dependent switching of regulative action could be implemented by allowing genetic interconnection parameters to switch sign at certain regulator concentration thresholds. The current model explicitly does not include such a possibility. Nevertheless, circuits have been obtained that reproduce Kr expression faithfully, suggesting that a dual role of Hb is not required for proper Kr expression. Moreover, activation of Kr by Hb was ever observed in any of the circuits. Therefore, the results support a mechanism in which the activation of Kr by Hb is indirect through derepression of kni (Jaeger, 2004b).

Repression by Tll: Only a few earlier theoretical approaches have considered terminal gap genes. Gap gene circuits accurately reproduce tll expression. However, in gene circuits, tll is subject to regulation by other gap genes, which is inconsistent with experimental evidence. In contrast, the correct expression pattern of tll in gap gene circuits allows its effect on other gap genes to be studied in great detail. Strong repressive effects of Tll on Kr, kni, and gt have been found. Tll binding sites have been found in the regulatory regions of Kr and kni. In tll mutants, Kr expression is normal, whereas expression of kni expands posteriorly, and the posterior gt domain fails to retract from the posterior pole. No expression of Kr, kni, or gt can be detected in embryos overexpressing tll under a heat-shock promoter (Jaeger, 2004b).

Reverse engineering the gap gene network of Drosophila

A fundamental problem in functional genomics is to determine the structure and dynamics of genetic networks based on expression data. A new strategy is described for solving this problem and it is applied to recently published data on early Drosophila development. The method is orders of magnitude faster than current fitting methods and allows fitting of different types of rules for expressing regulatory relationships. Specifically, this approach is sused to fit models using a smooth nonlinear formalism for modeling gene regulation (gene circuits) as well as models using logical rules based on activation and repression thresholds for transcription factors. The technique also allows inference of regulatory relationships de novo or testing network structures suggested by the literature. A series of models is fitted to test several outstanding questions about gap gene regulation, including regulation of and by hunchback and the role of autoactivation. Based on the modeling results and validation against the experimental literature, a revised network structure is proposed for the gap gene system. Interestingly, some relationships in standard textbook models of gap gene regulation appear to be unnecessary for, or even inconsistent with, the details of gap gene expression during wild-type development (Perkins, 2006).

The regulatory structure of the Combined model is itself sufficient to reproduce all six gap gene domains using either the gene circuit or logical formalisms for production rate functions. Support is cited for the Combined model, and then consider the results of the individual models in light of several outstanding questions about gap gene regulation are discussed (Perkins, 2006).

The maternal proteins Bcd and Cad are largely responsible for activating the trunk gap genes, with Bcd being more important for the anterior domains and Cad more important for the posterior domains. Bcd is a primary activator of the anterior hb domain, the anterior gt domain, and the Kr domain. Cad activates posterior gt. The kni domain is present in bcd mutants and in cad mutants, but not in bcd;cad double mutants. This suggests redundant activation by the two maternal factors. Such redundant activation of kni is present in the Unc-GC model. For the other models, the optimization selected one or the other as activators, but not both. Tll is crucial for activating the posterior hb domain, while it represses Kr, kni, and gt, preventing their expression in the extreme posterior. All the regulatory relationships between the gap genes in the Combined model are repressive. The complementary gap gene pairs, hb-kni and Kr-gt are known to be strongly mutually repressive, as was found in nearly all the models. [Repression of hb by Kni is not part of the Rivera-Pomar and Jäckle (RPJ) regulatory relationships (Rivera-Pomar, 1996b), but the unconstrained gene circuit (Unc-GC) model and Unc-Logic model (that employs the regulatory structure discovered by the unconstrained gene circuit fit, except that Gt activation of hb and Kni activation of gt were removed) included the link.] The models also suggest that mutual repression between hb and Kr helps to set the boundary between those two domains. A chain of repressive relationships, hb-gt-kni-Kr, causes the shifts in the Kr, kni, and posterior gt domains. Autoactivation by hb is well-established, and there is also some evidence for autoactivation by Kr and gt (Perkins, 2006).

Does Hb have a dual regulatory effect on Kr? There is a long-running debate about whether or not low levels of Hb activate Kr. In hb mutants, the Kr domain expands anteriorly, suggesting that Hb represses Kr. However, Kr expression in these mutants is lower than in wild-type and expands posteriorly in embryos overexpressing Hb. Further, in embryos lacking Bcd and Hb, the Kr domain is absent, but can be restored in a dosage-dependent manner by reintroducing Hb. These observations suggest that Hb activates Kr. It has been suggested, therefore, that low levels of Hb activate Kr while high levels repress it. An alternative explanation, however, is that the apparently activating effects of Hb are indirect, via Hb's repression of kni and Kni's repression of Kr. Optimization of the Unc-GC model, which could have resulted in activation or repression of Kr by Hb, but not both, resulted in repression. The RPJ models allow for a dual effect, but activation by Hb was eliminated during optimization of the RPJ-Logic model. The RPJ-GC model retained functional activation and repression of Kr by Hb. However, Kr expression in this model is defective. Kr is not properly repressed in the anterior. Further, Kr is ectopically expressed in a small domain in the posterior of the embryo. Thus, the current models provide no support for activation of Kr by Hb. The only support found, which is crucial in all models except Unc-Logic and also consistent with the mutant and overexpression studies, is for repression of Kr by Hb (Perkins, 2006).

What represses hb between the anterior and posterior domains? Another point of disagreement in the literature is what prevents the expression of hb between its two domains. In the model of Rivera-Pomar and Jäckle (1996b), repression by Kr is the explanation. The RPJ models confirm that this mechanism is sufficient. Specifically, in these models Kr repression prevents hb expression just to the posterior of the anterior hb domain. Between the Kr and posterior hb domains, there is no explicit repression of hb. Rather, Hb is not produced simply because of a lack of activating factors. In contrast, the models of Jaeger (2004a and b) detected no effect of Kr and attributed repression solely to Kni. The Unc-GC and Unc-Logic models found repression by Kni, but in addition to repression by Kr, not instead of it. Kr is more responsible for repression near the anterior hb domain and Kni is more responsible for repression near the posterior hb domain. This is consistent with observations of expression in mutant embryos. Embryos mutant for Kr show slight expansion of the anterior hb domain, while kni embryos show expansion of the posterior hb domain. In Kr;kni double mutants, hb is completely derepressed between its two usual domains. This suggests, as seen in the Unc-GC and Unc-Logic models, that Kr and Kni are both repressors of hb, that their activity is redundant in the center of the trunk, and that Kr and Kni are the dominant repressors for setting the boundaries of the anterior and posterior domains, respectively. This interpretation was also favored by Jaeger (2004a and b), on the basis of the mutant data, even though Jaeger's models did not find repression by Kr (Perkins, 2006).

The posterior hb domain. In all of the current models, the posterior hb domain is activated by Tll and sustained by Tll and hb autoactivation. Rivera-Pomar (1996b) did not consider the posterior hb domain, and did not include activation by Tll in his model. That link was added to the RPJ network structure because otherwise it was not possible to capture the posterior hb domain. The model of Jaeger (2004a and b) captured the domain without Tll activation by substituting activation from cad. However, there is no confirming evidence for such an interaction. The absence of posterior hb in tll mutants and the inability of the models to explain posterior hb by other means, leads to the straightforward hypothesis that Tll activates posterior hb. Posterior hb is unique in that the domain begins to form later than the other five domains modeled. In the RPJ models, this happens simply because high levels of Tll are needed to activate hb -- levels that are reached only at about t = 30 min. The Unc-GC and Unc-Logic models also employ repression by Cad to slightly delay Hb production in the posterior. However, there is no confirming evidence for such repression, and it is omitted from the Combined model (Perkins, 2006).

Shifting of the Kr, kni, and posterior gt domains. Domain shifting was first observed by Jaeger (2004a and b) and attributed to a chain of repressive regulatory relationships, hb-gt-kni-Kr. The current models largely support the importance of this regulatory chain, particularly the final two links. Repression of Kr by Kni was significant in all of the current models. Repression of kni by Gt was present in all models except RPJ-Logic, where it would be of little impact anyway, since RPJ-Logic has a defective posterior gt domain. Consistent with these findings, Kni binds to the regulatory region of Kr, and the Kr domain expands towards the posterior in kni mutants. Similarly, the kni domain expands posteriorly in gt mutants, while embryos overexpressing gt show reduced kni expression (Perkins, 2006).

Repression of gt by Hb is not as well supported by the current models. The Unc-GC model included the link, though the regulatory weight was the smallest of all those in the model. The link was eliminated from Unc-Logic and, of course, not present in the RPJ network structure. Instead, the models utilized decreasing activation by Cad (Unc-GC, Unc-Logic) and repression by Tll (Unc-GC, RPJ-GC) to shift the posterior gt domain. Even with these links, however, shifting of the domain is not well-captured. RPJ-GC appears to capture the posterior gt shift best (Figure 3E). However, it relies on its small ectopic Kr domain to repress gt, a completely incorrect mechanism. Interestingly, a gene circuit fit using the network structure of Sanchez and Thieffry (2001), captured the shift of posterior gt better than any of the other current models, and it did so using repression of gt by Hb, providing additional modeling support for the relationship. There also is strong mutant evidence in favor of the relationship. In hb mutants, the posterior gt domain does not retract from the posterior pole. Further, Gt is absent in embryos that have ubiquitous Hb, such as maternal oskar or nanos mutants or embryos expressing Hb ubiquitously under a heat-shock promoter. Thus, sufficient evidence was found to include a repressive link from hb to gt in the Combined model (Perkins, 2006).

Activating or repressing links that oppose the direction of the repressive chain were eliminated by optimization of the Unc-Logic, RPJ-GC, and RPJ-Logic models. In agreement with this result, the boundaries of the kni and posterior gt domains are correctly positioned in Kr and kni mutants, respectively. Thus, the simplest picture supported by the current models and consistent with the mutant studies is that there is no regulation from Kr, kni, or posterior gt to any of their immediate posterior neighbors, and that the repressive chain highlighted by Jaeger (2004a and b) is indeed responsible for domain shifting (Perkins, 2006).

Do gap genes autoregulate? All four of the current models include autoactivation by hb. This is supported by the observation that late anterior hb expression is absent in embryos lacking maternal and early zygotic Hb 47. The models suggest hb autoactivation also plays a crucial role in sustaining the posterior domain, once it has been initiated by Tll, a role not previously emphasized. Autoactivation for the other genes was found by the Unc-GC model, but is not part of the RPJ network structure. It included autoactivation only for Kr and gt in the Combined model, on the basis of a weakened and narrowed Kr domain in embryos producing defective Kr protein and a delay in gt expression in embryos producing defective gt protein. Interestingly, the gene circuit models of Jaeger (2004a and b) also found autoactivation for all four gap genes, but they considered autoactivation by gt to be the weakest and least certain. In contrast, the Unc-Logic model retained gt autoactivation while eliminating autoactivation for Kr and kni. The RPJ-Logic model was unable to reproduce the posterior gt domain. However, it was found that by adding gt autoactivation to the model, it was able to create and sustain posterior gt correctly, bringing the error of the model down to 15.34. This suggests that, after hb, gt is the most likely candidate for autoactivation. However, even this is not strictly necessary. The RPJ-GC model is able to reproduce and sustain the posterior gt domain without autoactivation by relying on cooperative activation from Bcd and Cad (Perkins, 2006).

Comparison of regulatory architectures. The regulatory relationships proposed by Rivera-Pomar and Jäckle (1996b) are not fully consistent with the data and require amending. Repression of gt by Kni, which contradicts the mechanism of domain shifts described by Jaeger (2004a and b), was eliminated by the optimization in both of the current models based on the RPJ regulators. Activation of kni by Kr was never observed. No support was found for a dual regulatory effect of Hb on Kr. Activation of Kr at low levels of Hb was eliminated in the RPJ-Logic model. It was retained in the RPJ-GC model, but resulted in serious patterning defects. Inclusion of Tll as an activator of hb was sufficient to produce the posterior hb domain. Based on the current fits and the primary experimental literature, there are likely other regulatory links missing from the model of Rivera-Pomar and Jäckle, though they are not strictly required to reproduce the wild-type gap gene patterns. Foremost is repression of hb by Kni, which appears important for eliminating hb expression anterior of the posterior domain. Fits based on the Sanchez and Thieffry (2001) regulatory relationships also support these conclusions (Perkins, 2006).

In contrast, the regulatory relationships in the Combined model and both the Unc-GC and Unc-Logic models are able to capture the wild-type gap patterns without gross defects. The relationships in the Unc-GC model are very similar to those obtained by Jaeger (2004a and b). For example, the regulation of Kr and kni is qualitatively equivalent in both models, and there is a single minor difference in the regulation of gt. The optimizations correctly identified activation of hb by Tll, which was missed by Jaeger (2004a and b), though the current models did less well at capturing shifting of the posterior gt domain. These regulatory relationships are also similar to those found by Gursky (2004), though that study was based on gap gene expression data with much lower accuracy and temporal resolution than the data used in this study. These similarities show that differences in the mathematical formulations of these models-as ordinary versus partial differential equations, how diffusion and nuclei doubling are modeled, and choice of boundary conditions and other simulation parameters-are not important for the reproduction of the gap gene patterns nor for the inference of regulatory relationships from the data (Perkins, 2006).

Hunchback regulation of pair-rule genes

A 480 bp region of the even-skipped promoter is both necessary and sufficient to direct a stripe of LacZ expression within the limits of the endogenous eve stripe 2. The maternal morphogen Bicoid and the gap proteins Hunchback, Krüppel and Giant all bind with high affinity to closely linked sites within this small promoter element. Activation appears to depend on cooperative interactions among BCD and HB proteins, since disrupting single binding sites causes catastrophic reductions in expression (Small, 1992).

An single enhancer sequence consisting of 500bp mapping 3.3kb upstream of the transciption start site is sufficient to direct eve expression in both stripes 3 and 7. There are 5 KNI binding sites in the 3 + 7 enhancer and 11 HB sites. HB and KNI act as repressors of stripe 3 expression, while the JAK kinase HOP, acting through the Drosophila STAT protein Marelle, is involved in activation, with the KNI and HB sites closely linked to two STAT binding sites. A model is presented in which the repressors provide short term quenching of widespread STAT activation (Small, 1996).

Analysis of the initial paired expression suggests that the gap genes hunchback, Krüppel, knirps and giant activate paired expression in stripes. Specifically, in hb mutants prd stripes 2 to 4 fail to form properly, and stripe 8 is never activated (Gutjahr, 1993).

A genetic and molecular analysis of two hairy pair-rule stripes has been carried out in order to determine how gradients of gap proteins position adjacent stripes of gene expression in the posterior of Drosophila embryos. Regulatory sequences of hairy have been identified that are critical for the expression of h stripes 5 and 6. hairy stripe 5 is repressed by Giant protein on its posterior border and h stripe 6 is repressed by Hunchback protein on its posterior border (Langeland, 1994).

The entire functional even-skipped locus of Drosophila is contained within a 16 kilobase region. As a transgene, this region is capable of rescuing even-skipped mutant flies to fertile adulthood. Detailed analysis of the 7.7 kb of regulatory DNA 3' of the transcription unit reveals ten novel, independently regulated patterns. Most of these patterns are driven by non-overlapping regulatory elements, including ones for syncytial blastoderm stage stripes 1 and 5, while a single element specifies both stripes 4 and 6. Expression analysis in gap gene mutants shows that stripe 5 is restricted anteriorly by Krüppel and posteriorly by giant, the same repressors that regulate stripe 2. Consistent with the coregulation of stripes 4 and 6 by a single cis-element, both the anterior border of stripe 4 and the posterior border of stripe 6 are set by zygotic hunchback, and the region between the two stripes is ‘carved out’ by knirps. Thus the boundaries of stripes 4 and 6 are set through negative regulation by the same gap gene domains that regulate stripes 3 and 7, but at different concentrations (Fujioka, 1999).

Gradients of regulatory factors are essential for establishing precise patterns of gene expression during development; however, it is not clear how patterning information in multiple gradients is integrated to generate complex body plans.Opposing gradients of two Drosophila transcriptional repressors, Hunchback (Hb) and Knirps (Kni), position several segments by differentially repressing two distinct regulatory regions (enhancers) of the pair-rule gene even-skipped (eve). Computational and in vivo analyses suggest that enhancer sensitivity to repression is controlled by the number and affinity of repressor-binding sites. Because the kni expression domain is positioned between two gradients of Hb, each enhancer directs expression of a pair of symmetrical stripes, one on each side of the kni domain. Thus, only two enhancers are required for the precise positioning of eight stripe borders (four stripes), or more than half of the whole eve pattern. These results show that complex developmental expression patterns can be generated by simple repressor gradients. They also support the utility of computational analyses for defining and deciphering regulatory information contained in genomic DNA (Clyde, 2003).

In Drosophila, the pair-rule gene eve is expressed in a pattern of seven stripes during the syncytial blastoderm stage of development. This pattern foreshadows the mature segmented body plan and is regulated by five enhancers. Three enhancers drive expression of single stripes (eve 1, eve 2 and eve 5), and the remaining two drive expression of pairs of stripes (eve 3 - 7 and eve 4 - 6). The best characterized eve enhancer drives the expression of stripe 2 (eve 2), which is activated in a broad anterior domain by the maternal morphogens Bicoid and Hb. Borders of the stripe are formed by repressive interactions involving the gap proteins Giant (Gt) and Kruppel (Kr), which are expressed in gradients anterior and posterior to the stripe, respectively. Activation and repression are mediated by the direct binding of all four proteins to discrete sites in the enhancer. Thus, this enhancer acts as a transcriptional switch that senses activator/repressor ratios in individual nuclei (Clyde, 2003).

Considerably less is known about the molecular regulation of the enhancers that drive two stripes. eve 3 - 7 is activated by ubiquitous factors including dSTAT92E, and activation of eve 4 - 6 requires the function of the fish-hook gene, but other activators are unknown. Genetic studies have shown that the gap genes hb and kni are required for forming the borders of all four of these stripes. kni is expressed in a broad posterior domain located between eve stripes 4 and 6. In kni mutants, the two-stripe patterns driven by eve 3 - 7-lacZ and eve 4 - 6-lacZ reporter genes are completely derepressed in the region between the stripes. By contrast, hb is expressed in an anterior domain that abuts eve 3 and a broad posterior stripe that overlaps eve 7. In zygotic hb mutants, there are marked derepressions of the outer borders of the stripes driven by both the eve 3 - 7 and eve 4 - 6 reporter genes (Clyde, 2003).

To test whether the eve 3 - 7 and eve 4 - 6 enhancers are differentially sensitive to Kni- and Hb-mediated repression, the snail(sna) promoter was used to misexpress these genes along the ventral surface of the embryo. The ectopic domain directed by this promoter is uniformly distributed along the anterior-posterior axis, and forms a ventral to dorsal gradient of protein diffusion. Since all seven eve stripes are subject to the same increase in protein concentration, differential sensitivities among stripes can be assayed directly. Weakly affected stripes will be repressed only in the ventral-most nuclei, whereas strongly affected stripes will show repression in more lateral or even dorsal regions. Ventral expression of either Kni (sna:kni) or Hb (sna:hb) is sufficient for repression of eve stripes 3, 4, 6 and 7 in ventral regions, but specific stripes require different quantities of ectopic protein for repression. One copy of the sna:kni transgene represses eve stripes 3 and 7, but has little effect on stripes 4 and 6. Two copies repress all four stripes, but stripes 3 and 7 are more strongly repressed than stripes 4 and 6. Misexpression of Hb shows the opposite effects. One copy of sna:hb causes a strong repression of stripes 4, 5 and 6, and an anterior weakening and posterior expansion of stripe 3. The posterior expansion is probably caused by Hb-mediated repression of kni (Clyde, 2003).

Two copies of sna:hb cause a stronger repression of stripes 4, 5 and 6, repress stripe 3 completely in ventral-most nuclei, and considerably affect stripe 7, which seems slightly weaker and expanded anteriorly, again toward the region normally occupied by kni. The weaker effect on stripe 7 suggests that higher concentrations of Hb are required to repress this stripe. This is consistent with the fact that the posterior hb stripe overlaps stripe 7, and that additional factors (including Tll) are required for activation of this stripe8. The strong repressive effect of ectopic Hb on stripe 5 is unexpected as this stripe seems to be normal in hb mutants. In addition, computational analysis shows that there are very few Hb-binding sites in the eve 5 enhancer region. These results suggest that Hb-mediated repression of this stripe is indirect (Clyde, 2003).

The above results suggest that the eve 3 - 7 and eve 4 - 6 enhancers respond autonomously to different amounts of the Hb and Kni repressors. To test this idea further, lacZ reporter genes driven by the minimal eve 3 - 7 or eve 4 - 6 enhancer were crossed into embryos carrying the sna:kni or sna:hb misexpression transgene. Embryos were also stained for endogenous sna expression, which forms a sharp ventral-lateral border, a landmark for measuring the extent of repression along the dorsalventral axis. Ventral repression of the eve 3 - 7-lacZ transgene by Kni (2 sna:kni) extends at least five nuclei above the sna border, but the eve 4 - 6-lacZ transgene is repressed only within the sna domain. Ventral expression of Hb (1 sna:hb) causes the opposite effects: the eve 4 - 6-lacZ transgene is more strongly repressed than eve 3 - 7-lacZ . These experiments are consistent with the effects observed for the endogenous eve stripes (Clyde, 2003).

To determine how these enhancers sense differences in repressor concentration, bioinformatics was used to analyse the distribution and affinity of Hb- and Kni-binding sites in the eve locus. Position-weighted matrices (PWMs) for each protein were generated by compiling and aligning the sequences of all known Hb- and Kni-binding sites, and a clustering algorithm was used to search the 20-kilobase (kb) region surrounding the eve locus. This analysis identified only two main clusters for each factor in this region, which overlap precisely with the positions of the eve 3 - 7 and 4 - 6 enhancers. The composition of sites within these clusters, however, is very different. The 3 - 7 enhancer contains considerably more Kni sites with higher PWM scores than does the 4 - 6 enhancer, consistent with its higher sensitivity to repression by Kni (Clyde, 2003).

For Hb, searching with a low-PWM-cutoff value (.4.0) identified 11 sites in the more sensitive 4 - 6 enhancer and, unexpectedly, 16 sites in the 3 - 7 enhancer. These results are similar to previous findings; however, 10 of the 11 Hb sites in the 4 - 6 enhancer have very high PWM scores, as compared with the 3 - 7 enhancer. Also, six of the ten high-scoring sites in the 4 - 6 enhancer are very tightly clustered in a 130-base-pair (bp) interval, whereas those in the 3 - 7 enhancer are evenly distributed across the sequence. These results suggest that binding-site affinity and distribution may be crucial parameters in determining enhancer sensitivity to Hb-mediated repression (Clyde, 2003).

Next, whether the clustering algorithm could predictably change enhancer sensitivity was tested using the Kni-binding sites in the 3 - 7 enhancer as a test case. The PWM search identified 12 Kni-binding sites in the minimal 3 - 7 enhancer; six of these sites were mutated so that the cluster significance score of the mutated enhancer (denoted 3 - 7m6K) was intermediate between those of the wild-type 3 - 7 and 4 - 6 enhancers. Reporter expression driven by 3 - 7m6K shows a derepression of the inner borders of stripes 3 and 7, suggesting that Kni-mediated repression has been compromised by these mutations. The stripe 3 response of the mutated enhancer extends throughout the interstripe region posterior to eve 3 to the anterior border of, but not through the region occupied by, eve 4. Thus, the 3 - 7m6K enhancer is less sensitive to Kni than is the wild- type 3 - 7 enhancer, but is still more sensitive than the 4 - 6 enhancer. This suggests that the precise positioning of these stripes is controlled by the strength of Kni site clusters (Clyde, 2003).

Since the normal Hb and Kni gradients set several expression boundaries in the region between their domains, it is essential that their relative positions in the embryo are precisely established and maintained. This could be achieved by mutual repression. To test this, the effects of ventrally expressed Kni on the expression of hb messenger RNA was analyzed, and vice versa. Misexpression of Kni causes a strong reduction in hb mRNA in ventral regions. Similarly, misexpressed Hb causes a strong repression of kni (Clyde, 2003).

Loss-of-function experiments lend further weight to the mutual repression hypothesis. In hb mutants, there is a substantial expansion of the posterior kni domain. In kni mutants, there is a slight anterior expansion of the posterior hb domain, but no effect on the anterior domain. Double mutant embryos that lack kni and the central gap gene Krüppel (Kr) show, however, a marked expansion of zygotic hb expression throughout the posterior half of the embryo. Because misexpression of Kr alone has no effect on the hb expression pattern, this observation suggests that Kr and Kni may cooperate in repression of hb. In conclusion, the principle elements of a simple repression system have been demonstrated that greatly increases pattern complexity in the Drosophila embryo. Strong reciprocal repression between kni and hb positions a symmetrical Kni domain between two opposing gradients of Hb. This arrangement permits a single enhancer to make two stripes, one on both sides of the Kni domain. Two differentially sensitive enhancers effectively double the patterning information in each gradient, leading to the establishment of eight expression boundaries. A similar antagonistic relationship exists between the gap genes gt and Kr, which are expressed in nonoverlapping domains, with the central Kr domain positioned between two gt domains. The eve 2 and eve 5 stripes are formed on either side of the Kr domain by Kr- and Gt-mediated repression, but in this case each stripe is regulated by a separate enhancer, probably because the activators of these stripes are expressed in localized patterns (Clyde, 2003).

Previous studies have shown that activator gradients are crucial for differential positioning of target gene expression patterns along the anterior-posterior and dorsal-ventral axes. This study suggests that repressor gradients can also specify several gene expression boundaries by interacting with differentially sensitive regulatory elements. At the molecular level, repression mechanisms are flexible: enhancer activation can be prevented by direct repression or by interfering with the binding or activity of even a single activator protein. It is proposed that repressor gradients, owing to this flexibility, are inherently more effective than activator gradients at providing developmental patterning information (Clyde, 2003).

The sensitivity of an enhancer is likely to be determined by several parameters including the number, affinity and arrangement of repressor-binding sites, but predicting the relative importance of each of these parameters for a given enhancer is difficult. For the Kni repressor gradient, the different responses of the 3 - 7 and 4 - 6 enhancers seem to depend on different numbers of binding sites. By contrast, the different responses of the same enhancers to Hb repression seem to depend on the affinity and/or arrangement of sites. Thus, it may be impossible to formulate simple rules that describe the functional characteristics of most enhancers. However, future studies that combine computational analyses with experimental tests will undoubtedly increase the ability to identify and to characterize the genomic elements that regulate transcription (Clyde, 2003).

Regulation of segment polarity genes by Hunchback

Gap proteins Krüppel and Hunchback to function as transcriptional regulators in cultured cells. Both proteins bind to specific sites in a 100-bp DNA fragment located upstream of the segment polarity gene engrailed, which also contains functional binding sites for a number of homeo box proteins. The Hunchback protein is a strikingly concentration-dependent activator of transcription, capable of functioning both by itself and also synergistically with the pair-rule proteins Fushi tarazu and Paired. In contrast, Krüppel is a transcriptional repressor that can block transcription induced either by Hunchback or by several different homeo box proteins (Zuo, 1991).

Regulation of homeotic genes by Hunchback

Segment polarity genes are not the only genes to be expressed in a parasegmental register. The abdominal-A and Abdominal-B genes of the bithorax complex specify the identity of most of the Drosophila abdomen. Six different classes of infraabdominal mutations within the BX-C transform a subset of the parasegments affected by the lack of these two genes. These mutations define parasegmental cis-regulatory regions that control the expression of abd-A and Abd-B. (Sanchez-Herrero, 1991).

Expression of the abdominal-A and Abdominal-B genes is controlled by a cis-regulatory promoter and by distal enhancers called infraabdominal regions. The activation of these regions along the anteroposterior axis of the embryo determines where abdominal-A and Abdominal-B are transcribed. The gap genes hunchback, Krüppel, tailless and knirps control abdominal-A and Abdominal-B expression early in development. The gradients of the Hunchback and Krüppel products seem to be key elements in this restricted activation (Casares, 1995).

Krüppel and Knirps act to limit anterior Abd-B expression and regulate the graded ABD-B domain respectively. Both Hunchback and Polycomb are required for Abd-B silencing (Busturia, 1993).

The activation of Deformed is dependent on combinatorial input from at least three levels of the early hierarchy. The simplest activation code sufficient to establish Deformed expression, given the absence of negative regulators such as Fushi-tarazu, consists of a moderate level of expression from the coordinate gene bicoid, in combination with expression from both the gap gene hunchback, and the pair-rule gene even-skipped. In addition, the activation code for Deformed is redundant; other pair-rule genes in addition to even-skipped can apparently act in combination with bicoid and hunchback to activate Deformed (Jack, 1990).

As a segmentation gene, hunchback, acts as a direct repressor or "silencer" of the homeotic gene Ultrabithorax and thus prevents ectopic activity of this gene. HB protein binding sites are capable of repressing at a distance the activity of an embryonic Ubx enhancer outside the Ubx expression domain. This silencing activity is observed at advanced embryonic stages, at a time when the HB gene product is no longer detectable or required, and is dependent on the function of Polycomb . HB protein, in a "hit-and-run" fashion, may effect stable and heritable silencing of the Ubx gene throughout advanced stages of development, thus mediating repression of this homeotic gene outside its realm of function (Zhang, 1992).

The second promoter of the Antennapedia gene (Antp P2)is regulated by the zygotically-active segmentation genes ftz, hb, Kr, gt and kni acting as activators or repressors of Antp P2 in a combinatorial fashion. The timing of these events, and their positive versus negative nature, is critical for generating the expression patterns normal for Antp (Riley, 1991).

Spatial boundaries of homeotic gene expression are initiated and maintained by two sets of transcriptional repressors: the gap gene products and the Polycomb group proteins. DNA elements and trans-acting repressors that control spatial expression of the Abdominal-A (ABD-A) homeotic protein have been investigated. Analysis of a 1.7-kb enhancer element [iab-2(1.7)] from the iab-2 regulatory region shows that both Hb and Kruppel (Kr) are required to set the Abd-A anterior boundary in parasegment 7. DNase I footprinting and site-directed mutagenesis show that Hb and Kr are direct regulators of this iab-2 enhancer. The single Kr site can be moved to a new location 100 bp away and still maintain repressive activity, whereas relocation by 300 bp abolishes activity. These results suggest that Kr repression occurs through a local quenching mechanism. The gap repressor Giant (Gt) initially establishes a posterior expression limit at PS9, which shifts posteriorly after the blastoderm stage. This iab-2 enhancer contains multiple binding sites for the Polycomb group protein Pleiohomeotic (Pho). These iab-2 Pho sites are required in vivo for chromosome pairing-dependent repression of a mini-white reporter. However, the Pho sites are not sufficient to maintain repression of a homeotic reporter gene anterior to PS7. Full maintenance at late embryonic stages requires additional sequences adjacent to the iab-2(1.7) enhancer (Shimell, 2000).

The gap gene product Kr is required to set the iab-2(1.7) anterior expression border. However, since Kr is not expressed anterior to PS5, some other factor must also be required to repress the iab-2(1.7) enhancer in anterior regions. A likely candidate is the Hb protein, which has been shown to be important for repressing the bx and pbx enhancers anterior to PS6. To examine whether Hb plays a role in setting the iab-2(1.7) anterior expression boundary, this construct was crossed into both hb and osk mutant backgrounds. Loss of zygotic hb caused a slight broadening of the initial expression band, indicating an anterior shift in the expression pattern of this enhancer. The presence of maternal Hb likely minimizes the anterior shift in these zygotic hb mutant embryos. Consistent with this view, it has been found that, in an osk mutant background, in which the maternal level of Hb is uniform throughout the embryo, expression from the iab-2(1.7) enhancer is completely abolished. These findings suggest that, as with the bx and pbx enhancers, Hb is important for setting the initial anterior limit of iab-2 enhancer function (Shimell, 2000).

Hb, Kr, and Gt have been classified as short-range repressors whose range of action is limited to approximately 50 to 150 bps. Two major mechanisms of short-range repression are: competitive binding to an overlapping activator binding site, and quenching, which entails interference with function of locally bound activators. Since studies on Hb, Kr, and Gt action have focused primarily on their control of pair-rule genes such as eve, it was of interest to address mechanisms used by these repressors in the alternative context of a homeotic gene regulatory region. The in vitro binding analysis identified five discrete Hb sites on the iab-2(1.7) fragment. One of these sites, Hb2, overlaps extensively with one of the Eve binding sites. Since Eve acts as an activator of iab-2(1.7) expression, Hb may repress by competing with Eve for direct binding to this site. Evidence for a direct competition mechanism has been described for Hb repression through the bx and bxd/pbx control regions of the Ubx homeotic gene. In these cases, the anterior boundary is in PS6 rather than PS7, and Hb competes with Ftz rather than Eve. However, mutational analysis shows that Hb sites other than Hb2 also contribute to iab-2 repression. These additional sites could promote Hb competition with Eve by assisting Hb binding at Hb2 through cooperative interactions. Similarly, the single Gt binding site in iab-2(1.7) overlaps another Eve binding site, suggesting that Gt may also repress by direct competition with Eve in posterior parasegments. In contrast, the single Kr binding site (Kr1) does not overlap Eve sites. A distinct Kr mechanism is also supported by the ability of Kr1 to repress even when relocated 100 bp away from its normal position in the iab-2(1.7) fragment. This flexibility, together with failure of Kr repression when Kr1 is further relocated by 300 bp, is consistent with a short-range quenching mechanism. These results argue against Kr repression by direct interference with basal transcription factors, since 300 bp is small compared to the 20-kb distance between the iab-2 enhancer and the abd-A promoter. Previous studies using a synthetic regulatory region have shown that Kr can repress by a quenching mechanism in vivo (Shimell, 2000).

Any proposed mechanism for Kr action through iab-2, however, must account for the variability of Kr repression within its own expression domain. Specifically, Kr represses the iab-2 enhancer in PS3 and PS5 where Kr concentrations are low, but it does not repress in PS7 where Kr concentrations are high. This observation suggests that simple occupancy of the Kr1 site is not sufficient for iab-2 repression and that another factor acts in concert with Kr. The likely partner is Hb since Kr repression of iab-2 is limited to parasegments that accumulate significant levels of both Kr and Hb. In this view, repression just anterior to PS7 requires both Kr and Hb, whereas repression in more anterior parasegments, where Hb levels are highest, is mediated by Hb alone. Kr-Hb synergy could involve direct contact since the two proteins have been shown to interact when bound to DNA. Whether Kr synergizes with Hb by augmenting Hb binding to DNA in a cooperative manner or by recruiting additional corepressors is not clear. Kr, but not Hb, functions together with the corepressor dCtBP (Shimell, 2000 and references therein).

After Hb and Kr decay during early gastrulation, the repressed state is propagated through later stages of development by the PcG proteins. How the transition from early gap repressors to long-term PcG repressors occurs at the molecular level is not known. Two basic models have been proposed: (1) direct recruitment, and (2) chromatin recognition. Model (1): The gap gene products, especially Hb, have been proposed to help recruit PcG proteins directly to specific DNA sites. Based upon its early time of action, a role for the PcG protein Extra sex combs (Esc) as a molecular bridge between the two sets of repressors has been suggested. However, direct interactions between Esc and gap repressors have not been reported. A better candidate for such a molecular link is dMi-2, which binds directly to Hb and behaves genetically as an enhancer of PcG repression. In its simplest form the direct recruitment model is unlikely because the iab-2, bx, and pbx enhancers all contain Hb sites but do not effectively recruit PcG proteins. These elements fail to maintain A-P boundaries of expression and are unable to attract PcG proteins to sites on chromosomes. Furthermore, the continuous requirement for PRE sequences during development shows that DNA site recognition by PcG proteins can occur long after Hb and Kr have decayed. Model (2):The second model proposes that PcG proteins recognize some feature of silenced chromatin, rather than particular gap repressors. This model is supported by patterns of PcG-dependent silencing that reflect patterns of early gene activity rather than the distributions of gap proteins. In this view, PcG proteins sense the transcriptional off state and then assemble locally to imprint this state through later stages. These two models are not mutually exclusive. Both the Hb-interacting protein dMi-2 and the Kr-interacting protein dCtBP have mammalian homologs that interact with histone deacetylases. Perhaps the gap repressors work by targeting these deacetylases, whose action alters the local acetylation state of the histone tails. This could provide a feature of silenced chromatin that is recognized by PcG proteins and that promotes their association at nearby PREs (Shimell, 2000 and references therein).

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).

Many of the genes that are members of either the gap, pair rule, or segment polarity genes have some effect on the pattern of pb accumulation. For the most part, mutations in genes of these classes reduce the number of cells expressing pb but do not eliminate Pb entirely from the affected segments. In no case do they cause pb to accumulate ectopically. The most striking results were caused by zygotic mutations in the genes buttonhead (btd), giant (gt), and hunchback (hb). btd is a head gap gene required for formation of the mandibular segment. In btd mutants, no mandibular structures are seen and no pb accumulation occurs anterior of the maxillary segment. pb accumulation is normal in the other gnathal segments. Mutations in both gt and hb disrupt the formation of the labial lobe and result in concomitant loss of pb expression therein. When the pb reporter #7 was tested in a hb mutant background no lacZ expression in the presumptive labial segment was found. In the case of gt, pb expression is not entirely extinguished. Weak pb accumulation can sometimes be seen in the most dorsal and posterior cells of the presumptive labial segment, overlapping with the few remaining cells of the engrailed stripe in the labial segment. For both gt and hb, this reduction or loss of pb expression in the labial lobe cannot be attributed to alterations in the Scr pattern because Scr accumulates in the cells posterior to the maxillary segment (Rusch, 2000).

The expression of the homeotic gene Antennapedia (Antp), which is first activated in late blastoderm embryos as a strong stripe that overlaps PS4, and in the ventral-most cells of PS5, was examined. Loss-of-function Antp mutants show homeotic transformations of T2 and T3 toward a T1 fate, suggesting that Antp is critical for specifying pattern in these segments. The PS4-Antp stripe is abolished in zygotic hb mutants and in ftz mutants, suggesting that these genes are required for its activation. In st2-kni embryos, the PS4-Antp stripe is completely missing, but the ventral expression at PS5 is not affected. When the st2DeltaK-hb-1 transgene is added to these embryos, there is a reactivation of the PS4-Antp stripe, which is then expressed at wild-type levels by the beginning of germ band elongation. The recovery of the Antp stripe suggests that activation of Antp occurs as a direct response to the increase of Hb protein at PS4, since there is no detectable augmentation of the levels of ftz in the rescued embryos (Wu, 2001).

The preceding experiments suggest that Antp can be efficiently activated with a reduced level of ftz activity. To test this further, the eve stripe 2 enhancer was used to misexpress the pair-rule gene hairy (h), which is a potent repressor of ftz transcription. In wild-type embryos, h is expressed in seven stripes that overlap the anterior portions of the eve stripes. Misexpressing h using the stripe 2 enhancer (st2-h) expands accumulation of H mRNA into the interstripe between h stripes 2 and 3 and causes a deletion of T2. As with the st2-kni deletion, the effect is stronger on the ventral side of the embryo. At the molecular level, st2-h misexpression represses ftz stripe 2 in a dose-dependent manner. High levels completely abolish the stripe, especially in ventral regions, and prevent the activation of en stripe 4. This result is consistent with a direct role for ftz in activation of en. In contrast, the ectopic h does not cause a significant change in activation of PS4-Antp, suggesting that PS4-Antp expression can be activated in the absence of ftz activity. The PS4-hb stripe is also unaffected in these embryos, consistent with the hypothesis that Antp is specifically activated by Hb (Wu, 2001).

Involvement of Hunchback in gene silencing

Silencers from the Drosophila homeotic gene Ultrabithorax require Hunchback and Polycomb to suppress the activity of embryonic enhancers outside the Ubx domain. Embryonic silencing is initiated by HB protein which binds to the silencers to repress Ubx, thereby defining the Ubx domain. Silencing during subsequent development was studied by examining expression patterns in imaginal discs conferred by individual Ubx fragments and pair-wise combinations thereof. Fragments that mediate silencing in anterior regions of imaginal discs contain embryonic silencers and HB target sites. One exception is a fragment called BXD; while not under HB control itself, its silencing activity depends on combination with fragments containing HB protein binding sites. Since silencing by BXD also requires Polycomb function, this suggests that BXD contains target sites for PC or for PC-like proteins (Christen, 1994).

A regulatory element in the Ubx gene responds to PC-G and TRX-G genes. Transposons, genetically engineered pieces of DNA carrying the regulatory element to new ectopic sites, create new binding sites for PC-G products at the new sites to which they integrate. The transposons carry PC-G maintenance elements (PRE), DNA regions responsive to the repressive effects of PC-G genes. PREs and PC-G proteins establish a repressive complex that keeps itself and other distal enhancers repressed in cells where they were first active and then repressed, and maintains this repressed state for many cell divisions. PRE functions to silence these remote enhancers or to maintain expression regulated by TRX-G. Hunchback mediates repression at the PRE. TRX-G products stimulate the expression of separate and distinct enhancers, active in imaginal discs (Chan, 1994).

Newer evidence maintains that silencing of late Ubx enhancers by PRE is Hunchback-independent. If a PRE is combined with late enhancers, including a Wingless responsive CNS element or imaginal disc enhancers, repression of a reporter gene can be established everywhere in the embryo, irrespective of the presence or absence of Hunchback protein. If, however, these late enhancers are combined with a Ubx early enhancer, one that is silenced early, as well as with a PRE, then repression is established only where the reporter gene was inactive at early stages. These results imply that the Polycomb complex is not dependent on Hunchback, and suggest that the pattern of silencing reflects rather the level of activity of the gene at the time the Polycomb repressive protein complex is formed (Poux, 1996).

GAGA facilitates binding of Pleiohomeotic to a chromatinized Polycomb response element - Involvement of Hunchback

Polycomb response elements (PREs) are chromosomal elements, typically comprising thousands of base pairs of poorly defined sequences that confer the maintenance of gene expression patterns by Polycomb group (PcG) repressors and trithorax group (trxG) activators. Genetic studies have indicated a synergistic requirement for the trxG protein GAGA and the PcG protein Pleiohomeotic (PHO) in silencing at several PREs. However, the molecular basis of this cooperation remains unknown. Using DNaseI footprinting analysis, a high-resolution map is provided of sites for the sequence-specific DNA-binding PcG protein PHO, trxG proteins GAGA and Zeste and the gap protein Hunchback (HB) on the 1.6 kb Ultrabithorax (Ubx) PRE. Although these binding elements are present throughout the PRE, they display clear patterns of clustering, suggestive of functional collaboration at the level of PRE binding. While GAGA can efficiently bind to a chromatinized PRE, PHO alone is incapable of binding to chromatin. However, PHO binding to chromatin, but not naked DNA, is strongly facilitated by GAGA, indicating interdependence between GAGA and PHO already at the level of PRE binding. These results provide a biochemical explanation for the in vivo cooperation between GAGA and PHO and suggest that PRE function involves the integrated activities of genetically antagonistic trxG and PcG proteins (Mahmoudi, 2003).

This study has determined the precise distribution within the Ubx PRE of the recognition elements for four sequence-specific DNA-binding proteins that have all been implicated in Ubx regulation in vivo: PcG protein PHO, gap protein HB and trxG proteins GAGA and Zeste. The results indicate that, rather than a random collection, the binding site distribution within the Ubx PRE reflects a functional arrangement, allowing cooperation between distinct PRE binding proteins. Of particular interest is the observation that chromatin binding by the PcG protein PHO is strongly facilitated by the trxG protein GAGA. This finding provides a molecular mechanism for the requirement for both factors during PRE-directed silencing in vivo, and suggests that PHO and GAGA elements together may form a functional module (Mahmoudi, 2003).

Several independent genetic studies have pointed to a concurrent requirement for GAGA and PHO during gene silencing directed by distinct PREs. The PcG-dependent silencing conferred by a 230 bp fragment of the iab-7 PRE is dependent on both GAGA and PHO binding. Similarly, a 138 bp fragment of the MCP silencer, which was found to be sufficient for maintenance of embryonic silencing, contains PHO and GAGA sites. Mutations in either PHO or GAGA sites compromised silencing and revealed cooperation between both proteins. Particularly relevant for the current study are results that support a critical role in PcG silencing for GAGA and PHO sites within the Ubx PRE (Mahmoudi, 2003).

Functional dissection of the Ubx PRE has revealed that a Pc-dependent PRE silencer is contained in the central 567 bp fragment from position 577 to 1143, which includes all PHO and the highest density of GAGA sites. Another study showed that an oligomerized subfragment, corresponding to positions 890-1079 within PRE C, harboring two PHO and five GAGA elements, is able to confer PcG silencing in vivo. Finally, deletion of a 160 bp region corresponding to positions 851-1011 within PRE C impairs maintenance of silencing. The large extent of overlap between the DNA fragments identified in these independent studies strongly suggests that the common region within PRE C represents the critical core of the Ubx PRE. The most noticeable feature of this region is the many alternating GAGA and PHO binding elements. Moreover, it is of interest to note that footprinting analysis revealed the presence of Zeste as well as HB sites within this region, which may also contribute to the in vivo maintenance of repression (Mahmoudi, 2003).

The identification of Zeste as a component of the PRC1 PcG complex, suggests that it may play a direct role in PcG complex recruitment to the Ubx PRE. Further evidence for the involvement of Zeste in the maintenance of Ubx repression as well as activation has been provided by transgene experiments. Finally, the presence of HB sites within the Ubx PRE suggests a potential role for HB, not only during the initiation of Ubx repression, but also during the transition from establishment to maintenance. One attractive possibility is that this transition involves dMi-2 recruitment by HB. It should be noted that in the absence of initiating activation and repression elements, HB-independent PcG repression of the Ubx promoter has been documented (Mahmoudi, 2003).

Although there is substantial evidence for the notion that the proteins discussed above are involved in PcG silencing of homeotic genes, it remains unclear whether they can be sufficient for targeting or whether additional factors are required. One way to determine a minimal set of protein recognition sequences that can mediate PcG silencing will be the generation of synthetic PREs, which should be tested in vivo. The results suggest that, within such a PRE, PHO sites will need to be flanked by GAGA sites in order to facilitate chromatin binding. The proteins GAGA and Zeste may be particularly well adapted for such a purpose. Both GAGA and Zeste form large homo-oligomers that bind cooperatively to the multiple sites present in their natural response elements, such as the Ubx PRE and promoter. This cooperative mode of DNA-binding may allow these proteins to first bind an accessible site within a nucleosomal array and then progressively displace histones during binding to flanking sites. In addition, GAGA and Zeste have both been shown to recruit selective ATP-dependent chromatin remodeling factors. The process of targeting of remodelers to specific DNA elements may enable GAGA and Zeste to create nucleosome-free or remodeled areas, thus facilitating binding of other regulators. It is considerede likely that the remodeling complexes present in the chromatin preparations used in assays, are involved in the observed synergistic binding between PHO and either GAGA or Zeste (Mahmoudi, 2003).

GAGA oligomerization may also promote the communication between the Ubx PRE and promoter. Both elements, which are separated by ~24 kb of intervening DNA, contain a preponderance of binding sites for GAGA. GAGA oligomerization through its POZ domain allows it to form a protein bridge that directs long-range enhancer-promoter association. In fact, GAGA could even mediate enhancer function in trans by simultaneous binding of two separate DNA fragments. Thus, it is tempting to speculate that GAGA may link the Ubx PRE to the Ubx promoter. It should be noted that both the chromatin remodeling and long-range bridging functions of GAGA might accommodate PRE-mediated activation as well as repression (Mahmoudi, 2003).

The interdependence between proteins belonging to antagonistic genetic groups for efficient chromatin binding described it this study will have to be taken into account when interpreting mutational analysis of PRE function. Thus, removal of recognition sequences for the trxG protein GAGA may block its activation function but could also affect binding of the PcG protein PHO. Moreover, recent results suggest additional opportunities for cross-talk during recruitment of non-DNA-binding PcG complexes. Although a clear consensus between different studies is still lacking, there is experimental evidence for PcG complex recruitment by PHO, GAGA and Zeste. Because binding sites for either one of these proteins alone do not confer PRE function, it appears likely that they work in a combinatorial fashion. Depending on their context, the multitude of distinct binding elements that constitute a PRE might be redundant, cooperative or antagonistic to each other. Furthermore, distinct PREs may require different sets of PRE-binding proteins, and additional recruiters may be involved in PcG-silencing. Attractive candidates are GAGA-related factors batman and the PHO-related factor PHO-like (Mahmoudi, 2003).

In conclusion, current evidence suggests that PRE-directed maintenance of gene activation or repression is not achieved by a simple binary switch set by competing trxG and PcG proteins. Although their relative ratios vary considerably and correlate with transcription levels, they coexist at PREs during gene activation as well as repression. Likewise, genetic suppressor studies indicated extensive cross-talk between PcG and trxG proteins. This study has shown that, already at the level of PRE binding, there is strong interdependence between trxG protein GAGA and PcG protein PHO. The results demonstrate a direct biochemical mechanism for the cooperation between PcG and trxG proteins during PRE binding (Mahmoudi, 2003).

Precise registration of gene expression boundaries by a repressive morphogen in Drosophila

Morphogen gradients are thought to create concentration thresholds that differentially position the expression boundaries of multiple target genes. Despite intensive study, it is still unclear how the concentration profiles within gradients are spatially related to the critical patterning thresholds they generate. This study used a combination of quantitative measurements and ectopic-misexpression experiments to examine the transcriptional-repression activities of the Hunchback (Hb) protein gradient in Drosophila embryos. The results define five expression boundaries that are set primarily by differences in Hb concentration and two boundaries that are set by combinatorial mechanisms involving Hb and at least one other repressor. Hb functions as a repressive morphogen, but only within a specific range of concentrations (~40% to ~4.4% of maximum Hb concentration), within which there are at least four distinct concentration thresholds. The lower limit of the range reflects a position where the slope of the gradient becomes too shallow for resolution by specific target genes. Concentrations above the upper limit do not contribute directly to differential-repression mechanisms, but they provide a robust source that permits proper functioning of the gradient in heterozygous embryos that contain only one functional hb gene (Yu, 2008).

This study measured the relative Hb concentrations associated with the positions of seven expression boundaries and tested whether different Hb concentrations can account for the differential positioning of these boundaries along the AP axis of the Drosophila embryo. These experiments lead to the following conclusions. 1. The Hb gradient functions as a bona fide repressive morphogen for five target-gene expression boundaries, eve 3, nub, pdm2, eve 4, and kni, all of which appear to be positioned primarily, if not exclusively, by specific thresholds of Hb concentration. These boundaries move anteriorly in concert with the dynamic changes of the Hb gradient in wild-type embryos, they shift anteriorly in zygotic hb mutants, and their sensitivities to repression by ectopically expressed Hb are consistent with their relative positions in wild-type embryos. Two other boundaries, the anterior boundary of Kr and the anterior boundary of the posterior gt domain, are established by combinatorial mechanisms involving Hb and Gt, and Hb and Kr, respectively. 2. There is a specific concentration range (~40% to ~4.4% [Hb]max) that mediates the major morphogenetic activities of the Hb repression gradient. Within this range, four thresholds were detected, one at ~40% [Hb]max that sets the anterior boundary of eve 3, one at ~12% [Hb]max that sets the anterior boundaries of both nub and pdm2, one at ~8% [Hb]max that sets the anterior boundary of eve 4, and one at ~4.4% [Hb]max that sets the anterior boundary of kni. These results suggest that these five target genes are exquisitely sensitive to small changes in Hb concentration. Hb also acts as a direct repressor to position the anterior boundary of the Hox gene Ultrabithorax (Ubx), which is first activated in late cycle 14 just before the initiation of gastrulation. The anterior Ubx boundary is positioned between the eve 3 and eve 4 boundaries and thus may represent a fifth threshold within the morphogenetic range described in this study. Ventral misexpression of Hb causes a strong repression of Ubx. However, it was not possible to directly compare the sensitivity of Ubx with the other target genes because the patterns of these genes have begun to decay when Ubx is first activated (Yu, 2008).

Previous studies have identified discrete regulatory elements that recapitulate the five expression patterns within the morphogenetic range described in this study. All of these elements contain multiple Hb binding sites, and one attractive model is that differences in sensitivity are determined by the quantity and/or quality of their Hb binding sites. The more sensitive eve 4+6 enhancer seems to contain a stronger cluster of Hb binding sites than the less sensitive eve 3+7 enhancer, which is consistent with this hypothesis. However, in preliminary experiments, it was found that this simple model cannot be applied to the five target genes shown in this study to be differentially sensitive. For example, the kni expression pattern is more sensitive to Hb-mediated repression than either eve 3 or eve 4, but its enhancer sequence does not appear to have a stronger cluster of Hb binding sites than either the eve 3+7 or the eve 4+6 enhancer. Similarly, two enhancer elements have been found to be associated with the pdm locus, which contains both nub and pdm2. When tested in reporter genes, both enhancers drive patterns of gene expression similar to the endogenous nub and pdm2 patterns, but they do not appear to contain similar clusters of Hb sites (Yu, 2008).

If differential sensitivity cannot be linked to differences in the number or affinity of Hb binding sites for this set of regulatory elements, other architectural features may control the level of Hb required for repression. These features may include changes in spacing between adjacent Hb sites, or specific site orientations that affect cooperative binding. Also, specific arrangements between repressor and activator sites may influence the apparent sensitivities. Consistent with this, it has been shown that specific arrangements between Dl and Twi sites are critical for Dorsal-dependent target-gene expression in the prospective neuroectodermal region along the DV axis. A careful analysis of the enhancer elements that respond to Hb-mediated repression will be required to fully understand the molecular rules that govern differential sensitivity (Yu, 2008).

At the low end of the effective morphogenetic range, there is a ~2-fold difference between the Hb concentration at the eve 4 boundary (~8% [Hb]max) and the amount at the kni boundary (~4.4% [Hb]max). Moving farther posteriorly, from the kni boundary to the gt boundary, does not correlate with a significant drop in the relative Hb concentration (~4.4% [Hb]max to ~3.7% [Hb]max). It is proposed that the slope of the gradient in this region is too shallow for differential target-gene positioning. However, by participating in a combinatorial mechanism with Kr, the very low concentrations of Hb in this region can set the gt boundary in a more posterior position than the kni boundary. Hb and Kr both bind to the regulatory element that drives posterior expression of gt, suggesting that these interactions may be direct (Yu, 2008).

Within the morphogenetic range, the anterior-most boundary is that of eve 3, which corresponds to ~40% [Hb]max. Outside this range on the anterior side is the Kr boundary, which was previously shown to expand anteriorly in zygotic hb mutants. In these experiments, Kr appeared to be quite resistant to repression by ectopic Hb, which seemingly contradicts a previous study that showed that high levels of Hb were sufficient for repression. However, in the previous study, ectopic Hb was provided maternally, significantly before the sna-hb transgene used in this study would be activated. Together, the two studies support the idea that the Kr boundary is initially set independently by Hb, and that maintenance of the boundary requires both Hb and Gt activities. The results suggest that maintenance is mediated primarily by Gt, but that Gt is an effective repressor only in the presence of Hb. The potentiating effect of Hb on Gt-mediated repression may involve direct binding of Hb and Gt to the Kr promoter, which contains binding sites for both proteins (Yu, 2008).

One of the most important findings from this study is that the effective range of Hb's morphogenetic activity is between 40% [Hb]max and 4.4% [Hb]max. This range may seem surprising in light of the fact that Hb is expressed at much higher levels throughout the anterior half of the embryo. Previous studies suggest that anteriorly expressed Hb is required for activation of most Bcd-dependent target genes, which are expressed in a variety of anterior patterns, and that the zygotic stripe of Hb expressed at the position of PS4 is required for the activation of the Hox gene Antennapedia. It is proposed that the high level of Hb protein in anterior regions also provides a reservoir, or buffer, that ensures that the repressive gradient, with all of its thresholds, remains intact in individual embryos that vary in their absolute levels of Hb expression. Such a buffering mechanism could explain how heterozygous embryos, which contain roughly half the concentration of Hb, can nonetheless develop normally (Yu, 2008).

It is proposed further that most other morphogens will function via concentration ranges similar to the one measured in this study. The two best-studied morphogens in Drosophila are Bcd and Dorsal (Dl), both of which are viable and fertile in the heterozygous state. In embryos laid by bcd/+ females, there are dramatic shifts in the positioning of target genes in the early embryo, but the order of gene positioning remains unchanged, the embryos survive to adulthood, and the adults are fertile. Survival would not be possible if activation of a critical target gene required a threshold greater than 50% of the maximum concentration of Bcd (Yu, 2008).

Molecular dissection of cis-regulatory modules at the Drosophila bithorax complex reveals critical transcription factor signature motifs

At the Drosophila bithorax complex (BX-C) over 330kb of intergenic DNA is responsible for directing the transcription of just three homeotic (Hox) genes during embryonic development. A number of distinct enhancer cis-regulatory modules (CRMs) are responsible for controlling the specific expression patterns of the Hox genes in the BX-C. While it has proven possible to identify orthologs of known BX-C CRMs in different Drosophila species using overall sequence conservation, this approach has not proven sufficiently effective for identifying novel CRMs or defining the key functional sequences within enhancer CRMs. This study demonstrates that the specific spatial clustering of transcription factor (TF) binding sites is important for BX-C enhancer activity. A bioinformatic search for combinations of putative TF binding sites in the BX-C suggests that simple clustering of binding sites is frequently not indicative of enhancer activity. However, through molecular dissection and evolutionary comparison across the Drosophila genus it was discovered that specific TF binding site clustering patterns are an important feature of three known BX-C enhancers. Sub-regions of the defined IAB5 and IAB7b enhancers were both found to contain an evolutionarily conserved signature motif of clustered TF binding sites which is critical for the functional activity of the enhancers. Together, these results indicate that the spatial organization of specific activator and repressor binding sites within BX-C enhancers is of greater importance than overall sequence conservation and is indicative of enhancer functional activity (Starr, 2011).

The clustered organization of TF binding sites has been shown to be crucially important to the functional activity of enhancers. However, despite detailed studies of a small set of enhancers in Drosophila, including the eve stripe 2 (S2E) enhancer, the precise rules of cis-regulatory grammar have yet to be fully elucidated. In an effort to investigate the role of clustering of predicted TF binding sites for the identification of enhancers in the 330 kb Drosophila BX-C, a search for simple clusters of HB and KR binding sites was performed. The search algorithm returned 26 putative enhancers (PCRMs), of which 6 (23%) overlapped previously identified enhancers. The overlapping regions for four of these confirmed enhancers (BRE, IAB2, IAB5 and IAB8) were tested in transgenic reporter gene assay and recapitulated the known domains of regulatory activity in the embryo. Furthermore, the 1037 bp R10 region that was tested, that is able to recapitulate IAB2 enhancer functional activity, refines the boundaries of the previously characterized 1970 bp IAB2 sequence. The search also identified 20 additional PCRM sequences. Twelve of these previously uncharacterized genomic regions were analyzed for enhancer activity and only one (R8 from the bxd/pbx region) was found to be a novel embryonic enhancer capable of driving expression in a pattern indicative of Ubx gene expression. This result indicates that the approach of searching for novel enhancers in the BX-C using simple clustering may have significant limitations (Starr, 2011).

A key question is why 11 of the 16 PCRMs tested (69%) are false positives. Two possibilities include; a) that the PCRMs may in fact be actively regulating expression of the BX-C genes at later stages of development or in very specific patterns in post-embryonic tissues, and b) that in testing a specific ~ 1 kb genomic fragment from each PCRM critical regulatory sequences in neighboring regions may have been removed. However, the recent availability of in vivo TF binding data may also offer some potential answers. The binding of anterio-posterior restricted gap/terminal and pair-rule transcription factors in stages 4-5 embryos appears to correlate strongly with the functional activity of the PCRMs. When scored for ten specific TFs which are potential regulators of the BX-C enhancers, all the PCRMs tested in the transgenic assay that had chromatin immunoprecipitation (ChIP) binding peaks for 6 or more of the protein factors function as embryonic enhancers. For each of these confirmed enhancers, both KR and HB demonstrate in vivo binding at the endogenous genomic region corresponding to the enhancer. In contrast, all the false positive PCRMs do not have binding peaks for more than 5 of the TFs and most have less than 3, often reflecting an absence of binding for KR or HB (Starr, 2011).

One interpretation of this data is that the predicted TF binding sites in many of the false positive PCRMs do not represent actual in vivo embryonic binding sites and, as a result, the PCRM is not functional. In addition to KR and HB repressor binding sites, it is also important to consider the presence of potential binding sites for an appropriate activator (FTZ or EVE) necessary for the functional activity of the enhancer. Analysis of the 5 PCRMs that demonstrate in vivo activity reveals that each contains at least 3 strong predicted binding sites for the appropriate pair-rule activator. However, in many cases the false positive PCRMs tested also appear to contain putative activator binding sites. In these cases it is possible that additional architectural requirements (for example, close spacing between multiple activator and/or repressor binding sites) may be necessary for in vivo embryonic activity to occur. In support of this idea, analysis of the genomic fragments that were tested from the iab-2 to iab-8 genomic regions (R10, 11, 12, 13, 14, 15, 17, 20, and 21), predicts that R15 (overlapping IAB5) has a closely-spaced cluster of FTZ-KR sites and that R10 (overlapping IAB2) and R20 (overlapping IAB8) possess a closely spaced cluster of EVE-KR sites within 150 bp of one another, whereas the other regions do not appear to harbor pair-rule activator (FTZ or EVE) and repressor (KR) clusters in such close proximity. A third possibility is that additional protein factors may be involved which may affect the ability of TFs to access the binding sites within the predicted enhancer sequence. Such proteins, which control the recruitment of chromatin components and nucleosome positioning, are thought to be critical to the regulation of embryonic gene expression through the modulation of TF binding affinity at enhancers (Starr, 2011).

The presence of a simple cluster of KR and HB binding sites in many of the enhancers of the BX-C argues that certain precise patterns of TF binding site clusters may be responsible for functional activity among similarly-regulated enhancers. In the IAB8 enhancer, a distinct cluster of EVE-KR binding sites (one KR, two EVE sites) is highly conserved across different Drosophila species. The 3' third of IAB8 harboring the EVE-KR motif (minIAB8) is able to drive reporter gene expression in the characteristic IAB8-pattern in the presumptive A8 segment of transgenic Drosophila. Deletion of the pair of EVE binding sites (∆EVE) significantly weakens enhancer activity in A8, suggesting that while the these two EVE sites are important, cryptic weak EVE binding sites in the remaining sequence of the enhancer (which are sufficiently low scoring to escape computational prediction at the ln(p-value) cutoff of - 6.0) are capable of partially compensating for the loss of the two strong predicted EVE sites. In support of this idea is the recent discovery that even weak affinity binding sites contribute to TF occupancy at regulatory regions in Drososphila embryos. In that study it was found that the level of factor occupancy in vivo correlates more strongly with the degree of chromatin accessibility at a given site, rather than in vitro measurements of the affinity of a factor for a particular DNA sequence (Li, 2011). This observation may be especially relevant in the case of pair-rule factors (such as EVE), where a high localized concentration of the protein in each stripe may also facilitate the increased occupancy of low affinity binding sites (Starr, 2011).

A 141 bp fragment (EK) from within the minIAB8 region containing only the EVE-KR cluster drives strong reporter gene expression in A8, but also ectopic expression immediately posterior of A8 and weaker expression immediately anterior of A8. Ectopic reporter gene expression is also observed in the anterior head domain of the embryo. This result indicates that the EK fragment by itself lacks important binding sites responsible for repression in the anterior head domain of the embryo (such as HB) and for the region immediately anterior of A8 (such as KNI). Several predicted HB and KNI repressor sites capable of performing this role are present within the 602 bp minIAB8 enhancer. Importantly, in the C3-A4 domain of the embryo where the KR repressor protein is expressed, there is a lack of enhancer-driven reporter gene expression from the EK fragment, suggesting that the single KR site within the EVE-KR cluster is sufficient to allow KR-mediated repression in that domain of the embryo. The continued presence of the EVE-KR cluster within the IAB8 enhancer, despite extensive reorganization of TF binding sites across the Drosophila orthologs, is reminiscent of the architectural constraints in the Drosophila and Sepsid eve S2E orthologs, which possess a highly conserved cluster of overlapping BCD activator and KR repressor binding sites necessary for enhancer function (Starr, 2011).

To extend the analysis of the functional role of clustered TF binding sites the IAB5 and IAB7b enhancers from the Drosophila BX-C were also analyzed. Chimeric enhancers assembled from the D. melanogaster and D. pseudoobscura IAB5 orthologs appear to have their functional activity entirely preserved and drive reporter gene expression in presumptive abdominal segments A5, A7 and A9. This result contrasts with an earlier study in which chimeric enhancers assembled from reciprocal halves of D. melanogaster and D. pseudoobscura S2E orthologs did not accurately recapitulate enhancer activity. It is possible that the regulatory output for the chimeric IAB5 enhancers may be subject to very subtle modifications. Such modifications may result in changes to expression patterns that are beyond the detection of the reporter gene assay. However, one explanation for the difference in functional output between these two examples is that in the case of the S2E the organization of TF binding sites within the chimeric enhancer was sufficiently modified so as to destroy the functional activity of the enhancer, whereas for IAB5 this was not the case (Starr, 2011).

To further dissect the organization of TF binding sites in IAB5 the predicted TF binding sites in the sequence were examined. This approach reveals a highly evolutionarily conserved signature TF binding site motif consisting of two strong FTZ activator sites close to two strong KR repressor sites in the center of the defined 1 kb enhancer. The FTZ-KR signature motif is present and intact in both the functional IAB5 chimeric enhancers, cMP and cPM. In the case of the cMP enhancer, the signature motif is present in the IAB5.2 half from D. pseudoobscura, while in the case of the reciprocal cPM enhancer, the signature motif is present in the IAB5.2 half from D. melanogaster. Molecular dissection of IAB5 shows that the individual IAB5.2 halves from Drosophila and D. pseudoobscura each show functional enhancer activity, while the corresponding IAB5.1 halves that lack the FTZ-KR signature motif do not. Furthermore, the 424 bp region containing the center peak of sequence conservation of IAB5 (cIAB5) and the FTZ-KR signature motif drives reporter gene expression in the characteristic three-stripe IAB5-pattern in transgenic Drosophila. In support of the critical functional role of this region, previous functional studies showed that the strongest predicted KR binding site within this signature motif is in fact critical to regulate the spatially restricted expression directed by IAB5 to the posterior presumptive A5, A7, and A9 segments in the Drosophila embryo. In the context of the endogenous gene complex a single point mutation in this KR repressor binding site (Superabdominal mutation) causes an anterior expansion of the embryonic domain of Abd-B expression and results in a homeotic transformation of the A3 segment into the more posterior A5 segment. This result confirms that the strong KR binding site in the signature motif is essential for the in vivo functional activity of the IAB5 enhancer (Starr, 2011).

The IAB7b enhancer, which is expressed in the presumptive A7 segment of the Drosophila embryo, is thought to be regulated by many of the same activators and repressors as IAB5. Bioinformatic analysis reveals that a highly conserved FTZ-KR signature motif, very similar to the one identified in IAB5, is also present in the IAB7b enhancer. Molecular dissection of IAB7b to test the role of the signature motif in the activity of the enhancer demonstrates that a 154 bp region containing the motif (2F2K, with two FTZ and two KR sites) from within the Drosophila IAB7b enhancer is able to drive reporter gene expression in the presumptive A5, A7 and A9 segments of transgenic Drosophila, with notably stronger expression in A7. This expression pattern is very similar to that driven by the IAB5 enhancer. A 114 bp region (2F1K, containing two FTZ and one KR site) from within the Drosophila IAB7b enhancer also drives this same pattern of reporter gene expression, suggesting that the 3' KR site is dispensable for repression of enhancer activity in the central domains of the embryo. Despite the fact that the 3' KR site also overlaps predicted BCD and HB repressor binding sites, no ectopic anterior enhancer-driven expression is observed in the 2F1K construct when tested in transgenic embryos, suggesting that the single remaining 5' KR binding site is sufficient for repression. In fact, in more distantly related Drosophila species, the presence of two KR sites positioned near the pair of FTZ sites is lost, and only a single KR site remains (Starr, 2011).

A 110 bp region (1F2K, containing 1 FTZ and two KR sites) from IAB7b does not drive gene expression, demonstrating that the outer FTZ site is required for activation of the enhancer. One possible molecular explanation for the necessity of the outer FTZ binding site is that FTZ may be acting as a dimer in order to activate IAB5 and IAB7b. In both enhancers a pair of strong FTZ sites are present in the FTZ-KR signature motif. While the ability of FTZ to dimerize has not been reported in the literature, other homeodomain-leucine zipper proteins have been shown to function as dimers. In many such cases the protein factors are also able to bind DNA target sequences as monomers, albeit with comparatively lower affinity. There is also evidence that FTZ is capable of interaction with other proteins, namely the orphan nuclear receptor FTZ-F1 through its LXXLL leucine zipper motif. In this case the heterodimer is capable of co-activation of target genes. Given that the consensus binding sites for the two factors are very different; FTZ (NNYAATTR), FTZ-F1 (BSAAGGDKRDD, it is perhaps to be expected that none of the predicted FTZ and FTZ-F1 binding sites in the IAB5 or IAB7b enhancers directly overlap. However, in future studies it will be of interest to explore the role of FTZ homo- and hetero-dimerization in regulating IAB5 and IAB7b activity (Starr, 2011).

The ability of the 2F2K and 2F1K regions to drive reporter gene expression in an IAB5-like manner in the presumptive A5, A7 and A9 segments of transgenic Drosophila suggests that additional inputs into IAB7b are required to spatially restrict endogenous enhancer-driven gene expression to only the A7 segment. A likely candidate for repression of IAB7b activity in the A5 segment of the embryo is KNI, which is expressed in the presumptive A1-A6 segments. Bioinformatic analysis predicts several candidate KNI binding sites in the full length 728 bp IAB7b enhancer, whereas the 2F2K and 2F1K regions lack any such predicted KNI sites. Previous studies revealed that the repression of the IAB7b enhancer in A5 is mediated by sequences in the 728 bp fragment and does not require additional flanking 5' or 3' regions. In addition, while disruption of the two KR sites in the signature motif does result in reporter gene activation by IAB7b in anterior regions of the embryo, repression persists in the A5 segment. This result indicates that a factor other than KR is responsible for repression in A5. In the entire 728 bp enhancer only three strong KNI sites are predicted, all located in the ~ 300 bp region on the abd-A side of the signature motif. These sites all lie within an evolutionarily conserved genomic region and some of the sites are conserved in distantly related Drosophila species. The significance of these KNI sites in restricting the IAB7b mediated-expression pattern is currently under investigation (Starr, 2011).

A key question in understanding cis-regulatory grammar is why certain arrangements of TF binding sites confer functional enhancer activity while others fail to do so. The turnover of binding sites is common during the evolution of enhancers in different species, yet the functional activity of rapidly-evolving enhancer orthologs from different species is often robust, even across several hundred million years of evolutionary divergence. In the case of the BX-C, bioinformatic analysis demonstrates that there is extensive binding site turnover in the IAB5, IAB7b, and IAB8 enhancers across the Drosophila genus, particularly in more distantly related species. Despite this turnover of TF binding sites, the newly identified FTZ-KR signature motif present in both IAB5 and IAB7b and the functionally important EVE-KR cluster within IAB8 are composed of similar patterns of conserved binding site architecture. Specifically, the organization of sites is such that a pair of strong activator (FTZ or EVE) binding sites and at least one strong repressor (KR) site are in close proximity (< 116 bp) to each other. Notably, the spacing between the FTZ and KR sites in the signature motif is largely unchanged across IAB5 and IAB7b enhancer orthologs in distantly related Drosophila species, although in the case of IAB7b there is the loss of the secondary KR binding site in species more distantly related to Drosophila. Conservation of genomic architecture of these TFBSs in the BX-C enhancers does not directly indicate that the specific spacing between sites is essential. However, the functional activity of genomic regions containing these motifs supports previous findings that closely spaced activator and repressor binding sites are critical for enhancer function and suggests that the architecture of binding sites within an enhancer is subject to significant evolutionary constraint (Starr, 2011).

It has recently been suggested through computational synthetic evolution studies that the inherent bias for deletions over insertions in the genome of Drosophila (and many other species) may result in the gradual loss of nucleotide space between TF binding sites. In effect, this deletion bias helps to artificially cluster binding sites together. In this case, although clustering of TF binding sites may not itself be a feature originally selected for in evolution on the basis of its functional significance, once established in the genome it may still play a functional role in enhancer activity. Molecular dissection of IAB5, IAB7b, and IAB8 enhancer function argues that specific clusters of activator and repressor binding sites do play a key role in enhancer activity. As a result, such clusters, once present in enhancers, may well be under positive evolutionary selective pressure, as evidenced by the largely invariant organization of the binding sites in the IAB5 and IAB7b FTZ-KR signature motif. This selection does not preclude the possibility that if binding sites arise nearby in the genome de novo, these new binding sites may also contribute to enhancer functional activity. In this scenario, the original TF binding site cluster may no longer be necessary for enhancer function. Indeed, in the case of the IAB8 enhancer, the ∆EVE region tested in s transgenic assay may be an example of this phenomenon. This fragment is able to exhibit a weak IAB8-like enhancer function even with the deletion of the pair of strong predicted EVE binding sites, potentially through the activity of weaker EVE binding sites that are present in the remaining sequence (Starr, 2011).

Although the precise spatial arrangement of TF binding sites within an enhancer may not exactly mirror the ancestral arrangement, computational predictions suggest that functional clusters of TF binding sites are likely to result from the spatial re-organization of older pre-existing sites during evolution. Such clusters therefore also likely indicate genomic regions with robust enhancer activity. The fact that enhancer activity in the BX-C appears to be dependent on signature motifs that represent specific spatial arrangements of TF binding sites in minimal modular regions, indicates that the physical patterns of binding site clustering are functionally significant in terms of enhancer architecture (Starr, 2011).

Combinatorial activation and concentration-dependent repression of the Drosophila even skipped stripe 3+7 enhancer

Despite years of study, the precise mechanisms that control position-specific gene expression during development are not understood. This study analyzed an enhancer element from the even skipped (eve) gene, which activates and positions two stripes of expression (stripes 3 and 7) in blastoderm stage Drosophila embryos. Previous genetic studies showed that the JAK-STAT pathway is required for full activation of the enhancer, whereas the gap genes hunchback (hb) and knirps (kni) are required for placement of the boundaries of both stripes. The maternal zinc-finger protein Zelda (Zld) is absolutely required for activation, and evidence is presented that Zld binds to multiple non-canonical sites. A combination of in vitro binding experiments and bioinformatics analysis was used to redefine the Kni-binding motif, and mutational analysis and in vivo tests to show that Kni and Hb are dedicated repressors that function by direct DNA binding. These experiments significantly extend understanding of how the eve enhancer integrates positive and negative transcriptional activities to generate sharp boundaries in the early embryo (Struffi, 2011).

The experiments described in this study significantly refine understanding of how the eve 3+7 enhancer functions in the early embryo. In particular, it was shown that the maternal zinc-finger protein Zld is absolutely required for STAT-mediated enhancer activation, and that the gap proteins Kni and Hb establish stripe boundaries by directly binding to multiple sites within the enhancer (Struffi, 2011).

When first activated in late nuclear cycle 13, the minimal eve 3+7 enhancer drives weak stochastic expression in a broad central pattern, which refines in cycle 14 to a stripe that is about four nuclei wide. By contrast, stripe 7 expression, which is visible by enzymatic staining methods, is nearly undetectable using fluorescence in situ hybridization (Struffi, 2011).

Previous work showed that stripe 7 shares regulatory information with stripe 3 but is also controlled by sequences located between the minimal stripe 3+7 and stripe 2 enhancers, and possibly by sequences within and downstream of the stripe 2 enhancer. Thus, stripe 7 is unique among the eve stripes in that it is not regulated by a discrete modular element (Struffi, 2011).

Previous work showed that the terminal gap gene tailless (tll) is required for activation of eve 7. However, since the Tll protein probably functions as a dedicated repressor, it is likely that activation of eve 7 by Tll occurs indirectly, through repression of one or more repressors (Struffi, 2011).

The ubiquitous maternal protein Zld is required for the in vivo function of both the eve 3+7 and eve 2 enhancers, which are activated by the JAK-STAT pathway and Bicoid (Bcd), respectively. Zld was previously shown to bind to five sequence motifs that are over-represented in the regulatory regions of early developmental genes. Mutations of the single TAGteam site in the eve 3+7 enhancer caused a reduction in expression, but zld M- embryos, mutant for maternal zld expression, showed complete abolishment of eve 3+7-lacZ reporter gene expression. Also, the eve 2 enhancer, which does not contain any canonical TAGteam sites, is nonetheless inactive in zld M- embryos. This study showed that this enhancer contains at least four variants of the TAGteam sites, which suggests that Zld binding to non-canonical sites is crucial for its function in embryogenesis. ChIP-Chip data show that Zld binding extends throughout much of the eve 5' and 3' regulatory regions (Struffi, 2011).

The implication of such broad binding and the requirement for Zld for activation of two eve enhancers are consistent with its proposed role as a global activator of zygotic transcription. How might this work? One possibility is that there are cooperative interactions between Zld and the other activators of these stripes. A non-exclusive alternative is that Zld binding creates a permissive environment in broad regions of the genome, possibly by changing the chromatin configuration and making it more likely that the other activator proteins can bind. However, it is important to note that eve expression is not completely abolished in zld M- embryos, so at least some eve regulatory elements could function in the absence of Zld. Future experiments will be required to further characterize the role of Zld in the regulation of the entire eve locus (Struffi, 2011).

The genetic removal of kni causes a broad expansion of eve 3+7- lacZ expression in posterior regions of the embryo, and ectopic Kni causes a strong repression of both stripes. Interestingly, the posterior boundary of eve stripe 3 is positioned in regions with extremely low levels of Kni protein. If the stripe 3 posterior boundary is solely formed by Kni, the enhancer must be exquisitely sensitive to its repression, possibly through the high number of sites in the eve 3+7 enhancer. Previous attempts to mutate sites based on computational predictions failed to mimic the genetic loss of kni, so this study used a biochemical approach to identify Kni sites in an unbiased manner. EMSA analyses identified 11 Kni sites, and the PWM derived from these sites alone is very similar to the Kni matrix derived in a bacterial one-hybrid study. Thus, these studies provide biochemical support for the bacterial one-hybrid method as an accurate predictor of the DNA-binding activity of this particular protein (Struffi, 2011).

It was further shown that specific point mutations abolish binding to nine of the 11 sites, and when these mutations were tested in a reporter gene they caused an expansion that is indistinguishable from that detected in kni mutants. This result strongly suggests that Kni-mediated repression involves direct binding to the eve 3+7 enhancer, and that Kni alone can account for all repressive activity in nuclei that lie in the region between stripes 3 and 7. However, this work does not address the exact mechanism of Kni-mediated repression. The simplest possibility is that Kni competes with activator proteins for binding to overlapping or adjacent sites. This mechanism is considered unlikely because only one of the 11 Kni sites overlaps with an activator site. Also, the in vivo misexpression of a truncated Kni protein (Kni 1-105) that contains only the DNA-binding domain and the nuclear localization signal has no discernible effect on the endogenous eve expression pattern, whereas a similar misexpression of Kni 1-330 or Kni 1-429 strongly represses eve 3+7 (Struffi et al., 2004) (Struffi, 2011).

Whereas Kni-mediated repression forms the inside boundaries of the eve 3+7 pattern, forming the outside boundaries is dependent on Hb, which abuts the anterior boundary of stripe 3 and overlaps with stripe 7. Both stripes expand towards the poles of the embryo in zygotic hb mutants, and these expansions are mimicked by mutations in four or all nine Hb sites within the eve 3+7 enhancer. Further anterior expansions of the pattern are prevented by an unknown Bcd-dependent repressor (X) and the Torso (Tor)-dependent terminal system. Indeed, eve 3+7-lacZ expression expands all the way to the anterior tip in mutants that remove bcd and the terminal system (Struffi, 2011).

The mutational analyses suggest that Hb is a dedicated repressor of the eve 3+7 enhancer, and argue against a dual role in which high Hb levels repress, whereas lower concentrations activate, transcription. One caveat is that activation of the stripe might occur via maternal Hb in the absence of zygotic expression. However, triple mutants that remove zygotic hb, kni and tor, a terminal system component, show eve 3+7 enhancer expression that extends from ~75% embryo length (100% is the anterior pole) to the posterior pole. It is extremely unlikely that the maternal Hb gradient, which is not perturbed in this mutant combination, could activate expression throughout the posterior region. It is proposed that any activating role for Hb on this enhancer is indirect and might occur by repressing kni, which helps to define a space where the concentrations of both repressors are sufficiently low for activation to occur. kni expands anteriorly in hb mutants and is very sensitive to repression by ectopic Hb, consistent with an indirect role in activation. A similar mechanism has been shown to be important for the correct positioning of eve stripe 2. In this case, the anterior Giant (Gt) domain appears to be required for eve 2 activation, but it does so by strongly repressing Kr, thus creating space for activation in the region between Gt and Kr (Struffi, 2011).

The correct ordering of gene expression boundaries along the AP axis is crucial for establishing the Drosophila body plan. All gap genes analyzed so far seem to function as repressors that differentially position multiple boundaries. However, it is still unclear how differential sensitivity is achieved at the molecular level. Simple correlations of binding site number and affinity with boundary positioning cannot explain the exquisite differences in the sensitivity of individual enhancers, suggesting that they do more than 'count' binding sites and that specific arrangements of repressor and activator sites might control this process. The experiments described here better define the binding characteristics of both Hb and Kni and provide a firm foundation for future experiments designed to decipher the regulatory logic that controls differential sensitivity (Struffi, 2011).

The Drosophila gap gene network is composed of two parallel toggle switches

Drosophila gap genes provide the first response to maternal gradients in the early fly embryo. Gap genes are expressed in a series of broad bands across the embryo during first hours of development. The gene network controlling the gap gene expression patterns includes inputs from maternal gradients and mutual repression between the gap genes themselves. In this study a modular design is proposed for the gap gene network, involving two relatively independent network domains. The core of each network domain includes a toggle switch corresponding to a pair of mutually repressive gap genes, operated in space by maternal inputs. The toggle switches present in the gap network are evocative of the phage lambda switch, but they are operated positionally (in space) by the maternal gradients, so the synthesis rates for the competing components change along the embryo anterior-posterior axis. Dynamic model, constructed based on the proposed principle, with elements of fractional site occupancy, required 5-7 parameters to fit quantitative spatial expression data for gap gradients. The identified model solutions (parameter combinations) reproduced major dynamic features of the gap gradient system and explained gap expression in a variety of segmentation mutants (Papatsenko, 2011).

Fertilized eggs of Drosophila contain several spatially distributed maternal determinants - morphogen gradients, initiating spatial patterning of the embryo. One of the first steps of Drosophila embryogenesis is the formation of several broad gap gene expression patterns within first 2 hrs of development. Gap genes are regulated by the maternal gradients, so their expression appears to be hardwired to the spatial (positional) cues provided by the maternal gradients; in addition, gap genes are involved into mutual repression. How the maternal positional cues and the mutual repression contribute to the formation of the gap stripes has been a subject of active discussion (Papatsenko, 2011).

Accumulated genetics evidence and results of quantitative modeling suggest the occurrence of maternal positional cues (position-specific activation potentials), contributing to spatial expression of four trunk gap genes: knirps (kni), Kruppel (Kr), hunchback (hb) and giant (gt). Existing data suggest that the central Knirps domain stripe is largely the result of activation by Bicoid (Bcd) and repression by Hunchback. Central domain Kruppel stripe is the result of both activation and repression from Hunchback, which acts as a dual transcriptional regulator on Kr. Hunchback is one of the most intriguing among the segmentation genes. Maternal hb mRNA is deposited uniformly, but its translation is limited to the anterior, zygotic anterior expression of hb is under control of Bcd and Hb itself. Zygotic posterior expression of Hunchback (not included in the current model) is under the control of the terminal torso signaling system. Giant is activated by opposing gradients of Bicoid and Caudal and initially expressed in a broad domain, which refines later into anterior and posterior stripes. This late pattern appears to be the consequence of Kruppel repression (Papatsenko, 2011).

Predicting functional properties of a gene network combining even a dozen genes may be a difficult task. To facilitate the functional exploration, gene regulatory networks are often split into network domains or smaller units, network motifs with known or predictable properties. The network motif based models can explain dynamics of developmental gradients and even evolution of gradient systems and underlying gene regulatory networks. The gene network leading to the formation of spatial gap gene expression patterns is an example, where simple logic appeared to be far behind the system's complexity. Gap genes provide first response to maternal gradients in the early fly embryo and form a series of broad stripes of gene expression in the first hours of the embryo development. While the system has been extensively studied in the past two decades both in vivo and in silico a simple and comprehensive model explaining function of the entire network has been missing (Papatsenko, 2011).

In the current study, a modular design has been proposed for the gap gene network; the network has been represented as two similar parallel modules (or two sub networks). Each module involved three network motifs, two for maternal inputs (one for one gap gene) and a toggle switch describing mutual repression in the pair of the gap genes. Formally, the toggle switches present in the gap gene network are evocative of the bistable phage lambda switch; however, they are operated by maternal inputs and their steady state solutions depend on spatial position in embryo, not environmental variables. The proposed modular design accommodated 5-7 realistic parameters and reproduced major known features of the gap gene network (Papatsenko, 2011).

Response to the BMP gradient requires highly combinatorial inputs from multiple patterning systems in the Drosophila embryo

Pattern formation in the developing embryo relies on key regulatory molecules, many of which are distributed in concentration gradients. For example, a gradient of BMP specifies cell fates along the dorsoventral axis in species ranging from flies to mammals. In Drosophila, a gradient of the BMP molecule Dpp gives rise to nested domains of target gene expression in the dorsal region of the embryo; however, the mechanisms underlying the differential response are not well understood, partly owing to an insufficient number of well-studied targets. This study analyzed how the Dpp gradient regulates expression of pannier (pnr), a candidate low-level Dpp target gene. It was predicted that the pnr enhancer would contain high-affinity binding sites for the Dpp effector Smad transcription factors, which would be occupied in the presence of low-level Dpp. Unexpectedly, the affinity of Smad sites in the pnr enhancer was similar to those in the Race enhancer, a high-level Dpp target gene, suggesting that the affinity threshold mechanism plays a minimal role in the regulation of pnr. The results indicate that a mechanism involving a conserved bipartite motif that is predicted to bind a homeodomain factor in addition to Smads and the Brinker repressor, establishes the pnr expression domain. Furthermore, the pnr enhancer has a highly complex structure that integrates cues not only from the dorsoventral axis, but also from the anteroposterior and terminal patterning systems in the blastoderm embryo (Liang, 2012).

Most blastoderm genes are regulated primarily on either the DV or AP axis. For example, the gap genes are expressed in one or two domains of expression along the AP axis and, although some of them may exhibit regulation along the DV axis, they are nonetheless considered AP genes. pnr represents an interesting case because although it was originally reported as a DV gene, closer inspection of its expression pattern in wild-type and mutant embryos and detailed dissection of its cis-regulatory enhancers revealed that pnr is highly regulated by both AP and DV genes. Its pattern is a composite of two superimposed patterns that each exhibit AP and DV spatial regulation: a dorsal patch and six AP stripes, which are limited to the dorsal 30% of the embryo. The patch domain, but not the stripes, disappeared in dpp mutants, whereas both the patch and stripes expand ventrally in the absence of Brk. The stripes are more sensitive to Brk repression because activation of the patch domain is limited to the region where Dpp is present dorsally, whereas the stripes can be activated along the entire DV axis. Brk in the ventrolateral region and Sna in the ventral-most region repress stripe expression. Since pnr specifies dorsomedial fates, restricting its expression to the dorsal 30% of the circumference is crucial. Ectopic expression of pnr ventrally causes transformation of ventral epidermis into dorsomedial epidermis (Liang, 2012).

Competition between Brk and Smads for binding to overlapping DNA sequences is likely to set the border of the patch domain. Two Smad sites are particularly important for patch expression, and one of these, the M3 site, is a composite site that binds both Brk and Smads, raising the possibility that the patch border is established by competition between activating inputs from Smads in the dorsal region and repressive inputs from Brk emanating from the ventral region. Competition between Brk and Smads for overlapping binding sites has been observed for several Dpp target enhancers (Liang, 2012).

Repression of the AP stripes ventrally requires both Brk sites B1 and B2. The two posterior stripes driven by P3 expand to a lesser degree than the four anterior stripes driven by P4. This can be explained by the fact that P4 lacks Brk site B1, which is a stronger Brk site. Loss of both Brk sites would likely result in expansion to the edge of the mesoderm, as seen in embryos that lack Brk protein. Repression by Sna is likely to involve the Sna binding sites in the pnr enhancer, as genome-wide binding studies have shown that the pnr enhancer is bound by Sna (Liang, 2012).

The positioning of the stripes, as well as of the patch, along the AP axis is regulated by the gap genes. The results suggest that Hb, Gt and Tll set the anterior edge of the pnr domain, whereas Tll sets the posterior, and that direct and indirect interactions among the gap proteins establish the stripe borders relative to one another, as has been observed for eve. For example, the broad central stripe seen in kni- could be explained by the lack of direct Kni repression. However, owing to the complex cross-regulatory interactions among the gap genes, it is difficult to predict which gap proteins regulate the pnr stripes directly, although genome-wide binding data of the gap factors support their direct binding to the pnr enhancer. Although Bcd does not appear to bind directly to the pnr enhancer, its effects are mediated through its targets Gt and Hb (Liang, 2012).

In depth studies of three genes with different boundary positions in the dorsal region, Race, C15 and pnr, indicate that complex combinatorial mechanisms are employed to establish their expression domains, with each gene having a unique regulatory network of its own. Although they all respond to Dpp signaling, their borders of expression are not set by a simple threshold response to the Dpp gradient that depends on differential binding site affinity (Liang, 2012).

The feature that has been shown to be important for high-level Dpp target expression is the feed-forward motif involving Dpp and Zen. High levels of Dpp/Smads first activate zen expression in the dorsal-most region, the presumptive amnioserosa, and then both Zen and Smads bind and activate the Race enhancer. The intermediate-level target C15 has a different enhancer structure than high-level targets, containing many Smad sites that act in a cumulative manner to drive expression in regions of intermediate Dpp levels. Mutation analysis has shown that the number of intact Smad binding sites, rather than their affinity, is important for the C15 response. Nevertheless, the enhancer structure of C15 might promote high levels of Smad binding in vivo, and this may increase the response to Dpp. Do all intermediate-level Dpp targets have a similar enhancer structure? The enhancer that drives expression of the intermediate-level Dpp target gene tup was examined for putative Smad binding sites (SBEs and GC-rich regions), and observed multiple Smad sites across the enhancer, similar to that seen with C15. Thus, the multiple Smad site signature might be necessary for response to lower than peak levels of Dpp. In addition, intermediate-leveltargets may utilize repression mechanisms to help establish their borders of expression, as was shown for C15 (Liang, 2012).

These studies have revealed that the pnr enhancer resembles that of a high-level target in Smad site organization and Smad binding site affinity. In fact, it was surprisingly easy to convert the low-level target enhancer into a high-level target by mutating a single Smad site. This result could be easily explained if the M3 site had a higher affinity for Smads than those in Race; however, comparison of the binding sites by gel shift showed they have similar affinities. Furthermore, replacing the M3 Smad site with a Race Smad site had little effect on the expression pattern. These results suggest that activation of pnr in its broad domain has little to do with Smad binding affinity. How then does pnr respond to low levels of Dpp? One possible mechanism involves the highly conserved AGCAATTAA site that lies adjacent to the Smad sites. In the absence of this site, the P3 enhancer could not respond to low-level Dpp. It is possible that this site, when bound, leads to greater Smad binding, which would then promote pnr activation (Liang, 2012).

What factor(s) might bind to the AGCAATTAA site? ATTA is the core binding site for Antp class HD proteins. Although Zen binds to the ATTA site in vitro, neither the endogenous pnr pattern nor P3-lacZ expression is significantly affected in zen mutants. To identify candidate factors, the TOMTOM tool at FlyFactor Survey was used, and the best match was to the HD protein Hmx, which binds CAATTAA. However, Drosophila Hmx is expressed only in an anterior region that does not overlap with pnr (see FlyBase). Likewise, although several Antp class HD proteins were predicted to bind to the ATTA core sequence, their timing or domains of expression do not overlap ideally with those of pnr (Liang, 2012).

It has been proposed that the AGCAATTAA site in the Msx2 enhancer might bind a factor in addition to an HD protein via the 5' half of the site, perhaps a transcriptional partner such as FAST1, which was previously shown to function with Smads. Although the search did not reveal any candidates, if this is the case for pnr then the bipartite motif could potentially bind four proteins: Smads, Brk, HD and 'partner X', The combination of these proteins in a given cell along the DV axis would determine pnr transcriptional activity. The fact that the bipartite motif is not present in the enhancers of Race or C15, or in the other pnr enhancers identified, demonstrates the versatility of how Dpp uses different partners to establish multiple target gene domains (Liang, 2012).

Is the structure of the pnr enhancer typical for low-level Dpp targets? This is difficult to address owing to the lack of candidate low-level Dpp targets. Brk is considered a low-level Dpp target in imaginal disc development; however, Dpp represses brk, giving rise to a reciprocal gradient of the Brk repressor. Target gene borders are thus established by competition between Smad and Brk for overlapping binding sites, as mentioned above for pnr. The brk enhancer contains multiple enhancer/silencer modules consisting of activator and repressor (Mad/Medea/Schnurri) binding sites, which contribute to threshold responses to the Dpp gradient, and thus it does not resemble the pnr enhancer. Although good progress has been made in understanding how pnr is expressed in regions with low levels of Dpp, learning the general rules that control broad dorsal patterns will require the analysis of more enhancer elements (Liang, 2012).

What rules do target genes for other morphogens follow? Long before the 'feed-forward' term was it was shown that both the Dl and Bcd morphogens interact with their high-level targets, Twi and Hb, respectively, to activate downstream; thus, combinatorial motifs are generally utilized. Moreover, as more target genes of Dl and Bcd were identified and studied, it became apparent that the affinity threshold model could not explain all cases of differential response to the gradient. For example, analysis of several enhancers that drive Bcd-dependent expression in anterior regions of the embryo revealed a poor correlation between Bcd binding site affinity and the AP limits of the pattern. Also, although Dl targets remain archetypal examples of genes that utilize the affinity threshold mechanism, it was found that genes expressed in the lateral region also require input from the Zelda (Vielfaltig - FlyBase) transcription factor for expression in regions of low-level Dl. Zelda binding sites are present in target enhancers, and it was proposed that Zelda boosts Dl binding to help activate the neuroectodermal genes (Liang, 2012).

Downstream target gene interactions also shape domains of expression, in particular cross-

repression among the targets. In both the Drosophila neuroectoderm and the vertebrate neural tube, morphogen targets are expressed in discrete domains rather than nested overlapping domains due to the repression of one target by another. This mechanism establishes sharp boundaries among the target genes (Liang, 2012).

Thus, it is clear that additional factors help morphogens set threshold responses. Given that the pnr enhancer could potentially interact with four different factors along the DV axis and at least four factors along the AP axis, several combinations of inputs could regulate other Dpp target genes. More generally, depending on the number of different factors that interact with the cis-regulatory regions of target genes, morphogen gradients could elicit multiple threshold responses, as has been seen for morphogens such as Dl in Drosophila, Activin in the Xenopus blastula and Shh in the vertebrate neural tube, where up to seven threshold responses have been described. Only by dissecting enhancers can it be fully understood how target genes integrate diverse inputs (Liang, 2012).

Naturally occurring deletions of Hunchback binding sites in the Even-Skipped stripe 3+7 enhance

Changes in regulatory DNA contribute to phenotypic differences within and between taxa. Comparative studies show that many transcription factor binding sites (TFBS) are conserved between species whereas functional studies reveal that some mutations segregating within species alter TFBS function. Consistently, in this analysis of 13 regulatory elements in Drosophila melanogaster populations, single base and insertion/deletion polymorphism are rare in characterized regulatory elements. Experimentally defined TFBS are nearly devoid of segregating mutations and, as has been shown before, are quite conserved. For instance 8 of 11 Hunchback binding sites in the stripe 3+7 enhancer of even-skipped are conserved between D. melanogaster and Drosophila virilis. Oddly, a 72 bp deletion was found that removes one of these binding sites (Hb8), segregating within D. melanogaster. Furthermore, a 45 bp deletion polymorphism in the spacer between the stripe 3+7 and stripe 2 enhancers, removes another predicted Hunchback site. These two deletions are separated by approximately 250 bp, sit on distinct haplotypes, and segregate at appreciable frequency. The Hb8Delta is at 5 to 35% frequency in the new world, but also shows cosmopolitan distribution. There is depletion of sequence variation on the Hb8Delta-carrying haplotype. Quantitative genetic tests indicate that Hb8Delta affects developmental time, but not viability of offspring. The Eve expression pattern differs between inbred lines, but the stripe 3 and 7 boundaries seem unaffected by Hb8Delta. The data reveal segregating variation in regulatory elements, which may reflect evolutionary turnover of characterized TFBS due to drift or co-evolution (Palsson, 2014).

Analysis of functional importance of binding sites in the Drosophila gap gene network model

The statistical thermodynamics based approach provides a promising framework for construction of the genotype-phenotype map in many biological systems. Among important aspects of a good model connecting the DNA sequence information with that of a molecular phenotype (gene expression) is the selection of regulatory interactions and relevant transcription factor bindings sites. As the model may predict different levels of the functional importance of specific binding sites in different genomic and regulatory contexts, it is essential to formulate and study such models under different modeling assumptions. This study elaborates a two-layer model for the Drosophila gap gene network and includes in the model a combined set of transcription factor binding sites and concentration dependent regulatory interaction between gap genes hunchback and Kruppel. The new variants of the model are more consistent in terms of gene expression predictions for various genetic constructs in comparison to previous work. The functional importance of binding sites was quantified by calculating their impact on gene expression in the model, and how these impacts correlate across all sites were calculated under different modeling assumptions. The assumption about the dual interaction between hb and Kr leads to the most consistent modeling results, but, on the other hand, may obscure existence of indirect interactions between binding sites in regulatory regions of distinct genes. The analysis confirms the previously formulated regulation concept of many weak binding sites working in concert. The model predicts a more or less uniform distribution of functionally important binding sites over the sets of experimentally characterized regulatory modules and other open chromatin domains (Kozlov, 2015).

hunchback: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | Post-transcriptional Regulation | Effects of Mutation | References

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