Targets of Activity

The asymmetric distribution of the gap gene knirps (kni) in discrete expression domains is critical for striped patterns of pair-rule gene expression in the Drosophila embryo. To test whether these domains function as sources of morphogenetic activity, the stripe 2 enhancer of the pair-rule gene even-skipped was used to express kni in an ectopic position. Manipulating the stripe 2-kni expression constructs and examining transgenic lines with different insertion sites led to the establishment of a series of independent lines that display consistently different levels and developmental profiles of expression. Individual lines show specific disruptions in pair-rule patterning that are correlated with the level and timing of ectopic expression. No effect on the expression patterns of giant or Krüppel could be observed at any level of kni misexpression. However, the ectopic kni did significantly alter the hunchback pattern. Stripe 2-kni, centered on PS3, completely prevents the expression of the PS4 hb stripe. This expression occurs even in embryos that contain the lowest levels of ectopic kni (Kosman, 1997).

It is likely the KNI functions as a repressor to set the posterior border of eve stripe three. To test whether the early repression of eve stripe 3 is mediated through the eve stripe three enhancer, stripe 2-kni constructs were crossed with a line carrying lacZ under the control of this enhancer. Ectopic kni specifically represses the stripe 3 enhancer in a dose-dependent manner. Stripe 2-kni causes disruption of runt stripes 2 and 3, but has no effect on stripe 1. The repression of stripe 3 increases in proportion to the level of ectopic kni, a response similar to that seen for eve stripe 3. Different levels of ectopic kni cause disruptions of fushi tarazu stripes 2 and 3, but have no effect on the expression of ftz stripe 1. It is possible that these effects are indirect and may be mediated through other segmentation genes but this possibility is made unlikely by the fact that hairy expression is virtually unaffected in stripe 2-kni embryos. These results suggest that the ectopic domain of kni acts as a source for morphogenetic activity that specifies regions in the embryo where pair-rule genes can be activated or repressed. Evidence is presented that the level and timing of expression, as well as protein diffusion, are important for determining the specific responses of target genes (Kosman, 1997).

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).

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).

Knirps regulates Krüppel by acting as a competitive repressor of Bicoid-mediated activation of Krüppel. Knirps binds at a single site in a 730bp regulatory region located 3.4 kb upstream of Krüppel. The Krüppel site (Kr730) contains a single strong KNI binding site, a 16 base pair element. The KNI binding site overlaps with one BCD binding site (Hoch, 1992 and references).

Gradients of gap proteins position adjacent stripes of gene expression in the posterior of Drosophila embryos. How is this accomplished? Regulatory sequences of hairy have been identified that are critical for the expression of h stripes 5 and 6. Stripe 6 is dependent on Knirps for its activation, while stripe 5 likely requires a combination of both gap and non-gap activating proteins. The Knirps activator binds to the stripe 6 enhancer (Langeland, 1994).

Gap genes Kruppel (Kr), knirps (kni), and tailless (tll) control the expression of the pair-rule gene hairy (h) by activating or repressing independent cis-acting units that generate individual stripes. KR activates stripe 5 and represses stripe 6, KNI activates stripe 6 and represses stripe 7, and TLL activates stripe 7. KR and KNI proteins bind strongly to h control units that generate stripes in areas of low concentration of the respective gap gene products and weakly to those that generate stripes in areas of high gap gene expression. These results indicate that KR and KNI proteins form overlapping concentration gradients that generate the periodic pair-rule expression pattern (Pankratz, 1990).

The expression of the pair-rule gene hairy (h) in seven evenly spaced stripes along the longitudinal axis of the Drosophila blastoderm embryo is mediated by a modular array of separate stripe enhancer elements. The minimal enhancer element, which generates reporter gene expression in place of the most posterior h stripe 7 (h7-element), contains a dense array of binding sites for factors providing the trans-acting control of h stripe 7 expression as revealed by genetic analyses. The stripe seven enhancer is found in a minimal 932 bp region from a 1.5 kb DNA fragment of the h upstream region. The h7-element mediates position-dependent gene expression by sensing region-specific combinations and concentrations of both the maternal homeodomain transcriptional activators, Caudal and Bicoid, and of transcriptional repressors encoded by locally expressed zygotic gap genes. Zygotic caudal expression is not required for activation. Caudal and Bicoid, which form complementing concentration gradients along the longitudinal axis of the embryo, function as redundant activators, indicating that the anterior determinant Bicoid is able to activate gene expression in the most posterior region of the embryo. The spatial limits of the h stripe-7 domain are brought about by the local activities of repressors that prevent activation. The spatial limit of h7 is significantly altered in the gap mutants tailless, knirps and kruppel, but not in embryos lacking either hunchback, giant or huckebein. There are seven binding sites for Bcd, twenty-three for caudal, five for Kruppel, fourteen for Knirps, eight for Hunchback and five for Tailless. In the absence of both cad and bcd, activation still occurs. Thus, a third activator, likely to be Kr, must function in such embryos. It is thought that Kr acts as both a repressor and an activator within the h7 element depending on its concentration. The posterior border is set in response to Tll activity under the control of the terminal maternal organizer system. The anterior border of the expression domain is due to repression in response to Kni. The results suggest that the gradients of Bicoid and Caudal combine their activities to activate segmentation genes along the entire axis of the embryo (La Rosee, 1997).

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 knirps mutants, stripe 4 fails to separate from 5, and 6 from 7 (Gutjahr, 1993).

Expression of the abdominal-A and Abdominal-B genes of the bithorax complex (BX-C) of Drosophila is controlled by a cis-regulatory promoter and by distal enhancers called infraabdominal regions. The activation of these regions along the anterior/posterior axis of the embryo determines where abdominal-A and Abdominal-B are transcribed. There is spatially restricted transcription of the infraabdominal regions, reflective of this specific activation. 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).

The closely linked pair of POU domain genes pdm-1 and pdm-2 are first expressed in the presumptive abdomen early during cellularization, where they form 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).

To understand the nature of the regulatory signals impinging on the second promoter of the Antennapedia gene (Antp P2), analysis of its expression in mutants and in inhibitory drug injected embryos has been carried out. Products of the zygotically-active segmentation genes ftz, hb, Kr, gt and kni then activate or repress 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)

Transcriptional repression is a key mechanism operating at multiple levels to control Abd-B expression. The anterior Abdominal-B expression limit is apparently determined by Krüppel repression, whereas the knirps repressor may be responsible for the graded Abd-B expression within the Abd-B domain. iab-5 and two other fragments (MCP and FAB) show region-specific silencing activity: from a distance they suppress Abd-B expression, mediated by a linked heterologous enhancer. Silencing requires hunchback as well as Polycomb function and evidently provides maintenance of Abd-B expression limits throughout embryogenesis (Busturia, 1993).

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).

Precision of hunchback expression in the Drosophila embryo

Activation of the gap gene hunchback (hb) by the maternal Bicoid gradient is one of the most intensively studied gene regulatory interactions in animal development. Most efforts to understand this process have focused on the classical Bicoid target enhancer located immediately upstream of the P2 promoter. However, hb is also regulated by a recently identified distal shadow enhancer as well as a neglected 'stripe' enhancer, which mediates expression in both central and posterior regions of cellularizing embryos. This study employed BAC transgenesis and quantitative imaging methods to investigate the individual contributions of these different enhancers to the dynamic hb expression pattern. These studies reveal that the stripe enhancer is crucial for establishing the definitive border of the anterior Hb expression pattern, just beyond the initial border delineated by Bicoid. Removal of this enhancer impairs dynamic expansion of hb expression and results in variable cuticular defects in the mesothorax (T2) due to abnormal patterns of segmentation gene expression. The stripe enhancer is subject to extensive regulation by gap repressors, including Kruppel, Knirps, and Hb itself. It is proposed that this repression helps ensure precision of the anterior Hb border in response to variations in the Bicoid gradient (Perry, 2012).

hunchback (hb) is the premier gap gene of the segmentation regulatory network. It coordinates the expression of other gap genes, including Kruppel (Kr), knirps (kni), and giant (gt) in central and posterior regions of cellularizing embryos. The gap genes encode transcriptional repressors that delineate the borders of pair-rule stripes of gene expression. hb is activated in the anterior half of the precellular embryo, within 20-30 min after the establishment of the Bicoid gradient during nuclear cleavage cycles 9 and 10 (~90 min following fertilization). This initial hb mRNA transcription pattern exhibits a reasonably sharp on/off border within the presumptive thorax. This border depends on cooperative interactions of Bicoid monomers bound to linked sites in the proximal ('classical') enhancer. However, past studies and recent computational modeling suggest that Bicoid cooperativity is not sufficient to account for this precision in hb expression (Perry, 2012).

The hb locus contains two promoters, P2 and P1, and three enhancers. The 'classical' proximal enhance and distal shadow enhancer mediate activation in response to the Bicoid gradient. Expression is also regulated by a third enhancer, the 'stripe' enhancer, which is located over 5 kb upstream of P2. Each of these enhancers was separately attached to a lacZ reporter gene and expressed in transgenic embryos. As shown previously, the Bicoid target enhancers mediate expression in anterior regions of nuclear cleavage cycle (cc) 12-13 embryos, whereas the stripe enhancer mediates two stripes of gene expression at later stages, during cc14. The anterior stripe is located immediately posterior to the initial hb border established by the proximal and distal Bicoid target enhancers (Perry, 2012).

BAC transgenesis was used to determine the contribution of the stripe enhancer to the complex hb expression pattern. For some of the experiments, the hb transcription unit was replaced with the yellow (y) reporter gene, which contains a large intron permitting quantitative detection of nascent transcripts. The resulting BAC mimics the endogenous expression pattern, including augmented expression at the Hb border. However, removal of the stripe enhancer from an otherwise intact y-BAC transgene leads to diminished expression at this border and in posterior regions (Perry, 2012).

The functional impact of removing the stripe enhancer was investigated by genetic complementation assays. A BAC transgene containing 44 kb of genomic DNA encompassing the entire hb locus and flanking regulatory DNAs fully complements deficiency homozygotes carrying a newly created deletion that cleanly removes the hb transcription unit. The resulting adults are fully viable, fertile, and indistinguishable from normal strains. Embryos obtained from these adults exhibit a normal Hb protein gradient, including a sharp border located between eve stripes 2 and 3 (Perry, 2012).

The Hb BAC transgene lacking the stripe enhancer fails to complement hb/hb mutant embryos due to the absence of the posterior hb expression pattern, which results in the fusion of the seventh and eighth abdominal segments. In addition, the anterior Hb domain lacks the sharp 'stripe' at its posterior limit, resulting in an anterior expansion of Even-skipped (Eve) stripe 3 because the Hb repressor directly specifies this border. There is also a corresponding shift in the position of Engrailed (En) stripe 5, which is regulated by Eve stripe 3. The narrowing of En stripes 4 and 5, due to the anterior shift of stripe 5, correlates with patterning defects in the mesothorax (Perry, 2012).

Quantitative measurements indicate significant alterations of the anterior Hb expression pattern upon removal of the stripe enhancer. There is an anterior shift at the midpoint of the mature pattern, spanning two to three cell diameters. This boundary normally occurs at 47.2% egg length (EL; measured from the anterior pole). In contrast, removal of the stripe enhancer shifts the boundary to 45.6% EL. The border also exhibits a significant diminishment in slope. Normally, there is a decrease in Hb protein concentration of 20% over 1% EL. Removal of the stripe enhancer diminishes this drop in concentration, with a reduction of just 10% over 1% EL. The most obvious qualitative change in the distribution of Hb protein is seen in regions where there are rapidly diminishing levels of the Bicoid gradient. Normally, the transition from maximum to minimal Hb levels occurs over a region of 10% EL (43%-53% EL). Removal of the stripe enhancer causes a significant expansion of this transition, to 26% EL (27%-53% EL). It is therefore concluded that the stripe enhancer is essential for shaping the definitive Hb border (Perry, 2012).

The preceding studies suggest that the proximal and distal Bicoid target enhancers are not sufficient to establish the definitive Hb border at the onset of segmentation during cc14. Instead, the initial border undergoes a dynamic posterior expansion encompassing several cell diameters due to the action of the stripe enhancer. This enhancer is similar to the eve stripe 3+7 enhancer. Both enhancers mediate two stripes, one in central regions and the other in the posterior abdomen, and the two sets of stripes extensively overlap. Previous studies provide a comprehensive model for the specification of eve stripes 3 and 7, whereby the Hb repressor establishes the anterior border of stripe 3 and the posterior border of stripe 7 while the Kni repressor establishes the posterior border of stripe 3 and anterior border of stripe 7. Whole-genome chromatin immunoprecipitation (ChIP) binding assays and binding site analysis identify numerous Hb and Kni binding sites in the hb stripe enhancer, along with several Kr sites (Perry, 2012).

Site-directed mutagenesis was used to examine the function of gap binding sites in the hb stripe enhancer. Since the full-length, 1.4 kb enhancer contains too many binding sites for systematic mutagenesis, a 718 bp DNA fragment was identified that mediates weak but consistent expression of both stripes, particularly the posterior stripe. Mutagenesis of all ten Hb binding sites in this minimal enhancer resulted in a striking anterior expansion of the expression pattern. This observation suggests that the Hb repressor establishes the anterior border of the central stripe, as seen for eve stripe 3. There is no significant change in the posterior border of the central stripe or the anterior border of the posterior stripe, and repression persists in the presumptive abdomen (Perry, 2012).

Mutagenesis of the Kni binding sites resulted in expanded expression in the presumptive abdomen, similar to that seen for the eve 3+7 enhancer. More extensive depression was observed upon mutagenesis of both the Kni and Kr binding sites. These results suggest that the Kr and Kni repressors establish the posterior border of the central Hb stripe and the anterior border of the posterior stripe. This depressed pattern is virtually identical to the late hb expression pattern observed in Kr1;kni10 double mutants. The reliance on Kr could explain why the Hb central stripe is shifted anterior of eve stripe 3, which is regulated solely by Kni (Perry, 2012).

The dynamic regulation of the zygotic Hb expression pattern can be explained by the combinatorial action of the proximal, shadow, and stripe enhancers. The proximal and distal shadow enhancers mediate activation of hb transcription in response to the Bicoid gradient in anterior regions of cc10-13 embryos. The initial border of hb transcription is rather sharp, but the protein that is synthesized from this early pattern is distributed in a broad and shallow gradient, extending from 30% to 50% EL. During cc14 the stripe enhancer mediates transcription in a domain that extends just beyond the initial hb border. Gap repressors, including Hb itself, restrict this second wave of zygotic hb transcription to the region when there are rapidly diminishing levels of the Bicoid gradient, in a stripe that encompasses 44%-47% EL. The protein produced from the stripe enhancer is distributed in a sharp and steep gradient in the anterior thorax. It has been previously suggested that the steep Hb protein gradient is a direct readout of the broad Bicoid gradient. However, the current studies indicate that this is not the case. It is the combination of the Bicoid target enhancers and the hb stripe enhancer that produces the definitive pattern (Perry, 2012).

It has been proposed that Hb positive autofeedback is an important feature of the dynamic expression pattern. However, the mutagenesis of the hb stripe enhancer is consistent with past studies suggesting that Hb primarily functions as a repressor. The only clear-cut example of positive regulation is seen for the eve stripe 2 enhancer. Mutagenesis of the lone Hb-3 binding site results in diminished expression from a minimal enhancer. It was suggested that Hb somehow facilitates neighboring Bicoid activator sites, and attempts were made to determine whether a similar mechanism might apply to the proximal Bicoid target enhancer. The two Hb binding sites contained in this enhancer were mutagenized, but the resulting fusion gene mediates an expression pattern that is indistinguishable from the normal enhancer). It is therefore likely that the reduction of the central hb stripe in hb/hb embryos is the indirect consequence of expanded expression of other gap repressors, particularly Kr and Kni (Perry, 2012).

The hb stripe enhancer mediates expression in a central domain spanning 44%-47% EL, which coincides with the region exhibiting population variation in the distribution of the Bicoid gradient. Despite this variability, the definitive Hb border was shown to be relatively constant among different embryos. Previous studies suggest that the Kr and Kni repressors function in a partially redundant fashion to ensure the reliability of this border. This paper has presented evidence for direct interactions of these repressors with the hb stripe enhancer, and suggest that a major function of the enhancer is to 'dampen' the variable Bicoid gradient. Indeed, removal of this enhancer from an otherwise normal Hb BAC transgene results in variable patterning defects in the mesothorax, possibly reflecting increased noise in the Hb border (Perry, 2012).

Protein Interactions

Krüppel can associate with the transcription factors encoded by the gap genes knirps and hunchback which affect Krüppel-dependent gene expression in Drosophila tissue culture cells. The association of DNA-bound HB protein or free KNI protein with distinct but different regions of KR results in the formation of DNA-bound transcriptional repressor complexes (Sauer, 1995a).

Various mutant and vitro-modified versions of KNI were tested in various assay systems to identify essential domains of KNI protein. KNI contains several functional domains arranged in a modular fashion. The N-terminal 185-amino-acid region (including the DNA-binding domain and a functional nuclear location signal) fails to provide KNI activity to the embryo. However, a truncated KNI protein that contains additional 47-amino-acids exerts rather strong kni activity, functionally defined by a weak kni mutant embryo phenotype. The additional 47-amino-acid stretch includes a transcriptional repressor domain (Gerwin, 1994).

KNI can quench, or locally inhibit artificial upstream enhancer regions. The range of KNI repression if 50 to 100 base pairs, so that neighboring enhancers in a modular promoter are free to interact with the transcription complex, thereby exhibiting enhancer autonomy. KNI can also repress the transcription complex when bound in promoter-proximal regions. In this position, KNI functions as a dominant repressor and blocks multiple enhancers in a modular promoter. The lack of specificity of KNI repression indicates that KNI may not quench upstream activators through direct protein-protein interactions. It is conceivable that KNI, and other short-range repressors such as Krüppel and Snail recruit 'corepressors', which cause local changes in chromatin structure (e.g. positioning a nucleosome) or in some other way interfere with access to the DNA by activators or basal transcription factors. Alternatively, short-range repressors do not interact with neighboring activators, but instead might 'hitchhike' with neighboring activators, looping to contact the basal promoter, and then inhibit components of the transcription complex (Arnosti, 1996).

Human CtBP attenuates transcriptional activation and tumorigenesis mediated by the adenovirus E1A protein. The E1A sequence motif that interacts with CtBP, Pro-X-Asp-Leu-Ser-X-Lys (P-DLS-K), is present in the repression domains of two unrelated short-range repressors in Drosophila (Knirps and Snail) and is essential for the interaction of these proteins with Drosophila CtBP (dCtBP). A P-element-induced mutation in dCtBP exhibits gene-dosage interactions with a null mutation in knirps, which is consistent with the occurrence of Knirps-dCtBP interactions in vivo. These observations suggest that CtBP and dCtBP are engaged in an evolutionarily conserved mechanism of transcriptional repression, which is used in both Drosophila and mammals (Nibu, 1998a).

The pre-cellular Drosophila embryo contains 10 well characterized sequence-specific transcriptional repressors, which represent a broad spectrum of DNA-binding proteins. Two of the repressors, Hairy and Dorsal, are known to recruit a common co-repressor protein, Groucho. Evidence is presented that three different repressors, Knirps, Krüppel and Snail, recruit a different co-repressor, dCtBP. Mutant embryos containing diminished levels of maternal dCtBP products exhibit both segmentation and dorsoventral patterning defects, all of which can be attributed to loss of Krüppel, Knirps and Snail activity. In contrast, the Dorsal and Hairy repressors retain at least some activity in dCtBP mutant embryos. dCtBP interacts with Krüppel, Knirps and Snail through a related sequence motif, PXDLSXK/H (also termed P-DLS-R). This motif is essential for the repression activity of these proteins in transgenic embryos. It is proposed that dCtBP represents a major form of transcriptional repression in development, and that the Groucho and dCtBP co-repressors mediate separate pathways of repression (Nibu, 1998b).

A Gal4-Knirps fusion protein containing the C-terminal third of the Knirps protein (amino acid residues 255-429) has been shown to be able to repress a modified eve stripe 2-lacZ reporter gene in transgenic embryos. The fusion protein contains the Knirps P-DLS-K motif, and mutations in this sequence (PMDLSMK to AAAASMK) inactivate its repression activity. These results suggest that dCtBP is an important component of Knirps-mediated repression, but do not exclude the possibility that additional sequences in Knirps are also important for repression. To address this issue of sufficiency, the function of the P-DLS-K motif was examined in the context of the full-length, wild-type protein. Knirps is normally expressed in two domains, one anterior to eve stripe 1 and the other in the presumptive abdomen, spanning eve stripes 4, 5 and 6. The posterior border of stripe 3 is thought to depend on repression by Knirps. Ectopic expression of knirps with the eve stripe 2 enhancer results in the loss of stripe 3 expression and dominant lethality. It has been suggested that the endogenous stripe 3 pattern is repressed by the diffusion of ectopic Knirps products from stripe 2. A mutant form of Knirps that lacks the P-DLS-K motif does not repress stripe 3 expression. The mutant protein is identical to native Knirps except for four changes in the P-DLS-K motif (PMDLSMK to AAAASMK). The mutant protein is expressed at the same levels as the wild-type protein, but does not mediate efficient repression. Moreover, while the ectopic expression of the wild-type Knirps protein results in embryonic lethality, transgenic strains that misexpress similar levels of the mutant protein are fully viable. These results suggest that P-DLS-K represents the primary repression motif in the Knirps protein, although high levels of the mutant protein cause weak and variable disruptions in the stripe 3 pattern (Nibu, 1998b).

The mechanism by which dCtBP mediates transcriptional repression is unknown. However, the current study provides evidence against a previously proposed mechanism for Krüppel (Sauer, 1995b). Krüppel activity is shown to be lost in dCtBP mutants, and the C-terminal region of the protein contains an essential P-DLS-H repression motif. Moreover, preliminary studies suggest that ectopic expression of the native Krüppel protein causes patterning defects in early embryos, which are reversed when the P-DLS-H motif is mutagenized. These results strongly suggest that Krüppel-mediated repression depends on the recruitment of the dCtBP co-repressor. The earlier study provided evidence that repression depends on the direct interactions of Krüppel with the beta-subunit of the TFIIE general transcription factor (Sauer, 1995b). It is conceivable that this mechanism of repression is employed in other tissues at later stages in the Drosophila life cycle, although it is noted that a recent study provides strong evidence that a mammalian Krüppel-like protein also employs a CtBP co-repressor (Nibu, 1998b and references).

Transcriptional repressor proteins play essential roles in controlling the correct temporal and spatial patterns of gene expression in Drosophila embryogenesis. Repressors such as Knirps, Krüppel, and Snail mediate short-range repression and interact with the dCtBP corepressor. The mechanism by which short-range repressors block transcription is not well understood; therefore, a detailed structure-function analysis of the Knirps protein has been undertaken. To provide a physiological setting for measurement of repression, the activities of endogenous or chimeric Knirps repressor proteins were assayed on integrated reporter genes in transgenic embryos. Two distinct repression functions have been identified in Knirps. One repression activity depends on dCtBP binding, and this function maps to a C-terminal region of Knirps that contains a dCtBP binding motif. In addition, an N-terminal region was identified that represses in a CtBP mutant background and does not bind to the dCtBP protein in vitro. Although the dCtBP protein is important for Knirps activity on some genes, one endogenous target of the Knirps protein, the even-skipped stripe 3 enhancer, is not derepressed in a CtBP mutant. These results indicate that Knirps can utilize two different pathways to mediate transcriptional repression and suggest that the phenomenon of short-range repression may be a combination of independent activities (Keller, 2000).

Knirps has been shown to repress transcription when bound adjacent to either basal promoters or activators within enhancer elements. These studies of Knirps activity when the protein binds close to the basal promoter reveals additional properties of the endogenous protein. (1) Repression by Knirps does not appear to be sensitive to phasing effects, as shown by equivalent activity of constructs with Knirps binding sites offset by 5 bp at -70 and -75 bp. (2) In this series of genes, the transcriptional repression activity appears to be directed at the basal promoter element, because the repression weakens as the distance from Knirps sites to the basal promoter is increased while the distance to the enhancer element is held constant. (3) While Knirps repression is limited to a relatively short distance, there is a measurable interval (from 100 to 130 bp) over which Knirps activity is attenuated but not entirely abolished (Keller, 2000).

This intermediate level of repression might be useful in adjusting the amount of repression imposed on a target gene or setting a target gene threshold, as has been demonstrated for the Drosophila Giant short-range repressor. With Giant, a less-than-twofold difference in posterior versus anterior protein levels is sufficient to switch a gene from on to off. Thus, two features of short-range repressors may allow for flexibility in genetic regulatory circuits: (1) short-range repressors allow modular enhancers to act independently, by avoiding regulatory cross talk, and (2) the exquisite distance dependence may contribute to the differential response of endogenous target genes to repressor gradients (Keller, 2000).

This study demonstrates that the Knirps protein contains two functionally distinct repression activities. The C-terminal region appears to mediate repression through recruitment of the dCtBP protein: it consists of a region contained within residues 202 to 358 (minimally, residues 248 to 291 and 313 to 358 including the PMDLSMK dCtBP binding motif. In contrast, the N-terminal repression region (minimally, residues 139 to 330) appears to function independently of dCtBP. Although this region contains some of the amino acid residues that are present in the dCtBP binding constructs, the two activities are clearly distinct based on dCtBP dependence. The N-terminal region does not bind to dCtBP and it can repress in a mutant embryo that lacks maternal dCtBP. Any residual amounts of dCtBP from maternal or zygotic expression are likely to be very low, because the loss of maternal dCtBP expression causes a loss of activity of Snail, Knirps, and Krüppel on a number of target genes, producing severe embryonic defects and early developmental arrest (Keller, 2000).

Knirps repression domains have been defined in the context of Gal4 fusion proteins, but several lines of evidence suggest that the native Knirps protein can also repress target genes independently of dCtBP. Most compellingly, an eve stripe 3 lacZ reporter gene that is derepressed in a knirps mutant background is not derepressed in a CtBP mutant. In addition, a frameshift mutation (kni14F) that produces a protein lacking the dCtBP interaction motif retains partial activity, perhaps via the N-terminal repression activity that has been defined in this study. Finally, a study of ectopically expressed Knirps protein that lacks a dCtBP binding motif found that the protein has weak repression on eve stripe 3 (Keller, 2000).

A region of the Knirps protein containing an alanine-rich tract had been identified in earlier studies as a repression domain in cell culture studies but is neither necessary nor sufficient for repression in the embryo. The repression function of 189-254 protein may be specific to transfection assays, similar to findings for the non-Groucho binding region of the Engrailed repressor protein (Keller, 2000).

It is not yet clear whether repression by the N-terminal and C-terminal regions of Knirps contribute to quantitative or qualitative differences in repression, or if these two aspects of repression are indeed entirely separable. The eve stripe 3 enhancer is clearly repressed in the region of kni expression in the absence of maternal dCtBP, yet in previous experiments, ectopically expressed Knirps was able to repress the eve stripe 3 element effectively only when the dCtBP binding motif of the protein was still intact. The most likely explanation for these apparently contradictory results is that dCtBP contributes to a portion of the Knirps-mediated repression of stripe 3. Endogenous Knirps is abundant enough to repress expression of eve stripe 3 in dCtBP mutant embryos, but the levels of ectopically produced Knirps protein are apparently insufficient to repress effectively when binding to dCtBP is abolished. dCtBP may also have an effect on Knirps protein stability or targeting, which might contribute to the reduced activity of the mutant protein. Previous studies have indicated that in the absence of dCtBP, repression of a synthetic rho lacZ reporter gene by endogenous Knirps is reduced. However, close examination of the data indicates that some anterior repression is apparently present, consistent with the idea that Knirps retains a measurable level of activity in the dCtBP mutant (Keller, 2000).

The Gal4-Knirps chimeras containing only the N-terminal repression domain appear to have higher levels of activity on lacZ reporters than does full-length Knirps protein lacking the dCtBP binding motif. A test was performed to see whether this difference might be attributed to masking of the N-terminal repression region by the C terminus in the absence of dCtBP. The data indicate that this model is not correct; Gal4-Knirps chimeras containing the N-terminal repression domain linked to a C-terminal region lacking a dCtBP binding activity are highly effective repressors. Gal4-Knirps chimeras may be inherently more effective repressors if one role of dCtBP is to facilitate dimerization of Knirps proteins. With chimeras, this function would be provided by the Gal4 DNA binding domain, because Gal4 binds DNA as a dimer. Alternatively, autoinhibition of the Knirps DNA binding domain, similar to that seen with Ets-1, AML-1, and Pitx2, may be relieved by dCtBP binding, but Gal4 chimeras would not be subject to such regulation. However, the effective regulation of eve stripe 3 lacZ in a CtBP mutant argues for a simpler quantitative effect model. Loss of dCtBP binding might simply reduce the total repression activity of Knirps protein, so that the low levels of misexpressed Knirps would be unable to effect repression. The Gal4-Knirps repressor utilizing only one repression region might be more functional due to increased effectiveness of dimerized repressor proteins or to a greater sensitivity of the lacZ reporters used (Keller, 2000).

Multiple repression activities in a protein may allow for qualitative or quantitative effects on gene expression. Qualitatively, a repressor may operate selectively in distinct tissue types or on different promoters. Loss of maternal dCtBP protein does not affect eve stripe 3 regulation, but it does abolish repression of the eve stripe 4+6 enhancer element, suggesting that this element is dCtBP dependent. Quantitatively, dual activities may increase the overall level of repression, much as transcriptional activators have been suggested to employ multiple paths to achieve synergistic activation (Keller, 2000).

Examples of both qualitative and quantitative effects are seen with the ZEB repressor, a protein that contains two repression domains. One domain blocks activation by Myb and Ets factors of lymphocyte-specific promoters, while the second domain, which contains a conserved CtBP binding motif, blocks the activity of the muscle cell-specific MEF2C factor. In contrast to these activator-specific effects, a quantitative contribution of multiple repression domains has been observed with the murine ZEB homolog deltaEF-1. When CtBP binding residues are mutated in deltaEF-1, repression of a MyoD-activated promoter is impaired but not abolished (Keller, 2000 and references therein).

Other repressor proteins may also possess both CtBP-dependent and dCtBP-independent activities. In Drosophila, the Krüppel protein contains a C-terminal dCtBP binding repression domain and an N-terminal repression domain. The latter domain has only been characterized in cell culture assays, but genetic evidence indicates that Krüppel can repress hairy in a CtBP mutant, possibly by means of this N-terminal domain. The Wnt signaling pathway transcription factor Tcf-3 can interact with both the Groucho and CtBP proteins through separate repression domains in Xenopus laevis, and the CtBP-binding portion of XTcf-3 has potent repression activity in the frog embryo. The Rb retinoblastoma protein has been shown to interact with both histone deacetylases and CtBP, although the physiological relevance of the CtBP interactions is not yet clear. Net, an Ets protein family member that can repress transcription of the c-fos promoter, has also been shown to possess two independent repression domains, one of which interacts with CtBP1. Loss of the CtBP binding motif from Net reduces the repression activity of the protein in cell culture assays. Finally, the BKLF transcription factor, which can interact with CtBP2 to repress transcription in Drosophila cell culture, contains an additional CtBP-independent activity detectable in NIH 3T3 cells (Keller, 2000).

dCtBP and its homologs appear to be able to mediate repression directly when recruited to promoters by a heterologous DNA binding domain, both in cell culture systems and in the embryo. The dCtBP corepressor has homology to alpha-hydroxy acid dehydrogenases and contains a conserved NAD-binding domain. The protein binds to NAD, but no dehydrogenase activity has been detected in vitro, and mutation of a conserved histidine in the putative active site does not compromise the repression activity of a chimeric CtBP2 protein in cell culture assays. The dCtBP protein may contain other uncharacterized enzymatic activities. Recently it was reported that the Sir2 transcriptional repressor possesses ADP ribosylation activity, and furthermore, that NAD is important for histone deacetylase activity of the protein. Some evidence suggests that CtBP may function through histone deacetylase pathways, but pair-rule gene repression by gap proteins such as Knirps and Krüppel is not compromised by mutations in the Rpd3 histone deacetylase (Keller, 2000).

The physiological relevance of CtBP binding is not yet known for a number of proteins that have been found to interact in yeast two-hybrid assays, but genetic evidence from Drosophila clearly indicates that dCtBP is an important repression cofactor. These data demonstrate that for at least one Knirps target gene, another pathway of repression is also utilized. A considerable body of evidence, including genetic and biochemical data, indicates that repressors may have multiple lines of communication with the transcriptional machinery, just as transcriptional activators have been found to contain multiple activation domains that act on multiple targets. Further genetic and biochemical characterization of Knirps will help elucidate the pathways utilized by this short-range repressor (Keller, 2000).

C-Terminal binding protein (CtBP) interacts with a highly conserved amino acid motif (PXDLS) at the C terminus of adenovirus early region 1A (AdE1A) protein. This amino acid sequence has recently been demonstrated in the mammalian protein C-terminal interacting protein (CtIP) and a number of Drosophila repressors including Snail, Knirps and Hairy. The structures of synthetic peptides identical to the CtBP binding sites on these proteins have been investigated using NMR spectroscopy. Peptides identical to the CtBP binding site in CtIP and at the N terminus of Snail form a series of beta-turns similar to those seen in AdE1A. The PXDLS motif towards the C terminus of Snail forms an alpha-helix. However, the motifs in Knirps and Hairy did not adopt well-defined structures in TFE/water mixtures as shown by the absence of medium range NOEs and a high proportion of signal overlap. The affinities of peptides for Drosophila and mammalian CtBP were compared using enzyme-linked immunosorbent assay. CtIP, Snail (N-terminal peptide) and Knirps peptides all bind to mammalian CtBP with high affinity [K(i) of 1.04, 1.34 and 0.52 microM, respectively]. However, different effects were observed with dCtBP, most notably the affinity for the Snail (N-terminal peptide) and Knirps peptides are markedly reduced [K(i) of 332 and 56 microM, respectively] whilst the Hairy peptide binds much more strongly [K(i) for dCtBP of 6.22 compared to 133 microM for hCtBP]. In addition peptides containing identical PXDLS motifs but with different N and C terminal sequences have appreciably different affinities for mammalian CtBP and different structures in solution. It is concluded that the factors governing the interactions of CtBPs with partner proteins are more complex than simple possession of the PXDLS motif. In particular the overall secondary structures and amino acid side chains in the binding sites of partner proteins are of importance as well as possible global structural effects in both members of the complex. These data constitute evidence for a multiplicity of CtBPs and partner proteins (Molloy, 2001).

The transcription factors knirps (kni) and knirps-related (knrl) are expressed in overlapping patterns during tracheal development in which they share redundant functions. They are both required in two phases of tracheal development. The initial tracheal expression of kni/knrl appears around stage 10 and their activity is required early in the tracheal placode for primary branching outgrowth. Subsequently, kni/knrl expression becomes restricted to some branches, among them the visceral branches, and they are required for the directed outgrowth of these tracheal branches. Since kni and knrl share redundant functions, an examination was made of mutant embryos homozygous for a deficiency that uncovers both genes and whose effect on tracheal development is due solely to the lack of kni and knrl. In this mutant background, it was found that alphaPS1 is no longer expressed in the tracheal cells, indicating that these genes act as positive regulators of alphaPS1. To further assess the specific role of the late expression of kni/knrl in the expression of alphaPS1 in the visceral branch, a mutant combination was examined that provides the segmentation and early tracheal kni expression, but not the later branch-specific expression of kni. In these mutant embryos, there are also defects in visceral branch development and expression of alphaPS1 is either absent or very much reduced. Thus, the late phase of kni/knrl branch-specific expression is specifically required for the proper expression of alphaPS1 in the cells of the visceral branches. However, kni/knrl are not sufficient to drive alphaPS1 expression, as they are expressed in other branches that do not express alphaPS1. Similarly, the alphaPS1 gene is not the only target of these transcription factors, since GAL4-driven expression of alphaPS1 is not able to rescue the phenotype of the deficiency (Boube, 2001).

Transcriptional repressors can be classified as short- or long-range, according to their range of activity. Functional analysis of identified short-range repressors has been carried out largely in transgenic Drosophila, but it is not known whether general properties of short-range repressors are evident in other types of assays. To study short-range transcriptional repressors in cultured cells, chimeric tetracycline repressors were created based on Drosophila transcriptional repressors Giant, Drosophila C-terminal-binding protein (dCtBP), and Knirps. Giant and dCtBP are found to be efficient repressors in Drosophila and mammalian cells, whereas Knirps is active only in insect cells. The restricted activity of Knirps, in contrast to that of Giant, suggests that not all short-range repressors possess identical activities, consistent with recent findings showing that short-range repressors act through multiple pathways. The mammalian repressor Kid is more effective than either Giant or dCtBP in mammalian cells but is inactive in Drosophila cells. These results indicate that species-specific factors are important for the function of the Knirps and Kid repressors. Giant and dCtBP repress reporter genes in a variety of contexts, including genes that are introduced by transient transfection, carried on episomal elements, or stably integrated. This broad activity indicates that the context of the target gene is not critical for the ability of short-range repressors to block transcription, in contrast to other repressors that act only on stably integrated genes (Ryu, 2002).

The regulation of inducible promoters via chimeric tetracycline repressor (TetR) proteins has attracted considerable interest for use in ectopic expression systems in cell culture, microbes, plants, and whole animals. In these systems, a chimeric protein consisting of the Escherichia coli TetR protein fused to an activation domain binds to promoters containing Tet response elements (TREs). On addition of tetracycline or doxycycline, the chimeric protein is released from the promoter and the gene is inactivated. TetR DNA-binding domains with reverse specificity have been developed to permit activation of target genes on addition of the drug. Although this system can be highly regulated, low-level basal expression can be a problem in the case of potentially toxic gene products. To overcome this problem, higher specificity Tet DNA-binding domains have been recently developed. Many endogenous genes accomplished tight regulation by the coordinated action of repressors and activators. To mimic such composite systems, a Tet repressor can be combined with a Tet activator to give repression and activation in the absence and presence of doxycycline, respectively. Such combined Tet-based activation/repression systems have been developed for yeast and mammalian systems. Most of these systems use the KRAB repressor domain. Whether KRAB repressors can work in nonvertebrate cell types has not been reported, however. In this study, a panel of transcriptional repressors has been created based on well characterized short-range repressors from Drosophila. The chimeric proteins show reproducible repression activity in the Tet system in a variety of cell types and on stably integrated or transiently introduced reporter genes. Compared with the mammalian Kid repressor, these repressors may be the preferred alternative for regulation of expression in some cell types and with certain transgene configurations (Ryu, 2002).

CtBP is essential when Krüppel and Knirps repressor sites do not overlap activator sites but are instead located adjacent to either activators or the core promoter

There are three mechanisms of transcriptional repression in eukaryotes. The first is quenching, whereby repressors and activators co-occupy closely linked sites and then the repressor inhibits adjacent activators. The second is direct repression, in which repressors block the function of the core transcription complex. The third is competition, in which repressors compete with activators for a common DNA-binding site. Previous studies have shown that the Drosophila CtBP corepressor (dCtBP) is essential for the quenching activity of three short-range sequence-specific repressors in the early Drosophila embryo: Krüppel, Knirps, and Snail. This study demonstrates that dCtBP is dispensable for target enhancers that contain overlapping activator and repressor binding sites. However, it is essential when Krüppel and Knirps repressor sites do not overlap activator sites but are instead located adjacent to either activators or the core promoter. These findings provide evidence that competition is distinct from quenching and direct repression. Quenching and direct repression depend on dCtBP, whereas competition does not (Nibu, 2003 ).

Krüppel is a zinc finger DNA-binding protein that is composed of 502 aa residues. The quenching activity of the C-terminal repression domain (aa 402 to 502) requires a dCtBP interaction motif located at amino acids (aa) 464 to 470. Another repression domain has been identified in cultured cells. It is located between aa 62 and 92 and does not contain a dCtBP interaction motif. A transgenic embryo assay was used to determine whether this N-terminal repression domain might be a source for CtBP-independent repression in early embryos (Nibu, 2003).

A Gal4-Krüppel fusion protein containing aa 402 to 502 created gaps in the staining patterns directed by st2.UAS-st3-lacZ, NEE.UAS-lacZ, and NEE.UAS-twi-lacZ. The st2.UAS-st3-lacZ reporter gene contains Gal4 UAS binding sites near the distal eve stripe 2 enhancer (st2). NEE.UAS-lacZ reporter gene is driven by a modified 200-bp rhomboid rhomboid lateral stripe neurectoderm (NEE) enhancer that contains three Gal4 binding sites and three Dorsal activator sites. This reporter gene is normally activated in the ventral mesoderm. For the st2.UAS-st3-lacZ (st3 is the eve stripe 3 enhancer) and NEE.UAS-twi-lacZ reporter genes, repression was observed only for the staining pattern produced by the enhancer containing UAS binding sites. For example, the binding of the Gal4-Krüppel fusion protein to the stripe 2 enhancer does not alter expression from the neighboring stripe 3 enhancer. Similarly, the binding of the fusion protein to the rhomboid NEE enhancer does not alter expression from the twist enhancer. Substitutions in three of the amino acid residues within the dCtBP interaction motif (PEDLSMH to AAALSMH) eliminate the repression activity of an otherwise normal Gal4-Krüppel fusion protein (Nibu, 2003).

There is a second potential dCtBP interaction motif, located between aa 414 and 420 (PLDLSED), that weakly binds dCtBP in vitro. However, this second motif is not sufficient to support discernible repression activity in vivo. These results suggest that most or all of the repression activity of the Gal4-Krüppel 402-502 fusion protein resides within the major dCtBP interaction motif between amino acid residues 464 and 470. Moreover, repression is not observed for a Gal4-Krüppel fusion protein that contains the N-terminal repression domain (aa 62 to 92). These results suggest that the C-terminal dCtBP motif mediates most or all of the quenching activity in the early embryo (Nibu, 2003).

The proximal UAS site within the NEE.UAS-lacZ reporter gene is located 120 bp 5' of the core promoter, slightly beyond the range of Krüppel-mediated repression. In contrast, the UAS sites map within 50 bp of critical Dorsal sites within the NEE. Thus, repression of the reporter gene is most likely due to quenching rather than the direct repression of the core promoter. Another lacZ reporter was created to investigate this issue, NEE-5xUAS-lacZ. The most distal UAS site is located 250 bp 5' of the most proximal Dorsal binding site within the modified 700-bp NEE enhancer, while the most proximal UAS site is located just 57 bp 5' of the transcription start site of the hsp70 promoter. The Gal4-Krüppel 402-502 fusion protein attenuates lacZ expression. This direct repression is not obtained with the mutagenized fusion protein lacking the dCtBP interaction motif or with a fusion protein containing the N-terminal repression domain. These results suggest that the C-terminal dCtBP interaction motif is essential for both quenching and direct repression (Nibu, 2003).

Previous studies suggest that Krüppel mediates quenching by recruiting dCtBP to distal enhancers, such as the eve stripe 2 enhancer. An NEE-lacZ reporter gene that contains two synthetic Krüppel recognition sequences located 50 bp 5' of the most distal Dorsal binding site and 50 bp 3' of the most proximal site was created. This enhancer lacks the native Snail repressor sites and therefore directs lacZ expression in both lateral and ventral regions of early embryos. lacZ staining was diminished in central regions due to the localized expression of the Krüppel repressor. This gap in the pattern was eliminated in Kr1/Kr1 mutant embryos. Krüppel also failed to repress the reporter gene in mutant embryos derived from dCtBP germ line clones. These results indicate that dCtBP+ gene activity is required for the quenching activity of the Krüppel repressor (Nibu, 2003).

Subsequent experiments were done to determine whether dCtBP is required for the direct-repression activity of Krüppel and another short-range repressor, Knirps. lacZ transgenes with either Krüppel or Knirps binding sites located near the core promoter were examined. Both transgenes contain two tandem copies of the 250-bp twist proximal enhancer placed either upstream or downstream of rhomboid lateral stripe enhancers (NEE). In wild-type embryos, the enhancers direct additive patterns of expression in the lateral neurogenic ectoderm and ventral mesoderm. A single Krüppel binding site located 75 bp 5' of the transcription start site was sufficient to create a central gap in both staining patterns. Staining directed by the tandem twist enhancers was nearly eliminated, whereas the lateral stripe produced by the rhomboid NEE was diminished. Repression of the twist pattern is almost certainly due to direct repression, since the solo Krüppel site maps more than 800 bp from the nearest Dorsal activator site in the twist enhancer. Krüppel-mediated repression is lost when the transgene is introduced into embryos obtained from dCtBP germ line clones. There is no longer a central gap in the staining pattern. Moreover, there is a fusion of the expression patterns directed by the twist and NEE enhancers due to a loss in the activity of the Snail repressor. Normally, Snail binds to the NEE enhancer and represses expression in the ventral mesoderm, thereby restricting the staining pattern to lateral stripes in the neurogenic ectoderm. The broad uniform staining pattern obtained in dCtBP mutants suggests that the dCtBP corepressor is required for the direct repression of the core promoter (Nibu, 2003).

Similar results were obtained with the Knirps repressor. In this case, two tandem Knirps binding sites were placed 55 bp 5' of the transcription start site. In wild-type embryos, there is a clean gap in both the NEE-mediated lateral stripes and the twist-mediated staining pattern in the ventral mesoderm. This gap coincides with the site of Knirps expression in the presumptive abdomen. As seen for Krüppel, the gap in the staining patterns disappears in dCtBP mutant embryos. These results suggest that dCtBP is required for the direct repression activities of both Krüppel and Knirps (Nibu, 2003).

The preceding experiments suggest that dCtBP is required for both quenching and the direct repression of the core promoter. A synthetic lacZ reporter gene was prepared to determine whether Krüppel can mediate repression by competition and, if so, whether dCtBP is required for this repression. A 14-bp oligonucleotide that contains overlapping Dorsal and Krüppel binding sites was synthesized. Each subunit of the Dorsal homodimer binds to an inverted half-site: GGG...CCC. Krüppel binds DNA as a monomer, and the core recognition sequence includes the CCC Dorsal half-site. This short sequence also contains an optimal Bicoid binding site (GGATTA). This motif is located between the two half-sites of the Dorsal recognition sequence and overlaps the Krüppel consensus sequence (Nibu, 2003).

Gel shift assays were done to determine whether Dorsal and Krüppel bind the synthetic 14-bp sequence in a mutually exclusive manner. A 30-bp fragment that contains the 14-bp sequence along with 8 bp of flanking sequence at each end was synthesized. In the first set of experiments, a full-length Krüppel protein produced in E. coli was mixed with the 30-bp fragment and fractionated on an agarose gel. A shifted Krüppel-DNA complex was observed. The addition of increasing amounts of the Dorsal DNA-binding domain (Dl DBD; aa 1 to 403) resulted in the gradual loss of this complex. A new complex that is identical in size to those obtained with the Dorsal protein alone was observed. These results suggest that high concentrations of the Dorsal DNA-binding domain can displace Krüppel (Nibu, 2003).

Similar results were obtained in reciprocal DNA-binding assays. In this case, the shifted Dorsal-DNA complex was formed in the absence of Krüppel. The addition of increasing amounts of the Krüppel protein resulted in the gradual loss of the Dorsal-DNA complex. A new complex was obtained that has the same size as the one observed with increasing amounts of Krüppel in the absence of the Dorsal protein. These results suggest that increasing amounts of Krüppel can displace Dorsal-DNA complexes. Thus, the gel shift assays indicate mutually exclusive binding of Dorsal and Krüppel to the overlapping binding sites contained within the 14-bp fragment (Nibu, 2003).

Transient-transfection assays were used to determine whether the Krüppel DNA-binding domain is sufficient to mediate transcriptional repression. Six tandem copies of the synthetic oligonucleotide used in the preceding DNA-binding assays were attached to an eve-luciferase reporter gene containing the minimal eve promoter. This reporter gene was introduced into mbn-2 cultured cells (a Drosophila blood cell line) along with various expression vectors containing Dorsal or Krüppel coding sequences. An expression vector containing the full-length Dorsal coding sequence (Dl FL) produced a 6 fold induction in luciferase activity. However, an expression vector containing the Krüppel DNA-binding domain (Kr DBD; aa 217 to 401) reduced luciferase activity to background levels. This reduction in reporter gene expression was not obtained with a Krüppel expression vector that contained a single amino acid substitution in the zinc finger DNA-binding domain (Kr9 DBD). These results suggest that Krüppel can repress the synthetic enhancer by simply binding DNA and excluding the Dorsal activator. Repression does not depend on Krüppel protein sequences that map outside the DNA-binding domain. Subsequent experiments were done to determine whether Krüppel can mediate repression by competition in transgenic embryos (Nibu, 2003).

Either 6 or 14 tandem copies of the 14-bp synthetic enhancer sequence were attached to a lacZ reporter gene containing the minimal, 42-bp eve promoter region. Similar results were obtained with both fusion genes, and most of the following results were obtained with individual strains carrying the transgene with six copies attached. The transgene exhibits a combinatorial pattern of lacZ staining in wild-type (yw) embryos. Staining is first detected in the anterior 40% of 120-min embryos, presumably in response to the broad Bicoid activator gradient and is also detected in both anterior regions and along the entire length of the ventral mesoderm. Mesoderm expression was first seen at the time when the maternal Dorsal protein is released from the cytoplasm and enters nuclei. During cellularization, staining is lost in central regions, presumably due to the onset of Krüppel expression. In addition, there is a refinement in the anterior staining pattern, so that it becomes restricted to the anterior one-fourth of the embryo and exhibits a reasonably sharp posterior border. This staining pattern persists during gastrulation and germ band elongation (Nibu, 2003).

The transgene was introduced into different mutant backgrounds in order to confirm that the synthetic enhancer is regulated by Bicoid, Dorsal, and Krüppel. The anterior staining pattern is eliminated when the transgene is introduced into embryos derived from females homozygous for a null mutation in bicoid. However, staining persists in ventral regions in response to the Dorsal gradient. The loss of staining in the anterior regions correlates with an anterior expansion of the Krüppel expression pattern in bicoid mutants. The maternal Dorsal gradient is eliminated in embryos derived from females that are homozygous for a null mutation in gastrulation defective (gd7/gd7). lacZ staining in the ventral mesoderm of these mutants is lost. However, staining persists in anterior regions, presumably in response to the Bicoid gradient, which is unaffected in gd mutants. The transgene was also crossed into Kr1/Kr1 mutant embryos. The central gap of repression seen in wild-type embryos is essentially abolished in Kr mutants. There may be a subtle attenuation in central regions due to the low levels of Krüppel protein that are retained in this mutant (Kr1 is not quite a null allele. The anterior staining pattern directed by the Bicoid gradient may be a bit broader in Kr mutants than in wild-type embryos, suggesting that the Krüppel repressor might help refine the pattern. These results indicate that the artificial enhancer is activated by Bicoid and Dorsal but repressed by Krüppel. Competition is the likely form of repression since the Krüppel repressor sites directly overlap the Bicoid and Dorsal activator sites (Nibu, 2003).

One of the central goals of this study was to determine whether Krüppel requires dCtBP when it mediates repression by competition. This issue was investigated by crossing the transgene into mutant embryos derived from germ line clones produced in dCtBP/+ females. Krüppel continues to induce a central gap of repression in these mutants. In fact, the repression obtained in dCtBP mutants is comparable to that observed in wild-type embryos. These results provide a clear example of Krüppel-mediated repression in the absence of the dCtBP corepressor. In contrast, Krüppel fails to repress transcription in dCtBP mutants when Krüppel and Dorsal sites do not overlap (Nibu, 2003).

This study provides evidence for two distinct mechanisms of short-range repression, corepressor-dependent (quenching and direct repression) and corepressor-independent (competition) repression. In addition, this is the first demonstration that transcriptional repression by competition does not require a corepressor in transgenic Drosophila embryos. dCtBP is dispensable when Krüppel binding sites directly overlap Dorsal activator sites. However, dCtBP is essential for repression when the Krüppel and Dorsal sites are nonoverlapping and can be coordinately occupied. The previous analysis of eve stripe 2 regulation led to the proposal that the Krüppel repressor establishes the posterior stripe 2 border via competition. Two of the Krüppel repressor sites contained within the stripe 2 enhancer overlap Bicoid activator sites. Subsequent studies led to the surprising observation that Krüppel binding sites need not overlap activator sites in order to mediate transcriptional repression (Nibu, 2003).

There are three Krüppel binding sites in the minimal, 480-bp eve stripe 2 enhancer. Two of the sites directly overlap Bicoid activator sites. In both cases, it is likely that the binding of the Krüppel repressor precludes the binding of Bicoid. This type of simple competition is probably not restricted to the regulation of eve stripe 2. For example, one of the mixed Bicoid/Krüppel binding sites in the stripe 2 enhancer is conserved in a newly identified ftz enhancer, which appears to be activated by Bicoid but repressed by Krüppel (V. Calhoun and M. Levine, unpublished data reported in Nibu, 2003). The two enhancers contain the same composite recognition sequence, ACGGATTAA. Repression by competition probably governs, in part, the regulation of the rhomboid lateral stripe enhancer (NEE) since some of the Snail repressor sites directly overlap critical Dorsal and basic helix-loop-helix activator sites (Nibu, 2003).

An implication of this study is that the residual activity of the Krüppel repressor observed in dCtBP mutants might be due to repression by competition. For example, Krüppel can repress the hairy stripe 7 enhancer when misexpressed throughout early embryos using the heat-inducible hsp70 promoter. This repression is retained in dCtBP mutants. Moreover, a mutant form of Krüppel that lacks the dCtBP interaction motif can repress hairy stripe 7 expression. hairy stripe 7 is activated, at least in part, by Caudal and repressed by Krüppel. Interestingly, five Krüppel binding sites directly overlap Caudal activator sites within the hairy stripe 7 enhancer. Similar arguments apply to the Knirps repressor, which helps establish the posterior border of eve stripe 3. The stripe 3 pattern expands in kni-/kni- mutant embryos but is essentially unchanged in dCtBP mutants. Knirps repressor sites might overlap critical activator sites, such as binding sites for D-Stat or an unknown activator(s) within the stripe 3 enhancer. Previous studies suggest that Brinker can also function independently of corepressors when bound to sites that directly overlap critical Smad activator sites within cis regulatory regions of Dpp target genes. Direct evidence for simple competition was obtained in transient-transfection assays. The Krüppel DNA-binding domain is sufficient to inhibit activation of the synthetic enhancer by Dorsal in cultured mbn-2 cells (Nibu, 2003).

The results reported in this study exclude another possible explanation for the residual activity of the Krüppel and Knirps repressors in dCtBP mutants: direct repression of the core promoter. In principle, direct repression could involve distinct corepressor proteins. If so, then target genes that contain promoter-proximal Krüppel and Knirps binding sites might be repressed in dCtBP mutants. However, the lacZ fusion genes containing either a single Krüppel site or two tandem Knirps sites located near the transcription start site are no longer repressed in dCtBP mutants. Thus, the possibility is favored that the residual Krüppel and Knirps repression activities depend on competition between overlapping activator and repressor binding sites within selected target enhancers (Nibu, 2003).

The demonstration that both quenching and direct repression require dCtBP raises the possibility that these two seemingly distinct forms of repression employ similar mechanisms. At least three types of models come to mind. First, dCtBP could disrupt physical interactions between upstream activators and the RNA polymerase II transcription machinery/mediator complex at the core promoter. Perhaps dCtBP masks or modifies the activation domains of upstream activators. However, this model can account for quenching but not direct repression. A second type of model involves local chromatin modification. dCtBP contains a well-conserved dehydrogenase catalytic center and binds NADH. Perhaps dCtBP modifies proteins such as histones and helps condense DNA within the limits of a nucleosome. In Saccharomyces cerevisiae, the Rpd3 histone deacetylase (HDAC) causes histone deacetylation over a distance of just two nucleosomes. A third model is that dCtBP 'poisons' the RNA polymerase II transcription machinery and impedes its binding, assembly, or function at the core promoter. This poisoning can be accomplished by placing dCtBP-dependent repressors near the core promoter or by looping distal enhancers to the promoter. According to the latter model, the linkage requirement seen for short-range repressors (they must bind within 100 bp of adjacent activators) might reflect a reliance of the repressors on linked activators in order to loop to the core promoter (Nibu, 2003).

Quantitative contributions of CtBP-dependent and -independent repression activities of Knirps

The Drosophila Knirps protein is a short-range transcriptional repressor that locally inhibits activators by recruiting the CtBP co-repressor. Knirps also possesses CtBP-independent repression activity. The functional importance of multiple repression activities is not well understood, but the finding that Knirps does not repress some cis-regulatory elements in the absence of CtBP suggested that the co-factor may supply a unique function essential to repress certain types of activators. CtBP-dependent and -independent repression domains of Knirps were assayed in Drosophila embryos; the CtBP-independent activity, when provided at higher than normal levels, can repress an eve regulatory element that normally requires CtBP. Dose response analysis has revealed that the activity of Knirps containing both CtBP-dependent and -independent repression activities is higher than that of the CtBP-independent domain alone. The requirement for CtBP at certain enhancers appears to reflect the need for overall higher levels of repression, rather than a requirement for an activity unique to CtBP. Thus, CtBP contributes quantitatively, rather than qualitatively, to overall repression function. The finding that both repression activities are simultaneously deployed suggests that the multiple repression activities do not function as cryptic 'backup' systems, but that each contributes quantitatively to total repressor output (Struffi, 2004).

The expression of the endogenous eve gene is strongly perturbed by a loss of CtBP, consistent with the important role of this co-repressor in the activity of gap repressors Giant, Krüppel, and Knirps. To study the effectiveness of Knirps repression of individual eve regulatory elements, the expression of eve-lacZ reporter genes was examined. Knirps is required for correct regulation of the eve stripe 3/7 and 4/6 enhancers, as demonstrated by the expression patterns of lacZ reporter genes in kni mutant embryos. The posterior border of eve stripe 3 was not derepressed in a CtBP mutant, consistent with the CtBP-independent activity of Knirps on this enhancer. By contrast, Knirps repression of eve stripe 4/6 is compromised in a CtBP mutant background, indicating that the CtBP-independent repression activity of Knirps is insufficient to regulate this enhancer. Therefore, depending on which part of the eve gene is bound by the Knirps protein, its repression activity is either dependent or independent of the CtBP co-factor (Struffi, 2004).

Previous studies of Krüppel, Giant and Knirps have indicated that CtBP dependence or independence of their repression activities varies according to the specific cis regulatory element involved, suggesting that there are particular enhancer architectures that necessitate CtBP activity. The clearest example of enhancer specific requirements for CtBP is shown in the case of eve enhancers. In nuclei situated between eve stripes 4 and 6, the stripe 4/6 and 3/7 enhancers are both repressed by Knirps in the same nuclei, yet this repression is independent of CtBP on the 3/7 element and dependent on CtBP on the 4/6 element. By expressing increasing levels of the CtBP-independent form of Knirps, the requirement for CtBP is obviated. These results suggest that distinct requirements for the CtBP co-factor at different genes or cis regulatory elements can be based on the quantitative levels of repression activity. Indeed, the combination of the CtBP-dependent and CtBP-independent activities make a particularly powerful repressor, as judged by comparison of repression activities of Knirps 1-429 versus Knirps 1-330 on eve and other pair-rule genes. These results suggest that both repression domains can be simultaneously engaged on a given cis regulatory element, rather than a particular repression activity being selectively engaged at particular enhancers. Consistent with this picture, when they are assayed separately as Gal4 fusion proteins in embryos, both CtBP-dependent and CtBP-independent repression domains of Knirps have equal, modestly effective repression activities. By contrast, a Gal4 protein containing both domains is much more effective at repressing a strongly activated promoter (Struffi, 2004).

A model is presented that explains the quantitative contribution of the CtBP co-repressor to Knirps repression activity. At a relatively low level of Knirps protein activity, the eve 3/7 enhancer is repressed, and this level of repression activity is achieved at similar levels of Knirps, regardless of whether or not CtBP contributes to repression. Thus, in the absence of CtBP, the positions at which the stripe 3/7 boundaries form shift very little. The much higher level of repression required by the stripe 4/6 element is achieved only near the peak of Knirps protein levels. If CtBP is not complexed with Knirps, the intercept shifts sharply to the right, to a level of Knirps not normally present in the embryo. The sufficient level of repression in the absence of CtBP activity or protein is only achieved under conditions where Knirps is overexpressed (Struffi, 2004).

The threshold model explains how the contributions of separate repression activities act in a quantitative fashion to meet given thresholds, but what is the basis for distinct repression thresholds? There are at least two variables involved in dictating a threshold, namely, regulatory protein levels and the nature (number, affinity, and placement) of the relevant binding sites within a regulatory element. Varying intranuclear activator levels can influence repression thresholds, as suggested by regulation of the Krüppel gene: Giant requires CtBP for repression of this gene only in nuclei containing peak levels of the Bicoid activator. Varying intranuclear repressor levels will dictate how easily those thresholds are met, with or without multiple repression activities. Gap genes, including knirps, generate protein gradients that have properties of morphogens, i.e., they trigger differential responses at different threshold levels. The stripe 4/6 and 3/7 modular enhancers of the even-skipped gene are designed to respond to different levels of Knirps protein, allowing the embryo to establish multiple stripe boundaries with a single protein gradient. The short-range activity of Knirps allows the two enhancers to act independently, so that activators bound to the stripe 4/6 enhancer activate the gene in nuclei where the levels of Knirps are already sufficiently high to inhibit the stripe 3/7 enhancer (Struffi, 2004).

Binding site affinity and number have been clearly established to influence threshold responses in the case of transcriptional activators, such as Bicoid and Dorsal. A similar effect is likely to be true for repressors. Sequence analysis of the eve gene indicates that there are more high-affinity Knirps binding sites within the eve stripe 3/7 element than in the 4/6 enhancer, consistent with relative sensitivities of these elements that were determined experimentally. Removal of some of the Knirps binding sites in the eve stripe 3/7 enhancer reduces the sensitivity of this element to the Knirps gradient. However, the number of predicted high-affinity binding sites alone is not sufficient information to predict relative sensitivity to Knirps. If it were, one would expect the eve stripe 2 enhancer, with three predicted Knirps sites, to be more sensitive to Knirps than eve stripe 4/6, with only a single site, yet the reverse is true. This lack of correlation might be partly attributable to errors in the prediction of binding sites; however, additional factors, such as affinity of binding sites and relative placement with respect to other proteins, are likely to make the decisive difference in determining enhancer sensitivity to Knirps. In the case of the Giant repressor, small shifts in the placement of the binding site allows detection of less than two-fold differences in repressor concentrations, a 'gene tuning' mechanism that seems to have been invoked during internal evolution of the eve stripe 2 enhancer. The stoichiometry of activators to repressors has also been suggested to be a crucial factor in determining repression levels, and direct tests indicate that Giant and Knirps respond sensitively to differences in activator binding site number and affinity on defined regulatory elements (Struffi, 2004).

eve stripe 1 lies just posterior to the weak anterior domain of knirps expression, suggesting a possible role for Knirps in regulating that element, but it is not clear whether the relative sensitivity of other eve stripe enhancers normally active outside of the main posterior domain of Knirps expression is of physiological significance. The eve stripe 2 pattern lies outside of the normal area of Knirps expression, and is only repressed at the highest levels of Knirps, suggesting that repression might be through cryptic Knirps sites in the element. The robust activity of the eve stripe 5 enhancer even under conditions of high levels of Knirps misexpression emphasizes that this regulatory element has been designed to function in nuclei containing peak levels of Knirps protein. Similarly, runt stripe 5 also resists peak levels of ectopic Knirps. Both of these regulatory elements have few or no predicted Knirps-binding sites. These elements would provide a useful platform to test the number and placement of novel Knirps binding sites required to bring the element under the control of this repressor (Struffi, 2004).

The effects of Knirps misexpression on other endogenous pair rule genes reinforce the lessons learned from eve, regarding the relative potency of the Knirps repression domains and the sensitivity of different enhancers. Both the CtBP-independent region of Knirps as well as the intact protein are capable of repressing the hunchback parasegment 4 stripe, a highly sensitive target of Knirps. However, hairy, runt and ftz, which have been previously noted to have a higher threshold to Knirps repression, are noticeably less affected by Knirps 1-330 compared with Knirps 1-429. Thus, it is likely that CtBP activity contributes quantitatively to repression of other Knirps target genes in addition to eve (Struffi, 2004).

Repression of central runt stripes is consistent with previous findings of direct repression by Knirps and the greater sensitivity of stripes 2-4 relative to stripe 1. A greater effect of ectopic expression of Knirps is observed on hairy than noted in previous experiments, probably on account of higher levels of expression. Knirps expressed under the control of an eve stripe 2 enhancer was previously found to have little effect on anterior hairy expression, except for a delay in stripe 3/4 separation. Heat shock expression of full-length Knirps 1-429, by contrast, results in strong repression of hairy stripes 3, 4 and 7. The hairy stripe 3, 4 and 7 enhancers are predicted to contain Knirps-binding sites, in contrast to the unrepressed stripe 1 and 5 enhancers. The weaker Knirps 1-330 protein had an effect similar to that of full-length Knirps expressed from an eve stripe 2 expression construct, i.e., a delay of stripe 3/4 separation. Interestingly, knirps is important for activation of hairy stripe 6, and the protein can bind to the stripe 6 enhancer directly in vitro. No evidence of activation is seen upon overexpression, however, suggesting that such activation might be indirect (Struffi, 2004).

The derepression of ftz observed between stripes 2-4 and 6-7 is likely due to indirect effects of repression of hairy and eve expression; both of these genes are thought to repress ftz directly. By contrast, previous work involving lower levels of anteriorly expressed Knirps observed only weakened ftz stripes 2 and 3, rather than stripe fusion. This lower level of Knirps had a much less profound effect on upstream regulators hairy and eve, suggesting that Knirps might be a direct gap gene input to this pair-rule gene (Struffi, 2004).

This study suggests that the multiple repression activities of Knirps can be simultaneously mobilized to provide quantitatively correct levels of repression activity, and that the design of cis regulatory elements can elicit CtBP dependence. CtBP-independent activity can in some cases be directly attributed to direct competition with activator for DNA binding; however, the CtBP-independent activity of Knirps can repress activators on elements where sites are not overlapping, and overexpression of the DNA-binding domain of Knirps (Knirps1-105) is insufficient to mediate repression of endogenous eve enhancers. Cell culture and transgenic embryo assays indicate that both CtBP-dependent and independent repression activities of Knirps have very similar characteristics with respect to activator specificity, distance dependence and overall potency, thus the targets and molecular mechanisms might well be similar in each case. Key to a deeper understanding of the molecular circuitry controlled by short-range repressors such as Knirps will be biochemical knowledge of the mechanisms of repression employed on these developmentally regulated enhancers (Struffi, 2004).

Long- and short-range transcriptional repressors induce distinct chromatin states on repressed genes

Transcriptional repression is essential for establishing precise patterns of gene expression during development. Repressors governing early Drosophila segmentation can be classified as short- or long-range factors based on their ranges of action, acting either locally to quench adjacent activators or broadly to silence an entire locus. Paradoxically, these repressors recruit common corepressors, Groucho and CtBP, despite their different ranges of repression. To reveal the mechanisms underlying these two distinct modes of repression, chromatin analysis was performed using the prototypical long-range repressor Hairy and the short-range repressor Knirps. Chromatin immunoprecipitation and micrococcal nuclease mapping studies reveal that Knirps causes local changes of histone density and acetylation, and the inhibition of activator recruitment, without affecting the recruitment of basal transcriptional machinery. In contrast, Hairy induces widespread histone deacetylation and inhibits the recruitment of basal machinery without inducing chromatin compaction. This study provides detailed mechanistic insight into short- and long-range repression on selected endogenous target genes and suggests that the transcriptional corepressors can be differentially deployed to mediate chromatin changes in a context-dependent manner (Li, 2011).

To directly compare functional aspects of Hairy- and Knirps- mediated repression in the Drosophila embryo, these proteins’ interactions were studied with two segmentally expressed pair-rule genes. Hairy directly represses fushi tarazu (ftz), a secondary pair-rule gene expressed in the blastoderm embryo in a seven-stripe pattern. ftz is regulated by both regionally acting gap genes and the segmentally expressed hairy pair-rule gene. Chromatin immunoprecipitation (ChIP) experiments have revealed dense clusters of peaks around the ftz gene for key transcription factors active in the blastoderm embryo, including Caudal, Hunchback, Knirps, Giant, Huckebein, Krüppel, and Tailless. These transcription factors bind to the promoter-proximal Zebra element, the stripe 1+5 enhancer located 3' of ftz, and a presumptive 5' regulatory region located between 23 kbp and 28 kbp. Hairy has been found to bind in vivo to all of these regions. This repressor is expressed in a striped pattern in the blastoderm embryo; therefore, the ftz gene is active in some nuclei and repressed in others. In order to obtain a homogeneous population of nuclei for chromatin studies, Hairy protein was overexpressed in embryos using a heat-shock driver, which results in complete repression of ftz. This repression requires the recruitment of the Groucho corepressor, because a mutant version of Hairy that does not bind to Groucho fails to repress ftz (Li, 2011).

Interestingly, a titration of heat-shock induction resulted in a nonuniform, progressive loss of specific ftz stripes, with stripe 4 being the most sensitive and stripe 1+5 the least. This result points to the intriguing possibility that Hairy can act locally on specific enhancers, at least very transiently, although the end result of Hairy repression is complete silencing of all enhancer elements. The asynchronous repression of the ftz locus also suggests that Hairy-mediated long-range repression does not act solely by direct targeting the basal promoter, as suggested by a previous model for this class of repressor, because this mechanism should cause uniform inhibition of stripe elements. Similar to ftz, the pair-rule gene even skipped (eve) is also expressed in a seven-stripe pattern and is regulated by multiple modular enhancers. eve is a well-characterized target of the short-range repressor Knirps, which sets posterior boundaries of eve stripe 3 and 4 and anterior borders of eve stripe 6 and 7. After substantial overexpression of Knirps (20 min heat-shock induction), the repressor is able to repress all of the eve stripe enhancers except for the stripe 5 enhancer. When the induction is titrated, Knirps represses individual enhancers in a stepwise manner, with the most sensitive enhancers downregulated earliest, at a low dose of Knirps. Together, these experiments indicate that Hairy can initially act locally but ultimately acts in a globally dominant fashion, whereas Knirps acts in a restricted manner (Li, 2011).

To compare the effects of repression by Hairy and Knirps, chromatin changes associated with repression of ftz and eve were studied via ChIP. No significant change of histone H3 occupancy were detected at regions sampled throughout the ftz locus after Hairy overexpression (although some regions showed modest differences. In contrast, Knirps repression of eve resulted in significantly increased histone H3 density, particularly in two of the three regions corresponding to the Knirps-sensitive enhancers, namely stripe 4+6 and stripe 2. Little change was noted in the promoter region, transcribed region, or the stripe 1 and 5 enhancers, which are not readily repressed by Knirps. An apparent increase in histone H3 density on the repressed stripe 3+7 enhancer, although of low statistical significance, correlates with other alterations common to repressed enhancers, noted below (Li, 2011).

To provide a more detailed picture of chromatin structure, a micrococcal nuclease (MNase) mapping protocol used in yeast and cultured cells was adapted for Drosophila embryos. MNase mapping showed that Hairy repression had little effect on chromatin accessibility throughout the ftz locus, whereas Knirps induced a significant increase in MNase insensitivity specifically at the eve stripe 3+7, 2, and 4+6 enhancers and a minor increase in stripe 1 protection. The promoter and the eve stripe 5 enhancer were little changed, mirroring the patterns noted for overall histone H3 occupancy. The changes noted for the eve locus appear to be specific, because Knirps did not induce any change of a nontargeted intergenic site on the third chromosome. Hairy also had no effect at this locus. The similar results from overall histone H3 density and MNase mapping suggest that Hairy-mediated long-range repression does not involve a general compaction of chromatin on the ftz locus. In contrast, repression by Knirps is associated with an increase in the histone density of targeted enhancer regions, which may result either from Knirps recruitment of factors that mediate chromatin condensation or the blocking of proteins responsible for loosening of chromatin. Recruitment of Groucho by other repressor proteins is also associated with distinct effects: Runt-dependent repression of slp1 does not involve changes in H3 density, but Brinker repression of the vgQ enhancer does. The distance dependence of these repressors has not been established, but in light of the current results, it is apparent that the Groucho corepressor can be involved in distinct effects depending on the context of recruitment (Li, 2011).

Histone acetylation is dynamically regulated on transcribed genes in eukaryotes, with histone acetylation generally correlated with active loci. The histone deacetylase Rpd3 is a component of both Hairy and Knirps corepressor complexes; therefore, histone acetylation levels were assayed across the eve and ftz genes before and after repression. Hairy repression resulted in widespread histone H4 deacetylation throughout the ftz locus. The ectopically expressed Hairy protein itself was not observed to spread but remained restricted to regions of the gene previously observed to bind endogenous Hairy. Using anti-H3-acetylation antibodies, similar widespread H3 deacetylation was also noted. This distributed effect on the ftz locus correlates with prior observations that Hairy-mediated long-range repression might involve a Groucho-mediated 'spreading' mechanism. By this means, Rpd3 may be delivered to extensive areas of a gene. To test whether a spreading of histone deacetylation might correlate with the successive inhibition of ftz enhancers, histone acetylation levels were investigated across ftz after a brief 5 min heat shock followed by immediate fixing, before the entire complement of enhancers can be repressed. In this setting, deacetylation was mostly concentrated around the stripe 1+5 enhancer and the immediate 5' regulatory region, areas that show Hairy occupancy in vivo. More distal 5' regulatory regions and the transcription unit itself showed little initial change, consistent with a spreading action of this repressor during the more extensive repression period (Li, 2011).

A different picture emerged from studies of Knirps acting on eve. Here, repression led to selective decreases in H3 and H4 acetylation levels, concentrated over the eve stripe 4+6 and stripe 2 enhancers, with lesser decreases noted at stripe 3+7 and stripe 1 enhancers. A local change in acetylation was also noted near the transcriptional initiation site, but not immediately 5' and 3' of this area. The reductions in histone acetylation levels seen on both eve and ftz are consistent with Hairy and Knirps recruiting deacetylases to their target genes. However, it is striking that the broad deacetylation mediated by Hairy on ftz is not associated with dramatic changes in histone density or resistance to nuclease accessibility, whereas increased histone density and resistance to nuclease digestion are associated with Knirps repression on eve. It is possible that in addition to inducing deacetylation, Knirps triggers additional histone modifications or interacts with nucleosome-remodeling complexes to further alter chromatin at the enhancers. H3 lysine 27 methylation is one chromatin signature associated with silenced genes; however, no significant change in this modification was noted at ftz or eve upon repression (Li, 2011).

Previous studies indicated that Hairy can effectively repress a reporter gene without displacing the activators. Attempts were made to test whether this was the case on an endogenous gene, ftz, by examining occupancy by Caudal, a transcription factor that also activates eve. Caudal activates the posterior stripes of both ftz and eve, and it was found that Caudal binds the ftz 5' regulatory region and the promoter-proximal Zebra element. Repression of the locus by Hairy did not affect the Caudal binding pattern, similar to the results obtained with a Hairy-regulated reporter gene. In contrast, Knirps repression decreased Caudal occupancy specifically at the eve 3+7 and 4+6 enhancers, bringing overall protein occupancy down to near baseline levels. This decrease is not an effect of global decrease of Caudal occupancy, because the Caudal binding peak at the eve promoter was not affected. A similar decrease in Caudal occupancy was also observed on a hunchback enhancer after repression by Knirps. Interestingly, Bicoid occupancy of the eve stripe 2 and stripe 1 enhancers was not altered by Knirps, although these enhancers were repressed. Clearly, loss of transcription factor occupancy is not required for short-range repression of a cis-regulatory element. It is possible that different transcriptional activators exhibit differential sensitivity to chromatin changes induced during repression (Li, 2011).

New insights have suggested that many developmental genes, including those regulated by short-range repressors such as Snail, feature RNA polymerase paused in the promoter region even in their inactive state, suggesting postrecruitment levels of regulation. Components of the core machinery were analyzed before and after repression by Hairy and Knirps. Upon Hairy repression, a marked decrease of RNA polymerase II (Pol II) occupancy was observed at the ftz locus. The same trend was observed for the preinitiation, initiation, and elongation forms of Pol II. These results suggest that Hairy directly or indirectly blocks recruitment of Pol II. Similar decreases were noted with levels of TATA box-binding protein (TBP) at the promoter (Li, 2011).

In contrast, induction of Knirps did not change Pol II occupancy at the eve transcription unit, even under condition where most enhancers were repressed. (Under conditions tested in this study, over three-quarters of the embryos had shut down expression of all but stripe 1 and/or 5.) Similarly, TBP occupancy remained at a comparable level before and after Knirps repression. The constant level of RNA polymerase on the eve transcription unit was a surprise in light of the sharp reduction in mRNA production as measured by in situ hybridization. However, there is precedence for this effect: Runt repression of slp1 appears to act through elongation control, which causes no change of the concentration of Pol II on slp1. Knirps may produce a similar effect by inducing a slower transit rate of Pol II on the repressed eve locus. Similar observations have been made at the hsp70 gene upon depletion of elongation factors such as Spt6 or Paf1 (Li, 2011).

The differential distance dependence of short- and long- range repressors such as Hairy and Knirps has been observed in many contexts. However, the mechanisms by which these proteins function have been poorly understood. With the recent demonstration that transcriptional factors considered to be short- and long-range repressors utilize shared cofactors, namely CtBP and Groucho, there has been a question of whether long-range repression is actually functionally distinct from short-range repression (Payankaulam, 2009). The current study provides evidence that the chromatin states associated with long- and short-range repressors are distinct in several ways. It is not yet knowm whether the effects seen on ftz are observed for all Hairy targets, although the similarity of changes observed on the lacZ reporter subject to Hairy repression suggests that they are conserved (Martinez, 2008). Similarly, the reproducibility of Knirps-induced changes at different eve enhancers indicates that this protein can effect related chromatin changes on cis-regulatory modules bound by different activators. Snail, another short-range repressor, also appears to mediate localized deacetylation and activator displacement; thus, this mechanism may be a common feature of this entire class of repressors (Qi, 2009; Y. Nibu, personal communication to Li, 2011). It will be interesting to determine how general are the observations made in this study for long- and short-range repression, a question that can be approached using genome-wide methods. In any event, the highly divergent activities of Knirps and Hairy demonstrated in this study not only underscore the fact that these proteins can mediate biochemically divergent events but also raise interesting questions about how similar cofactors can participate in such distinct effects in a context-dependent manner. It is possible that the corepressors adopt distinct conformations when recruited by different repressors, or the corepressor may form distinct complexes with unique activities. In addition to determining how cis- and trans-acting factors affect repression pathways, these mechanistic insights will provide important contextual information for interpretation of genome-wide transcription factor binding and chromatin modifications and will inform quantitative modeling of cis-regulatory elements for the aim of understanding the activity and evolution of enhancers (Li, 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).

knirps: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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