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Proc Natl Acad Sci U S A. Aug 16, 2011; 108(33): 13570–13575.
Published online Aug 8, 2011. doi:  10.1073/pnas.1109873108
PMCID: PMC3158186
Developmental Biology

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


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.

Keywords: cis-regulation, development, robustness

Recent studies identified “shadow” enhancers for genes engaged in the dorsal-ventral patterning of the early Drosophila embryo (1). These enhancers are sometimes located within neighboring genes, and along with conventional, proximal enhancers, they produce robust patterns of gene expression in early embryos under stress (2, 3). For example, the snail gene exhibits erratic patterns of activation in embryos raised at 30 °C when either 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 network (e.g., refs. 5 and 6). For example, the gap genes hunchback (hb), Kruppel (Kr), and knirps (kni) are each regulated by two distinct enhancers that control the initial bands of gene expression within the presumptive head, thorax, and abdomen. Evidence is presented that the two enhancers work together (enhancer synergy) to ensure uniform expression within correct spatial limits.

Previous studies have documented examples of enhancer autonomy 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 separate enhancers (two located 5′ of the eve transcription unit and three located downstream of the gene) (79). Sometimes, multiple enhancers interfere with one another when placed within a common regulatory region. For example, ventral repressors that delineate 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 discuss potential mechanisms for such enhancer synergy and suggest that minimal enhancers producing aberrant patterns of gene expression might nonetheless contribute to authentic expression 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 (1217). 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).

Fig. 1.
Activities of gap enhancers identified by in situ hybridization. (A and B) The hb/lacZ transgenes containing the (A) proximal (classic) or (B) newly identified distal enhancer. The locations of these enhancers are shown in C. (D and E) Kr/lacZ transgenes ...

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” enhancer 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). Subsequent lacZ fusion assays identified each ChIP-chip peak and underlying binding sites as separable proximal and distal enhancers (Fig. 1 D–F). Similarly, more refined limits were determined for the kni intronic enhancer (Fig. 1 G and I, and Fig. S1B) (12), in addition to the previously identified 5′ distal enhancer (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 additional 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 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 Drosophila 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 complete hb 5′ and 3′ UTRs. Additional BACs were created by inactivating 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).

Fig. 2.
Function of hb enhancers via BAC transgenesis. An ~20-kb BAC containing genomic DNA encompassing the hb locus was modified to remove specific proximal or distal enhancers. The hb transcription unit was replaced with the yellow reporter gene to ...

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 response 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 enhancer is removed (Fig. 2B). Control transgenic embryos containing both enhancers exhibit more uniform patterns of transcription, whereby only an average of ~3% of nuclei fail to match the endogenous pattern of transcription (Fig. 2C). The distribution 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 removal 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 statistical analyses indicate that the loss of either the proximal or distal enhancer results in a significant change in yellow transcription patterns compared with the control BAC transgene (P = 4E-8).

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).

Fig. 3.
Enhancer synergy produces correct spatial limits. Discordance of yellow (A–C) or lacZ (E–G, I–K) transgenes and endogenous gap gene nascent transcripts. Nuclei exhibiting ectopic transgene expression are indicated in red. Sites ...

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, including 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 median 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” because 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 attenuate 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 Huckebein, which have been shown to function as long-range dominant repressors (3032). 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 expression 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 and kni/lacZ fusion 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 abnormally 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 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 combined 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. Alternately, 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 enhancer might function in a similar manner to sharpen the expression 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. 3436). The kni intronic enhancer 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 dominant manner. If the action of short-range repressors is also affected 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 defined 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 produces 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 regulating 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 embryos, 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 environmental changes. Despite these apparent differences, it is possible that dominant repression is also used as a mechanism of synergy for the regulation of snail expression. The distal enhancer contains repressor elements (e.g., Huckebein) that inhibit the expression of the proximal enhancer at the termini (3).

Different mechanisms can be envisioned to account for enhancer synergy. Perhaps the simplest is that there are fewer inactive 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 cooperative 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 together 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.

Fig. 4.
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 activating expression and transcription factor binding or enhancer looping is rate limiting, then the two ...

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 repressor elements to appear in both enhancers to achieve normal spatial limits of gene expression. It has been suggested that long-range repressors, such as Hairy, mediate the assembly of positioned nucleosomes at the core promoter (3840). Such repressive nucleosomes should block productive enhancer–promoter interactions, even for enhancers lacking repressor sites (Fig. 4 C and D).

Regardless of the detailed molecular mechanisms, the combined action of multiple enhancers helps explain why an individual enhancer sometimes fails to recapitulate an authentic expression pattern when taken from its native context. Enhancers 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 ClusterDraw2 (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× maternal Bicoid copy number background using the BB9+16 fly line (19).

Recombineering and Transgenesis.

Recombineering was performed as described previously in ref. 3 (see also refs. 23, 24, 43, and 44). The yellow reporter (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 intact. The bcd binding-site clusters and surrounding regions of the primary or shadow enhancers were removed via replacement with an ampicillin resistance 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 Confocal microscope as described in ref. 3. Image segmentation and analysis was performed as described in ref. 3, with minor modification. Nuclei were segmented using a difference-of-Gaussians filter optimized with size selection (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 segmentation. Nascent transcripts were localized for both the reporter and the endogenous 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. Segmentation results were curated by the user. This segmentation enabled the nucleus-by-nucleus analysis of transcriptional state of reporter and endogenous gene as described in the text and shown in Figs. 2 and and3.3. These analysis scripts were wrapped in a Graphical User Interface implemented through Matlab’s software GUI Design Environment (GUIDE). The source code for this analysis is available in the supplemental material of ref. 3. Updated versions of our image segmentation routines can be found on the authors’ Github page for image processing tools: https://github.com/AlistairBoettiger/Image_Analysis. All of the source codes used to compute and plot the results from this publication are available on the Github source page for this project on “shadow enhancers”: https://github.com/AlistairBoettiger/Shadow-Enhancers.

Supplementary Material

Supporting Information:


The authors thank Jacques Bothma, Nipam Patel, and Vivek Chopra for helpful suggestions; Chiahao Tsui and Emilia Esposito for technical support; and Steve Small for providing the tll (+4) enhancer line and Gary Struhl for providing the BB9+16 bcd extra copy line. M.W.P. and A.N.B are the recipients of National Science Foundation predoctoral fellowships. This work was funded by Grant GM34431from the National Institutes of Health (to M.L.).


The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1109873108/-/DCSupplemental.


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