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Caenorhabditis elegans is a powerful model for studying gene regulation, as it has a compact genome and a wealth of genomic tools. However, identification of regulatory elements has been limited, as DNA-binding motifs are known for only 71 of the estimated 763 sequence-specific transcription factors (TFs). To address this problem, we performed protein binding microarray experiments on representatives of canonical TF families in C. elegans, obtaining motifs for 129 TFs. Additionally, we predict motifs for many TFs that have DNA-binding domains similar to those already characterized, increasing coverage of binding specificities to 292 C. elegans TFs (∼40%). These data highlight the diversification of binding motifs for the nuclear hormone receptor and C2H2 zinc finger families and reveal unexpected diversity of motifs for T-box and DM families. Motif enrichment in promoters of functionally related genes is consistent with known biology and also identifies putative regulatory roles for unstudied TFs. DOI: http://dx.doi.org/10.7554/eLife.06967.001, eLife digest Many scientists use ‘model’ species—such as the fruit fly or a nematode worm called Caenorhabditis elegans—in their research because these organisms have useful features that make it easier to carry out many experiments. For example, C. elegans has a smaller genome compared to many other animals, which is useful for studying the roles of individual genes or stretches of DNA. Transcription factors are a type of protein that can bind to specific stretches of DNA and help to switch certain genes on or off. These ‘motifs’ may be close to the gene or further away in the genome, and therefore, must stand out amongst the rest of the DNA, like lights on a landing strip. However, the motifs for only 10% of the estimated 763 transcription factors in C. elegans have been identified so far. In this study, Narasimhan, Lambert, Yang et al. used a technique called a ‘protein binding microarray’ to identify the motifs for many more of the C. elegans transcription factors. These findings were then used to predict motifs for other transcription factors. Together, these methods increased the proportion of C. elegans transcription factors with known DNA-binding motifs from 10% to around 40%. Now that more DNA motifs have been identified, it is possible to look for similarities and differences between them. For example, Narasimhan, Lambert, Yang et al. found that transcription factors with similar sequences can bind to very varied motifs. On the other hand, some transcription factors that are very different are able to recognize very similar motifs. The experiments also indicate that motifs found very close to genes—in sequences known as ‘promoters’—may be able to interact with many proteins to influence the activity of genes. Narasimhan, Lambert, Yang et al.'s findings increase the number of C. elegans transcription factors with a motif, bringing the knowledge of these proteins more in line with the better-studied transcription factors of humans and fruit flies. The next challenge is to identify DNA motifs for the remaining 60% of transcription factors. DOI: http://dx.doi.org/10.7554/eLife.06967.002

Gateway-compatible yeast one-hybrid (Y1H) assays provide a convenient gene-centered (DNA-to-protein) approach to identify the repertoire of transcription factors that can bind a DNA sequence of interest. We present a set of Y1H resources, including clones for 988 of 1,434 (69%) predicted human transcription factors, for the interrogation of interactions using either low or high-throughput settings. These approaches detect both known and novel interactions between human DNA regions and transcription factors.

BackgroundHox genes encode master regulators of regional fate specification during early metazoan development. Much is known about the initiation and regulation of Hox gene expression in Drosophila and vertebrates, but less is known in the non-arthropod invertebrate model system,C. elegans. TheC. elegansHox genelin-39is required for correct fate specification in the midbody region, including the Vulval Precursor Cells (VPCs). To better understandlin-39regulation and function, we aimed to identify transcription factors necessary forlin-39expression in the VPCs, and in particular sought factors that initiatelin-39expression in the embryo.ResultsWe used the yeast one-hybrid (Y1H) method to screen for factors that bound to 13 fragments from thelin-39region: twelve fragments contained sequences conserved betweenC. elegansand two other nematode species, while one fragment was known to drive reporter gene expression in the early embryo in cells that generate the VPCs. Sixteen transcription factors that bind to eightlin-39genomic fragments were identified in yeast, and we characterized several factors by verifying their physical interactionsin vitro, and showing that reduction of their function leads to alterations inlin-39levels andlin-39::GFPreporter expressionin vivo. Three factors, the orphan nuclear hormone receptor NHR-43, the hypodermal fate regulator LIN-26, and the GATA factor ELT-6 positively regulatelin-39expression in the embryonic precursors to the VPCs. In particular, ELT-6 interacts with an enhancer that drives GFP expression in the early embryo, and the ELT-6 site we identified is necessary for proper embryonic expression. These three factors, along with the factors ZTF-17, BED-3 and TBX-9, also positively regulatelin-39expression in the larval VPCs.ConclusionsThese results significantly expand the number of factors known to directly bind and regulatelin-39expression, identify the first factors required forlin-39expression in the embryo, and hint at a positive feedback mechanism involving GATA factors that maintainslin-39expression in the vulval lineage. This work indicates that, as in other organisms, the regulation of Hox gene expression inC. elegansis complicated, redundant and robust.

Pax genes encode a family of transcription factors, many of which play key roles in animal embryonic development but whose evolutionary relationships and ancestral functions are unclear. To address these issues, we are characterizing the Pax gene complement of the coral Acropora millepora , an anthozoan cnidarian. As the simplest animals at the tissue level of organization, cnidarians occupy a key position in animal evolution, and the Anthozoa are the basal class within this diverse phylum. We have identified four Pax genes in Acropora : two ( Pax-Aam and Pax-Bam ) are orthologs of genes identified in other cnidarians; the others ( Pax-Cam and Pax-Dam ) are unique to Acropora. Pax-Aam may be orthologous with Drosophila Pox neuro , and Pax-Bam clearly belongs to the Pax-2 / 5 / 8 class. The Pax-Bam Paired domain binds specifically and preferentially to Pax-2/5/8 binding sites. The recently identified Acropora gene Pax-Dam belongs to the Pax-3 / 7 class. Clearly, substantial diversification of the Pax family occurred before the Cnidaria/higher Metazoa split. The fourth Acropora Pax gene, Pax-Cam , may correspond to the ancestral vertebrate Pax gene and most closely resembles Pax-6 . The expression pattern of Pax-Cam , in putative neurons, is consistent with an ancestral role of the Pax family in neural differentiation and patterning. We have determined the genomic structure of each Acropora Pax gene and show that some splice sites are shared both between the coral genes and between these and Pax genes in triploblastic metazoans. Together, these data support the monophyly of the Pax family and indicate ancient origins of several introns.

Vertebrate Pax-6 and its Drosophila homolog eyeless play central roles in eye specification, although it is not clear if this represents the ancestral role of this gene class. As the most "primitive" animals with true nervous systems, the Cnidaria may be informative in terms of the evolution of the Pax gene family. For this reason we surveyed the Pax gene complement of a representative of the basal cnidarian class (the Anthozoa), the coral Acropora millepora. cDNAs encoding two coral Pax proteins were isolated. Pax-Aam encoded a protein containing only a paired domain, whereas Pax-Cam also contained a homeodomain clearly related to those in the Pax-6 family. The paired domains in both proteins most resembled the vertebrate Pax-2/5/8 class, but shared several distinctive substitutions. As in most Pax-6 homologs and orthologs, an intron was present in the Pax-Cam locus at a position corresponding to residues 46/47 in the homeodomain. We propose a model for evolution of the Pax family, in which the ancestor of all of the vertebrate Pax genes most resembled Pax-6, and arose via fusion of a Pax-Aam-like gene (encoding only a paired domain) with an anteriorly-expressed homeobox gene resembling the paired-like class.

Yeast one-hybrid (Y1H) assays are used to identify which transcription factor (TF) “prey” molecules can bind a DNA fragment of interest that is used as “bait”. Y1H assays involve introducing plasmids that encode TFs into a yeast “bait strain” in which the DNA fragment of interest is integrated upstream of one or more reporters, and activation of these reporters indicates that a TF–DNA interaction has occurred. These plasmids express each TF as a hybrid protein (hence the “one-hybrid” name) fused to the activation domain (AD) of the yeast TF Gal4. The AD moiety activates reporter expression even if the TF to which it is fused typically functions as a repressor. Here, we describe how to perform a Y1H screen of a library of cDNA fragments cloned into a pPC86 plasmid expressing the protein encoded by the cDNA as an AD fusion. The method assumes availability of either commercially available libraries or libraries generated in house using mRNA extracted from a tissue of interest. We also assume that users have access to a yeast bait strain that possesses the DNA fragment of interest integrated upstream of two different reporters—HIS3, an auxotrophic marker, and LacZ, a colorimetric marker that changes colorless X-gal into a blue compound. Briefly, the screen involves transforming the AD-cDNA library into the yeast bait strain, identifying colonies that show activation of both reporters, retesting the interaction in a freshly grown bait strain, and sequencing the cDNA insert to identify the interacting TF.

Here, we present a protocol for amplifying DNA fragments from the genome of, or plasmids transformed into, yeast strains that require the use of the lytic enzyme zymolyase to break open the yeast cells by digesting the cell wall. Yeast strains requiring such treatment include YM4271 and Y1HaS2, whereas other yeast strains (e.g., MaV103) might not require treatment with Zymolyase.

Yeast one-hybrid (Y1H) assays are used to identify which transcription factor (TF) “preys” can bind a DNA fragment of interest that is used as the “bait.” Undertaking Y1H assays requires the generation of a yeast “bait strain” for each DNA fragment of interest that features the DNA bait coupled to a reporter(s). Plasmids encoding TFs fused to the Gal4 activation domain (AD) are then introduced into the bait strain, and activation of the reporter(s) indicates that a TF–DNA interaction has occurred. Here, we present a protocol for the first part of the strategy—the generation of a bait strain for Y1H assays. We assume that the DNA bait has already been cloned into two different reporter constructs: One places the fragment of interest upstream of HIS3, an auxotrophic growth marker, whereas the other places the DNA bait upstream of LacZ, a colorimetric marker that changes colorless X-gal into a blue compound. Briefly, generation of the bait strain involves using homologous recombination to integrate the two reporters into the genome of the yeast strain, screening individual integrants for background reporter expression (i.e., expression in the absence of a TF), and using polymerase chain reaction (PCR) and sequencing to confirm the DNA bait identity from both integrated reporter cassettes.

Gene duplication results in two identical paralogs that diverge through mutation, leading to loss or gain of interactions with other biomolecules. Here, we comprehensively characterize such network rewiring for C. elegans transcription factors (TFs) within and across four newly delineated molecular networks. Remarkably, we find that even highly similar TFs often have different interaction degree and partners. In addition, we find that most TF families have a member that is highly connected in multiple networks. Further, different TF families have opposing correlations between network connectivity and phylogenetic age, suggesting that they are subject to different evolutionary pressures. Finally, TFs that have similar partners in one network generally do not in another, indicating a lack of pressure to retain cross-network similarity. Our multiparameter analyses provide an unprecedented glimpse into the evolutionary dynamics that shaped TF networks.

Since its development about two decades ago, the yeast one-hybrid (Y1H) assay has become an important technique for detecting physical interactions between sequence-specific regulatory transcription factor proteins (TFs) and their DNA target sites. Multiple versions of the Y1H methodology have been developed, each with technical differences and unique advantages. We will discuss several of these technical variations in detail, and also provide some ideas for how Y1H assays can be further improved.

Transcription is regulated by sequence-specific transcription factors (TFs) that bind to short genomic DNA elements that can be located in promoters, enhancers and other cis-regulatory modules. Determining which TFs bind where requires techniques that enable the ab initio identification of TF-DNA interactions. These techniques can either be "TF-centered" (protein-to-DNA), where regions of DNA bound by a TF of interest are identified, or "gene-centered" (DNA-to-protein), where TFs that bind a DNA sequence of interest are identified. Here, we describe gene-centered yeast one-hybrid (Y1H) assays. Briefly, in Y1H assays, a DNA fragment is cloned upstream of two different reporters, and these reporter constructs are integrated into the genome of a yeast strain. Next, plasmids expressing TFs as hybrid proteins (hence the name of the assay) fused with the strong transcriptional activation domain (AD) of the yeast TF Gal4 are introduced into the yeast strain. When a TF interacts with the DNA fragment of interest, the AD moiety activates reporter expression in yeast regardless of whether the TF is an activator or repressor in vivo. Sequencing the plasmid in the colonies that exhibit reporter activation reveals the identity of the TFs that can bind the DNA fragment. We have shown Y1H to be a robust method for detecting interactions between a variety of DNA elements and multiple families of TFs.

We present an Arabidopsis thaliana full-length transcription factor resource of 92% of root stele–expressed transcription factors and 74.5% of root-expressed transcription factors. We demonstrate its use with enhanced yeast one-hybrid (eY1H ) screening for rapid, systematic mapping of plant transcription factor–promoter interactions. We identified 158 interactions with 13 stele-expressed promoters, many of which occur physically or are regulatory in planta.

Insulin/IGF-1 signaling controls metabolism, stress resistance and aging in Caenorhabditis elegans by regulating the activity of the DAF-16/FoxO transcription factor (TF). However, the function of DAF-16 and the topology of the transcriptional network that it crowns remain unclear. Using chromatin profiling by DNA adenine methyltransferase identification (DamID), we identified 907 genes that are bound by DAF-16. These were enriched for genes showing DAF-16-dependent upregulation in long-lived daf-2 insulin/IGF-1 receptor mutants (P=1.4e(-11)). Cross-referencing DAF-16 targets with these upregulated genes (daf-2 versus daf-16; daf-2) identified 65 genes that were DAF-16 regulatory targets. These 65 were enriched for signaling genes, including known determinants of longevity, but not for genes specifying somatic maintenance functions (e.g. detoxification, repair). This suggests that DAF-16 acts within a relatively small transcriptional subnetwork activating (but not suppressing) other regulators of stress resistance and aging, rather than directly regulating terminal effectors of longevity. For most genes bound by DAF-16::DAM, transcriptional regulation by DAF-16 was not detected, perhaps reflecting transcriptionally non-functional TF 'parking sites'. This study demonstrates the efficacy of DamID for chromatin profiling in C. elegans.

The Caenorhabditiselegans genome encodes more than 100 microRNAs (miRNAs). Genetic analyses of miRNA deletion mutants have only provided limited insights into miRNA function. To gain insight into the function of miRNAs, it is important to determine their spatiotemporal expression pattern. Here, we use miRNA promoters driving the expression of GFP as a proxy for miRNA expression. We describe a set of 73 transgenic C. elegans strains, each expressing GFP under the control of a miRNA promoter. Together, these promoters control the expression of 89 miRNAs (66% of all predicted miRNAs). We find that miRNA promoters drive GFP expression in a variety of tissues and that, overall, their activity is similar to that of protein-coding gene promoters. However, miRNAs are expressed later in development, which is consistent with functions after initial body-plan specification. We find that miRNA members belonging to families are more likely to be expressed in overlapping tissues than miRNAs that do not belong to the same family, and provide evidence that intronic miRNAs may be controlled by their own, rather than a host gene promoter. Finally, our data suggest that post-transcriptional mechanisms contribute to differential miRNA expression. The data and strains described here will provide a valuable guide and resource for the functional analysis of C. elegans miRNAs.

Background The C. elegans Promoterome is a powerful resource for revealing the regulatory mechanisms by which transcription is controlled pan-genomically. Transcription factors will form the core of any systems biology model of genome control and therefore the promoter activity of Promoterome inserts for C. elegans transcription factor genes was examined, in vivo, with a reporter gene approach. Results Transgenic C. elegans strains were generated for 366 transcription factor promoter/gfp reporter gene fusions. GFP distributions were determined, and then summarized with reference to developmental stage and cell type. Reliability of these data was demonstrated by comparison to previously described gene product distributions. A detailed consideration of the results for one C. elegans transcription factor gene family, the Six family, comprising ceh-32, ceh-33, ceh-34 and unc-39 illustrates the value of these analyses. The high proportion of Promoterome reporter fusions that drove GFP expression, compared to previous studies, led to the hypothesis that transcription factor genes might be involved in local gene duplication events less frequently than other genes. Comparison of transcription factor genes of C. elegans and Caenorhabditis briggsae was therefore carried out and revealed very few examples of functional gene duplication since the divergence of these species for most, but not all, transcription factor gene families. Conclusion Examining reporter expression patterns for hundreds of promoters informs, and thereby improves, interpretation of this data type. Genes encoding transcription factors involved in intrinsic developmental control processes appear acutely sensitive to changes in gene dosage through local gene duplication, on an evolutionary time scale.

Transcription regulatory networks consist of physical and functional interactions between transcription factors (TFs) and their target genes. The systematic mapping of TF-target gene interactions has been pioneered in unicellular systems, using "TF-centered" methods (e.g., chromatin immunoprecipitation). However, metazoan systems are less amenable to such methods. Here, we used "gene-centered" high-throughput yeast one-hybrid (Y1H) assays to identify 283 interactions between 72 C. elegans digestive tract gene promoters and 117 proteins. The resulting protein-DNA interaction (PDI) network is highly connected and enriched for TFs that are expressed in the digestive tract. We provide functional annotations for approximately 10% of all worm TFs, many of which were previously uncharacterized, and find ten novel putative TFs, illustrating the power of a gene-centered approach. We provide additional in vivo evidence for multiple PDIs and illustrate how the PDI network provides insights into metazoan differential gene expression at a systems level.

A compendium of 934 transcription factor genes in C. elegans and identified by computational searches and extensive manual., Background Transcription regulatory networks are composed of interactions between transcription factors and their target genes. Whereas unicellular networks have been studied extensively, metazoan transcription regulatory networks remain largely unexplored. Caenorhabditis elegans provides a powerful model to study such metazoan networks because its genome is completely sequenced and many functional genomic tools are available. While C. elegans gene predictions have undergone continuous refinement, this is not true for the annotation of functional transcription factors. The comprehensive identification of transcription factors is essential for the systematic mapping of transcription regulatory networks because it enables the creation of physical transcription factor resources that can be used in assays to map interactions between transcription factors and their target genes. Results By computational searches and extensive manual curation, we have identified a compendium of 934 transcription factor genes (referred to as wTF2.0). We find that manual curation drastically reduces the number of both false positive and false negative transcription factor predictions. We discuss how transcription factor splice variants and dimer formation may affect the total number of functional transcription factors. In contrast to mouse transcription factor genes, we find that C. elegans transcription factor genes do not undergo significantly more splicing than other genes. This difference may contribute to differences in organism complexity. We identify candidate redundant worm transcription factor genes and orthologous worm and human transcription factor pairs. Finally, we discuss how wTF2.0 can be used together with physical transcription factor clone resources to facilitate the systematic mapping of C. elegans transcription regulatory networks. Conclusion wTF2.0 provides a starting point to decipher the transcription regulatory networks that control metazoan development and function.

The phylum Cnidaria is the closest outgroup to the triploblastic metazoans and as such offers unique insights into evolutionary questions at several levels. In the post-genomic era, a knowledge of the gene complement of representative cnidarians will be important for understanding the relationship between the expansion of gene families and the evolution of morphological complexity among more highly evolved metazoans. Studies of cnidarian development and its molecular control will provide information about the origins of the major bilaterian body axes, the origin of the third tissue layer, the mesoderm, and the evolution of nervous system patterning. We are studying the cnidarian Acropora millepora, a reef building scleractinian coral, and a member of the basal cnidarian class, the Anthozoa. We review ourwork on descriptive embryology and studies of selected transcription factor gene families, where our knowledge from Acropora is particularly advanced relative to other cnidarians. We also describe a recent preliminary whole genome initiative, a coral EST database.

The caudal-related (Cdx) homeodomain transcription factors have a conserved role in the development of posterior structures in both vertebrates and invertebrates. A particularly interesting finding is that Cdx proteins have an important function in the regulation of expression from a subset of Hox genes. In this study, we report the cloning of cDNAs from the Cdx genes of the amphibian Xenopus tropicalis. Xenopus tropicalis is a diploid species, related to the commonly used laboratory animal Xenopus laevis, and has attracted attention recently as a potential genetic model for animal development. The Xenopus tropicalis cDNAs, Xtcad1, Xtcad2, and Xtcad3, show between 88 and 94% sequence identity with their Xenopus laevis orthologues. This finding corresponds to between 90 and 95% identity at the level of derived amino acid sequence. We also present a detailed description of Xtcad1, Xtcad2, and Xtcad3 expression during normal development. In common with the Cdx genes of other vertebrates, the Xenopus tropicalis Cdx genes show overlapping and dynamic patterns of expression in posterior regions of the embryo through the early stages of development. © 2001 Wiley-Liss, Inc.

Arabidopsis root development provides a remarkably tractable system to delineate tissue-specific, developmental gene regulatory networks and to study their functionality in a complex multicellular model system over developmental time. Tightly controlled gene expression within tissues is a hallmark of multicellular development and is accomplished by transcription factors (TFs) and microRNAs (miRNAs). We present an automated, enhanced yeast one hybrid (eY1H) assay using a tissue-specific TF resource to comprehensively map gene regulatory networks in the Arabidopsis root stele. These gene regulatory networks are robust and highly combinatorial in nature. Using these methods and computational modeling, we have additionally modeled a gene regulatory network that regulates distinct transcriptional events in developmental time. Distinct regulatory modules were identified that temporally drive the expression of genes involved in xylem specification and in the subsequent synthesis of secondary cell wall metabolites associated with xylem differentiation.

Background: Transcription regulatory networks are composed of interactions between transcription factors and their target genes. Whereas unicellular networks have been studied extensively, metazoan transcription regulatory networks remain largely unexplored. Caenorhabditis elegans provides a powerful model to study such metazoan networks because its genome is completely sequenced and many functional genomic tools are available. While C. elegans gene predictions have undergone continuous refinement, this is not true for the annotation of functional transcription factors. The comprehensive identification of transcription factors is essential for the systematic mapping of transcription regulatory networks because it enables the creation of physical transcription factor resources that can be used in assays to map interactions between transcription factors and their target genes. Results: By computational searches and extensive manual curation, we have identified a compendium of 934 transcription factor genes (r eferred to as wTF2.0). We find that manual curation drastically reduces the number of both false positive and false negative transcription factor predictions. We discuss how transcription factor splice variants and dimer formation may affect the total number of functional transcription factors. In contrast to mouse transcription factor genes, we find that C. elegans transcription factor genes do not undergo significantly more splicing than other genes. This difference may contribute to differences in organism complexity. We identify candidate redundant worm transcription factor genes and orthologous worm and human transcription factor pairs. Finally, we discuss how wTF2.0 can be used together with physical transcription factor clone resources to facilitate the systematic mapping of C. elegans transcription regulatory networks.

Cdx homeodomain transcription factors have multiple roles in early vertebrate development. Furthermore, mis-regulation of Cdx expression has been demonstrated in metaplasias and cancers of the gut epithelium. Given the importance of Cdx genes in development and disease, the mechanisms underlying their expression are of considerable interest. We report an analysis of the upstream regulatory regions from the amphibian Xenopus laevis Cdx4 gene. We show that a GFP reporter containing 2.8 kb upstream of the transcription start site is expressed in the posterior of transgenic embryos. Deletion analysis of the upstream sequence reveals that a 247-bp proximal promoter fragment will drive posterior expression in transgenic embryos. We show that 63 bp of upstream sequence, that includes a consensus site for POU-domain octamer-binding proteins, retains significant promoter activity. Co-expression of the octamer-binding protein Oct1 induces expression from a Cdx4 reporter and mutation of the octamer site abolishes activity of the same reporter. We show that the octamer site is highly conserved in the promoters of the human, mouse, chicken, and zebrafish Cdx4 genes and within the promoters of amphibian Cdx1 and Cdx2. These data suggest a conserved function for octamer-binding proteins in the regulation of Cdx family members.

Homeobox proteins are critical regulators of developmental gene transcription and cell specification. Many insights into transcriptional regulation have been gained from studies in the nematode Caenorhabditis elegans. We investigated the expression and regulation of the C. elegans homeobox gene ceh-63, which encodes a single-homeodomain transcription factor of 152 amino acids. ceh-63 is expressed in the interneuron DVC in both sexes, from late embryogenesis through adulthood, and two pairs of uterine cells in reproductive hermaphrodites only. A reporter gene fusion, encoding GFP fused to the full-length CEH-63, also drove weak inconsistent expression in additional unidentified cells in the head and tail. A potential ceh-63 null mutant had no obvious abnormalities, except for a possible increase in subtle defects of the DVC axon projection. No behavioural responses were observed upon either laser ablation of DVC or activation of DVC through light stimulation of channelrhodopsin-2 specifically expressed in this neuron. The function of DVC therefore remains enigmatic. A transcriptional regulatory cascade operating in DVC was defined from the LIM-homeodomain protein CEH-14 through CEH-63 to the helix–turn–helix transcription factor MBR-1. Both CEH-14 and CEH-63 individually bound the mbr-1 promoter in a yeast one-hybrid assay. A model is proposed suggesting that CEH-14 activates ceh-63 and then along with CEH-63 co-ordinately activates mbr-1., Highlights ► The C. elegans homeobox gene ceh-63 is expressed in a single interneuron, DVC. ► Although a highly conserved gene, a ceh-63 null mutant had no major phenotype. ► A transcription factor regulatory cascade operating in DVC was defined.

SummaryTranscription factor (TF) DNA sequence preferences direct their regulatory activity, but are currently known for only ∼1% of eukaryotic TFs. Broadly sampling DNA-binding domain (DBD) types from multiple eukaryotic clades, we determined DNA sequence preferences for >1,000 TFs encompassing 54 different DBD classes from 131 diverse eukaryotes. We find that closely related DBDs almost always have very similar DNA sequence preferences, enabling inference of motifs for ∼34% of the ∼170,000 known or predicted eukaryotic TFs. Sequences matching both measured and inferred motifs are enriched in chromatin immunoprecipitation sequencing (ChIP-seq) peaks and upstream of transcription start sites in diverse eukaryotic lineages. SNPs defining expression quantitative trait loci in Arabidopsis promoters are also enriched for predicted TF binding sites. Importantly, our motif “library” can be used to identify specific TFs whose binding may be altered by human disease risk alleles. These data present a powerful resource for mapping transcriptional networks across eukaryotes.

Cnidarians are animals with a single (oral/aboral) overt body axis and with origins that nominally predate bilaterality. To better understand the evolution of axial patterning mechanisms, we characterized genes from the coral, Acropora millepora (Class Anthozoa) that are considered to be unambiguous markers of the bilaterian anterior/posterior and dorsal/ventral axes. Homologs of Otx/otd and Emx/ems, definitive anterior markers across the Bilateria, are expressed at opposite ends of the Acropora larva; otxA-Am initially around the blastopore and later preferentially toward the oral end in the ectoderm, and emx-Am predominantly in putative neurons in the aboral half of the planula larva, in a domain overlapping that of cnox-2Am, a Gsh/ind gene. The Acropora homologs of Pax-3/7, NKX2.1/vnd and Msx/msh are expressed in axially restricted and largely non-overlapping patterns in larval ectoderm. In Acropora, components of both the D/V and A/P patterning systems of bilateral animals are therefore expressed in regionally restricted patterns along the single overt body axis of the planula larva, and two ‘anterior’ markers are expressed at opposite ends of the axis. Thus, although some specific gene functions appear to be conserved between cnidarians and higher animals, no simple relationship exists between axial patterning systems in the two groups.