Genome-wide analyses of Shavenbaby target genes reveals distinct features of enhancer organization
- Delphine Menoret†1, 2,
- Marc Santolini†3,
- Isabelle Fernandes1, 2, 4,
- Rebecca Spokony5,
- Jennifer Zanet1, 2,
- Ignacio Gonzalez6, 7,
- Yvan Latapie1, 2,
- Pierre Ferrer1, 2,
- Hervé Rouault3, 8,
- Kevin P White5,
- Philippe Besse6, 7,
- Vincent Hakim3,
- Stein Aerts9,
- Francois Payre1, 2Email author and
- Serge Plaza1, 2Email author
© Menoret et al.; licensee BioMed Central Ltd. 2013
Received: 7 March 2013
Accepted: 23 August 2013
Published: 23 August 2013
Developmental programs are implemented by regulatory interactions between Transcription Factors (TFs) and their target genes, which remain poorly understood. While recent studies have focused on regulatory cascades of TFs that govern early development, little is known about how the ultimate effectors of cell differentiation are selected and controlled. We addressed this question during late Drosophila embryogenesis, when the finely tuned expression of the TF Ovo/Shavenbaby (Svb) triggers the morphological differentiation of epidermal trichomes.
We defined a sizeable set of genes downstream of Svb and used in vivo assays to delineate 14 enhancers driving their specific expression in trichome cells. Coupling computational modeling to functional dissection, we investigated the regulatory logic of these enhancers. Extending the repertoire of epidermal effectors using genome-wide approaches showed that the regulatory models learned from this first sample are representative of the whole set of trichome enhancers. These enhancers harbor remarkable features with respect to their functional architectures, including a weak or non-existent clustering of Svb binding sites. The in vivo function of each site relies on its intimate context, notably the flanking nucleotides. Two additional cis-regulatory motifs, present in a broad diversity of composition and positioning among trichome enhancers, critically contribute to enhancer activity.
Our results show that Svb directly regulates a large set of terminal effectors of the remodeling of epidermal cells. Further, these data reveal that trichome formation is underpinned by unexpectedly diverse modes of regulation, providing fresh insights into the functional architecture of enhancers governing a terminal differentiation program.
Many studies have established that transcriptional networks control development, through determining specific programs of genome expression . These gene regulatory networks (GRNs) are implemented by transcription factors (TFs) that bind to regulatory DNA sequences, known as enhancers or cis-regulatory modules (CRMs), to control the transcription of nearby genes. Although recruited to target genes via their DNA binding properties , TFs recognize only short and often degenerate motifs (reviewed in [3, 4]). Consequently, thousands of putative binding sites (BSs) are scattered throughout the genome, hampering efficient prediction of CRMs [3, 5, 6]. The fine structure of enhancers as well as putative general rule(s) underlying their organization remain, however, poorly understood.
Although animals encode hundreds of TFs, only a few of them have been studied in detail to elucidate the regulatory logic of their target enhancers [7, 8]. In Drosophila, current knowledge of enhancer structure mainly comes from works on early development - for example, TFs controlling segmentation and mesoderm specification [9–12]. Within these early acting networks, several studies have shown that the local enrichment for BSs (homotypic or heterotypic clustering) in evolutionarily conserved regions is a general signature of active enhancers [13–15]. Functionally related enhancers (driving similar expression patterns) often share a combination or code of cis-regulatory motifs, together defining a specific program of expression [11, 16–18]. Whether enhancers rely on a constrained organization of cis-regulatory motifs or can accommodate flexibility in their number, composition and positioning is still debated (reviewed in [4, 19, 20]). While several studies have shown that regulatory codes are efficient to predict expression pattern [9, 11, 16], recent large-scale work suggests that developmental enhancers may have a more flexible architecture [10, 20]. However, in-depth analyses of individual enhancers [21–24] have revealed an unexpected level of functional constraint in their intimate architecture. It has been proposed that constrained enhancers could be critical when TFs display limiting concentrations  - for example, to accurately integrate gradients . On the other hand, enhancers that do not hold integrative properties might be of simpler architecture [27, 28]. Distinguishing between these possibilities thus requires detailed analyses of the structure and regulatory logic of CRM-TF interactions that occur at late developmental stages.
Here, we focus on a GRN that controls cell morphogenesis during terminal differentiation of the Drosophila embryonic epidermis. The subset of epidermal cells that express the TF Ovo/Shavenbaby (Svb)  undergo localized changes in cell shape leading to the formation of dorsal hairs and ventral denticles, collectively referred to as trichomes . Svb triggers the expression of various classes of cellular effectors in trichome cells. Developmental and genetic analyses have established that trichome formation relies on their collective action, acting together as a developmental module to promote cell shape reorganization [31–33]. The mechanisms underlying the co-expression of Svb-regulated genes in trichome cells remained yet poorly understood. A first level of regulation resides in the activity of Svb itself, which is controlled in a post-translational manner in response to small peptides encoded by the gene polished-rice (pri) . Pri peptides trigger amino-terminal truncation of the Svb protein, switching its activity from a full-length repressor to a cleaved activator , therefore providing temporal control to the program of trichome formation . However, little is known concerning how this TF recognizes and selects its target genes. Besides definition of DNA-binding specificity in vitro  and the identification of a few targets regulated by Ovo germline-specific isoforms [35, 36], only a single epidermal enhancer dependent on Svb has been identified so far . Thus, whether or not Svb targets genes that are co-expressed in trichome cells and have similar cis-regulatory elements remained an open question.
To address this question, we designed a set of computational modeling coupled to experimental approaches to identify and investigate the cis-regulatory logic of Svb-dependent enhancers. By systematic in vivo assays, we first identified a robust set of Svb target effectors, specifically expressed in trichome cells at the time of their morphological differentiation. We then searched for and identified 14 Svb-dependent epidermal enhancers driving their expression in trichome cells and investigated their functional organization. Computational analyses and experimental dissection led to a refinement of the Svb BSs bound in vivo and the identification of two additional motifs required for enhancer activity. Our studies further reveal that the distribution of these cis-regulatory motifs does not follow a stereotypical organization. Coupled to chromatin immunoprecipitation (ChIP)-seq and microarray profiling, the models built from these fine scale experiments allow efficient genome-wide identification of new enhancers that drive the specific expression of trichome effectors. In summary, our results show that enhancers driving co-expression in cells of a late GRN have variable composition and respective organization of cis-regulatory motifs, extending the idea that co-expressed developmental enhancers can have diverse cis-regulatory architectures [11, 37], including for those mediating terminal stages of cell differentiation.
Enrichment of conserved binding sites in Svb downstream genes
Distribution of Svb binding site clusters poorly correlates with enhancer activity
We noticed that OvoQ6 clusters failed to predict a number of active enhancers. This was the case for sha1 (Figure 2) and Emin, an epidermal enhancer previously identified in the gene miniature . Examination with Cluster-Buster  and Swan  did not detect supplementary OvoQ6 in sha1 or Emin sequences (even in D. melanogaster only), explaining why these enhancers, containing a single Svb BS, are not included in in silico predictions. Six additional enhancers identified during initial stages of this study using alternative prediction criteria (Figure S1C in Additional file 1) were not highly ranked by cisTargetX because they lack BS clustering and/or evolutionary conservation. These data therefore show that BS clustering is not an absolute requisite for Svb regulation (Figure 2c), suggesting that additional sites are required to discriminate between enhancers and inactive regions.
De novo motif discovery identifies a specific signature of Svb binding sites active in vivo
The ACHGTTAK motif, hereafter called svbF7, was sufficient to detect 10 out of 14 enhancers (Figure 3b). The proportion of svbF7-positive enhancers reached 13/14, when relaxing the penalty imposed for poor conservation . In contrast, svbF7 was found in only 6/25 negative regions (Figure 3b), even when lowering the threshold (data not shown). Once added to the cisTargetX library, svbF7 is the most significant motif found in the set of 39 Svb downstream genes (Figure S1C, D in Additional file 1). It also increased the accuracy of enhancer predictions, with 3 additional positives (32159, Emin and EminB) while 9 negatives were removed from the top 100 cisTargetX regions (Figure S1C in Additional file 1). Hence, svbF7 performs better than OvoQ6 or any other related motifs  (Figure 3b; Figure S1D in Additional file 1). To evaluate whether this slight extension of the Svb BS was relevant for activity, we substituted nucleotides flanking the core CnGTTa in the single svbF7 of Emin - that is, we altered the svbF7 motif without disrupting the OvoQ6 consensus sequence (Figure 3c). When assayed in vivo, different patterns of flanking substitutions, including a single point mutation of the 5' A residue, were sufficient to strongly reduce Emin expression (Figure 3c). This demonstrates the functional importance of flanking nucleotides within the svbF7 motif for CRM activity. Hence, our computational analysis of Svb-dependent enhancers has discovered a refined nucleotide sequence required for in vivo regulation.
Trichome enhancers use different combinations of cis-regulatory motifs
Taken together, these data support that svbF7 is a main feature of Svb targets, this motif being shared by the vast majority (13/14) of active enhancers. Our analyses have discovered two additional cis-regulatory elements, the blue and yellow motifs, present in overlapping subsets of trichome enhancers (9/14 and 7/14, respectively). While the three motifs are present in various patterns and combinations (Figure 5B; Figure S2 in Additional file 1), functional assays demonstrated that each of them contributes to the in vivo activity of this sample of trichome enhancers.
Genome-wide prediction of Shavenbaby target enhancers
With the large number of Svb bound regions detected by ChIP-seq, it was unlikely that all of them were functional in the regulation of target genes [5, 15]. Therefore, in order to identify the entire set of genes regulated by Svb, we performed microarray profiling, comparing wild-type to mutant embryos. In mRNA samples prepared from svb whole embryos, we often detected only a modest reduction in the levels of validated targets (Figure 6b; Figure S3 in Additional file 1), challenging unambiguous identification of Svb downstream genes. In the absence of pri, Svb behaves as a dominant repressor  and consistently we observed a stronger decrease in the levels of known Svb targets in pri mutants (Figure 6b, c; Figure S3 in Additional file 1), therefore providing an additional criterion to identify genes regulated by Svb. Henceforth, we selected the genes down-regulated in svb mutants and that also displayed a further (more than two-fold) reduction in their expression in pri mutants, as benchmarked for known Svb targets. This defined a set of 150 genes encompassing 16/39 Svb targets validated in vivo (Figure S1A in Additional file 1), as well as 42 additional epidermal candidates (Figure S3 in Additional file 1). Among these, we examined 23 genes by in situ hybridization and confirmed that 21 of them required Svb to be expressed in trichome cells (Figure 6b, c; Figure S4 in Additional file 1). These results therefore show that microarray profiling has defined a representative set of genes activated by Svb in trichome cells.
Focusing on this genomic set of Svb-regulated genes, we found 172 peaks associated with 85 genes (Figure S3 in Additional file 1), including 11 out of 14 active enhancers (Figure S7 in Additional file 1). Within the whole set of relevant Svb-bound regions, we retrieved the characteristic features of cis-regulatory motifs as defined previously. Although retrieved in many Svb-bound regions (Figure 6a; Figure S5 in Additional file 1), the enrichment of yellow motifs within ChIP peaks associated with Svb-regulated genes does not reliably reach a significant threshold, consistent with a broad genomic distribution . In contrast, we found clear association of svbF7 motifs and to a lesser extent of blue motifs (Figure S5 in Additional file 1). Importantly, these motifs were not detected in peaks associated with a control set of genes independent of Svb (Figure S5 in Additional file 1), strongly supporting that they are hallmarks of Svb-target enhancers. As an independent way to evaluate this conclusion, we used ab initio analysis of ChIP peaks using PeakMotif . This identified the motif ACAGTTA, which is characteristic of peaks associated with Svb downstream genes and extensively matches svbF7 (Figure S6 in Additional file 1). A second sequence (TGAAAAG), partly matching the blue motif, was also detected in about 50% of peaks, again only in Svb-regulated genes and not among control genes (Figure S6 in Additional file 1).
Therefore, the regulatory signatures derived from modeling and experimental dissection of a subset of enhancers helps in understanding how the Svb TF selects the genomic set of its direct targets. Furthermore, they collectively allow efficient identification of CRMs that specify the program of trichome-specific expression in response to Svb.
It is well established that the Shavenbaby TF determines trichome fate [29, 32, 52]; however, little was known on the repertoire of its direct target genes and mechanistic insights into the functional organization of trichome enhancers were lacking. Combining functional dissection, computational modeling and genome-wide profiling, we provide here a molecular map of the ultimate repertoire of genes and cis-regulatory elements implementing the network of trichome differentiation.
Physical elements of the GRN governing trichome formation
Our results identify a high-confidence set of more than 150 genes activated by Svb in trichome cells. We confirmed 60 of these, showing complete or partial down-regulation in the absence of active Svb protein. While most genes are expressed in all trichome cells, some are restricted to trichome subsets, suggesting that they can contribute to the diversity of trichome shape and organization observed along the body . Functional annotation (Gene Ontology and manual curation) indicates that Svb controls terminal players of trichome differentiation. In addition to novel factors of F-actin organization [31, 39], extracellular matrix remodeling [31, 33], cuticle formation [31, 38] and pigmentation , we identify enzymes involved in oxidation-reduction, proteolysis and cell trafficking, further extending the repertoire of cellular functions involved in the terminal differentiation of trichome cells. Hence, a major role of Svb in trichome formation is to directly activate the expression of a battery of cell morphogenesis effectors. In support of this, ChIP-seq peaks are present in >70% of these Svb-dependent effector genes. Experimental assays further validated 22 functional enhancers driving the expression of genes encoding factors involved in cytoskeletal or extracellular matrix reorganization, sugar binding, proteolysis and additional enzymes.
Recent work has established that apparently redundant, or shadow, enhancers ensure robust expression of TFs [53, 54]. For example, the transcription of svb itself involves separate enhancers that buffer the trichome pattern against variations in the genetic background and external conditions . It has been proposed that shadow enhancers are required to drive acute expression of some key developmental regulators . We define within both shavenoid and miniature separable enhancers (sha1, sha3, Emin, EminB, EminC) that mediate Svb regulation. These data indicate that apparently redundant enhancers may not be limited to regulatory factors operating at high hierarchic positions in gene networks. Instead, we provide evidence that several 'blue collar' effector genes display a similar regulatory architecture, suggesting that multiple enhancers represent an overlooked feature of the successive tiers of gene networks.
Binding site clustering as a general signature of active enhancers?
Early acting enhancers often comprise multiple BSs for a given TF [56, 57]. For example, conserved BS clusters have identified target enhancers of Dorsal  or Bicoid  and feature functional Twist-bound regions . Of note, most algorithms developed for enhancer detection extensively use motif clustering as an important predictor . We found a clear enrichment in putative Svb BSs (OvoQ6 motif) in its downstream genes; however, only a small proportion of these motifs mediate in vivo regulation. There is very limited, if any, clustering of Svb BSs in ChIP peaks associated with Svb target genes, and even genome-wide. Within the trichome enhancers we validated experimentally, 13 out of 22 display a single Svb site. Furthermore, for the enhancers tyn2, sha3 and dyl2, which contain two to three Svb BSs, the inactivation of individual sites has often limited consequences, as also reported for other TFs . Even if some sites have been missed by computational approaches, the presence of multiple BSs within a short region is not a deterministic feature of active Svb-dependent enhancers.
These findings highlight a paradoxical discrepancy between the enrichment of putative BSs accumulated in Svb downstream genes and the limited number of those acting as cis-regulatory elements. Is there a role for this evolutionary accumulation of Svb-like motifs in Svb targets? For example, these sites with presumably weaker affinity (at least in vivo) can increase the local concentration of the TF facilitating regulation through a few BSs stably bound in vivo, as it has been suggested on thermodynamics grounds  or to explain the existence of thousands of binding events that are transcriptionally inactive [5, 15].
Trichome enhancers rely on diverse combinations of cis-regulatory motifs
We found that the motif bound by Svb in vivo is more constrained than the consensus defined from in vitro  or one-hybrid approaches . This shows that slight sequence differences, not detected in vitro, can play a key role within genomic context , such as revealing the influence of co-factors .
In addition, other motifs influence which Svb BSs are functional as regulatory elements, a notion well in line with recent results on the in vivo specificity of Hox factors . Our statistical approaches identified a more widely spread 'blue' motif. Importantly, only half of the enhancers comprise blue motifs, indicating that there are several ways to build Svb-responsive enhancers. Indeed, the systematic dissection of Emin disclosed an additional motif (TTATGCAA) ultra-conserved across Drosophilidae  and contributing to its activity. This 'yellow' motif is retrieved in half of the trichome enhancers, with or without blue motifs. It is, however, barely specifically enriched in Svb-bound regions and therefore was not predicted by our computational analyses (positives versus negative regions), showing the importance of unbiased functional dissection to disclose the full spectrum of cis-regulatory elements. Indeed, the disruption of either blue or yellow motifs strongly affects enhancer function in all tested cases, providing experimental evidence of their cis-regulatory activity.
Trichome enhancers thus display various combinations of motifs, from those containing only Svb BSs (5/22), Svb plus yellow (4/22), Svb plus blue (6/22) or all three together (7/22). These different motif compositions do not appear to correlate with distinct subclasses of gene function (DM, unpublished data). Furthermore, multiple enhancers from the same gene can harbor distinct combinations, as exemplified by shavenoid and to a lesser extent by miniature (Figure 8; Figure S6 in Additional file 1). Several studies have shown that motif composition may correlate with a given spatio-temporal pattern - for example, for neurogenic or muscular GRNs [11, 16]. Since most trichome enhancers are often active in the very same population of cells, with highly similar dynamics, it is surprising to observe such diversity in their motif compositions. There are four enhancers restricted to dorsal trichome cells, but again they accommodate different motif compositions, with EminB and 4702B, which contain blue motifs, versus cyrA and 31559, which do not. These data thus indicate that trichome enhancers display diverse distributions of functional motifs, supporting that distinct cis-regulatory architectures drive highly similar spatio-temporal expression.
Flexibility in cis-regulatory motifs among enhancers versus across species
Although highly constrained sequences, such as the interferon-β enhanceosome, do not seem widely spread , developmental enhancers may yet require some 'grammar' for motif positioning  - for example, with an optimal pair-wise spacing of motifs  that could reflect the cooperative binding of TFs. For trichome enhancers we did not detect any obvious bias in the number or respective arrangement of the cis-regulatory motifs they rely on (Figure S2 in Additional file 1). Likewise, recent results from the analysis of Drosophila cardiac enhancers support that similar expression patterns can be generated from divergent compositions and positioning of motifs [10, 65].
That several different inputs lead to similar enhancer outputs does not, however, formally rule out the existence of constraints, even though they are not detected by 'horizontal' comparison of different enhancers within the same species. An independent way to evaluate this possibility is to look at the evolution of individual regulatory regions throughout species [15, 21]. Across Drosophilidae, trichome enhancers often display similar numbers and organization of cis-regulatory motifs (Figure 8; Figure S6 in Additional file 1). Furthermore, besides turnover of some motifs, svbF7, blue and yellow motifs are often embedded within short-sized islands of high evolutionary conservation, when compared to neighboring sequences (Figure 8). Similar strong evolutionary conservation was also noticed for the binding site of Twist  and its partner TFs , although these studies did not examine evolution of the detailed pattern of motif positioning. These data therefore suggest that despite diverse arrangements of motifs, patterns of evolutionary conservation likely represent the signature of functional constraints that locally shape the architecture of individual enhancers.
Materials and methods
Fly strains and transgenic constructs
We used btd, svb 1 or svb R9 [30, 31] and pri 1  stocks kept over green fluorescent protein (GFP) balancers. To delineate the epidermal enhancer of sn and sha, transgenic lines were initially generated using P-element-mediated transformation (Fly Facility) and at least three independent insertions were analyzed for each construct. We then switched to the PhiC31 system (Bestgene, Chino Hills, CA, USA) to quantify effects of mutations, with all constructs integrated at the same location (zh-86F), except for sha1, sha3 and snE1, for which mutant versions were assayed in P-elements for homogeneity (Additional file 3). Genomic regions were amplified and cloned into pCasper or pAttB lacZ derivatives. QuikChange II XL site-directed mutagenesis (Agilent Technologies, Santa Clara, CA, USA) was used to introduce point mutations in enhancers, or CCGCCGGCGG stretches for linker scanning of Emin. All constructs were verified by sequencing. Details (genomic position) of the CRM are given in Additional file 3.
Dig- or biotin-labeled antisense RNA probes were used for in situ hybridization following standard protocols and embryos imaged using a Nikon Eclipse90i microscope. For immunodetection of lacZ reporter expression, 10- to 14-h embryos were stained using anti-β-galactosidase (1/1,000; Cappel, MP Biomedical, Solon, OH USA) and Alexafluor488 (Molecular Probes, Life Technologies, Carlsbad, CA, USA). Pictures were taken with a Leica SP2 confocal microscope, using the same settings to allow quantitative comparisons.
We hand selected 13- to 15-h svb R9 or pri 1 embryos using GFP balancers. We subjected 200 embryos to trizol (Invitrogen, Life Technologies, Carlsbad, CA, USA) extraction and RNA quality was monitored using Agilent Chip. Five independent samples of each genotype were used for microarrays (Affymetrix, Santa Clara, CA, USA; IGBMC, Strasbourg, France). Data extraction and normalization were performed using Affymetrix software and statistical analyses with R. A more than two-fold difference in expression levels between mutant genotypes was the most efficient criterion to retrieve Svb downstream genes (with a false discovery rate of 0.01 for pri). The top 150 genes down-regulated in both pri and svb mutants defined the set of Svb-regulated genes. One-hundred genes showing irrelevant variation of their expression (P-value > 0.8, false discovery rate >0.99) were used as a negative control set. The data discussed in this article have been deposited in NCBI's Gene Expression Omnibus  and are accessible through GEO series accession number GSE48997. Details are given in Additional file 3.
A svb rescue construct (RSQ8)  was used for ChIP-seq experiments. It expresses a Svb-GFP protein under the control of two svb enhancers (medial and proximal) driving specific expression in epidermal trichome cells. Stocks were expanded to fill three population cages. Adults were allowed to lay eggs for 2 hours on apple juice plates covered with yeast. Embryos deposited on the plates were aged for 12 h at 25°C. Chromatin was collected from approximately 100 mg of whole embryos for each replicate chromatin collection. ChIP was done with an anti-GFP antibody as described . Data presented are from two independent replicates. Peaks were called for single replicates using MACS P < 0.00001 for downstream computational analyses. MACS was used to call loose criteria peaks for two replicates of RSQ8 12- to 14-h embryos. Those peaks were then used for an IDR (Irreproducible Discovery Rate) analysis (IDR = 0.02). DNA sequencing libraries were generated with Nextera DNA Sequencing Library kits (VWR, Radnor, PA, USA) Details are given in Additional files 3 and 4. The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus  and are accessible through GEO series accession number GSE48791.
Motif detection and genome analysis
Detection of motifs enriched in Svb-dependent and Svb-independent epidermal genes was performed using cisTargetX . For de novo motif discovery, genomic sequences of enhancer and negative regions were processed through a C++ program and statistical operations performed within the R software, as described . To compute the cross-correlation between conserved motif instances and Svb ChIP-Seq data, we defined a 10 kb window centered around each ChIP peak, collected distances of each motif to the peak center and plotted these values using a 500 bp bin. In the cases of Svb-regulated and control genes, each ChIP peak was associated with the nearest transcription start site. Further details are available in Additional files 3 and 4.
List of abbreviations used
green fluorescent protein
gene regulatory network
We are grateful to the Bloomington Drosophila Stock Center and Drosophila Genomic Resource Center for providing us with flies and molecular clones. We are indebted to B Ronsin (Toulouse RIO Imaging), P Valenti and O Bohner for excellent technical assistance. We also thank C Hermann, N Negre, E Preger-Ben Noon, A Vincent and members of the SP/FP lab for critical reading of the manuscript. This work was supported by grants from ANR Blanc 'Netoshape', Association pour la Recherche contre le Cancer (n°3832, n°1111, SFI20101201669 and fellowships to IF and DM) and University Paul Sabatier (cisdecode). SA was supported by grants from FWO (G.0704.11N and G.0640.13) and HFSP (RGY0070/2011).
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