Ian A. Hope

Functional dissection of a eukaryotic transcriptional activator protein, GCN4 of Yeast

Yeast GCN4 protein binds specifically to the promoters of amino acid biosynthetic genes and coordinately induces their transcription. Serially deleted GCN4 and hybrid LexA-GCN4 proteins were assayed for specific DNA binding activity in vitro, and for stimulation of transcription in vivo. The specific DNA binding activity resides in the 60 C-terminal amino acids, a basic region of GCN4. However, certain deletions containing the entire DNA binding region are unable to activate transcription and instead act as repressors in vivo. The activation function appears to critically involve just 19 amino acids that are centrally located in an acidic region of GCN4. In addition to their functional separation, the DNA binding and transcriptional activation regions of the protein can be separated physically by elastase cleavage. The implications of these results for the mechanisms of DNA sequence recognition and transcription activation are discussed.

GCN4 protein, synthesize in vitro, binds HIS3 regulatory sequences: Implications for general control of amino acid biosynthetic genes in yeast

The yeast GCN4 gene product is necessary for the transcriptional induction of many amino acid biosynthetic genes in response to conditions of amino acid starvation. We synthesized radioactively pure GCN4 protein by in vitro translation of mRNA produced by in vitro transcription with SP6 RNA polymerase. GCN4 protein binds specifically to the 20 bp region of the HIS3 gene that is critical for transcriptional regulation in vivo and contains the TGACTC sequence common to coregulated genes. A synthetic GCN4 mutant protein lacking the 40 C-terminal amino acids fails to bind DNA; this correlates with a gcn4 mutant gene that is nonfunctional in vivo. Finally, GCN4 protein binds to the promoter regions of coordinately regulated genes, but not to analogous regions of other genes. We suggest that GCN4 protein is a specific transcription factor, and we describe a molecular model for the general control of amino acid biosynthetic genes.

GCN4, a eukaryotic transcriptional activator protein, binds as a dimer to target DNA.

The eukaryotic transcriptional activator protein, GCN4, synthesized in vitro from the cloned gene, binds specifically to the promoters of yeast amino acid biosynthetic genes. Previous analysis of truncated GCN4 derivatives localized the DNA binding domain to the C‐terminal 60 amino acids and revealed that the size of the GCN4 derivative and the electrophoretic mobility of the protein‐DNA complex were inversely related. This observation was utilized here to develop a novel method for determining the subunit structure of DNA binding proteins. A mixture of wild‐type GCN4 protein and a smaller GCN4 derivative generated three complexes with DNA, two corresponding to those observed when the proteins are present individually and one new complex of intermediate mobility. This extra complex results from the heterodimer of the two GCN4 proteins of different sizes, demonstrating that GCN4 binds DNA as a dimer. The contacts sufficient for dimerization were localized to the 60 C‐terminal amino acid, DNA binding domain, suggesting that dimerization of GCN4 is a critical aspect of specific DNA binding. Furthermore, stable GCN4 dimers were formed in the absence of target DNA. These observations suggest a structural model of GCN4 protein in which a dimer binds to overlapping and non‐identical half‐sites, explaining why GCN4 recognition sites act bidirectionally in stimulating transcription.

A compendium of Caenorhabditis elegans regulatory transcription factors: a resource for mapping transcription regulatory networks

BackgroundTranscription 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.ResultsBy 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.ConclusionwTF2.0 provides a starting point to decipher the transcription regulatory networks that control metazoan development and function.

Insight into transcription factor gene duplication from Caenorhabditis elegans Promoterome-driven expression patterns

BackgroundThe 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.ResultsTransgenic 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.ConclusionExamining 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.

A simplified counter-selection recombineering protocol for creating fluorescent protein reporter constructs directly from C. elegans fosmid genomic clones

BackgroundRecombineering is a genetic engineering tool that enables facile modification of large episomal clones, e.g. BACs, fosmids. We have previously adapted this technology to generate, directly from fosmid-based genomic clones, fusion gene reporter constructs designed to investigate gene expression patterns in C. elegans. In our adaptation a rpsL-tet(A) positive/negative-selection cassette (RT-cassette) is first inserted and then, under negative selection, seamlessly replaced with the desired sequence. We report here on the generation and application of a resource comprising two sets of constructs designed to facilitate this particular recombineering approach.ResultsTwo complementary sets of constructs were generated. The first contains different fluorescent protein reporter coding sequences and derivatives while the second set of constructs, based in the copy-number inducible vector pCC1Fos, provide a resource designed to simplify RT-cassette-based recombineering. These latter constructs are used in pairs the first member of which provides a template for PCR-amplification of an RT-cassette while the second provides, as an excised restriction fragment, the desired fluorescent protein reporter sequence. As the RT-cassette is flanked by approximately 200 bp from the ends of the reporter sequence the subsequent negative selection replacement step is highly efficient. Furthermore, use of a restriction fragment minimizes artefacts negating the need for final clone sequencing. Utilizing this resource we generated single-, double- and triple-tagged fosmid-based reporters to investigate expression patterns of three C. elegans genes located on a single genomic clone.ConclusionsWe describe the generation and application of a resource designed to facilitate counter-selection recombineering of fosmid-based C. elegans genomic clones. By choosing the appropriate pair of ‘insertion’ and ‘replacement’ constructs recombineered products, devoid of artefacts, are generated at high efficiency. Gene expression patterns for three genes located on the same genomic clone were investigated via a set of fosmid-based reporter constructs generated with the modified protocol.

Forward locomotion of the nematode C. elegans is achieved through modulation of a single gait

The ability of an animal to locomote through its environment depends crucially on the interplay between its active endogenous control and the physics of its interactions with the environment. The nematode worm Caenorhabditis elegans serves as an ideal model system for studying the respective roles of neural control and biomechanics, as well as the interaction between them. With only 302 neurons in a hard‐wired neural circuit, the worms apparent anatomical simplicity belies its behavioural complexity. Indeed, C. elegans exhibits a rich repertoire of complex behaviors, the majority of which are mediated by its adaptive undulatory locomotion. The conventional wisdom is that two kinematically distinct C. elegans locomotion behaviors—swimming in liquids and crawling on dense gel‐like media—correspond to distinct locomotory gaits. Here we analyze the worms motion through a series of different media and reveal a smooth transition from swimming to crawling, marked by a linear relationship between key locomotion metrics. These results point to a single locomotory gait, governed by the same underlying control mechanism. We further show that environmental forces play only a small role in determining the shape of the worm, placing conditions on the minimal pattern of internal forces driving locomotion.

Gene expression markers for Caenorhabditis elegans vulval cells

The analysis of cell fate patterning during the vulval development of Caenorhabditis elegans has relied mostly on the direct observation of cell divisions and cell movements (cell lineage analysis). However, reconstruction of the developing vulva from EM serial sections has suggested seven different cell types (vulA, vulB1, vulB2, vulC, vulD, vulE, and vulF), many of which cannot be distinguished based on such observations. Here we report the vulval expression of seven genes, egl-17, cdh-3, ceh-2, zmp-1, B0034.1, T04B2.6 and F47B8.6 based on gfp, cfp and yfp (green fluorescent protein and color variants) reporter fusions. Each gene expresses in a specific subset of vulval cells, and is therefore useful as a marker for vulval cell fates. Together, expressions of markers distinguish six cell types, and reveal a strict temporal control of gene expression in the developing vulva.

DamID in C. elegans reveals longevity-associated targets of DAF-16/FoxO

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.

Caenorhabditis elegans reporter fusion genes generated by seamless modification of large genomic DNA clones

By determining spatial-temporal expression patterns, reporter constructs provide significant insights into gene function. Although additionally providing information on subcellular distribution, translational reporters, where the reporter is fused to the gene coding sequence, are used less frequently than simpler constructs containing only putative promoter sequences. Because these latter constructs may not contain all necessary regulatory elements, resulting expression patterns must be interpreted cautiously. To ensure inclusion of all such elements and provide details of subcellular localization, construction of translational reporters would, preferably, utilize genomic clones, containing the complete locus plus flanking regions and permit seamless insertion of the reporter anywhere within the gene. We have developed such a method based upon λ Red-mediated recombineering coupled to a robust two-step counter-selection protocol. We have inserted either gfp or cfp precisely at the C-termini of three Caenorhabditis elegans target genes, each located within different fosmid clones, and examined previously with conventional reporter approaches. Resulting transgenic lines revealed reporter expression consistent with previously published data for the tagged genes and also provided additional information including subcellular distributions. This simple and straightforward method generates reporters highly likely to recapitulate endogenous gene expression and thus represents an important addition to the functional genomics toolbox.