Meyers, Stephany G. et al. (2009) International Worm Meeting "Regulating the transcription of a transcription factor."

Corsi, Ann K., Meyers, Stephany G.

[International Worm Meeting, 2009]

An important issue in developmental biology is understanding the temporospatial regulation of genes encoding transcription factors. The hlh-8 gene encodes a basic helix-loop-helix transcription factor called Twist that is involved in mesoderm development in C. elegans and other metazoa. Specifically, hlh-8 is expressed in the undifferentiated M lineage, 2 coelomocytes, sex muscles, enteric muscles and head mesodermal cell (hmc). In humans, misregulation of Twist is implicated in cancer metastasis and mutations cause an autosomal dominant craniosynostotic disorder. Studies in multiple organisms, including C. elegans, reveals that Twist also provides an interesting paradigm of transcriptional control, functioning as both a homo- and heterodimer and often in a dose-dependent manner. Consequently, understanding how hlh-8 expression is regulated is key to understanding its function in tissue-specific processes. Extensive analysis has revealed that the upstream promoter region of hlh-8 only controls expression in a subset of tissues where it functions; namely in the coelomocytes and undifferentiated M lineage. Thus, important cis-acting elements must exist elsewhere. We are exploring the large first intron of hlh-8 for additional regulatory elements using gfp reporter genes and a mutant, hlh-8 (tm726), with a large deletion (646 bp) in the first intron of the gene. In contrast to hlh-8 null mutants, the intron deletion allele displays a subset of phenotypes affecting only certain tissues, suggesting intron sequences are involved in regulating expression. Through an analysis of intron 1 sequence elements, we have found two elements that appear to control hlh-8 autoregulation, implicating Twist dosage effects that seem to be restricted to the differentiated tissues of the hmc, sex and enteric muscles. These results underscore the complexity underlying the regulation of a transcription factor with multiple modes of regulation that directly impact its function. Such complexity would be difficult to discern from high throughput, genome-wide studies.

Olsen PH et al. (1993) International C. elegans Meeting "Structure and expression of a Tc5-encoded transcript."

Olsen PH, Collins JJ

[International C. elegans Meeting, 1993]

Yi W et al. (1998) Midwest Worm Meeting "MAB-3 is a direct transcriptional repressor of vitellogenin transcription"

Zarkower D, Yi W

[Midwest Worm Meeting, 1998]

Nematodes, insects, and possibly mammals control sexual development using a class of transcription factors containing a DNA binding motif called a DM domain (see abstract by Raymond, et al.). We have compared the DNA binding properties of two DM domain proteins: the C. elegans male-promoting protein MAB-3 and the Drosophila sex determining protein DSX. MAB-3 has two DM domains, while DSX has one. Surprisingly, despite the different structures of the proteins, the preferred in vitro DNA binding sites are similar. We found potential MAB-3 binding sites in the vit-1, vit-2 and vit-6 promoters. The vit-2 and vit-6 sites are completely conserved between C. elegans and C. briggsae. A short (250 bp) region of the vit-2 promoter is sufficient for sex-, tissue- and stage-specific transcription of vit-2. We found that MAB-3 binds this promoter region in vitro. We tested the in vivo relevance of the MAB-3 DNA binding site by examining the role of MAB-3 in regulating vitellogenin transcription. Mutating the MAB-3 binding site in the vit-2 promoter eliminates sex-specific regulation without affecting tissue- or stage-specificity. This strongly suggests that MAB-3 is a direct translational repressor of vitellogenins in the male intestine. Strikingly, the site bound by MAB-3 in vit-2 and the sites bound by DSX in the Drosophila YP1 (yolk protein 1) promoter are very similar, despite the fact that the vitellogenins and the YPs themselves are unrelated at the protein sequence level. Thus it appears in this case that the regulators and their regulatory sites are much more highly conserved than the structural proteins they control. What regulates mab-3? mab-3 mRNA levels are controlled by tra-1, a zinc finger putative transcription factor. We find that the mab-3 promoter directs male-specific transcription in the intestine and in various male sensory neurons, and that two positively acting tissue-specific promoter elements are required for this expression. While mab-3 is transcriptionally regulated by tra-1, no TRA-1 DNA binding sites are required for this regulation, and thus it appears that regulation by tra-1 is indirect.

Chen, Ron et al. (2011) International Worm Meeting "Identification of transcription start sites and novel transcripts in C. elegans."

Chen, Ron, Down, Thomas, Ahringer, Julie

[International Worm Meeting, 2011]

Transcription start sites (TSSs) for most C. elegans genes are not known because the majority (>70 %) are trans-spliced. The 5' end of primary transcripts are spliced off and degraded, being replaced by a 22nt spliced-leader RNA. This lack of knowledge of transcription initiation sites has hampered research in defining promoters and mapping regulatory elements in C. elegans. In theory, mRNAs that have initiated but not elongated fully should not yet have undergone trans-splicing and so would retain the original 5' end with a methyl cap. To identify such nascent transcripts, we have isolated and sequenced 5' capped short nuclear RNAs (20-100 nt). We find that many of these sequences indeed mark transcription initiation sites for not only non-trans-spliced, but also trans-spliced transcripts. For trans-spliced genes, TSSs usually lie 30-150nt upstream of the trans-splice sites. Transcription initiation sites fall into three major classes: single prominent starts, major starts with a cluster of weaker starts, and clusters of weak starts. In addition, we detect shared sequence motifs around TSSs allowing an analysis of promoter architecture in C. elegans. Combining data from the identified TSSs and sequences from long capped RNA (>200 nt), we also discovered novel transcripts. The functions of these novel transcripts in gene expression regulation are being investigated. The identification of transcript start sites should facilitate analyses of genomic regulatory features in C. elegans.

Lancaster, B. et al. (2017) International Worm Meeting "How transcription factor literacy influences target gene transcription."

McGhee, J., Goszczynski, B., Lancaster, B.

[International Worm Meeting, 2017]

Transcriptional activation is a complex multifactorial process that centres on two fundamental properties of transcription factor proteins: sequence specific DNA binding and the subsequent recruitment of RNA polymerase to a target gene promoter. We have established an experimental system to investigate, inside the living animal, how transcription factor binding affinity at a promoter quantitatively influences transcriptional activity of a target gene. In C. elegans, the major intestinal transcription factor ELT-2 binds to a core TGATAA motif. The intestine-specific asp-1 protease gene is a direct target of ELT-2 and is controlled by two TGATAA sites. We have created two versions of asp-1 by introducing a novel KpnI restriction site at different locations in the coding region, thereby producing two reporters distinguishable by restriction digestion. The two reporters are combined into multicopy extrachromosomal transgenic arrays in the same animal, one reporter controlled by a wildtype promoter, the other by a promoter with altered ELT-2 binding sites. RNA isolation followed by RT-PCR, restriction digestion, and electrophoresis, allows the levels of both reporter transcripts to be measured simultaneously. Promoter swaps have confirmed that there is little reporter bias. We have measured relative affinities of ELT-2 for TGATAA variants in vitro by competitive band shifts, by both direct competition of two target sequences and by high-throughput competition of a library of target sequences (Spec-Seq). As an example, the in vitro ELT-2 binding affinity to the core sequence CATGATAATC is ten-fold lower than to ACTGATAAGA. When this low affinity sequence is introduced into both of two TGATAA sites of the asp-1 promoter, the level of asp-1 transcripts measured in vivo are reduced five-fold. Overall, the relation between transcription factor binding affinity at the promoter and mRNA production by the target gene appears to be roughly linear, with a plateau at the maximum transcriptional output. We have measured the production of nascent transcripts by nuclear run-on and confirm that the reduction in transcripts seen with variant promoters is the effect of decreased transcriptional initiation/elongation as opposed to mRNA processing and/or stability. We now aim to use this experimental system to investigate how chromatin factors might influence the relation between ELT-2 binding and target gene transcription.

Yana Miteva et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "The transcriptional response to osmotic stress overlaps with transcriptional responses to infection and is regulated by the GATA transcription factor elt-2"

Anne-Katrin Rohlfing, Todd Lamitina, Yana Miteva, Sridhar Hannenhalli

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

Proper cellular responses to osmotic dyshomeostasis are important for numerous physiological and pathophysiological processes. While many responses to osmotic disruption are regulated at the level of transcription, the global suite of genes regulated by osmotic stress in animals are poorly defined. Using the nematode C. elegans, we profiled gene expression changes following a time course of exposure to hypertonicity. 330 genes that exhibit robust and highly significant transcription changes were identified, with over 50 genes exhibiting significant changes within 15 minutes of exposure to hypertonicity. Osmotically regulated gene expression does not overlap with other types of stress induced gene expression (heat shock, hypoxia, ethanol, heavy metals, or xenobiotics) but does show a significant overlap with genes regulated by pathogen infection. We found that 48% (160/330) of osmotically regulated genes are also transcriptionally regulated in at least one model of infection. Like pathogen regulated genes, osmotically regulated genes are enriched for the GATA transcription factor binding motif and RNAi knockdown of the GATA factor elt-2 blocked osmotically induced expression of the glycerol biosynthetic enzyme gpdh-1 and decreased survival in worms exposed to hypertonic, but not isotonic, environments. However, inhibition of the nsy-1/sek-1/pmk-1 p38 MAP kinase signaling pathway that is required for induction of pathogen regulated gene expression did not effect osmotically regulated gene expression, suggesting that the signaling mechanisms controlling infection and osmotically regulated gene expression are molecularly distinct. Together, these data support a model in which the GATA factor elt-2 functions downstream from multiple signaling pathways as a co-factor for stress-inducible gene expression.

Vieux, Karl-Frederic et al. (2021) International Worm Meeting "Novel approaches to studying maternal transcript regulation in C. elegans"

McJunkin, Katherine, Vieux, Karl-Frederic

[International Worm Meeting, 2021]

In most model organisms of sexual reproduction, transcription is interrupted before the end of oogenesis, only to resume in the early embryo, after several rounds of mitosis. In the meantime, during the maternal-to-zygote transition (MZT), the fully differentiated oocyte must clear away maternal transcripts before the zygote becomes totipotent and autonomous. In the absence of transcription, post-transcriptional gene regulation is the dominant form of gene expression. RNA regulation is therefore critical for early embryogenesis during the MZT. Recent single molecule imaging methods have provided great insight into different aspects of RNA biology. One such method images nascent peptides on individual transcripts. Briefly, a reporter transcript is comprised of a coding sequence containing repeated SunTag motifs and a 3'UTR containing 24 tandem MS2-stem-loop hairpin structures. A GFP-tagged antibody fragment that binds to SunTag motifs is co-expressed, identifying sites of translation. An MS2 binding protein tagged with mCherry is also co-expressed, allowing for visualization of the transcript. The multiplexed nature of the SunTag and stem-loop motifs amplifies the corresponding signals, allowing for single-molecule resolution. Various features of translation can be dissected from the quantification of the signal intensities, the number of corresponding foci, and their localization. We are adapting this approach to study post-transcriptional control of maternal transcripts in germlines and embryos during the MZT of C. elegans. First, we reduced the repetitive nature of the 24 SunTag cassette sequence while optimizing codons for expression in C. elegans. Next, this cassette was inserted upstream of an endogenous coding sequence of interest (nos-2), using a CRISPR approach. In these tagged strains, single molecule FISH and immunofluorescence will be used to label transcripts and nascent SunTag-containing peptides, respectively, to characterize translation across tissues and developmental stages. Combined, this system will provide new perspectives and tools to dissect the dynamics of maternal transcripts and deepen our understanding of RNA regulation in the germline and the early embryo in C. elegans.

Tang LZ et al. (1991) International C. elegans Meeting "THE UNC-44 SITE B TRANSCRIPT ENCODES A PUTATIVE ACIDIC PROTEIN."

Franco R, Otsuka AJ, Tang LZ

[International C. elegans Meeting, 1991]

Mutations in the unc-44 gene result in defective targeting of several axons to their appropriate partners [Hedgecock, E. M. et. al., Devel. Biol., 111, 158-170 (1985)]. The six characterized insertion mutations are found in two transcriptional regions, designated sites A and B [Otsuka, A. et al., WBG, 11(5) 39 (1991)]. Four alleles (mn259, mn339, rhlO42, and st200) are insertions at site B. Three independently isolated cDNA clones (approx. 3.4 kb) with similar restriction maps were obtained from the Barstead and Waterston cDNA clone bank. One of these, DD#PA013, has been sequenced. The 821-amino acid open reading frame present on DD#PA013 appears to extend beyond the cloned cDNA. The predicted protein product of DD#PA013 is acidic (pI = approx. 4). The central portion of the predicted protein (612 amino acid residues) lacks cysteine residues. The protein contains two copies of the sequence SL(Q/A)EFERLEKE. The Tcl insertion in the rhlO42 allele occurs between these repeats. A region of 9 related tandem repeats, including the two just noted, can be surmised from the sequence, although the similarity is very poor in some of the repeats. The limited number of cysteine residues, the hydrophilic character of the predicted protein, and the high predicted alpha-helical content suggest an acidic protein with an extended central domain. In searches of Genbank with the predicted DD#PA013 protein sequence, the intermediate filament proteins are selected, but the core domain of the intermediate filament proteins [Steinert, P. M. and Roop, D. R., Ann. Rev. Biochem., 57, 593-625 (1988)] is not obvious in the sequence of the predicted DD#PA013 product.

Craig, Hannah L. et al. (2009) International Worm Meeting "Investigation of alternative transcription factor gene transcripts in Caenorhabditis elegans by recombineering."

Craig, Hannah L., Wirtz, Julia, Hope, Ian A., Bamps, Sophie

[International Worm Meeting, 2009]

Of about 1000 potential transcription factor genes in the C. elegans genome, 126 are thought or confirmed to have alternative transcripts that would be translated to give different protein isoforms. These isoforms differ due to alternative initiation or termination of transcription, or alternative splicing. Whether these modifications in protein sequence reflect functional variation is not yet clear for the majority of transcription factor genes. To this end, recombineering has been employed to seamlessly tag individual isoforms of selected transcription factors with GFP to examine any differences in expression pattern. The forkhead transcription factor genes fkh-7 and fkh-9 both have 2 transcripts with alternative 5'' ends, based on EST data, and may therefore be transcribed under the control of alternative promoters. Examination of expression patterns generated from N- and C-terminal gfp-tagged fusions suggests that for both these genes, the downstream promoter is responsible for driving the majority of expression. Knock out of individual transcripts for the reporter tagged genes is underway. HLH-30 is reputedly a helix loop helix transcription factor and has an orthologue in the human genome. The hlh-30 gene encodes at least 4 alternative isoforms due to 2 or 3 alternative promoters and an optional internal exon. Previous experiments showed an hlh-30 promoter reporter fusion to be expressed in larvae and adults in many cells types. A C-terminal gfp fusion generated, by fosmid recombineering, to tag all HLH-30 isoforms, gave the same expression pattern. Tagging of transcripts specifically generated from the alternative promoters gave similar expression patterns. Interestingly, the fusion proteins encoded by recombineered fosmids were cytoplasmic rather than nuclear, suggesting that the function of HLH-30 may not be regulation of transcription. Previous experiments on the homeodomain transcription factor UNC-62 focused on its functions in development and locomotion, but its expression pattern is yet to be fully characterized. The unc-62 locus encodes at least 4 isoforms due to 2 alternative promoters and 2 alternate internal exons. The upstream promoter drives GFP expression in the developing vulva and uterus, several nerve cells in the posterior of larvae and in the VC neurons in late L3s. A recombineered fosmid reporter fusion, which should tag all UNC-62 isoforms at the C-terminus, drove visible GFP expression only in a single posterior nerve cell, identified as DVA, plus faint vulval expression. Further data from tagging of individual transcripts is being generated.

Lancaster, Brett et al. (2013) International Worm Meeting "A quantitative system to define the role of transcription factor binding affinity in transcriptional activation."

Lancaster, Brett, McGhee, James

[International Worm Meeting, 2013]

We are developing a quantitative experimental system to investigate, inside a living animal, how the affinity of a transcription factor for its cis-acting binding sites influences the rate of target gene transcription. In C. elegans, the zinc finger transcription factor ELT-2 binds to a core TGATAA DNA sequence to regulate the majority (possibly all) of the genes transcribed in the differentiating intestine. Our objective is to quantitatively measure the binding affinity of ELT-2 to a series of binding site variants (e.g. NTGATAAN), and, at the same time, measure the transcriptional output (mRNA) of the target gene with these variants in the promoter, all inside the living worm. The asp-1 gene encodes the major intestinal-specific aspartic protease of the worm, has two TGATAA sites in its 1kb promoter and is under direct ELT-2 control. We have constructed two versions of the asp-1 gene that differ only in the location of a KpnI site introduced by silent mutation. Reporter "A" is regulated by the wildtype asp-1 promoter; reporter "B" is regulated by a mutant reporter in which the wildtype CTGATAAG site has been replaced by, for example, an ATGATAAT site. Equimolar mixtures of the two constructs are introduced into worms (unc-119(-); asp-1(-); elt-4/7(-)) as multicopy transgenic arrays, which guarantees that each transgene, on average, will be exposed to the same cellular environment. RNA isolation followed by RT-PCR, KpnI digestion, and microfluidic gel electrophoresis (Agilent Bioanalyzer) yields distinct and easily quantitated profiles that allow us to estimate the levels of transcript produced by each reporter. Expression of a GFP reporter driven by the asp-1 1kb promoter requires both TGATAA sequences and I have quantitatively replicated this observation using this new system. In the future, binding affinity of purified ELT-2 protein to each binding site variant will be determined by fluorescence anisotropy. I will present our results on the reproducibility and dynamic range of the experimental system, together with possible limitations.