- Open Access
cis-Decoder discovers constellations of conserved DNA sequences shared among tissue-specific enhancers
© Brody et al.; licensee BioMed Central Ltd. 2007
- Received: 29 September 2006
- Accepted: 9 May 2007
- Published: 09 May 2007
A systematic approach is described for analysis of evolutionarily conserved cis-regulatory DNA using cis-Decoder, a tool for discovery of conserved sequence elements that are shared between similarly regulated enhancers. Analysis of 2,086 conserved sequence blocks (CSBs), identified from 135 characterized enhancers, reveals most CSBs consist of shorter overlapping/adjacent elements that are either enhancer type-specific or common to enhancers with divergent regulatory behaviors. Our findings suggest that enhancers employ overlapping repertoires of highly conserved core elements.
- Additional Data File
- Sequence Element
- Conserve Sequence Block
- Coordinate Gene Expression
- Conserve Sequence Element
Tissue-specific coordinate gene expression requires multiple inputs that involve dynamic interactions between sequence specific DNA-binding transcription factors and their target DNAs. The enhancer or cis-regulatory module is the focal point of integration for many of these regulatory events. Enhancers, which usually span 0.5 to 1.0 kb, contain clusters of transcription factor DNA-binding sites (reviewed by [1–3]). DNA sequence comparisons of different co-regulating enhancers suggest that many may rely on different combinations of transcription factors to achieve coordinate gene regulation. For example, the Drosophila pan-neural genes deadpan, scratch and snail all have distinct central nervous system (CNS) enhancers that drive expression in the same embryonic neuroblasts, yet comparisons of these enhancers reveal that they have few sequences in common [4, 5].
Comparative genomic analysis of orthologous cis-regulatory regions reveals that many contain multi-species conserved sequences (MCSs; reviewed by [6–8]). Close inspection of enhancer MCSs reveals that these sequences are made up of smaller blocks of conserved sequences, designated here as 'conserved sequence blocks' (CSBs). EvoPrint analysis of enhancer CSBs reveals that many have remained unchanged for over 160 million years (My) of collective divergence  (and see below). CSBs that are over 10 base-pairs (bp) long are likely to be made up of adjacent or overlapping sequence-specific transcription factor DNA-binding sites. For example, DNA-binding sites for transcription factors that play essential roles in the regulation of the previously characterized Drosophila Krüppel central domain enhancer [10–12] are found adjacent to or overlapping one another within enhancer CSBs . Although transcription factor consensus DNA-binding sites are detected within CSBs, searches of 2,086 CSBs (27,996 total bp) curated from 35 mammalian and 99 Drosophila characterized enhancers reveal that well over half of the sequences do not correspond to known DNA-binding sites and, as yet, have no assigned function(s) (this paper).
To demonstrate the efficacy of cis-Decoder analysis in identifying shared enhancer sequence elements, we show how cDT-library scans of different EvoPrinted mammalian and Drosophila enhancers accurately identify shared sequences within enhancers involved in similar regulatory behaviors. The cis-regulatory regions of the mammalian Delta-like 1 (Dll1) and Drosophila snail genes, which contain closely associated neural and mesodermal enhancers, were selected to highlight cis-Decoder's ability to differentiate between enhancers with different regulatory functions. We show how a cDT-library generated from both mammalian and Drosophila enhancer CSBs can be used to identify enhancer type-specific elements that have been conserved during the evolutionary diversification of metazoans. Finally, we show how cis-Decoder analysis can be used to examine novel putative enhancer regions.
Generation of EvoPrintsand CSB-libraries
atonal F:2.6 PNS
eve neuronal CNS
eve EL CNS
eve stripe 1
eve stripe 2
eve stripe 4+6
eve stripe 5
eve stripe 3+7
eyeless 12 PNS
ftz neuro CNS
hairy stripe 0
hairy stripe 1
hairy stripe 5
hairy stripe 3+4
hairy stripe 6+2
AK (pers. com.)
paired stripe P
paired stripe 1
paired stripe 2P
pdp1 intron 1
pdp1 intron 2
runt stripe 1E+6
runt stripe 1+7
runt stripe 3+7
runt stripe 5
runt 15G CNS
string b-5.8 CNS
vnd A CNS
bagpipe Hox 1
Dll1 HI CNS
Dll1 HII CNS
Dll1 msd II
forkhead box f1
H. domain only
Otx 2 CNS
Serum response f
Sox-2 #2 CNS
Sox 9p CNS
Stem cell leukemia
cis-Decoder tag libraries
Neural and segmental
For Drosophila, three CSB-libraries, neural, segmental and mesodermal, were generated from CSBs identified by EvoPrinting (Tables 1 and 2): neural enhancers included those regulating both CNS and peripheral nervous system (PNS) determinants; segmental enhancers included those regulating both pair-rule and gap gene expression; and mesodermal enhancers included those regulating both presumptive and late expression. Many of the D. melanogaster reference sequences used to initiate the EvoPrints were curated from the regulatory element database REDfly , while others were identified from their primary reference (Table 1). The collection of neural enhancers includes both those that direct expression during early development, such as the snail , scratch, and deadpan CNS and PNS enhancers , and late nervous system regulators, such as the eyeless enhancer ey12 , which confers expression in the adult brain. The early embryonic segmental enhancers represent pair-rule regulators such as the hairy stripe 1  and even-skipped stripe 1  enhancers, and gap expression regulators, such as the hunchback enhancers [19, 20]. The mesodermal enhancers include those directing mesodermal anlage expression of snail  and tinman , and late expressing enhancers, such as those directing serpent fat body expression  and mesodermal expression of Sex combs reduced . The collective evolutionary divergence of all of the EvoPrints was greater than 100 My and in most cases EvoPrints represented over approximately 160 My of additive divergence. The average CSB length for both the Drosophila and mammalian CSBs is 13 bp; the longest identified CSBs were 99 bp from the giant (-10) segmental enhancer [15, 24] and 95 bp from the Paired-like homeobox-2b mammalian neural enhancer . Complete lists of all CSBs identified in this study are given at the cis-Decoder website .
Identification and use of cis-Decoder tags
As an initial step toward understanding the nature of the CSB substructure, we have developed a set of DNA sequence alignment tools, known collectively as cis-Decoder, that allow identification of 6 bp or greater perfect match identities, called cDTs, within two or more CSBs from either similar or divergent enhancers. The cDTs, which range in size from 6 to 14 bp with an average of 7 or 8 bp, are organized into cDT-libraries that identify sequence elements within CSBs of the same CSB-library. In addition, common cDT-libraries that represent sequence elements aligning to CSBs of two or more different CSB-libraries were also organized.
Mammalian CSB alignments, using the CSB-aligner program, yielded 336 neural specific and 60 neural-enriched cDTs and analysis of the mammalian mesodermal CSBs yielded 258 mesodermal specific and 55 mesodermal enriched cDTs (Table 2). The CSB alignments also produced 137 cDTs that are common to both neural and mesodermal CSBs. Alignments of the Drosophila enhancer CSBs yielded 444 neural specific cDTs (showing no hits on mesodermal or segmental enhancer CSBs), 284 segmental enhancer specific cDTs and an additional 451 cDTs found in neural and segmental enhancers but not part of mesodermal CSBs (Table 2). We also identified 451 cDTs that were enriched in neural and/or segmental CSBs but were also found at a lower frequency in mesodermal enhancer CSBs. From the mesodermal CSBs analyzed, 169 mesodermal specific cDTs (not in neural or segmental enhancer CSBs) were identified along with 104 additional cDTs enriched in mesodermal enhancers but also found at a lower frequency among neural and/or segmental enhancer CSBs. A common cDT-library was also generated that contains 993 cDTs that represent common sequence elements found in CSBs of both neural and mesodermal enhancers.
To search for enhancer sequence element conservation between taxa, we generated neural and mesodermal cDT-libraries from the combined alignments of mammalian and fly CSBs (Table 2) and many of the cDTs in these libraries align to both mammalian and fly CSBs. For example, the 11 bp neural specific cDT (CAGCTGACAGC) aligns with CSBs in the vertebrate Math-1  and Drosophila deadpan  early CNS enhancers. All CSB-, cDT-libraries and alignment tools are available at the cis-Decoder website.
The constituent sequence elements of the different cDT-libraries are dependent on the enhancers used to identify them. As additional CSBs are included in the cDT-library construction, certain cDTs may be re-designated. For example, some that are currently considered neural specific will be discovered to be neural enriched, and others that are part of enriched libraries may be reassigned to common cDT-libraries.
Although each mammalian and fly cDT is present in at least two or more enhancers, most are not found as repeated sequences in any of the enhancers. In addition, one of the principle observations of our analysis is that enhancers of similarly regulated genes share different combinatorial sets of elements that are enhancer-type specific (see below).
Cross-library CSB alignments revealed that nearly all CSBs contain cDTs that are either shared by CSBs from divergent enhancer types or found only in CSBs from enhancers with related regulatory functions. For example, the 37 bp neural mastermind #10 CSB (TATTATTACTATATACAATATGGCATATTATTATTAC) contains a 9 bp sequence (first underlined sequence) also found in the 20 bp #8 CSB from the dpp mesodermal enhancer [15, 28] and it also contains a 14 bp sequence (second underlined sequence) that constitutes the entire 14 bp #33 CSB from the neural enhancer region of nerfin-1 ( and unpublished results).
The analysis of both the mammalian and fly common cDT-libraries reveals that many cDTs contain core recognition sequences for known transcription factors. However, when additional flanking CSB sequences are considered, many common transcription factor binding sites become tissue specific cDTs. For example, the DNA-binding site for basic helix-loop-helix (bHLH) transcription factors, the E-box motif CAGCTG (reviewed by ) is present 22 times in different neural CSBs, and 2 and 4 times within the CSBs of segmental and mesodermal enhancers, respectively. However, when flanking sequences are included in the analysis, such as the sequences CAGCTGG, CAGCTGAT, CAGCTGTG, CAGCTGCA, CAGCTGCT and ACAGCTGCC, all are neural specific cDTs (E-box underlined). It has been previously shown that different E-boxes bind different bHLH transcription factors to regulate different neural target genes . Although transcription factor consensus DNA-binding sites are well represented in the cDT-libraries, greater than 50% of the cDTs in all of the libraries, both mammalian and fly, represent novel sequences whose function(s) are currently unknown. The fact that there exists such a high percentage of novel sequences within these highly conserved sequences indicates that the identity, function and/or the combinatorial events that regulate enhancer behavior are as yet unknown.
cis-Decoder analysis of the murine Delta-like 1 enhancers identifies multiple shared elements with other related vertebrate embryonic enhancers
Although the resolution of cis-Decoder analysis increases as more enhancers and/or enhancer types are included in the CSB and cDT alignments, our analysis of mammalian enhancers found that many shared sequence elements can be identified among related enhancers when as few as two different enhancer groups are used to generate specific cDT-libraries. This is a particularly useful feature of cis-Decoder, especially when studying a biological process or developmental event where relatively little is known about the participating genes and their controlling enhancers. To demonstrate the ability of cis-Decoder to analyze relatively small subsets of enhancers, we show how cDT-libraries generated from 14 neural and 21 mesodermal mammalian enhancers can be used to distinguish between the neural and mesodermal enhancers that regulate embryonic expression of Dll1.
Dll1 encodes a Notch ligand that is essential for cell-cell signaling events that regulate multiple developmental events (reviewed by ). Studies in the mouse reveal that Dll1 is dynamically expressed in specific regions of the developing brain, spinal cord and also in a complex pattern within the embryonic mesoderm [33, 34]. The 1.6 kb Dll1 cis-regulatory region, located 5' to its transcribed sequence, has been shown to contain distinct enhancers that direct gene expression in these different tissues . These studies have identified two highly conserved neural enhancers, designated Homology I (H-I) and Homology II (H-II), and two mesodermal enhancers termed msd and msd-II. The H-I enhancer directs expression to the ventral neural tube, while the H-II enhancer primarily drives Dll1 expression in the marginal zone of the dorsal region of the neural tube . The msd enhancer drives expression in paraxial mesoderm, and msd-II directs Dll1 expression to the presomitic and somitic mesoderm.
cis-Decoder identifies sequence elements within the Drosophila snail and hairystripe 1 enhancers that are also conserved in other functionally related tissue-specific enhancers
Within the presumptive mesodermal enhancer CSBs, 11 cDTs mesodermal specific aligned with 5 of the 12 CSBs, covering 40% of the CSBs (Figure 7). Like the neural cDTs, some of the mesodermal cDTs contain putative DNA-binding sites for classes of known transcription factor families. For example, the seventh cDT (TAATTGGA) contains a consensus core DNA-binding sequence (underlined) for Antennapedia class homeodomain factors  (reviewed by ).
In the snail early PNS enhancer region, 5 of the 7 CSBs aligned with a total of 15 different cDTs that cover 69% of the total PNS CSB sequence (Figure 7). Similar to the CNS enhancer CSB cDT alignments, close to half of the PNS cDT hits represent sequence elements within both neural and segmental enhancer CSBs, again most likely a reflection of the segmental structure of the PNS. The significant overlap in cDTs found in both CNS and PNS enhancer CSBs may reflect the likelihood that many early neural specific transcriptional regulatory factors are pan-neural.
Many of the snail enhancer CSB-cDT hits represent sequences found only in two CSBs, snail itself and one other. In these instances it appears that these elements, although specific for neural or mesodermal CSBs, are relatively rare when compared to others. Only through analysis of additional enhancers will it be clear whether these rare elements are indeed type-specific or only enriched in the type-specific CSBs. Nevertheless, the fact that the sequence elements identified by these rare cDTs are conserved in two distinct enhancer CSBs that have both been under positive selection for over 160 My of collective divergence merits their inclusion in the analysis.
As part of our study of Drosophila enhancers, we carried out cis-Decoder analysis of 38 segmentation enhancers responsible for both gap and pair-rule gene expression during Drosophila embryogenesis. Although the segmentation enhancer specific library consisted of only 284 cDTs, these cDTs aligned with over 70% of bases of the CSBs of segmentation enhancers. As an example of alignment of these cDTs with a segmental enhancer, we present an alignment of segmentation specific cDTs with the hairy stripe 1 enhancer (Additional data file 2). cis-Decoder recognizes highly conserved Abdominal-B, HOX, Hunchback, Kruppel and Tramtrack binding sites, as well as additional uncharacterized sites, as being shared by hairy stripe 1 enhancer and other segmentation enhancers.
Full-enhancer scanner identifies less conserved repeated cDTs and CSBs
Previous studies have demonstrated that certain enhancers, particularly those controlling the dynamic expression of developmental genes, contain clusters of DNA-binding site motifs for specific transcription factors (for example, see [44, 45]; reviewed by ). Comparative genomic studies of orthologous enhancers have also revealed that, within a binding site cluster, individual DNA-binding sites can undergo turnover (discussed in [47, 48]). This loss of and/or gain of transcription factor docking sites during evolution suggests that the repeated motifs may be functionally redundant and that the stability of any one binding site is most likely due to selective pressure(s) to maintain: total number of binding sites for tight spatial/temporal regulation; functional interactions between a bound factor and adjacent factors and/or; competition between antagonistic regulatory factors for overlapping binding sites. For example, overlapping/linked binding sites have been identified in the 3' most CSB of the Krüppel central domain enhancer [9, 10]. The 15 bp CSB (CTGAACTAAATCCG) contains overlapping sites for the transcriptional activator Bicoid and repressor Knirps proteins . In vivo experiments reveal that these interlocking sites are functionally important . Additional binding sites for both of these factors are also present in the Krüppel enhancer but not all are found in CSBs (data not shown).
Use of cis-Decoder to examine novel cis-regulatory sequences
During embryonic development, HLHmβ expression is activated in the ventral neurogenic ectoderm immediately prior to neuroblast delamination [50, 54] and enhancer-reporter constructs from the HLHmβ enhancer region  are expressed in proneural territories in the ventral ectoderm at the time of the first wave of neuroblast delamination(stages 9-10) and in neuroblasts (Figure 1 of ). Our EvoPrint analysis of the 883 bp enhancer region (Figure 10a) revealed that 338 bases were highly conserved, and over 90% of these were found in CSBs of 6 or more bases. Alignment of Drosophila neural-specific and mesodermal-specific cDTs revealed that 11 of the 15 HLHmβ CSBs aligned with a total of 28 neural specific cDTs, while only 1 of its CSBs aligned with a single mesodermal specific cDT (Figure 10b,c). Both proneural transcription factors and the Notch pathway, acting through the Su(H) transcription factor, are implicated in the regulation of E(spl) complex genes (reviewed by ). Among the cDTs aligning with the CSBs, one, GCATGTGC, contains an E-box (underlined), the focus of activity of proneural transcription factors, and two others, TTTCCCA and TCCCAC, align with the consensus Su(H) binding site.
Although higher specificity is obtained by alignment with cDTs of 7 bases or greater, we have found that it is not unusual for 80% of CSBs associated with neural expressed genes to align with neural-specific cDTs versus only 20% of the CSBs in the same putative enhancer regions aligning with mesodermal-specific cDTs even when 6 base long cDTs are included in the analysis (data not shown). As the size and specificity of these libraries grow, their use as predictors of enhancer function will most likely increase as well.
As an additional assessment of the specificity of cDT-library scans, we generated negative control CSB-libraries for alignment to cDTs. These datasets, both Drosophila and mammalian, consisted of conserved sequence blocks within exons of genes that are not predominantly expressed in the CNS (data not shown). For this analysis we use the percent coverage of CSBs by cDTs, as used above for the analysis of Dll1 enhancers in which we counted the percent of the bases in the CSBs that aligned with cDTs. Whereas Drosophila and mammalian neural-specific cDTs, including hexamers, cover approximately 56% and 70%, respectively, of CSBs from neural enhancers, alignment with control CSBs was 20% or less. Again, when the alignment was repeated with cDTs of 7 bp or greater the CSB coverage of neural sequence was 5-fold greater than that observed with the control datasets. Taken together, our cDT alignments demonstrate their utility in identifying enhancer type-specific conserved sequence elements.
Evaluation of the cis-Decoder method was also carried out by examining the contribution that each enhancer made to the cDT-libraries. As one adds new enhancer CSBs to a specific library, the number of cDTs increases, such that alignment coverage of enhancer type-specific CSBs also increases. We illustrate the contribution of each enhancer to the specific cDT-libraries in our study (Additional data file 4). Overall, for Drosophila enhancers, prior to their inclusion in a library, on average 41% of the conserved nucleotides of enhancers align with the tissue specific cDT-library appropriate for that enhancer, while after inclusion in a library, 65% of the conserved nucleotides align. For example, addition of the bearded proneural enhancer , consisting of 21 CSBs (a total of 303 bp), to the Drosophila neural-specific CSB library resulted in 26 new neural-specific cDTs that were shared with at least one other neural enhancer. Prior to its inclusion, coverage of the bearded CSBs by alignment of neural-specific cDTs was 43%, while after its inclusion in the cDT-library preparation the alignment coverage of its CSBs increased to 67%. Addition of new enhancers to the out-group, used to remove common cDTs from a specific library, also enhances the specificity of the type-specific library and frequently shifts cDTs from specific to enriched libraries. Taken together, increased specificity of an enhancer-type cDT-library can be achieved either by including new similarly regulated enhancers in the generation of the cDT-library or increasing the number of out-group CSBs used to remove non-specific cDTs. Ideally, both approaches should be pursued to increase the depth and resolution of a particular cDT-library.
This study describes a systematic approach for the identification and comparative analysis of highly conserved DNA sequences within enhancers. Because our approach focuses solely on conserved sequences, the probability that cis-Decoder analysis dissects functionally important DNA is greatly enhanced. Most of the 2,086 CSBs identified in this study have undergone negative selection during more than 160 My of collective evolutionary divergence. Alignment of hundreds of CSBs from both similarly regulating enhancers and functionally different enhancers assures that conserved cis-regulatory elements shared by as few as two enhancers are identified and included in the analyses. Our cDT-scans show that most CSBs have a modular organization made up of smaller overlapping/interlocking sequence elements that align with CSBs of other enhancers. A typical CSB is made up of both enhancer type-specific sequence elements and common elements that are found in enhancers with different regulatory functions and, surprisingly, more than half of all of the shared CSB sequence elements do not correspond to know transcription factor DNA-binding sites and, as of yet, are functionally novel.
cDT-library scans of EvoPrinted cis-regulatory DNA reveal that it is possible to differentiate between functionally different enhancer types before any experimental/expression data are known. For example, cDT-library scans of the mammalian Dll1 or Drosophila snail cis-regulatory DNA sequences accurately differentiate between neural and mesodermal enhancers (Figures 3 and 7). cDT-library scans of co-regulating enhancers, using multiple libraries, reveal the combinatorial complexity of the cis-regulatory sequence elements involved in coordinate gene expression. Our studies indicate that many co-regulating enhancers rely on different combinations of the tissue-specific cis-regulatory elements to achieve synchronous regulatory behaviors. Although not highlighted in this paper, information gleaned from the cDT-scans and subsequent cDT-cataloger analysis of multiple co-regulating enhancers can be used to construct 'higher resolution' cDT-libraries that harbor many, or most, of the sequence elements that direct coordinate gene expression.
For example, sub-libraries of the Drosophila neural specific library can be generated to identify neuroblast- and PNS-specific tags. Enhancer CSB analysis using cDT-libraries generated from the combined alignments of both mammalian and fly CSBs also suggests that many of the sequence elements represented by the different cDTs have been conserved across taxonomic divisions and may represent core elements used by many metazoans to direct tissue-specific gene expression patterns.
Although we have initially generated cDT-libraries from general classes of different enhancer types, this approach should be applicable to the analysis of gene co-regulation in any cell type involved in any biological event. As the variety and depth of the different cDT-libraries increase, we believe that cDT-library scans of EvoPrinted putative enhancer regions will have great utility for the identification and initial characterization of cis-regulatory sequences. Future efforts that address the role of individual enhancer CSBs and the dissection of their modular elements will undoubtedly yield new insights into the function of these 'evolutionarily hardened' sequences and ultimately produce a better understanding of the regulatory code underlying coordinate gene expression.
cis-Decoder  is a six-step integrated series of protocols and web-based algorithms that can be used to identify evolutionarily conserved DNA sequences that are shared among different enhancers (Figure 1). The following sections provide a detailed description of each step of the cis-Decoder procedure: EvoPrint analysis , for the discovery of MCSs; EvoPrint-parser, for CSB extraction and annotation; CSB-aligner, for the identification of shared elements between CSBs; cDT-scanner, to reveal cDT positions and their relations to other cDTs within CSBs; Full-enhancer scanner, for the discovery of less-conserved repeated cDTs or CSBs within enhancers; and cDT-cataloger for the identification of enhancers with shared sequence elements. A more detailed description of these steps is given at the cis-Decoder website. The Java applets CSB-aligner, cDT-scanner, Full-enhancer scanner and cDT-cataloger are available on-line at the cis-Decoder website and can be downloaded to the users computer to avoid Java-web browser incompatibilities. In our experience, a current version of the Mozilla browser avoids many potential incompatibilities.
The first step in the cis-Decoder analysis of an enhancer is preparing CSB-libraries from enhancers with related and/or divergent expression patterns. Enhancer CSBs were identified by the phylogenetic footprinting algorithm EvoPrinter . Unlike other multi-species alignment programs that identify CSBs by outputting multiple aligned sequences interrupted by sequence gaps to optimize alignments, EvoPrinter outputs a single uninterrupted sequence to reveal CSBs as they exist in a species of interest. In Drosophila, when 9 or more species are used to generate an EvoPrint, the combined mutagenic histories of all of the orthologous DNAs represent an excess of 160 My of collective evolutionary divergence, thus affording near base-pair resolution of the functionally important DNA within the species of interest (discussed in ). Likewise, EvoPrint analysis of orthologous DNAs that include placental mammals (human, chimpanzee, rhesus monkey, cow, dog, rat and mouse), and, optionally, the opossum, detects CSBs that have been maintained for over 200 My of collective divergence. The EvoPrinter and EvoDifference print analysis algorithms and companion protocols are described , and are found online at the EvoPrinter tutorial website.
CSB-aligner is a Java applet that allows one to identify short sequence elements shared between different CSBs. To generate a CSB-alignment, parsed CSBs from multiple enhancer regions are placed in the upper window of the CSB-aligner applet. Then, forward direction CSBs from one or more enhancers are placed in the lower window of the CSB-aligner. A box associated with the lower window of the CSB-aligner allows for the naming of the CSBs introduced into the lower box and selection of the minimum aligned length (6, 7 or 8 base windows have been routinely used). Output length of the alignments produced by CSB-aligner can be selected (default value 100 bases).
Output of the CSB-aligner consists of the CSBs that were input into the lower window aligned with the CSBs that were introduced into the upper window. The CSB-aligner does not record CSB self-alignments. A second output window, the results table, is a list of the aligned matches along with their positions. Each of the output columns of the results table can be sorted by selecting the column header of the column to be sorted. Contents of results tables can be copy-pasted into Microsoft Word.
The CSB-alignment can be saved as an HTML file. Saving the HTML file allows copy pasting from the saved file into Microsoft Word and, once in Word, the file can be reformatted and saved or printed as the original readout. The CSB-alignment program has functioned successfully with the introduction of thousands of CSBs in both windows. The following CSB-libraries were created from EvoPrints of enhancers listed in Table 1: mammalian neural, mammalian mesodermal, Drosophila neural, Drosophila mesodermal and Drosophila segmental.
Interpreting the CSB-aligner readout and generation of cDT-libraries
A cDT is a short sequence element of 6 bp or greater that is a perfect match to sequences within CSBs that are present in two or more enhancers. A cDT-library represents a collection of cDTs that are shared by the various enhancers examined. Two types of cDT-libraries have been generated in this study. First, a 'tissue-specific library' contains cDTs that are shared by a group of enhancers that regulate similar expression patterns but are absent from a second set of enhancers that direct expression in tissues outside of the first group. Second, a 'common cDT-library' contains cDTs that were shared between sets of enhancers of divergently regulated genes. A subset of common libraries included 'enriched' libraries that had a three-fold greater representation from one enhancer type (for example, neural) than from a second type (for example, mesodermal).
For Drosophila, segmental, neural (treating CNS and PNS specific enhancers together), and mesodermal specific cDT-libraries were generated. The out-group for neural and segmental cDT-libraries was the mesodermal CSB-library, and the out-group for the mesodermal cDT-library was neural CSBs. For mammals, neural and mesodermal cDT-libraries were generated. All cDT-libraries are listed in Table 2 and full libraries are available online .
Identification of shared elements within enhancers with the cDT-scanner
The function of cDT-scanner is to determine the relationship between any enhancer and any other group of MCSs used to generate the CSB libraries. cDT-scanner aligns the cDTs contained within various cDT-libraries with CSBs within an EvoPrint. cDT-scanner is a Java applet that uses a variant of the cis-Decoder aligner; it looks for only perfect matches between cDTs and CSB sequences. Alignment of cDTs using cDT-scanner is accomplished by first pasting a cDT-library in the upper window of cDT-scanner and then pasting the EvoPrint or CSBs to which they are to be aligned in the lower window. The output of cDT-scanner consists of perfect matches of cDTs aligned under the input CSBs. Since each library consists of cDTs shared by different enhancers, cDT-scanner portrays the shared elements within each CSB. A cDT-scanner alignment should be saved; information from saved files can be copy-pasted into Microsoft Word without loss of formatting features. For details on how to format cDT-alignments, see the website. A second output window for the cDT-scanner, a results table, is a list of the aligned matches along with their positions. Selecting the output column header sorts the results under that header. Contents of results tables can then be copy-pasted into Microsoft Word.
Finding less-conserved sequence elements
The 'Full-enhancer scanner' is a Java applet that identifies additional repeated cDT or CSB sequences within less conserved sequences flanking CSBs of enhancers. For this alignment, cDTs or CSBs present within an enhancer can be curated from the output of cDT-scanner termed 'Results from cDT-scan.' Curate both forward and reverse/complement sequences and paste into the upper window of Full-enhancer scanner. The EvoPrinted enhancer should be copy-pasted into the lower window. The program aligns to both conserved and non-conserved sequences of the EvoPrint.
Identification of enhancers that share conserved elements using cDT-cataloger
cDT-cataloger uses a variant of the CSB-aligner; it records only perfect matches between CSBs and cDTs of a specified size. The output lists those CSBs containing perfect sequence matches to the cDTs, and can be used to identify enhancers and count the number of times each cDT aligns with any CSB-library. Cataloguing is accomplished by copy-pasting the CSB-libraries (both forward and reverse directions) into the upper window of the cDT-cataloger and the selected cDTs of a single uniform size in the lower window. The size of the cDT(s) must be entered into the window provided.
The following additional data are available with the online version of this paper. Additional data file 1 contains the cDT-cataloger analysis of the murine Delta-like 1 Homology-II and msd-II enhancers supplemental to Figure 4. Additional data file 2 contains the cis-Decoder analysis of the Drosophila hairy stripe 1 enhancer. Additional data file 3 is a figure that contains cis-Decoder analysis of the human TIP39 5' proximal promoter. Additional data file 4 is a table that documents the contribution of each Drosophila and mammalian enhancer to the specific cDT-libraries generated in this study.
We thank Laura Elnitski and Brian Mozer for critically reading the manuscript and Anthonois Ekatomatis for technical assistance. We are also indebted to Judy Brody for help with the cis-Decoder website construction and editorial assistance. This research was supported by the Intramural Research Program of the NIH, NINDS and NIMH.
- Wray GA, Hahn MW, Abouheif E, Balhoff JP, Pizer M, Rockman MV, Romano LA: The evolution of transcriptional regulation in eukaryotes. Mol Biol Evol. 2003, 20: 1377-1419. 10.1093/molbev/msg140.PubMedGoogle Scholar
- Levine M, Davidson EH: Gene regulatory networks for development. Proc Natl Acad Sci USA. 2005, 102: 4936-4942. 10.1073/pnas.0408031102.PubMedPubMed CentralGoogle Scholar
- Istrail S, Davidson EH: Logic functions of the genomic cis-regulatory code. Proc Natl Acad Sci USA. 2005, 102: 4954-4959. 10.1073/pnas.0409624102.PubMedPubMed CentralGoogle Scholar
- Ip YT, Levine M, Bier E: Neurogenic expression of snail is controlled by separable CNS and PNS promoter elements. Development. 1994, 120: 199-207.PubMedGoogle Scholar
- Emery JF, Bier E: Specificity of CNS and PNS regulatory subelements comprising pan-neural enhancers of the deadpan and scratch genes is achieved by repression. Development. 1995, 121: 3549-3560.PubMedGoogle Scholar
- Wasserman WW, Palumbo M, Thompson W, Fickett JW, Lawrence CE: Human-mouse genome comparisons to locate regulatory sites. Nat Genet. 2000, 26: 225-228. 10.1038/79965.PubMedGoogle Scholar
- Yuh CH, Brown CT, Livi CB, Rowen L, Clarke PJ, Davidson EH: Patchy interspecific sequence similarities efficiently identify positive cis-regulatory elements in the sea urchin. Dev Biol. 2002, 246: 148-161. 10.1006/dbio.2002.0618.PubMedGoogle Scholar
- Berezikov E, Guryev V, Plasterk RH, Cuppen E: CONREAL: conserved regulatory elements anchored alignment algorithm for identification of transcription factor binding sites by phylogenetic footprinting. Genome Res. 2004, 14: 170-178. 10.1101/gr.1642804.PubMedPubMed CentralGoogle Scholar
- Odenwald WF, Rasband W, Kuzin A, Brody T: EVOPRINTER, a multigenomic comparative tool for rapid identification of functionally important DNA. Proc Natl Acad Sci USA. 2005, 102: 14700-14705. 10.1073/pnas.0506915102.PubMedPubMed CentralGoogle Scholar
- Hoch M, Schröder C, Seifert E, Jäckle H: cis-acting control elements for Krüppel expression in the Drosophila embryo. EMBO J. 1990, 9: 2587-2595.PubMedPubMed CentralGoogle Scholar
- Hoch M, Seifert E, Jackle H: Gene expression mediated by cis-acting sequences of the Kruppel gene in response to the Drosophila morphogens bicoid and hunchback. EMBO J. 1991, 10: 2267-2278.PubMedPubMed CentralGoogle Scholar
- Hoch M, Gerwin N, Taubert H, Jackle H: Competition for overlapping sites in the regulatory region of the Drosophila gene Kruppel. Science. 1992, 256: 94-97. 10.1126/science.1348871.PubMedGoogle Scholar
- Prabhakar S, Poulin F, Shoukry M, Afzal V, Rubin EM, Couronne O, Pennacchio LA: Close sequence comparisons are sufficient to identify human cis-regulatory elements. Genome Res. 2006, 16: 855-863. 10.1101/gr.4717506.PubMedPubMed CentralGoogle Scholar
- Kent WJ: BLAT - the BLAST-like alignment tool. Genome Res. 2002, 12: 656-664. 10.1101/gr.229202. Article published online before March 2002.PubMedPubMed CentralGoogle Scholar
- Gallo SM, Li L, Hu Z, Halfon MS: REDfly: a regulatory element database for Drosophila. Bioinformatics. 2006, 22: 381-383. 10.1093/bioinformatics/bti794.PubMedGoogle Scholar
- Adachi Y, Hauck B, Clements J, Kawauchi H, Kurusu M, Totani Y, Kang YY, Eggert T, Walldorf U, Furukubo-Tokunaga K, Callaerts P: Conserved cis-regulatory modules mediate complex neural expression patterns of the eyeless gene in the Drosophila brain. Mech Dev. 2003, 120: 1113-1126. 10.1016/j.mod.2003.08.007.PubMedGoogle Scholar
- Riddihough G, Ish-Horowicz D: Individual stripe regulatory elements in the Drosophila hairy promoter respond to maternal, gap, and pair-rule genes. Genes Dev. 1991, 5: 840-854. 10.1101/gad.5.5.840.PubMedGoogle Scholar
- Fujioka M, Emi-Sarker Y, Yusibova GL, Goto T, Jaynes JB: Analysis of an even-skipped rescue transgene reveals both composite and discrete neuronal and early blastoderm enhancers, and multi-stripe positioning by gap gene repressor gradients. Development. 1999, 126: 2527-2538.PubMedPubMed CentralGoogle Scholar
- Schröder C, Tautz D, Seifert E, Jäckle H: Differential regulation of the two transcripts from the Drosophila gap segmentation gene hunchback. EMBO J. 1988, 7: 2881-2887.PubMedPubMed CentralGoogle Scholar
- Margolis JS, Borowsky ML, Steingrimsson E, Shim CW, Lengyel JA, Posakony JW: Posterior stripe expression of hunchback is driven from two promoters by a common enhancer element. Development. 1995, 121: 3067-3077.PubMedGoogle Scholar
- Yin Z, Xu XL, Frasch M: Regulation of the twist target gene tinman by modular cis-regulatory elements during early mesoderm. Development. 1997, 124: 4971-4982.PubMedGoogle Scholar
- Miller JM, Oligino T, Pazdera M, Lopez AJ, Hoshizaki DK: Identification of fat-cell enhancer regions in Drosophila melanogaster. Insect Mol Biol. 2002, 11: 67-77. 10.1046/j.0962-1075.2001.00310.x.PubMedGoogle Scholar
- Gindhart JG, King AN, Kaufman TC: Characterization of the cis-regulatory region of the Drosophila homeotic gene Sex combs reduced. Genetics. 1995, 139: 781-795.PubMedGoogle Scholar
- Schroeder MD, Pearce M, Fak J, Fan H, Unnerstall U, Emberly E, Rajewsky N, Siggia ED, Gaul U: Transcriptional control in the segmentation gene network of Drosophila. PLoS Biol. 2004, 2: E271-10.1371/journal.pbio.0020271.PubMedPubMed CentralGoogle Scholar
- Samad OA, Geisen MJ, Caronia G, Varlet I, Zappavigna V, Ericson J, Goridis C, Rijli FM: Integration of anteroposterior and dorsoventral regulation of Phox2b transcription in cranial motoneuron progenitors by homeodomain proteins. Development. 2004, 131: 4071-4083. 10.1242/dev.01282.PubMedGoogle Scholar
- cis-Decoder. [http://evoprinter.ninds.nih.gov/cisdecoder/index.htm]
- Helms AW, Abney AL, Ben-Arie N, Zoghbi HY, Johnson JE: Autoregulation and multiple enhancers control Math1 expression in the developing nervous system. Development. 2000, 127: 1185-1196.PubMedGoogle Scholar
- Manak JR, Mathies LD, Scott MP: Regulation of a decapentaplegic midgut enhancer by homeotic proteins. Development. 1994, 120: 3605-3619.PubMedGoogle Scholar
- Kuzin A, Brody T, Moore AW, Odenwald WF: Nerfin-1 is required for early axon guidance decisions in the developing Drosophila CNS. Dev Biol. 2005, 277: 347-365. 10.1016/j.ydbio.2004.09.027.PubMedGoogle Scholar
- Murre C, McCaw PS, Baltimore D: A new DNA binding and dimerization motif in immunoglobulin enhancer daughterless, MyoD and myc proteins. Cell. 1989, 56: 777-783. 10.1016/0092-8674(89)90682-X.PubMedGoogle Scholar
- Powell LM, zur Lage PI, Prentice DRA, Senthinathan B, Jarman AP: The proneural proteins Atonal and Scute regulate neural target genes through different E-Box binding sites. Mol Cell Biol. 2004, 24: 9517-9526. 10.1128/MCB.24.21.9517-9526.2004.PubMedPubMed CentralGoogle Scholar
- Artavanis-Tsakonas S, Rand MD, Lake RJ: Notch signaling: cell fate control and signal integration in development. Science. 1999, 284: 770-776. 10.1126/science.284.5415.770.PubMedGoogle Scholar
- Bettenhausen B, Hrabe de Angelis M, Simon D, Guenet JL, Gossler A: Transient and restricted expression during mouse embryogenesis of Dll1, a murine gene closely related to Drosophila Delta. Development. 1995, 121: 2407-2418.PubMedGoogle Scholar
- Beckers J, Clark A, Wünsch K, De Angelis MH, Gossler A: Expression of the mouse Delta-1 gene during organogenesis and fetal development. Mech Dev. 1999, 84: 165-168. 10.1016/S0925-4773(99)00065-9.PubMedGoogle Scholar
- Beckers J, Caron A, Hrabe de Angelis M, Hans S, Campos-Ortega JA, Gossler A: Distinct regulatory elements direct Delta-1 expression in the nervous system and paraxial mesoderm of transgenic mice. Mech Dev. 2000, 95: 23-34. 10.1016/S0925-4773(00)00322-1.PubMedGoogle Scholar
- Rowitch DH, Echelard Y, Danielian PS, Gellner K, Brenner S, McMahon AP: Identification of an evolutionarily conserved 110 base-pair cis-acting regulatory sequence that governs Wnt-1 expression in the murine neural plate. Development. 1998, 125: 2735-2746.PubMedGoogle Scholar
- Bagheri-Fam S, Barrionuevo F, Dohrmann U, Gunther T, Schule R, Kemler R, Mallo M, Kanzler B, Scherer G: Long-range upstream and downstream enhancers control distinct subsets of the complex spatiotemporal Sox9 expression pattern. Dev Biol. 2006, 291: 382-397. 10.1016/j.ydbio.2005.11.013.PubMedGoogle Scholar
- Molkentin JD, Antos C, Mercer B, Taigen T, Miano JM, Olson EN: Direct activation of a GATA6 cardiac enhancer by Nkx2.5: evidence for a reinforcing regulatory network of Nkx2.5 and GATA transcription factors in the developing heart. Dev Biol. 2000, 217: 301-309. 10.1006/dbio.1999.9544.PubMedGoogle Scholar
- Awgulewitsch A, Jacobs D: Deformed autoregulatory element from Drosophila functions in a conserved manner in transgenic mice. Nature. 1992, 358: 341-344. 10.1038/358341a0.PubMedGoogle Scholar
- Malicki J, Cianetti LC, Peschie C, McGinnis W: A human HOX4B regulatory element provides head-specific expression in Drosophila. Nature. 1992, 358: 345-347. 10.1038/358345a0.PubMedGoogle Scholar
- Ip YT, Park RE, Kosman D, Yazdanbakhsh K, Levine M: dorsal-twist interactions establish snail expression in the presumptive mesoderm of the Drosophila embryo. Genes Dev. 1992, 6: 1518-1530. 10.1101/gad.6.8.1518.PubMedGoogle Scholar
- Odenwald WF, Garbern J, Arnheiter H, Tournier-Lasserve E, Lazzarini RA: The Hox-1.3 homeo box protein is a sequence-specific DNA-binding phosphoprotein. Genes Dev. 1989, 3: 158-172. 10.1101/gad.3.2.158.PubMedGoogle Scholar
- Gehring WJ: On the homeobox and its significance. Bioessays. 1986, 5: 3-4. 10.1002/bies.950050102.PubMedGoogle Scholar
- Markstein M, Zinzen R, Markstein P, Yee KP, Erives A, Stathopoulos A, Levine MA: A regulatory code for neurogenic gene expression in the Drosophila embryo. Development. 2004, 131: 2387-2394. 10.1242/dev.01124.PubMedGoogle Scholar
- Ochoa-Espinosa A, Yucel G, Kaplan L, Pare A, Pura N, Oberstein A, Papatsenko D, Small S: The role of binding site cluster strength in Bicoid-dependent patterning in Drosophila. Proc Natl Acad Sci USA. 2005, 102: 4960-4965. 10.1073/pnas.0500373102.PubMedPubMed CentralGoogle Scholar
- Markstein M, Markstein P, Markstein V, Levine MS: Genome-wide analysis of clustered Dorsal binding sites identifies putative target genes in the Drosophila embryo. Proc Natl Acad Sci USA. 2002, 99: 763-768. 10.1073/pnas.012591199.PubMedPubMed CentralGoogle Scholar
- Ludwig M, Bergman C, Patel N, Kreitman M: Evidence for stabilizing selection in a eukaryotic enhancer element. Nature. 2000, 403: 564-567. 10.1038/35000615.PubMedGoogle Scholar
- Papatsenko D, Levine M: Quantitative analysis of binding motifs mediating diverse spatial readouts of the Dorsal gradient in the Drosophila embryo. Proc Natl Acad Sci USA. 2005, 102: 4966-4971. 10.1073/pnas.0409414102.PubMedPubMed CentralGoogle Scholar
- Fairall L, Schwabe JW, Chapman L, Finch JT, Rhodes D: The crystal structure of a two zinc-finger peptide reveals an extension to the rules for zinc-finger/DNA recognition. Nature. 1993, 366: 483-487. 10.1038/366483a0.PubMedGoogle Scholar
- Schrons H, Knust E, Campos-Ortega JA: The Enhancer of split complex and adjacent genes in the 96F region of Drosophila melanogaster are required for segregation of neural and epidermal progenitor cells. Genetics. 1992, 32: 481-503.Google Scholar
- Usdin TB, Hoare SR, Wang T, Mezey E, Kowalak JA: TIP39: a new neuropeptide and PTH2-receptor agonist from hypothalamus. Nat Neurosci. 1999, 2: 941-943. 10.1038/14724.PubMedGoogle Scholar
- Dobolyi A, Palkovits M, Usdin TB: Expression and distribution of tuberoinfundibular peptide of 39 residues in the rat central nervous system. J Comp Neurol. 2003, 455: 547-566. 10.1002/cne.10515.PubMedGoogle Scholar
- Usdin TB, Dobolyi A, Ueda H, Palkovits M: Emerging functions for tuberoinfundibular peptide of 39 residues. Trends Endocrinol Metab. 2003, 14: 14-19. 10.1016/S1043-2760(02)00002-4.PubMedGoogle Scholar
- de Celis JF, de Celis J, Ligoxygakis P, Preiss A, Delidakis C, Bray S: Functional relationships between Notch, Su(H) and the bHLH genes of the E(spl) complex: the E(spl) genes mediate only a subset of Notch activities during imaginal development. Development. 1996, 122: 2719-2728.PubMedGoogle Scholar
- Nellesen DT, Lai EC, Posakony JW: Discrete enhancer elements mediate selective responsiveness of enhancer of split complex genes to common transcriptional activators. Dev Biol. 1999, 213: 33-53. 10.1006/dbio.1999.9324.PubMedGoogle Scholar
- Bray SJ: Expression and function of Enhancer of split bHLH proteins during Drosophila neurogenesis. Perspect Dev Neurobiol. 1997, 4: 313-323.PubMedGoogle Scholar
- Lai EC, Posakony JW: The Bearded box, a novel 3' UTR sequence motif, mediates negative post-transcriptional regulation of Bearded and Enhancer of split Complex gene expression. Development. 1997, 124: 4847-4856.PubMedGoogle Scholar
- EvoPrinter. , [http://evoprinter.ninds.nih.gov/]
- Puzzamatic. [http://www.mcn.org/c/rsurratt/Puzzamatic/WordSearchJar.html]
- cDT-cleaner. , [http://evoprinter.ninds.nih.gov/cisdecoder/Cdt_cleaner.htm]
- Ramos E, Price M, Rohrbaugh M, Lai ZC: Identifying functional cis-acting regulatory modules of the yan gene in Drosophila melanogaster. Dev Genes Evol. 2003, 213: 83-89.PubMedGoogle Scholar
- Sun Y, Jan LY, Jan YN: Transcriptional regulation of atonal during development of the Drosophila peripheral nervous system. Development. 1998, 125: 3731-3740.PubMedGoogle Scholar
- Lee HH, Frasch M: Nuclear integration of positive Dpp signals antagonistic Wg inputs and mesodermal competence factors during Drosophila visceral mesoderm induction. Development. 2005, 132: 1429-1442. 10.1242/dev.01687.PubMedGoogle Scholar
- Bush A, Hiromi Y, Cole M: Biparous: a novel bHLH gene expressed in neuronal and glial precursors in Drosophila. Dev Biol. 1996, 180: 759-772. 10.1006/dbio.1996.0344.PubMedGoogle Scholar
- Reeves N, Posakony JW: Genetic programs activated by proneural proteins in the developing Drosophila PNS. Dev Cell. 2005, 8: 413-425. 10.1016/j.devcel.2005.01.020.PubMedGoogle Scholar
- McDonald JA, Fujioka M, Odden JP, Jaynes JB, Doe CQ: Specification of motoneuron fate in Drosophila: integration of positive and negative transcription factor inputs by a minimal eve enhancer. J Neurobiol. 2003, 57: 193-203. 10.1002/neu.10264.PubMedGoogle Scholar
- Jiang J, Hoey T, Levine M: Autoregulation of a segmentation gene in Drosophila: combinatorial interaction of the even-skipped homeo box protein with a distal enhancer element. Genes Dev. 1991, 5: 265-277. 10.1101/gad.5.2.265.PubMedGoogle Scholar
- Small S, Blair A, Levine M: Regulation of even-skipped stripe 2 in the Drosophila embryo. EMBO J. 1992, 11: 4047-4057.PubMedPubMed CentralGoogle Scholar
- Small S, Blair A, Levine M: Regulation of two pair-rule stripes by a single enhancer in the Drosophila embryo. Dev Biol. 1996, 175: 314-324. 10.1006/dbio.1996.0117.PubMedGoogle Scholar
- Pick L, Schier A, Affolter M, Schmidt-Glenewinkel T, Gehring WJ: Analysis of the ftz upstream element: germ layer-specific enhancers are independently autoregulated. Genes Dev. 1990, 4: 1224-1239. 10.1101/gad.4.7.1224.PubMedGoogle Scholar
- Berman BP, Pfeiffer BD, Laverty TR, Salzberg SL, Rubin GM, Eisen MB, Celniker SE: Computational identification of developmental enhancers: conservation and function of transcription factor binding-site clusters in Drosophila melanogaster and Drosophila pseudoobscura. Genome Biol. 2004, 5: R61-10.1186/gb-2004-5-9-r61.PubMedPubMed CentralGoogle Scholar
- Hiromi Y, Kuroiwa A, Gehring WJ: Control elements of the Drosophila segmentation gene fushi tarazu. Cell. 1985, 43: 603-613. 10.1016/0092-8674(85)90232-6.PubMedGoogle Scholar
- Li X, Gutjahr T, Noll M: Separable regulatory elements mediate the establishment and maintenance of cell states by the Drosophila segment-polarity gene gooseberry. EMBO J. 1993, 12: 1427-1436.PubMedPubMed CentralGoogle Scholar
- Bouchard M, St-Amand J, Cote S: Combinatorial activity of pair-rule proteins on the Drosophila gooseberry early enhancer. Dev Biol. 2000, 222: 135-146. 10.1006/dbio.2000.9702.PubMedGoogle Scholar
- La Rosée A, Häder T, Taubert H, Rivera-Pomar R, Jäckle H: Mechanism and Bicoid dependent control of hairy stripe 7 expression in the posterior region of the Drosophila embryo. EMBO J. 1997, 16: 4403-4411. 10.1093/emboj/16.14.4403.PubMedPubMed CentralGoogle Scholar
- Langeland JA, Carroll SB: Conservation of regulatory elements controlling hairy pair-rule stripe formation. Development. 1993, 117: 585-596.PubMedGoogle Scholar
- Howard KR, Struhl G: Decoding positional information: regulation of the pair-rule gene hairy. Development. 1990, 110: 1223-1231.PubMedGoogle Scholar
- Stathopoulos A, Tam B, Ronshaugen M, Frasch M, Levine M: pyramus and thisbe: FGF genes that pattern the mesoderm of Drosophila embryos. Genes Dev. 2004, 18: 687-699. 10.1101/gad.1166404.PubMedPubMed CentralGoogle Scholar
- Hader T, Wainwright D, Shandala T, Saint R, Taubert H, Bronner G, Jäckle H: Receptor tyrosine kinase signaling regulates different modes of Groucho-dependent control of Dorsal. Curr Biol. 2000, 10: 51-54. 10.1016/S0960-9822(99)00265-1.PubMedGoogle Scholar
- Driever W, Thoma G, Nusslein-Volhard C: Determination of spatial domains of zygotic gene expression in the Drosophila embryo by the affinity of binding sites for the bicoid morphogen. Nature. 1989, 340: 363-367. 10.1038/340363a0.PubMedGoogle Scholar
- Biemar F, Zinzen R, Ronshaugen M, Sementchenko V, Manak JR, Levine MS: Spatial regulation of microRNA gene expression in the Drosophila embryo. Proc Natl Acad Sci USA. 2005, 102: 15907-15911. 10.1073/pnas.0507817102.PubMedPubMed CentralGoogle Scholar
- Nguyen HT, Xu X: Drosophila mef2 expression during mesoderm development is controlled by a complex array of cis-acting regulatory modules. Dev Biol. 1998, 204: 550-566. 10.1006/dbio.1998.9081.PubMedGoogle Scholar
- Gutjahr T, Vanario-Alonso CE, Pick L, Noll M: Multiple regulatory elements direct the complex expression pattern of the Drosophila segmentation gene paired. Mech Dev. 1994, 48: 119-128. 10.1016/0925-4773(94)90021-3.PubMedGoogle Scholar
- Kambadur R, Koizumi K, Stivers C, Nagle J, Poole SJ, Odenwald WF: Regulation of POU genes by castor and hunchback establishes layered compartments in the Drosophila CNS. Genes Dev. 1998, 12: 246-260.PubMedPubMed CentralGoogle Scholar
- Reddy KL, Wohlwill A, Dzitoeva S, Lin MH, Holbrook S, Storti RV: The Drosophila PAR domain protein 1 (Pdp1) gene encodes multiple differentially expressed mRNAs and proteins through the use of multiple enhancers and promoters. Dev Biol. 2000, 224: 401-414. 10.1006/dbio.2000.9797.PubMedGoogle Scholar
- Klingler M, Soong J, Butler B, Gergen JP: Disperse versus compact elements for the regulation of runt stripes in Drosophila. Dev Biol. 1996, 177: 73-84. 10.1006/dbio.1996.0146.PubMedGoogle Scholar
- Culi J, Modolell J: Proneural gene self-stimulation in neural precursors: an essential mechanism for sense organ development that is regulated by Notch signaling. Genes Dev. 1998, 12: 2036-2047.PubMedPubMed CentralGoogle Scholar
- Lehman DA, Patterson B, Johnston LA, Balzer T, Britton JS, Saint R, Edgar BA: Cis-regulatory elements of the mitotic regulator, string/Cdc25. Development. 1999, 126: 1793-1803.PubMedGoogle Scholar
- McCormick A, Core N, Kerridge S, Scott MP: Homeotic response elements are tightly linked to tissue-specific elements in a transcriptional enhancer of the teashirt gene. Development. 1995, 121: 2799-2812.PubMedGoogle Scholar
- Wharton KA, Crews ST: CNS midline enhancers of the Drosophila slit and Toll genes. Mech Dev. 1993, 40: 141-154. 10.1016/0925-4773(93)90072-6.PubMedGoogle Scholar
- Buttgereit D: Redundant enhancer elements guide beta 1 tubulin gene expression in apodemes during Drosophila embryogenesis. J Cell Sci. 1993, 105: 721-727.PubMedGoogle Scholar
- Lin SC, Lin MH, Horvath P, Reddy KL, Storti RV: PDP1, a novel Drosophila PAR domain bZIP transcription factor expressed in developing mesoderm, endoderm and ectoderm, is a transcriptional regulator of somatic muscle genes. Development. 1997, 124: 4685-4696.PubMedGoogle Scholar
- Shao X, Koizumi K, Nosworthy N, Tan DP, Odenwald WF, Nirenberg M: Regulatory DNA required for vnd/NK-2 homeobox gene expression pattern in neuroblasts. Proc Natl Acad Sci USA. 2002, 99: 113-117. 10.1073/pnas.012584599.PubMedPubMed CentralGoogle Scholar
- Ashraf SI, Hu X, Roote J, Ip YT: The mesoderm determinant Snail collaborates with related zinc-finger proteins to control Drosophila neurogenesis. EMBO J. 1999, 18: 6426-6438. 10.1093/emboj/18.22.6426.PubMedPubMed CentralGoogle Scholar
- Rodrigo I, Bovolenta P, Mankoo BS, Imai K: Meox homeodomain proteins are required for bapx1 expression in the sclerotome and activate its transcription by direct binding to its promoter. Mol Cell Biol. 2004, 24: 2757-2766. 10.1128/MCB.24.7.2757-2766.2004.PubMedPubMed CentralGoogle Scholar
- Tou L, Quibria N, Alexander JM: Transcriptional regulation of the human Runx2/Cbfa1 gene promoter by bone morphogenetic protein-7. Mol Cell Endocrinol. 2003, 205: 121-129. 10.1016/S0303-7207(03)00151-5.PubMedGoogle Scholar
- Kim IM, Zhou Y, Ramakrishna S, Hughes DE, Solway J, Costa RH, Kalinichenko VV: Functional characterization of evolutionarily conserved DNA regions in forkhead box f1 gene locus. J Biol Chem. 2005, 280: 37908-37916. 10.1074/jbc.M506531200.PubMedGoogle Scholar
- McFadden DG, Charite J, Richardson JA, Srivastava D, Firulli AB, Olson EN: A GATA-dependent right ventricular enhancer controls dHAND transcription in the developing heart. Development. 2000, 127: 5331-5341.PubMedGoogle Scholar
- Bessho Y, Sakata R, Komatsu S, Shiota K, Yamada S, Kageyama R: Dynamic expression and essential functions of Hes7 in somite segmentation. Genes Dev. 2001, 15: 2642-2647. 10.1101/gad.930601.PubMedPubMed CentralGoogle Scholar
- Tabaries S, Lapointe J, Besch T, Carter M, Woollard J, Tuggle CK, Jeannotte L: Cdx protein interaction with Hoxa5 regulatory sequences contributes to Hoxa5 regional expression along the axial skeleton. Mol Cell Biol. 2005, 25: 1389-1401. 10.1128/MCB.25.4.1389-1401.2005.PubMedPubMed CentralGoogle Scholar
- Mühlfriedel S, Kirsch F, Gruss P, Stoykova A, Chowdhury K: A roof plate-dependent enhancer controls the expression of Homeodomain only protein in the developing cerebral cortex. Dev Biol. 2005, 283: 522-534. 10.1016/j.ydbio.2005.04.033.PubMedGoogle Scholar
- Breslin MB, Zhu M, Lan MS: Neurod1/E47 regulates the E-box element of a novel zinc finger transcription factor, IA-1, in developing nervous system. J Biol Chem. 2003, 278: 38991-3899. 10.1074/jbc.M306795200.PubMedPubMed CentralGoogle Scholar
- Jethanandani P, Kramer RH: α7 integrin expression is negatively regulated by δEF1 during skeletal myogenesis. J Biol Chem. 2005, 280: 36037-36046. 10.1074/jbc.M508698200.PubMedGoogle Scholar
- Wang DZ, Valdez MR, McAnally J, Richardson J, Olson EN: The Mef2c gene is a direct transcriptional target of myogenic bHLH and MEF2 proteins during skeletal muscle development. Development. 2001, 128: 4623-4633.PubMedGoogle Scholar
- Verma-Kurvari S, Savage T, Gowan K, Johnson JE: Lineage-specific regulation of the neural differentiation gene MASH1. Dev Biol. 1996, 180: 605-617. 10.1006/dbio.1996.0332.PubMedGoogle Scholar
- Buchberger A, Nomokonova N, Arnold HH: Myf5 expression in somites and limb buds of mouse embryos is controlled by two distinct distal enhancer activities. Development. 2003, 130: 3297-3307. 10.1242/dev.00557.PubMedGoogle Scholar
- Zimmerman L, Parr B, Lendahl U, Cunningham M, McKay R, Gavin B, Mann J, Vassileva G, McMahon A: Independent regulatory elements in the nestin gene direct transgene expression to neural stem cells or muscle precursors. Neuron. 1994, 12: 11-24. 10.1016/0896-6273(94)90148-1.PubMedGoogle Scholar
- Zhou B, Wu B, Tompkins KL, Boyer KL, Grindley JC, Baldwin HS: Characterization of Nfatc1 regulation identifies an enhancer required for gene expression that is specific to pro-valve endocardial cells in the developing heart. Development. 2005, 132: 1137-1146. 10.1242/dev.01640.PubMedGoogle Scholar
- Simmons AD, Horton S, Abney AL, Johnson JE: Neurogenin2 expression in ventral and dorsal spinal neural tube progenitor cells is regulated by distinct enhancers. Dev Biol. 2001, 229: 327-339. 10.1006/dbio.2000.9984.PubMedGoogle Scholar
- Lien CL, McAnally J, Richardson JA, Olson EN: Cardiac-specific activity of an Nkx2-5 enhancer requires an evolutionarily conserved Smad binding site. Dev Biol. 2002, 244: 257-266. 10.1006/dbio.2002.0603.PubMedGoogle Scholar
- Kurokawa D, Takasaki N, Kiyonari H, Nakayama R, Kimura-Yoshida C, Matsuo I, Aizawa S: Regulation of Otx2 expression and its functions in mouse epiblast and anterior neuroectoderm. Development. 2004, 134: 3307-3317. 10.1242/dev.01219.Google Scholar
- Brown CB, Engleka KA, Wenning J, Lu MM, Epstein JA: Identification of a hypaxial somite enhancer element regulating Pax3 expression in migrating myoblasts and characterization of hypaxial muscle Cre transgenic mice. Genesis. 2005, 41: 202-209. 10.1002/gene.20116.PubMedGoogle Scholar
- Barron MR, Belaguli NS, Zhang SX, Trinh M, Iyer D, Merlo X, Lough JW, Parmacek MS, Bruneau BG, Schwartz RJ: Serum response factor, an enriched cardiac mesoderm obligatory factor, is a downstream gene target for Tbx genes. J Biol Chem. 2005, 280: 11816-11828. 10.1074/jbc.M412408200.PubMedGoogle Scholar
- Kutejova E, Engist B, Mallo M, Kanzler B, Bobola N: Hoxa2 downregulates Six2 in the neural crest-derived mesenchyme. Development. 2005, 132: 469-478. 10.1242/dev.01536.PubMedGoogle Scholar
- Catena R, Tiveron C, Ronchi A, Porta S, Ferri A, Tatangelo L, Cavallaro M, Favaro R, Ottolenghi S, Reinbold R, et al: Conserved POU binding DNA sites in the Sox-2 upstream enhancer regulate gene expression in embryonic and neural stem cells. J Biol Chem. 2004, 279: 41846-41857. 10.1074/jbc.M405514200.PubMedGoogle Scholar
- Miyagi S, Nishimoto M, Saito T, Ninomiya M, Sawamoto K, Okano H, Muramatsu M, Oguro H, Iwama A, Okuda A: The Sox2 regulatory region 2 functions as a neural stem cell specific enhancer in the telencephalon. J Biol Chem. 2006, 281: 13374-13381. 10.1074/jbc.M512669200.PubMedGoogle Scholar
- Gottgens B, Nastos A, Kinston S, Piltz S, Delabesse EC, Stanley M, Sanchez MJ, Ciau-Uitz A, Patient R, Green AR: Establishing the transcriptional programme for blood: the SCL stem cell enhancer is regulated by a multiprotein complex containing Ets and GATA factors. EMBO J. 2002, 21: 3039-3050. 10.1093/emboj/cdf286.PubMedPubMed CentralGoogle Scholar
- Yamagishi H, Maeda J, Hu T, McAnally J, Conway SJ, Kume T, Meyers N, Yamagishi C, Srivastava D: Tbx1 is regulated by tissue-specific forkhead proteins through a common Sonic hedgehog-responsive enhancer. Genes Dev. 2003, 17: 269-281. 10.1101/gad.1048903.PubMedPubMed CentralGoogle Scholar
- Carroll SB, Laughon A, Thalley BS: Expression, function, and regulation of the hairy segmentation protein in the Drosophila embryo. Genes Dev. 1988, 2: 883-890. 10.1101/gad.2.7.883.PubMedGoogle Scholar
- Stanojevic D, Hoey T, Levine M: Sequence-specific DNA-binding activities of the gap proteins encoded by hunchback and Kruppel in Drosophila. Nature. 1989, 341: 331-335. 10.1038/341331a0.PubMedGoogle Scholar
- Treisman J, Desplan C: The products of the Drosophila gap genes hunchback and Kruppel bind to the hunchback promoters. Nature. 1989, 341: 335-337. 10.1038/341335a0.PubMedGoogle Scholar
- Ekker SC, Jackson DG, von Kessler DP, Sun BI, Young KE, Beachy PA: The degree of variation in DNA sequence recognition among four Drosophila homeotic proteins. EMBO J. 1994, 13: 3551-3560.PubMedPubMed CentralGoogle Scholar
- Ohsako S, Hyer J, Panganiban G, Oliver I, Caudy M: Hairy function as a DNA-binding helix-loop-helix repressor of Drosophila sensory organ formation. Genes Dev. 1994, 8: 2743-2755. 10.1101/gad.8.22.2743.PubMedGoogle Scholar
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