University of Wisconsin-Madison, Howard Hughes Medical Institute, 1525 Linden Drive, Madison, WI 53706, USA
* Author for correspondence (e-mail: sbcarrol{at}wisc.edu)
Accepted 2 February 2005
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SUMMARY |
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Key words: Hox genes, Cis-regulatory element, Ultrabithorax, Drosophila, Evolution
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Introduction |
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Within insects, the Hox gene Ultrabithorax (Ubx) is
important for proper specification of the third thoracic segment. In
Drosophila lacking Ultrabithorax function, the third
thoracic segment is transformed to a second thoracic segment fate, resulting
in complete duplication of the wing and mesonotum. In butterflies, clonal loss
of Ubx also results in transformation of hindwing scales to a
forewing pattern (Weatherbee et al.,
1999). Thus, orthologous UBX proteins specify differences between
forewings and hindwings in these two morphologically distinct contexts. A
simple model postulates that Ubx modifies hindwing morphology by
regulating different sets of downstream target genes in these insect orders
(Weatherbee et al., 1999
).
Several genes that are differentially expressed in the forewing and the
haltere in Drosophila, and therefore are genetically downstream of
Ubx, have been identified
(Weatherbee et al., 1998).
However, direct regulation of only one gene, spalt (sal),
has been demonstrated (Galant et al.,
2002
). Through identification of additional UBX-regulated targets
and characterization of their regulatory elements, we may determine sequence
features that are required for UBX regulation, and better understand how
regulation by Hox proteins is integrated into a morphogenetic program,
together with regulation by signaling pathways, other selector proteins and
tissue-specific transcription factors.
The knot gene is a candidate for direct UBX regulation in the
haltere. Loss of knot function causes apposition of the L3 and L4
veins, and loss of the L3-L4 intervein region in the forewing
(Mohler et al., 2000;
Vervoort et al., 1999
).
knot is expressed at the anteroposterior compartment boundary in the
developing wing, where it is activated by Cubitus interruptus (Ci)
(Vervoort et al., 1999
), the
transcriptional effector of the Hedgehog signaling pathway. knot
expression is absent from the haltere, and knot is repressed
cell-autonomously in clones that overexpress UBX in the wing
(Galant et al., 2002
). In
addition, D-SRF (Drosophila serum response factor; bs
FlyBase), which is itself a target of UBX repression
(Weatherbee et al., 1998
),
requires knot for activation
(Vervoort et al., 1999
). Thus,
lack of D-SRF expression in the haltere may be due either to direct
action of UBX on D-SRF regulatory sequences or to UBX repression of
knot, its activator.
The knot gene is also required during embryonic development for
formation of embryonic muscle (Crozatier
and Vincent, 1999) and several head structures
(Crozatier et al., 1999
;
Seecoomar et al., 2000
).
knot is expressed in the lymph gland precursors, and is required for
the development of lamellocytes, large cells that encapsulate foreign bodies,
in response to parasitization (Crozatier et
al., 2004
). This multiplicity of functions suggests a multiplicity
of regulatory elements that control knot expression in its various
contexts.
We have identified a wing-specific regulatory element for the knot gene and demonstrate its direct regulation by the Hedgehog signaling pathway and the UBX Hox protein. We find that a minimal element for repression in the haltere is not conserved, but a second, apparently redundant, element is conserved, and is located more than 500 bp from the minimal region. This result suggests that UBX repression is distributed over a large regulatory region that may not have sharply bounded elements, as defined by sequence conservation. In addition, a second, novel UBX repression element appears to have evolved in the D. melanogaster lineage in the presence of a pre-existing functional element, suggesting that selection is acting on a larger region than the minimally defined regulatory module.
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Materials and methods |
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Immunohistochemistry
Third-instar imaginal discs were dissected, fixed and immunostained as
previously described (Galant et al.,
2002). Knot protein was detected with rabbit anti-Kn antibody
provided by Michèle Crozatier
(Crozatier and Vincent, 1999
).
Engrailed protein was detected with mouse monoclonal antibody 4F11 provided by
Nipam Patel (Patel et al.,
1989
).
Mutagenesis of Ci- and UBX-binding sites
Ci-binding sites were altered by PCR mutagenesis. Site Ci1047 was altered
from TGGGTGGCA to TGGGTAGGCA; site Ci1341 was altered
from GCGGTGGTC to GCGGTAGTC; site Ci1680 was altered
from TGTGTGGCC to TGTGTAGCC. UBX-binding sites were
altered or deleted by PCR mutagenesis. UBX site 1 was altered from
GCTTAATTTG to GCTGCGTTTG; UBX site 2 was altered from
AGAATTAAGC to AGAAGCGCGC; UBX site 3 was altered from
CCACTAATTA to CCACGCGCGC. The entire sequence of UBX
site 4 shown in Fig. 4C was
deleted by PCR sewing. The sequence of site 1835-1840 was altered from AACATGT
to GGCCTGT by PCR mutagenesis. UBX sites in aligned block 2 were
altered using the Stratagene Quickchange Mutagenesis kit following the
manufacturer's instructions.
|
Amplification of Drosophila spp. knot regulatory sequences
Genomic DNA was isolated from additional species of Drosophila
obtained from the Tucson Drosophila Stock Center: D. mauritiana,
D. malerkotliana, D. biarmipes, D. pseudoobscura and D. virilis.
Ten to fifteen flies suspended in homogenization buffer [10 mM Tris pH 7.8, 50
mM NaCl, 10 mM EDTA, 5% (w/v) sucrose] were crushed with a pestle. Lysis
buffer (300 mM Tris pH 9.5, 100 mM EDTA, 0.625% SDS, 5% sucrose) and RnaseA
(50 µg/ml final concentration) were added, and the homogenate was incubated
at 70°C for 15 minutes. One-tenth volume sodium acetate was added and the
mixture was incubated on ice 30 minutes. After pelleting debris, genomic DNA
was extracted with phenol:chloroform, precipitated in ethanol and resuspended
in TE. PCR amplification was performed with PfuTurbo polymerase:
forward primer 5'-GTCACTTGATCGCTGCATTG-3'; reverse primer
5'-GGATTTGCTTGGGGAATTG-3'. Amplified fragments were A-tailed and
cloned into pGEM-T-Easy for sequencing. Sequence alignments were generated
using CLUSTALW (Thompson et al.,
1994) and then adjusted by hand.
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Results |
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Based on the location of the knotSA2 lesion, we
generated reporter constructs with genomic DNA from the region 5-20 kb
5' of knot (Fig.
2A). We identified a 6.8 kb region of DNA 15 kb 5' of
knot that drove expression of lacZ in a stripe at the
anteroposterior compartment boundary in the wing imaginal disc (data not
shown), consistent with the expression pattern of the Knot protein. No
expression of lacZ was observed in the haltere, demonstrating that
this large region accurately recapitulates the expression and regulation of
the endogenous knot gene. To determine if both wing and haltere
regulation was confined to a single region within this 6.8 kb, we further
narrowed the activity to a 2.3 kb region that drove appropriate reporter gene
expression (data not shown). All subsequent numbering of constructs is in
reference to this 2.3 kb region, knMel1-2330.
|
A single Ci binding site mediates activation in the wing
Expression of the knot gene is dependent on Hedgehog (Hh)
activity, and overexpression of Hh can trigger ectopic knot
expression in the wing (Vervoort et al.,
1999). The transcriptional effector of Hh signaling is the Cubitus
interruptus (Ci) protein. Ci is a zinc-finger transcription factor of the Gli
family, and binds a 9 bp consensus sequence TGGG(T/A)GGTC
(Von Ohlen et al., 1997
). In
the 1.3 kb knMel701-1991 fragment, we identified three
potential Ci-binding sites that matched at least seven out of nine consensus
residues and that were conserved in D. pseudoobscura
(Fig. 3A). Two additional
potential sites were present, but were not conserved in D.
pseudoobscura. We mutagenized the three conserved binding sites
independently, converting a crucial guanine to an adenine
(Zarkower and Hodgkin, 1993
),
and re-introduced the mutagenized element into flies. Changes at two of the
three candidate sites had no effect on reporter gene expression (data not
shown), whereas the mutation of site Ci1680 almost completely abolished
reporter expression (Fig. 3B).
Mutation of all three sites did not have a more severe effect than mutation of
Ci1680 alone (data not shown). These results indicate that activation of the
wing-specific enhancer element by Hh signaling is dependent primarily on a
single Ci-binding site at position 1680 in the
knMel701-1991 element.
|
To determine which TAAT sequences might be functionally important for UBX repression, we removed sequences from each end of knMel701-1991 and observed the effect on reporter gene expression in vivo. Removal of the 5' end, with its small cluster of four core sequences, had no effect on expression. By contrast, removal of 156 bp from the 3' end, including nine putative UBX-binding sites (knMel701-1835), caused the reporter to be expressed at the AP compartment boundary in both the wing and the haltere (Fig. 4B). Therefore, knMel701-1991 does appear to be directly negatively regulated by UBX in the haltere, and removal of UBX-binding sites relieves repression in the haltere. In addition to the ectopic activation of expression in the haltere, we noted that the expression level in the wing is also elevated compared with knMel701-1991 (Fig. 2C, Fig. 4B), suggesting that additional repressor binding sites important for appropriate wing expression may have been removed in knMel701-1835. Importantly, the response to local spatial information within the wing field (encompassing both the wing and haltere) was maintained, as expression was appropriately observed at the AP compartment boundary in both tissues. Because the single deletion preserved the response to spatial information within the dorsal appendage wing field but altered the response to spatial information along the anteroposterior axis, we suggest that activation by Ci and repression by UBX are mediated through physically separable sites within knot cis-regulatory sequences.
To identify which potential binding sites could be occupied by UBX in vitro, we performed DNaseI footprinting on a 392 bp fragment (knMel1599-1991) that includes the functional Ci site and the 156 bp required for repression in the haltere. This fragment is itself capable of driving expression in the wing, although at a significantly lower level than that driven by the full knMel701-1991, and is repressed in the haltere (data not shown). We identified four regions protected from DNaseI digestion by binding of UBX (Fig. 4C,D, sites 1-4). These four regions include all TAAT core sequences present in the 392 bp fragment (10 in total).
Although UBX site 1 is located only 4 bp from the Ci-binding site, it is
still present in the knMel701-1835 construct that is
derepressed in the haltere, so this site alone is not sufficient to mediate
repression by UBX. To determine whether this site is necessary for repression
by UBX, we mutated UBX site 1 alone (knMel701-1991UBX1KO)
and did not observe any derepression of reporter gene expression in the
haltere (Fig. 5A). Therefore,
UBX Site 1, unlike individual UBX-binding sites in the spalt enhancer
(Galant et al., 2002), does
not appear to contribute significantly to repression of this element by UBX.
Of the other regions protected by UBX, the largest spans six TAAT core
sequences and
24 bp of sequence, and is located
250 bp from the Ci
binding site. Therefore, the DNA sequences necessary for repression in the
haltere appears to be comprised of multiple, functional UBX-binding sites that
do not overlap with the activating Ci-binding site. This organization suggests
that UBX does not repress knot in the haltere by competing for
activator binding sites.
|
To determine where additional potential regulatory sequences are located,
we restored sequence 3' of the knMel701-1835
construct. Addition of 43 bp (knMel701-1878) was
sufficient to partially restore repression in the haltere
(Fig. 5C), suggesting the
additional regulatory information was contained within this region. Deletion
of this block of sequence (knMel701-1991) resulted
in very weak, inconsistent de-repression in the haltere. By contrast, point
mutations introduced at positions 1834-1837
(knMel701-1991mut), the boundary of the derepressed
knMel701-1835 construct, resulted in consistent, partial,
de-repression (Fig. 5D). As
this position is not a UBX site, this result suggests that at least one
transcription factor acts in addition to UBX to repress knot in the
haltere through this regulatory element. Mutation of both positions 1834-1837
and all UBX TAAT core sequences (knMel701-1991KOmut)
resulted in full de-repression in the haltere
(Fig. 5E), suggesting that UBX
and another repressor act together to reduce expression in the haltere through
sequences located between knMel1835-1991. The DNA sequence
at knMel1834-1837 does not clearly match any binding sites
archived in transcription factor databases, and as yet we do not know the
identity of the factor that may act with UBX to repress knot in the
haltere.
Identification of a functional repressor element in D. pseudoobscura
To understand how UBX-regulated target gene networks evolve, it is crucial
to determine how UBX regulation of individual target genes evolves. We
combined our dissection of the knot wing regulatory element with
comparative genomics within Drosophila to establish how
UBX-responsive regulatory sequences in knot have evolved. We compared
the 156 bp knot repressor element from D. melanogaster to
D. pseudoobscura sequence, and did not observe either significant
sequence conservation or a comparable cluster of potential UBX-binding sites
in D. pseudoobscura. Because the expression pattern of knot
is the same between these two species (Fig.
1), these significant sequence differences suggest that regulation
by UBX is mediated through different regulatory sequences in D.
pseudoobscura. Therefore, we attempted to identify a functional
regulatory element from D. pseudoobscura that could regulate reporter
expression in the appropriate pattern.
Using blocks of sequence identity as relational anchor points, we amplified a fragment from D. pseudoobscura (knPse1-1935) that roughly corresponded to the knMel1-2330 D. melanogaster fragment (Fig. 6A). We introduced this fragment into D. melanogaster and found that it could properly drive expression in the wing while repressing expression in the haltere (Fig. 7A). The knPse1-1935 construct contained at its 3' end a cluster of 12 TAAT UBX core binding sites. To determine if this region is important for repression by UBX in D. pseudoobscura, we generated a truncation of knPse1-1935 that eliminated the TAAT core sequences. This knPse1-1643 construct appropriately drove expression in the wing, but now also drove haltere expression (Fig. 7B). Therefore, the region containing these putative UBX-binding sites acts as a repressor element in the haltere.
|
|
We next sought to determine whether UBX-binding sites in the knMel2499-2722 conserved element are sufficient to repress reporter expression, or whether this element also requires the action of a collaborating repressor. We mutated all UBX core binding sites in this sequence and attached the mutated knMel2499-2722KO sequence to the de-repressed knMel701-1835 (generating knMelcompositeKO). Whereas mutation of UBX sites alone in knMel701-1991KO did not fully de-repress in the haltere, mutation of UBX sites in knMelcompositeKO was sufficient for complete de-repression in the haltere (Fig. 7D). Thus, the knMel2499-2722 and knMel1835-1991 repressor elements appear to be organized differently the former with input only from UBX, and the latter with input from UBX and an additional trans-acting factor.
Does the presence of two elements in D. melanogaster indicate the
acquisition of a new element in this lineage or the loss of an element in
D. pseudoobscura? To analyze the distribution of these two regulatory
elements in other drosophilids, we amplified the knot regulatory
region from three additional Drosophila species D.
mauritiana, D. biarmipes and D. malerkotliana
phylogenetically intermediate between D. melanogaster and D.
pseudoobscura (Schawaroch,
2002). All three species have sequence similar to
knPse1643-1935 (Fig.
6B), but also possess sequence similar to
knMel1835-1991 in varying degrees. For example, the core
TAAT of UBX site 3 is shared by all three additional species (though sequence
surrounding the core is non-identical), whereas UBX site 2 is found only in
D. mauritiana. The most interesting pattern is observed for UBX site
4. D. malerkotliana has only a single core UBX sequence conserved
with D. melanogaster, D. biarmipes has two conserved core sequences
and two additional core sequences that are unique, and D. mauritiana
has five of the six core sequences present in D. melanogaster.
Therefore, in this sample of five drosophilid species, we observe the pattern
of an apparent accretion of UBX-binding sites in this region in the evolution
of the D. melanogaster lineage.
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Discussion |
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Mechanism of UBX repression
Owing to their low DNA-binding specificity and paucity of known direct
targets, mechanisms for the selection of specific target genes by Hox proteins
remain to be fully explained. Much work has focused on the role of co-factors
in increasing the binding specificity of their Hox partners. When Hox proteins
interact with PBC and MEIS proteins, represented in Drosophila by EXD
and HTH (Chan et al., 1997;
Gebelein et al., 2002
;
Ryoo and Mann, 1999
;
Ryoo et al., 1999
), the
resulting compound-binding sites are of sufficient size and information
content so as not to appear by random chance at high frequency in the genome.
However, neither EXD nor HTH are necessary for development of the haltere, so
the action of UBX in this tissue must be independent of these co-factors
(Azpiazu and Morata, 1998
;
Azpiazu and Morata, 2000
).
Repression of spalt gene expression by UBX in the haltere depends
upon multiple individual UBX monomer-binding sites,
(Galant et al., 2002
) rather
than compound binding sites. In addition, a DNA sequence that binds neither
Hox proteins nor Hox-PBC dimers determines specificity of Deformed or Labial
regulation of a Deformed autoregulatory element, but the identity of this
co-factor is unknown (Li et al.,
1999
).
Our functional analysis of the knot regulatory element is consistent with UBX repression occurring through monomer sites. UBX-binding sites in the sal1.1 and knot minimal enhancers cannot be aligned beyond the TAAT core, and so neither suggest the role of a common DNA binding co-factor. However, mutation of the identified UBX binding sites alone did not result in full de-repression of the knot minimal element in the haltere. Rather, full de-repression required mutation of additional sites not bound by UBX. This sequence may bind either a bona fide co-repressor that interacts with UBX to repress target genes or a protein that independently, but additively, contributes to repression. Because knMel701-1835 drives a higher level of expression in the wing than knMel701-1991, this putative repressor may act in both the wing and the haltere.
Analyses of both the sal and knot regulatory regions suggest that UBX may be a weak repressor that requires the collaboration of other factors, which may act in the wing and haltere to regulate other features of these tissues, in order to mediate full repression. However, as mutation of UBX sites alone in knMel2499-2722 is sufficient for de-repression, UBX may, in some contexts, be able to mediate full regulatory activity on its own. Flexibility in the organization of UBX-responsive enhancers may be due to the unsystematic, undesigned assembly of regulatory elements during evolution.
Regulatory elements that are cobbled together, incorporating binding sites
for multiple collaborating transcription factors to take advantage of an
existing landscape of developmental regulators, appear to be common. In the
developing Drosophila embryo, both UBX and ABD-A repress the target
gene Distalless (Dll) in abdominal segments, limiting leg
development to the thoracic segments
(Gebelein et al., 2002;
Vachon et al., 1992
).
Repression of Dll also requires the action of the
compartment-specific regulators, Engrailed and Sloppy-paired
(Gebelein et al., 2004
), in
collaboration with the Hox proteins. In addition, the Hox protein Labial
interacts with the Decapentaplegic (Dpp) signaling pathway to direct
appropriate expression of the lab550 autoregulatory enhancer element in the
Drosophila embryo (Marty et al.,
2001
), and Abdominal-A similarly collaborates with Dpp signaling
to regulate wingless expression
(Grienenberger et al., 2003
).
Collaboration may be a common requirement for Hox-regulated enhancers. Thus,
rather than being highly potent regulators, Hox proteins may be weak
regulators that employ a variety of collaborative factors in order to perform
their function. The ability of Hox proteins to act as either repressors or
activators of target genes may be regulated by interactions with different
collaborators (Li and McGinnis,
1999
; Li et al.,
1999
).
Furthermore, the weak activity of UBX and the potential requirement for collaborators for Hox repression of target genes may help to explain why it has not been possible to impart UBX regulation to a naïve cis-regulatory element by the addition of UBX monomer binding sites. Extensive efforts in this laboratory have placed multiple UBX-binding sites in various positions in cis-regulatory elements active in the wing and haltere, but with no effect (C. M. Walsh and S.B.C., unpublished). The separability of Ci activator binding sites from UBX repressor binding sites in the knot regulatory element demonstrates that in this enhancer UBX does not repress by direct competition for activator binding sites, and suggests that distance of UBX-binding sites from activator binding sites is not the cause for this failure. If UBX is such a weak repressor that UBX-binding sites alone, even in multiple copies, are not sufficient to impart repression, then the proximity of binding sites for collaborating repressor proteins may be a crucial determinant.
Conservation, redundancy, and the unit of selection in cis-regulatory elements
To better understand how UBX regulates morphology, we would ideally like to
know all target genes on which it acts and the DNA regulatory sequences
through which it exerts this control. Characterization of these regulatory
sequences would elucidate the rules governing transcriptional regulation and
how modification of regulatory sequences can occur during evolution. Our
knowledge of the organizational constraints on regulatory sequences and how
evolution operates within those constraints to maintain enhancer function is
limited. Several analyses indicate that sequence within regulatory elements
can vary even when function is maintained
(Hancock et al., 1999;
Ludwig et al., 1998
;
McGregor et al., 2001
;
Shaw et al., 2002
).
Nevertheless, sequence conservation between related organisms can successfully
identify regulatory sequences in some lineages
(Wasserman et al., 2000
;
Yuh et al., 2002
). However,
98% of non-exonic multi-species conserved sequences within mammals do not
correspond to known regulatory elements
(Thomas et al., 2003
). We can
either suppose that these sequences primarily represent regulatory elements
yet to be functionally characterized or that sequence conservation alone is
not an indicator of regulatory function.
Our dissection of the knot regulatory region provides examples of apparently redundant binding sites for individual transcription factors, apparently redundant functional repressor elements, and sequence conservation without obvious biological function. For example, of three conserved putative Ci-binding sites, each contained within larger blocks of sequence conservation, only Ci1680 is necessary for activation of knot in the wing field. This observation suggests several possible interpretations. First, the other Ci sites may be functioning in a different context a different tissue, for example than examined in our assay, and selection has maintained these sites for that additional role. However, even the large 6.8 kb knot regulatory fragment did not appear to drive lacZ reporter expression in a limited set of additional tissues surveyed (data not shown), so we do not have any positive evidence supporting its role elsewhere in development. Similarly, the conserved blocks could represent binding sites for other factors, with conservation a consequence of maintaining those regulatory sites rather than the Ci sites. Next, it is possible that the evolutionary distance between D. melanogaster and D. pseudoobscura is not appropriate for addressing the relationship between sequence conservation and functional consequence. However, as this distance is approximately equivalent to the distance of the human-mouse comparison, we must then infer significant differences in the dynamics of sequence evolution within these two lineages. Finally, the additional Ci sites may contribute to regulation in the context of the wing to a degree that we are unable to detect, but that purifying selection does act upon, and it is this view that we favor.
The apparent redundancy of UBX repressor elements in the D.
melanogaster knot regulatory region also requires explanation. The
accretion of UBX sites in the knot regulatory region in our sample of
species phylogenetically intermediate to D. pseudoobscura and D.
melanogaster suggests that a novel UBX-responsive element has evolved.
Given the presence of a pre-existing, functional sequence that is maintained
in both D. melanogaster and D. pseudoobscura, how has
selection maintained the conserved element and allowed expansion of the novel
element? Dissection of the eve stripe 2 regulatory element in both
D. melanogaster and D. pseudoobscura demonstrated that
compensatory evolution could lead to turnover of individual binding sites,
resulting in a regulatory element with conserved function in the absence of
sequence conservation (Ludwig et al.,
1998; Ludwig et al.,
2000
). However, compensatory evolution that maintains repression
of knot in the haltere does not seem to be the solution, as the
downstream element is still present and therefore presumably capable of
repressing knot expression. We conclude that the minimal element we
identified in our functional assay is not necessarily identical to the
functional unit upon which selection acts. That is, selection can detect and
select for organismal-level effects of regulatory changes that are not obvious
in our functional assay. Therefore, minimal functional regulatory elements
defined by molecular dissection may not reflect the full, complete enhancers
that selection has built. Rather than being sharply bounded and discrete,
regulatory elements may be more diffuse collections of transcription factor
inputs.
From an evolutionary standpoint, such a diffuse, flexible regulatory architecture seems a necessity. If particular precise arrangements of transcription factor binding sites are required to produce a transcriptional output, the probability of evolving a novel functional regulatory element by point mutation is exceptionally low. If, instead, a weak regulator, as UBX appears to be, collaborates with a factor already operating on an enhancer, then a novel output may be generated that may be reinforced by selection. This reinforcement may eventually result in a more precise, optimized arrangement of binding sites and a more robust regulatory output.
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ACKNOWLEDGMENTS |
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