Multiple enhancers ensure precision of gap gene-expression patterns in the Drosophila embryo.

Abstract

Segmentation of the Drosophila embryo begins with the establishment of spatially restricted gap gene-expression patterns in response to broad gradients of maternal transcription factors, such as Bicoid. Numerous studies have documented the fidelity of these expression patterns, even when embryos are subjected to genetic or environmental stress, but the underlying mechanisms for this transcriptional precision are uncertain. Here we present evidence that every gap gene contains multiple enhancers with overlapping activities to produce authentic patterns of gene expression. For example, a recently identified hunchback (hb) enhancer (located 5-kb upstream of the classic enhancer) ensures repression at the anterior pole. The combination of intronic and 5' knirps (kni) enhancers produces a faithful expression pattern, even though the intronic enhancer alone directs an abnormally broad expression pattern. We present different models for "enhancer synergy," whereby two enhancers with overlapping activities produce authentic patterns of gene expression.

Full text

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Multiple enhancers ensure precision of gap
gene-expression patterns in the Drosophila embryo
Michael W. Perrya, Alistair N. Boettigerb, and Michael Levinec,d,1
aDepartment of Integrative Biology,bBiophysics Graduate Group,cCenter for Integrative Genomics, Division of Genetics, Genomics, and Development, and
dDepartment of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200
Contributed by Michael Levine, June 22, 2011 (sent for review May 25, 2011)
Segmentation of the Drosophila embryo begins with the establish-
ment of spatially restricted gap gene-expression patterns in re-
sponse to broad gradients of maternal transcription factors, such
as Bicoid. Numerous studies have documented the fidelity of these
expression patterns, even when embryos are subjected to genetic
or environmental stress, but the underlying mechanisms for this
transcriptional precision are uncertain. Here we present evidence
that every gap gene contains multiple enhancers with overlapping
activities to produce authentic patterns of gene expression. For
example, a recently identified hunchback (hb) enhancer (located
5-kb upstream of the classic enhancer) ensures repression at the
anterior pole. The combination of intronic and 5′ knirps (kni) en-
hancers produces a faithful expression pattern, even though the
intronic enhancer alone directs an abnormally broad expression
pattern. We present different models for “enhancer synergy,”
whereby two enhancers with overlapping activities produce au-
thentic patterns of gene expression.
cis-regulation|development|robustness
R
embryo (1). These enhancers are sometimes located within
neighboring genes, and along with conventional, proximal en-
hancers, they produce robust patterns of gene expression in early
embryos under stress (2, 3). For example, the snail gene exhibits
erraticpatternsofactivationinembryosraisedat30°Cwheneither
the proximal or shadow enhancer is removed (3). It was proposed
that shadow enhancers represent a mechanism of “canalization”
(4), whereby populations of embryos develop normally even when
subject to variations in temperature or genetic background.
In the present study we provide evidence that many of the
genes controlling anterior-posterior patterning contain multiple
enhancers with overlapping activities, including head patterning
genes and gap genes, which initiate the segmentation gene net-
work (e.g., refs. 5 and 6). For example, the gap genes hunchback
(hb), Kruppel (Kr), and knirps (kni) are each regulated by two
distinctenhancersthatcontroltheinitialbandsofgeneexpression
within the presumptive head, thorax, and abdomen. Evidence is
presented that the two enhancers work together (enhancer syn-
ergy) to ensure uniform expression within correct spatial limits.
Previous studies have documented examples of enhancer au-
tonomy and enhancer interference. Multiple enhancers often
produce additive patterns of gene expression, as seen for the 7-
stripe even skipped (eve) expression pattern arising from five sep-
arate enhancers (two located 5′ of the eve transcription unit and
threelocateddownstreamofthegene)(7–9).Sometimes,multiple
enhancers interfere with one another when placed within a com-
mon regulatory region. For example, ventral repressors that de-
lineate the intermediate neuroblasts defective (ind) expression
pattern block the activities of a neighboring eve stripe-3 enhancer,
and conversely, repressors that establish the posterior limit of
the stripe-3 pattern interfere with ind (10).
In the present study, evidence is presented that combining
multiple enhancers in a common regulatory region can produce
sharper and more homogeneous patterns of gene expression. We
ecent studies identified “shadow” enhancers for genes en-
gaged in the dorsal-ventral patterning of the early Drosophila
discuss potential mechanisms for such enhancer synergy and
suggest that minimal enhancers producing aberrant patterns of
gene expression might nonetheless contribute to authentic ex-
pression profiles in the context of their native loci.
Results and Discussion
Every Gap Gene Contains Multiple Enhancers for a Single Gap Pattern.
Candidate gap enhancers were identified using ChIP-chip data
(11). Specifically, clustered binding sites for maternal and gap
proteins were identified within 100 kb of every gap gene (see
Materials and Methods). This survey identified each of the known
enhancers, as well as putative shadow enhancers (12–17). For
example, a potential distal shadow enhancer was identified for
hb, located 4.5-kb upstream of the proximal transcription start
site (designated “P2” in ref. 18) and upstream of the later-acting
distal promoter (designated “P1”) (Fig. 1C).
A 400-bp genomic DNA fragment from this newly identified
region was attached to a lacZ reporter gene and expressed in
transgenic embryos (Fig. 1B). The resulting hb/lacZ fusion gene
exhibits localized expression in anterior regions of the embryo
similar to that seen for the endogenous gene and “classic” en-
hancer identified over 20 y ago (13, 19) (Fig. 1B; compare with
A). The classic proximal and distal shadow enhancers exhibit
similar responses to increasing Bicoid copy number (Fig. S1A).
ChIP-chip data also identified potential pairs of enhancers for
Kr (Fig. 1 D–F) and kni (Fig. 1 G–I). There are two distinct
clusters of transcription factor binding sites upstream of Kr. The
previously identified Kr “CD2” enhancer contains the proximal
enhancer but also part of the distal binding cluster (14). Sub-
sequent lacZ fusion assays identified each ChIP-chip peak and
underlying binding sites as separable proximal and distal en-
hancers (Fig. 1 D–F). Similarly, more refined limits were de-
termined for the kni intronic enhancer (Fig. 1 G and I, and Fig.
S1B) (12), in addition to the previously identified 5′ distal en-
hancer (20). Both the distal Kr enhancer and the intronic kni
enhancer produce somewhat broader patterns of expression than
the endogenous gene (Fig. 1 E and G) (see below). Additional
gap enhancers were also identified for giant, including an addi-
tional distal enhancer located ∼35-kb downstream within a
neighboring gene (Fig. S2A and ref. 12).
The survey of gap and maternal binding clusters was extended
to include the so-called “head” and “terminal” gap genes, critical
for the differentiation of head structures and the nonsegmented
termini of early embryos (Fig. 1 J–O and Fig. S2 B–J). Additional
enhancers were identified for empty-spiracles (ems) (original
enhancer identified in ref. 21) (Fig. 1 M–O), huckebein (hkb)
(original enhancer in ref. 22) (Fig. S2 E–G), and forkhead (fkh)
(original enhancer in ref. 12) (Fig. S2 B–D). More refined limits
Author contributions: M.W.P., A.N.B., and M.L. designed research; M.W.P. and A.N.B.
performed research; M.W.P. and A.N.B. contributed new reagents/analytic tools;
M.W.P., A.N.B., and M.L. analyzed data; and M.W.P., A.N.B., and M.L. wrote the paper.
The authors declare no conflict of interest.
1To whom correspondence should be addressed. E-mail: mlevine@berkeley.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1109873108/-/DCSupplemental.
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were also determined for the previously identified ocelliless/
orthodenticle (oc/otd) intronic enhancer (12) (Fig. 1 J–L). For
simplicity, we will hereafter refer to the two enhancers regulating
a given gap gene as proximal and distal, based on their relative
locations to the transcription start site.
Multiple hb Enhancers Produce Authentic Expression Patterns. BAC
recombineering (23, 24), phiC31-targeted genome integration
(25, 26), and quantitative in situ hybridization assays (3, 27) were
used to determine the contributions of the proximal and distal
enhancers to the hb expression pattern (Fig. 2). BACs containing
∼20 kb of genomic DNA encompassing the hb gene and flanking
sequences were integrated into the same position in the Dro-
sophila genome. The hb transcription unit was replaced with the
yellow gene, which permits quantitative detection of nascent
transcripts using an intronic hybridization probe (3) (Materials
and Methods and Fig. S3). The modified BAC retains the com-
plete hb 5′ and 3′ UTRs. Additional BACs were created by in-
activating the proximal or distal enhancers by substituting critical
regulatory elements with “random” DNA sequences (see diagrams
above panels in Fig. 2 A–C and Materials and Methods).
BAC transgenes lacking either the distal (Fig. 2A) or proximal
(Fig. 2B) enhancer continue to produce localized patterns of
transcription in anterior regions of transgenic embryos in re-
sponse to the Bicoid gradient. However, the patterns are not as
faithful compared with the BAC transgene containing both
enhancers (Fig. 2C). Embryos were double-labeled to detect
both yellow and hb nascent transcripts. During nuclear cleavage
cycle (cc) 13, a substantial fraction of nuclei (14%) expressing
hb nascent transcripts lack yellow transcription upon removal
of the shadow enhancer (Fig. 2A). An even higher fraction of
nuclei (24%) lack yellow transcription when the proximal en-
hancer is removed (Fig. 2B). Control transgenic embryos con-
taining both enhancers exhibit more uniform patterns of trans-
cription, whereby only an average of ∼3% of nuclei fail to match
the endogenous pattern of transcription (Fig. 2C). The distri-
bution of “missing nuclei” across the population of cc13 embryos
is plotted in Fig. 2D.
The pairwise Wilcoxon rank sum test (also called the Mann-
Whitney u test) was used to determine the significance of the
apparent variation in gene expression resulting from the re-
moval of either the proximal or distal enhancer (Fig. 2D).
Control embryos containing the hb BAC transgene with both
enhancers exhibit some variation in the number of nuclei that
lack yellow nascent transcripts. Despite this variation, the sta-
tistical analyses indicate that the loss of either the proximal or
distal enhancer results in a significant change in yellow tran-
scription patterns compared with the control BAC transgene
(P = 4E-8).
1000 bp
hb
Kr proximal Kr distal
hb proximalhb distal
kni proximalkni distal
hb
CG8112
Kr
kni
1000 bp
1000 bp
P1
P2
ABC
DEF
GHI
JKL
MN
O
ems distal
ems
oc/otd
oc/otd distal
otd
ems
1000 bp
1000 bp
= proximal= distal
Fig. 1.
identified distal enhancer. The locations of these enhancers are shown in C. (D and E) Kr/lacZ transgenes containing the (D) proximal or (E) distal enhancer.
The locations of these enhancers are shown in F. (G and H) The kni/lacZ transgenes containing either the (G) proximal intronic enhancer or (H) the distal 5′
enhancer. The locations of these enhancers are shown in I. (J and K) Expression of endogenous oc/otd (J); oc/lacZ transgene containing an intronic enhancer
(K). The locations of the oc/otd enhancers are shown in L. (M and N) Expression of endogenous ems (M); ems/lacZ transgene containing a distal enhancer (N).
Locations of the enhancers shown in O.
Activities of gap enhancers identified by in situ hybridization. (A and B) The hb/lacZ transgenes containing the (A) proximal (classic) or (B) newly
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Distal hb Enhancer Mediates Dominant Repression. The preceding
analyses suggest that multiple enhancers produce more uniform
patterns of de novo transcription than individual proximal or
distal enhancers. Additional studies were done to determine
whether multiple enhancers also help produce authentic spatial
limits of transcription (Fig. 3).
The expression of hb normally diminishes at the anterior pole
of cc13 to 14 embryos. This loss in expression has been attributed
to attenuation of Bcd activity by Torso RTK signaling (e.g., ref.
28). However, the proximal enhancer fails to recapitulate this
loss (29) (Fig. 1A). In contrast, the distal enhancer is inactive at
the anterior pole (Fig. 1B), and the two enhancers together
produce a pattern that is similar to endogenous expression, in-
cluding reduced expression at the pole (Fig. 2C).
To examine the relative contributions of the proximal and
distal enhancers in this repression, yellow nascent transcripts
were measured in transgenic embryos expressing BAC reporter
genes containing one or both hb enhancers (Fig. 2). Particular
efforts focused on the early phases of cc14, when repression of
endogenous hb transcripts is clearly evident (Fig. 3). For the
transgene lacking the proximal, classic enhancer, but containing
the newly identified distal enhancer, a median of 6% (std 6%) of
nuclei exhibit expression of yellow nascent transcripts but lack
expression of the endogenous gene (Fig. 3A). In contrast, a me-
dian of 24% (std 11%) of nuclei displays a similar discordance
upon removal of the distal enhancer. In control embryos, 16%
(std 11%) of nuclei express yellow but lack hb nascent transcripts
(Fig. 3 B and C). It should be noted that the BAC transgene
lacking the proximal enhancer exhibits “super-repression” be-
cause of reduced activation at the anterior pole (P = 0.012)
(Fig. 3D).
These observations suggest that the distal enhancer contains
repression elements that function in a dominant manner to at-
tenuate the activities of the proximal enhancer at the anterior
pole. There is a loss of repression when the distal enhancer
driving lacZ is crossed into a torso mutant background (Fig. S4).
This observation implicates one or more repressors functioning
downstream of Torso signaling, including Tailless and Huck-
ebein, which have been shown to function as long-range domi-
nant repressors (30–32). Indeed, whole-genome ChIP assays
identify more potential Tll and Hkb binding sites in the distal vs.
proximal enhancer (11) (Fig. S5A). The persistence of hb ex-
pression in anterior regions has been shown to be detrimental,
causing defects in mouth parts and malformation of the gut (33).
Correction of the kni Expression Pattern.Kr/lacZ andkni/lacZfusion
genes containing either one or two enhancers were inserted into
the same position in the Drosophila genome (Fig. 3). Transgenic
embryos were double-labeled to detect the expression of the
transgene (lacZ) as well as the endogenous gap gene.
The kni proximal (intronic) enhancer alone produces an ab-
normally broad pattern of expression, especially in posterior
regions (Fig. 3F; but see also Fig. 1G and ref. 12). In contrast, the
kni distal (5′) enhancer produces erratic lacZ activation within
AB
CD
Fig. 2.
proximal or distal enhancers. The hb transcription unit was replaced with the yellow reporter gene to identify de novo transcripts via in situ hybridization
using a probe directed against the yellow intron (see diagram above images in A–C). (A) hb-BAC with distal enhancer inactivated (see X in diagram), (B) hb-
BAC with proximal enhancer inactivated, and (C) hb-BAC with both enhancers intact. The median ratio of “discordance” is indicated beneath each image. This
is the fraction of nuclei that express endogenous hb nascent transcripts, but not yellow transcripts. (D) Cumulative frequency distributions for the fraction of
“missing nuclei” in the three populations of embryos. The ordinate axis gives the probability of observing an embryo from this population, with fewer than
the abscissa fraction of nuclei transcribing the endogenous gene but not the reporter. Statistical comparisons between the distributions are presented above
the panel, with subscripts matching the panel labels [i.e., pCA is the P value from the pair wise comparison of the distribution of embryos with the hb control
BAC, (C), to those with the distal enhancer removed (A)].
Function of hb enhancers via BAC transgenesis. An ∼20-kb BAC containing genomic DNA encompassing the hb locus was modified to remove specific
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nearly normal spatial limits (Fig. 3E). An essentially normal
pattern of lacZ transcription is observed when both enhancers are
combined in a common transgene (intronic enhancer 5′ and distal
enhancer 3′ of lacZ) (Fig. 3G). It appears that lacZ transcription
is slightly broader than the endogenous pattern, but considerably
narrower than the pattern observed for the intronic enhancer
alone (Fig. 3J) (P = 1.8E-6), and not statistically different from
the expression limits of the distal enhancer alone (P = 0.72) (Fig.
3L). There is no significant narrowing of the Kr/lacZ expression
pattern when both the distal and proximal enhancers are com-
bined within the same transgene (Fig. 3 K and L) (P = 1.0).
Perhaps additional Kr regulatory elements are required for the
type of narrowing observed for the kni intronic enhancer. Alter-
nately, all of these transgenes use the eve basal promoter and it is
possible that promoter-specific interactions are important for
establishing the normal limits of the Kr expression pattern.
As discussed earlier, long-range repressors bound to the distal
hb enhancer might inhibit the activities of the proximal enhancer
at the anterior pole of precellular embryos. The distal kni en-
hancer might function in a similar manner to sharpen the ex-
pression limits of the intronic enhancer. The spatial limits of gap
gene-expression patterns have been shown to depend on cross-
repressive interactions (e.g., refs. 34–36). The kni intronic en-
hancer might lack critical gap repression elements because it
produces an abnormally broad expression pattern. Indeed, whole-
genome ChIP assays identify more putative Tailless binding sites
in the distal vs. intronic enhancer (11) (Fig. S5B). These Tailless
repression elements might function in a dominant fashion to
restrict the limits of the intronic enhancer.
The modest anterior expansion of the expression pattern
driven by the kni intronic enhancer is more difficult to explain
because this boundary is probably formed by the Hb repressor
(37), which is not known to function in a long-range and domi-
nant manner. If the action of short-range repressors is also af-
fected by stochastic processes (e.g., binding of the repressor to
enhancer or looping of a bound enhancer to promoter), perhaps
having two enhancers might improve the chances of maintaining
proper repression.
We have presented evidence that the robust and tightly de-
fined patterns of gap gene expression do not arise from the
unique action of individual enhancers. Rather, these patterns
depend on multiple and separable enhancers with similar, but
slightly distinct regulatory activities. This enhancer synergy pro-
duces more homogeneous patterns of transcriptional activity, as
well as more faithful spatial limits of expression.
The enhancer synergy documented in this study is somewhat
distinct from the proposed role of the shadow enhancer regu-
lating snail expression in the presumptive mesoderm (3). The
dual regulation of snail by the proximal and distal (shadow)
enhancers was shown to ensure homogenous and reproducible
expression in embryo after embryo in large populations of em-
bryos, even when they are subject to increases in temperature. In
contrast, dual regulation of hb expression by proximal and distal
enhancers appears to ensure homogenous activation in response
to limiting amounts of the Bicoid gradient. They are used as an
obligatory patterning mechanism rather than buffering environ-
mental changes. Despite these apparent differences, it is possible
that dominant repression is also used as a mechanism of synergy
Transcribe reporter AND endogenous Transcribe reporter but NOT endogenous
kni 2 enhancerkni distal kni proximal
EFG
ABC
D
Transcribe reporter AND endogenous Transcribe reporter but NOT endogenous (anterior pole)
X
X
hb controlhb proximal removed hb distal removed
Kr 2 enhancer Kr distal Kr proximal
IJK
L
Transcribe reporter AND endogenous Transcribe reporter but NOT endogenous
6% (σ=6%)24% (σ=11%)
20% (σ=9%)
7% (σ=7%)
7% (σ=5%)
7% (σ=7%)
H
7% (σ=4%)
1% (σ=4%)
16% (σ=11%)
0 0.2
fraction of ectopically active nuclei
0.40.60.81
0
0.2
0.4
0.6
0.8
1
cummulative frequency
pairwise Wilcoxon: pCB = 0.015 pCA = 0.012 pAB = 0.00044
control 22C N=44
no distal 22C N=47
no proximal 22C N=10
00.20.4 0.60.8 1
0
0.2
0.4
0.6
0.8
1
ectopic expression rate
cummulative frequency
pairwise Wilcoxon: pGF = 1.8e−06 pGE = 0.72 pEF = 2.3e−05
kni 2 enhancers, 22C N=21
kni distal, 22C N=28
kni proximal, 22C N=16
00.2 0.4 0.6 0.81
0
0.2
0.4
0.6
0.8
1
ectopic expression rate
cummulative frequency
pairwise Wilcoxon: pKI = 0.88 pKJ = 0.00074 pIJ = 0.00088
Kr 2 enhancers, 22C N=17
Kr distal, 22C N=12
Kr proximal, 22C N=14
Fig. 3.
transcripts. Nuclei exhibiting ectopic transgene expression are indicated in red. Sites of concordant expression are indicated in yellow. (A–C) BAC transgenes
lacking the proximal (A) or distal (B) enhancer, or containing both enhancers intact (C). Nuclei in the anterior third of the hb-expression region which
transcribe the reporter but not endogenous hb are shown in red. (D) Cumulative frequency of nuclei in the anterior third of the hb-expression domain
containing yellow, but not endogenous hb, nascent transcripts. Median and SDs are shown on the corresponding panels. (E and F) The kni/lacZ reporter genes
driven by (E) distal enhancer, (F) proximal enhancer, or (G) both enhancers. Median fractions of nuclei transcribing lacZ but not the endogenous gene are
indicated below each image. (H) Cumulative frequency distributions for the fraction of ectopically active nuclei. (I–L) Similar analysis of Kr/lacZ transgenes
containing the distal (I), proximal (J), or both (K) enhancers.
Enhancer synergy produces correct spatial limits. Discordance of yellow (A–C) or lacZ (E–G, I–K) transgenes and endogenous gap gene nascent
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for the regulation of snail expression. The distal enhancer con-
tains repressor elements (e.g., Huckebein) that inhibit the ex-
pression of the proximal enhancer at the termini (3).
Different mechanisms can be envisioned to account for en-
hancer synergy. Perhaps the simplest is that there are fewer in-
active nuclei within a given gap expression domain because of
the diminished failure rate of successful enhancer-promoter
interactions with two enhancers rather than one. If the rates at
which enhancers fail to activate transcription are completely
independent, then one would expect the combined action of two
enhancers to yield a multiplicative reduction in how often a given
cell fails to express the gene within a given window of time. This
sort of synergy does not require any direct physical or co-
operative interactions between the enhancers. Nonetheless, the
effect can be significant (as seen for hb). For example, two
enhancers, each with a 10% uncorrelated failure rate, may to-
gether be expected to have a 1% failure rate, a 10-fold reduction
(Fig. 4 A and B). For genes that produce strong bursts of mRNA
expression, this change in frequency of transcription may have
a dramatic effect on the variation of total mRNA levels.
A second but critical potential mechanism of enhancer synergy
concerns long-range, dominant repression. Repressors (such as
Tailless) bound to one enhancer are sufficient to restrict the spatial
limits of the other enhancer. There is no need for long-range re-
pressor elements to appear in both enhancers to achieve normal
spatial limits of gene expression. It has been suggested that long-
rangerepressors,suchasHairy,mediatetheassemblyofpositioned
nucleosomes at the core promoter (38–40). Such repressive nu-
cleosomes should block productive enhancer–promoter interac-
tions, even for enhancers lacking repressor sites (Fig. 4 C and D).
Regardless of the detailed molecular mechanisms, the com-
bined action of multiple enhancers helps explain why an in-
dividual enhancer sometimes fails to recapitulate an authentic
expression pattern when taken from its native context. Enhanc-
ers that produce abnormal patterns of expression (e.g., kni
intronic enhancer) can nonetheless contribute to homogeneous
and robust patterns of gene expression in conjunction with the
additional enhancers contained within the endogenous locus.
Materials and Methods
Enhancer Identification and Testing. Prospective enhancers were identified
near genes of interest using a combination of ChIP-chip data [provided for
various maternal, gap, and pair rule genes by the Berkeley Drosophila
Transcription Network Project (11)] and sequence-based binding-site cluster
analysis. The cluster analysis was performed using the software Cluster-
Draw2 (41). The program and binding motif models used are available on-
line at http://line.bioinfolab.net/webgate/submit.cgi.
Candidate regions (listed in Table S1) were tested in vivo using traditional
lacZ reporter assays combined with targeted phiC31 transgenesis as adapted
for use in Drosophila (25, 26). An nE2G backbone with insulators (42)
modified for targeted integration was used to test potential enhancers by
placing them upstream of an eve-lacZ fusion gene. The same construct was
used for the one vs. two enhancer experiments for Kr and kni; the second
enhancer for the two enhancer constructs was added into a BstBI restriction
site downstream of lacZ ∼5-kb away from the first enhancer. The landing
site 51D (26), Bloomington Stock Center number 24483, was used for
lacZ assays.
The two hb enhancer-lacZ constructs were crossed into a 4× or 6× ma-
ternal Bicoid copy number background using the BB9+16 fly line (19).
Recombineering and Transgenesis. Recombineering was performed as de-
scribedpreviously in ref. 3 (seealsorefs. 23, 24, 43, and 44).The yellowreporter
(used to detect sites of nascent transcript by using an intronic in situ probe)
was integrated as a yellow-kanamycin fusion that left the native hb UTRs in-
tact. The bcd binding-site clusters and surrounding regions of the primary or
shadow enhancers were removed via replacement with an ampicillin re-
sistance cassette taken from pBlueScript. Primers used for construct building
and recombineering are listed in Table S1. BAC CH322-55J23 (24) was the basis
for all subsequent modifications. All BACs were integrated into landing site
VK37 on chromosome 2 (23), Bloomington Stock Center number 24872.
Whole-Mount in Situ Hybridization. Embryos were fixed using standard
methods. Fluorescent or colormetric in situ hybridization was performed as
described in refs. 3 and 45. Probes were generated with the primers listed
in Table S1 and in vitro transcription. Reporter genes were labeled with
digoxigenin-tagged antisense probes, sheep anti-dig primary antibodies
(Roche), and donkey anti-sheep Alexa 555 secondary antibodies (Invitrogen).
Endogenous genes hb, Kr, and kni were labeled with biotin-tagged probes,
mouse anti-bio primary antibodies (Roche), and donkey anti-mouse Alexa
488 secondaries (Invitrogen). Nuclei were counterstained with DRAQ5
(Biostatus Ltd.).
Confocal Image Acquisition and Computational Image Processing. The 1,024 ×
1,024 3-color image stacks were acquired using a Leica SL Laser Scanning Con-
focal microscope as described in ref. 3. Image segmentation and analysis was
performed as described in ref. 3, with minor modification. Nuclei were seg-
mentedusing adifference-of-Gaussiansfilter optimized with sizeselection(Fig.
S3D). A space-filling, segment dilation algorithm was used to assign all pixels in
the embryo to one of the segmented nuclei, created a final nuclear mask (Fig.
S3E). All nuclear masks were manually checked to confirm accurate segmenta-
tion. Nascent transcripts were localized for both the reporter and the endoge-
nous genes, also using difference-of-Gaussians filters, this time optimized to
detect the bright transcripts corresponding to sites of transcription (Fig. S3).
Intensity thresholds and dot-size thresholds reduced spurious counts. Segmen-
X
Enhancer
[R]
10% failure
10% failure
1% failure
B
A
Activators presentRepressors present
10% failure
10% failure
X
10% failure
C
X
Long range repression
D
Ectopic expression
[R]
X
X
Fig. 4.
activating expression and transcription factor binding or enhancer looping is rate limiting, then the two enhancers should have a combined failure rate of
10% × 10% = 1% (A). Removing one enhancer increases the failure rate to 10% (B). (C and D) The binding of a long range, “dominant” repressor to one
enhancer is sufficient to inactivate the other (C). Removal of this enhancer results in “ectopic” expression (D).
Models for enhancer synergy. (A and B) Activation of one promoter by two enhancers. If two independent enhancers each have a 10% failure rate in
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