knirps
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).
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).
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).
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).
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