1 Department of Biochemistry and Molecular Biology, and Genetics Program,
Michigan State University, East Lansing, MI 48824-1319, USA
2 Department of Biology, New York University, New York, NY 10003, USA
* Author for correspondence (e-mail: arnosti{at}msu.edu)
Accepted 14 January 2004
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SUMMARY |
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Key words: CtBP, Knirps, Even-skipped, Enhancer, Repression, Transcription, Drosophila
![]() |
Introduction |
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The apparent impuissance of short-range repression actually provides a
highly flexible mechanism for specific gene regulation, allowing genes to be
`tuned' to respond to subtle differences in repressor protein concentration
and by small changes in the positions of factor binding sites
(Hewitt et al., 1999). Changes
in the spacing of short-range repressor binding sites correlate with
functional alterations observed during enhancer evolution
(Ludwig and Kreitman, 1998
;
Ludwig et al., 2000
).
The CtBP co-repressor is required for full activity of short-range
repressors such as Knirps, Krüppel, Giant and Snail that play important
roles in patterning the blastoderm embryo
(Nibu et al., 1998a;
Nibu et al., 1998b
). This
evolutionarily conserved co-factor also interacts with a number of vertebrate
transcriptional regulators, including the adenovirus E1A protein, Net, Ikaros,
Zeb and, indirectly, the Retinoblastoma tumor suppressor protein (reviewed by
Chinnadurai, 2002
).
Transcription factors typically bind to CtBP via a short peptide motif similar
to the PLDLS sequence originally identified in E1A
(Schaeper et al., 1995
). CtBP
is homologous to
-hydroxyacid dehydrogenases, and contains a conserved
NAD-binding domain as well as conserved residues in the putative active site
(reviewed by Chinnadurai, 2002
;
Turner and Crossley, 2001
).
Although not identified in previous studies, recent reports found a weak
dehydrogenase activity associated with CtBP
(Kumar et al., 2002
;
Balasubramanian et al., 2003
;
Shi et al., 2003
). CtBP has
been found to bind directly to histone deacetylases (HDACs), suggesting that
the corepressor may effect repression by chromatin remodeling (reviewed by
Turner and Crossley, 2001
;
Chinnadurai, 2002
). A recent
biochemical purification of CtBP identified additional proteins in a complex,
including histone methyltransferases, the CoREST repressor and a protein
homologous to polyamine oxidases (Shi et
al., 2003
). This additional complexity suggests that CtBP itself
may use multiple activities to effect transcriptional repression.
Drosophila factors functionally characterized as short-range
repressors all interact with CtBP, although it has not been established that
all factors that bind the co-factor are necessarily short-range repressors.
The long-range repressor protein Hairy, in particular, is thought to interact
with CtBP, although this might be in an antagonistic mode
(Poortinga et al., 1998
;
Zhang and Levine, 1999
).
CtBP-mediated repression is crucial for full activity of short-range
repressors; however, Drosophila short-range repressors also possess
CtBP-independent repression activities (La
Rosee-Borggreve et al., 1999;
Keller et al., 2000
;
Strunk et al., 2001
;
Nibu et al., 2003
). In the
case of Knirps, a form of the protein that lacks the CtBP-binding motif
exhibits weak activity when overexpressed
(Nibu et al., 1998b
). The CtBP
independent activity has been mapped to an N-terminal repression domain that
lacks a CtBP-binding motif and is able to repress in the absence of CtBP
(Keller et al., 2000
).
Although many transcriptional repressors have been found to possess multiple
activities, the functional relevance of such activities is not well
understood. Previous studies suggest that multiple repression activities
underlie both gene specific and activator specific effects. In the case of the
Zeb repressor, a protein with CtBP-dependent and -independent activities, it
was found that specific repression activities are directed at distinct classes
of transcriptional activators (Postigo and
Dean, 1999
). In another case, distinct mechanisms are used at
different promoters: the NRSF repressor mediates HDAC and DNA
methylation-dependent repression of the NaCh II gene and a distinct
form of repression of the Scg10 gene
(Lunyak et al., 2002
).
Previous studies also hint that the possession of CtBP-dependent and
-independent activities may confer important quantitative effects. For
example, the Krüppel promoter is activated by Bicoid in both
anterior and central regions of the blastoderm embryo, and is repressed by
Giant in either CtBP-independent or CtBP-dependent manners, depending on the
region of the embryo (Strunk et al.,
2001). The higher levels of Bicoid activator in anterior regions
of the embryo might necessitate additional repression activities beyond those
afforded by CtBP-independent pathways, suggesting that CtBP might contribute
quantitatively to overall repressor output. In cell culture studies,
CtBP-dependent and CtBP-independent repression activities of Knirps possess
similar functional attributes, including distance dependence, trichostatin A
insensitivity and activator specificity, suggesting that their quantitative
effects might be mediated through similar pathways
(Ryu and Arnosti, 2003
).
The Knirps protein is able to regulate at least one known target, the
stripe 3 enhancer of the even-skipped (eve) gene in a
CtBP-independent fashion, yet this CtBP-independent activity is not sufficient
to supply the full biological function of Knirps
(Keller et al., 2000;
Nibu et al., 1998b
). For
example, transheterozygous knirps and CtBP embryos have
disruptions in eve expression, suggesting that Knirps function is
partially impaired, and a frameshift mutation in knirps encoding a
protein lacking the CtBP binding motif is a strong hypomorph
(Gerwin et al., 1994
;
Nibu et al., 1998a
).
Furthermore, a point mutation in the CtBP binding motif results in a protein
that lacks the dominant phenotype of the wild-type protein when misexpressed
in a pattern of eve stripe 2
(Nibu et al., 1998b
). These
results suggest that Knirps requires CtBP for effective regulation of at least
some of its targets. Here, we examine the regulation of several enhancers
targeted by Knirps to test the possibility that the CtBP-dependent and
CtBP-independent repression activities of Knirps might be deployed to achieve
qualitatively or quantitatively distinct effects. Our results suggest that in
the case of the eve gene, the two activities are both required to
achieve quantitatively sufficient levels of repression.
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Materials and methods |
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Heat-shock experiments
To induce expression of recombinant Knirps proteins, 2- to 4-hour-old
embryos collected on apple-juice plates at room temperature (22-23°C) were
incubated for 5, 10, 20 or 30 minutes at 38°C in a 10 liter water bath to
ensure rapid and even heating. After induction, embryos were allowed to
recover in a water bath at room temperature for 30 minutes prior to fixation
or sonication. Heat-shock inductions of Knirps1-330 and Knirps1-429 were also
performed with no recovery. For the experiments described in
Fig. 6, hairy
expression pattern was monitored after 10 or 30 minutes of heat shock,
ftz expression pattern was determined after 5, 10, 15, 20 or 30
minutes of heat shock, runt expression pattern was determined after
15 or 30 minutes of heat shock and hb expression pattern was
determined after 30 minutes of heat shock. Thirty minutes of recovery after
heat shock was applied for all the experiments described in
Fig. 6.
|
Western blot analysis
Immunoblotting was performed according to standard protocols
(Harlow and Lane, 1999) using
a tank transfer system (Mini Trans-Blot Cell, Biorad). Sequi-BlotTM PVDF
membranes (BioRad) were used and antibody incubation was in TBST (20 mM
Tris-HCl, pH 7.5, 120 mM NaCl, 0.1% Tween-20) supplemented with 5% (w/v)
nonfat dry milk as blocking agent. The primary anti FLAG M2 monoclonal
antibody (Sigma) was used at 1:10,000 dilution. The secondary ImmunoPure®
Goat Anti-Mouse HRP-conjugated antibody (Pierce) was used at 1:20,000
dilution. Western blots were quantitated using a Fluor-S® MultiImager
(Biorad) set on high sensitivity and with an exposure time of 50 minutes. The
QuantityOne package (BioRad) was used to analyze the data. Four independent
quantitations of four gels were performed, analyzing lysates from an
experiment performed as described for Fig.
3 and Table 1.
|
|
lacZ reporters
The eve stripe 3/7 and 4/6 lacZ reporter genes used in
Fig. 1 were described elsewhere
(Small et al., 1996;
Fujioka et al., 1999
). Germline
mutants of CtBP were generated as previously described
(Keller et al., 2000
) using
CtBP03463/TM3, Sb (Bloomington stock no. P1590). The
even-skipped stripe 2/3 lacZ reporter used in
Fig. 4 contains
500 bp
minimal elements separated by a 340 bp spacer sequence and 2 UAS sites (not
used in this experiment) fused to the eve basal promoter
(Keller et al., 2000
). The
eve stripe 3/7 reporter used in
Fig. 4 (stock E9) was kindly
provided by Steve Small and the eve stripe 4/6 lacZ reporter
(stock B45C52-B) was kindly provided by Jim Jaynes
(Fujioka et al., 1999
).
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Results |
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Ectopic expression of Knirps proteins in embryos
To determine whether the eve stripe 4/6 enhancer is intrinsically
resistant to repression by the CtBP-independent activity of Knirps, or just
less sensitive to this activity, we overexpressed full-length (1-429) FLAG
epitope tagged Knirps or a truncated form of the protein that contains only
the N-terminal, CtBP-independent repression activity (1-330) in embryos
(Fig. 2A). As controls,
proteins lacking the N-terminal DNA-binding domain were also overexpressed to
test for specificity of repression. All proteins were expressed from a
hsp70 promoter construct introduced by germline transformation into
Drosophila. In situ analysis showed a uniform distribution of
knirps mRNA in embryos after heat shock, reaching levels comparable
with the endogenous knirps gene
(Fig. 2B). The different forms
of the Knirps protein were expressed at similar levels after heat shock
induction of the transgenes, with undetectable levels present before heat
shock (Fig. 2C; data not
shown). Heat-shock induction of full-length Knirps in the embryo was lethal
(data not shown), as is expected for this regulatory factor whose expression
usually exhibits tight temporal and spatial regulation.
|
Similar to the case with Knirps 1-429, stripes 3 and 7 were the first to be affected by misexpression of the Knirps 1-330 protein, which bears only the CtBP-independent repression activity. In this latter instance, however, the numbers of embryos showing repression was smaller (Fig. 3E,F; Table 1). Unexpectedly, overexpression of Knirps 1-330 also led to repression of eve stripes 4 and 6, indicating that this regulatory element is sensitive to the CtBP-independent activity of Knirps (Fig. 3G). Again, the relative number of affected embryos was smaller, indicative of a quantitative difference in repression between full-length Knirps and the CtBP-independent domain alone (Table 1). Unlike the case for Knirps 1-429, no embryos were observed that showed repression of stripes 1 and 2 by overexpression of Knirps 1-330. This result suggests that either these enhancers require still higher levels of Knirps 1-330 to be effectively repressed, or that there are qualitative as well as quantitative differences between the repressors. In a small percentage of cases, stripe 5 expression was also observed to be repressed in embryos misexpressing Knirps 1-330 and Knirps 1-429 (Table 1); however, a small percentage of nontransgenic controls also appear to show loss of stripe 5 expression (not shown), indicating that this phenotype may be a nonspecific heat shock effect.
Heat-shock experiments were also performed with no recovery time after induction to test whether Knirps might be repressing eve indirectly. We found that the order of repression of eve stripes was identical as in Fig. 3, although for each heat-shock regiment repression was not as complete (data not shown), possibly because the eve mRNA had less time to turn over. This result is consistent with a direct action of Knirps on eve enhancers.
The activity of Knirps 1-330, which contains the CtBP-independent domain of
Knirps may reflect the previously identified CtBP-independent autonomous
activity. Alternatively, some or all of the activity may be due to competition
of the DNA-binding domain for activator binding sites. Therefore, we
overexpressed the DNA-binding domain of Knirps (residues 1-105, containing the
previously defined DNA binding domain and nuclear localization signal)
(Gerwin et al., 1994) and
determined its effect on eve expression pattern. As measured by
quantitative western blotting, this protein was readily induced to levels
almost as great as Knirps 1-330. Even at high expression levels, however,
Knirps 1-105 was unable to perturb eve expression (data not shown),
suggesting that Knirps represses eve by means other than direct
competition for activator binding sites.
Differential effects of Knirps protein on minimal even-skipped stripe enhancers
Next, we tested the effects of overexpression of full-length Knirps and the
N-terminal, CtBP-independent domain of Knirps on minimal eve stripe
2-3, 3/7- and 4/6-lacZ reporters. We confirmed that the differential
susceptibility to repression of eve stripe 3 compared to stripe 2
(Fig. 3) was directly
associated with the previously defined regulatory regions using a
lacZ reporter gene coupled to the minimal 500 bp stripe 2 and 3
enhancers. Both full-length Knirps 1-429 and Knirps 1-330 preferentially
repressed stripe 3 over stripe 2 (Fig.
4B,F). The Knirps 1-429 protein was able to entirely repress
stripe 3 in almost all embryos, and stripe 2 in a majority of embryos
(Fig. 4A-C). By contrast, as
noted with the endogenous eve gene, the CtBP-independent 1-330
repression domain was less potent than 1-429, resulting in more embryos with
only partially repressed eve stripe 3, and fewer embryos in which
stripe 2 was repressed (Fig.
4D-F). Embryos in which both stripes 2 and 3 were repressed were
distinguishable from nontransgenic embryos by residual stripe 2 expression in
ventral regions and an anterior stripe driven by vector sequences
(Fig. 4C). The minimal stripe 2
enhancer is probably more sensitive to repression by Knirps 1-330 than the
endogenous stripe 2 enhancer because it does not contain all sequences
involved in stripe 2 regulation (M. Ludwig and M. Kreitman, personal
communication). A previous study found that the minimal stripe 2 element was
only slightly affected by Knirps overexpression
(Kosman and Small, 1997), but
here we are probably achieving higher levels of expression.
To further verify the relative activity of full-length Knirps versus the CtBP-independent activity of Knirps, we tested the effects of overexpression of Knirps proteins in embryos carrying eve stripe3/7- and 4/6-lacZ reporters (Fig. 4G-L). As observed on the endogenous eve gene, full-length Knirps was a more potent repressor than the CtBP-independent domain, causing complete repression of eve stripe 4 and 7 and almost complete repression of eve stripe 3 and 6 (Fig. 4H,K compare with 4G,J). A large decrease in the number of stained embryos also indicates that many lacZ reporter genes were completely repressed. Knirps 1-330 caused a similar repression pattern, but longer heat shocks were required to achieve comparable repression of the more sensitive eve stripe 4 and 7. After 30 minutes of heat shock, repression of eve stripe 3 and 6 was not as complete as that achieved by Knirps 1-429 after 15 minutes of heat shock (compare Fig. 4I with 4H, and compare 4L with 4K). Importantly, when expressed at high level, the CtBP-independent repression activity of Knirps was able to completely repress eve stripe 4, and partially repress stripe 6, confirming the results observed with the endogenous eve gene.
Higher specific activity of Knirps protein containing multiple repression activities
The lower activity of Knirps 1-330 protein relative to the full-length
Knirps protein might be due to a greater potency of the protein containing two
distinct repression activities, or it might merely reflect lower protein
expression levels. To directly compare levels of ectopically expressed Knirps
proteins, lysates from transgenic embryos were subject to western blot
analysis, using the same heat shock regime as that used for the in situ
analysis above (Fig. 5A).
Equivalent amounts of total protein from whole embryo lysates were separated
on SDS gels, transferred to membranes and probed with an antibody specific for
the C-terminal FLAG epitope. Quantitation of the signals from the blots
indicate that the weaker Knirps 1-330 repressor was actually expressed at
approximately twofold higher levels than Knirps 1-429 at each time point
tested (Fig. 5B). Therefore,
the greater potency of the full-length Knirps is not just a function of
greater expression or stability of this protein, but presumably reflects the
greater activity of the combined repression domains.
|
The stripe elements 3, 4, 6 and 7 of hairy have been found to be
affected by misexpression of Knirps protein or mutations in the
knirps gene (Pankratz et al.,
1990; Langeland et al.,
1994
; Kosman and Small,
1997
) and binding sites for Knirps protein have been mapped on the
hairy stripe 6 and 7 enhancer elements
(Langeland et al., 1994
;
Hader et al., 1998
).
Expression of Knirps 1-429 caused a strong repression of hairy stripe
3, 4, and 7 expression, while expression of Knirps 1-330 had no such
inhibitory effect (Fig. 6G-I).
The ftz pair-rule gene is also under control of gap gene regulators,
as well as primary pair-rule genes (Carroll
and Scott, 1986
; Yu and Pick,
1995
). In Knirps 1-429-overexpressing embryos, the central stripes
are fused, but overexpression of Knirps 1-330 had a much milder effect, with
partial weakening of ftz stripes 2 and 3
(Fig. 6J-K). As discussed
below, the effects of Knirps misexpression on ftz might well
represent secondary effects mediated through upstream regulators, in
particular eve and hairy. These effects on eve and
other endogenous pair-rule genes support the observation that the
CtBP-independent repression domain of Knirps is capable of mediating
repression on the most sensitive target genes, but is quantitatively less
potent than the full-length protein.
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Discussion |
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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 (Fig.
1). By expressing increasing levels of the CtBP-independent form
of Knirps, the requirement for CtBP is obviated
(Fig. 3). 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
(Fig. 3 and
Table 1) and other pair-rule
genes (Fig. 6). 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 (Sutrias-Grau and
Arnosti, 2004).
A model that explains the quantitative contribution of the CtBP co-repressor to Knirps repression activity is shown in Fig. 7. At the top, two lines depict the levels of repression activity generated by increasing Knirps concentrations, the top line illustrating the levels of repression achieved by the Knirps protein complexed with CtBP. Thresholds of repression required by the eve stripe 3/7 and 4/6 enhancers are depicted by horizontal lines. Below, relative levels of Knirps are shown with respect to position (egg length) in the embryo. 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, as in Fig. 3.
|
Binding site affinity and number have been clearly established to influence
threshold responses in the case of transcriptional activators, such as Bicoid
and Dorsal (Jiang and Levine,
1993; Szymanski and Levine,
1995
; Struhl et al.,
1989
). 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 we determined experimentally
(Fig. 3)
(Papatsenko et al., 2002
;
Berman et al., 2002
). 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
(Clyde et al., 2003
). 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
(Berman et al., 2002
)
(Fig. 3). This lack of
correlation might be partly attributable to errors in 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
(Ludwig et al., 2000
;
Hewitt et al., 1999
). 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
(Hader et al., 1998
;
Kulkarni and Arnosti,
2003
).
eve stripe 1 lies just posteriorly to the weak anterior domain of
knirps expression, suggesting a possible role of 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 highest levels of Knirps
(Table 1), suggesting that
repression might be through cryptic Knirps sites in the element
(Berman et al., 2002). The
robust activity of the eve stripe 5 enhancer even under conditions of
high levels of Knirps misexpression underlines that this regulatory element
has been designed to function in nuclei containing peak levels of Knirps
protein (Fig. 3). Similarly,
runt stripe 5 also resists peak levels of ectopic Knirps
(Fig. 6). Both of these
regulatory elements have few or no predicted Knirps-binding sites
(Berman et al., 2002
). 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.
Knirps regulation of hb, runt, h and ftz
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 were capable of repressing the hunchback parasegment 4
stripe, a highly sensitive target of Knirps
(Kosman and Small, 1997).
However, hairy, runt and ftz, which have been previously
noted to have a higher threshold to Knirps repression, were noticeably less
affected by Knirps 1-330 compared with Knirps 1-429
(Fig. 6). Thus, it is likely
that CtBP activity contributes quantitatively to repression of other Knirps
target genes in addition to eve.
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 (Kosman and
Small, 1997). We observed a greater effect of ectopic expression
of Knirps 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 (Kosman and Small,
1997
). Heat shock expression of full-length Knirps 1-429, by
contrast, resulted in strong repression of hairy stripes 3, 4 and 7
(Fig. 6I). 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
(Langeland et al., 1994
; La
Rosee et al., 1997; Berman et al.,
2002
). 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
(Fig. 6H). Interestingly,
knirps is important for activation of hairy stripe 6, and
the protein can bind to the stripe 6 enhancer directly in vitro
(Riddihough and Ish-Horowicz,
1991
; Langeland et al.,
1994
). We see no evidence of activation upon overexpression,
however, suggesting that such activation might be indirect.
The derepression of ftz we observe between stripes 2-4 and 6-7 is
likely to be due to indirect effects of repression of hairy and
eve expression; both of these genes are thought to repress
ftz directly (Jiménez et
al., 1996; Manoukian and
Krause, 1992
). 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, as suggested by earlier studies (Yu
and Pick, 1995
; Kosman and
Small, 1997
).
Repression mechanisms
Our 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
(Hoch et al., 1992;
Nibu et al., 2003
); however,
the CtBP-independent activity of Knirps can repress activators on elements
where sites are not overlapping (Keller et
al., 2000
; Ryu and Arnosti,
2003
), and overexpression of the DNA-binding domain of Knirps
(Knirps1-105) is insufficient to mediate repression of endogenous eve
enhancers (data not shown). 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 (Ryu
and Arnosti, 2003
;
Sutrias-Grau and Arnosti,
2004
). 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.
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ACKNOWLEDGMENTS |
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