Martinez NJ et al. (2008) Genome Res "Genome-scale spatiotemporal analysis of Caenorhabditis elegans microRNA promoter activity."

Walhout AJ, Ambros VR, Barrasa MI, Reece-Hoyes JS, Martinez NJ, Ow MC

[Genome Res, 2008]

The Caenorhabditis elegans 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.

Martinez NJ et al. (2008) Genes Dev "A C. elegans genome-scale microRNA network contains composite feedback motifs with high flux capacity."

Ow MC, Walhout AJ, Sequerra R, Doucette-Stamm L, Ambros VR, Roth FP, Barrasa MI, Hammell M, Martinez NJ

[Genes Dev, 2008]

MicroRNAs (miRNAs) and transcription factors (TFs) are primary metazoan gene regulators. Whereas much attention has focused on finding the targets of both miRNAs and TFs, the transcriptional networks that regulate miRNA expression remain largely unexplored. Here, we present the first genome-scale Caenorhabditis elegans miRNA regulatory network that contains experimentally mapped transcriptional TF --> miRNA interactions, as well as computationally predicted post-transcriptional miRNA --> TF interactions. We find that this integrated miRNA network contains 23 miRNA <--> TF composite feedback loops in which a TF that controls a miRNA is itself regulated by that same miRNA. By rigorous network randomizations, we show that such loops occur more frequently than expected by chance and, hence, constitute a genuine network motif. Interestingly, miRNAs and TFs in such loops are heavily regulated and regulate many targets. This "high flux capacity" suggests that loops provide a mechanism of high information flow for the coordinate and adaptable control of miRNA and TF target regulons.

Musset-Bilal F et al. (1994) Worm Breeder's Gazette "Characterization of the C. elegans Basement Membrane-Associated Protein SPARC"

Musset-Bilal F, Schwarzbauer JE, Ryan CS

[Worm Breeder's Gazette, 1994]

Characterization of the C. elegans Basement Membrane- Associated Frederique Musset-Bilal, Carol S. Ryan, and Jean E. Schwarzbauer Department of Molecular Biology, Princeton University, Princeton NJ 08544

Townsend SR et al. (1994) Worm Breeder's Gazette "C. elegans Molecular Genetics and Long PCR."

Finelli AL, Savage C, Xie T, Padgett RW, Townsend SR

[Worm Breeder's Gazette, 1994]

C. elegans Molecular Genetics and Long PCR Scott R. Townsend, Cathy Savage, Alyce L. Finelli, Ting Xie, and Richard W. Padgett, Waksman Institute and Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08855

Wilkinson HA et al. (1994) Worm Breeder's Gazette "Reciprocal Changes in Expression of lin-12 and lag-2 Prior to Commitment of Z1.ppp and Z4.aaa."

Greenwald IS, Wilkinson HA, Fitzgerald K

[Worm Breeder's Gazette, 1994]

Reciprocal changes in expression of lin-12 and lag-2 prior to commitment of Z1.ppp and Z4.aaa. Hilary A. Wilkinson*, Kevin Fitzgerald and Iva Greenwald, HHMI and Dept. of Biochemistry and Molecular Biophysics, Columbia University College of Physicians and Surgeons, New York, NY 10032; *Current address: Merck Research Laboratories, Rahway, NJ 07065

Ishii N et al. (1988) Worm Breeder's Gazette "partial sequence of the unc-6 gene."

Ishii N, Stern BD, Culotti JG, Hedgecock EM

[Worm Breeder's Gazette, 1988]

In the previous issue (WBG 10(1) p.35), we reported cloning a small DNA fragment containing the Tc1 insertion which caused the unc-6 ( rh1035) mutation. Since then, we have isolated an 11.5 kb EcoRI fragment including this region from N2 DNA (NJ#14, EMBL4 vector) Alan Coulson has kindly positioned this fragment on the MRC genome map. We have now sequenced both the original 1.25 kb XbaI fragment of unc-6( rh1035) DNA (plasmid NJ#6) and a 2.7 kb HindIII fragment of N2 DNA subcloned from phage NJ#14. The 2.7 kb HindIII fragment has five exons which encode a hypothetical protein fragment of 360 residues. This fragment is not closely related to any protein in the current PIR database The last exon in the 2.7 kb HindIII fragment is followed by an intron which continues beyond the fragment. The first exon is preceded by a non-coding region which may be part of an intron or the unc-6 promoter region. In fact, a possible 21 residue signal sequence with an initiator methionine occurs in the first exon shortly upstream of the Tc1::rh1035 insertion site. TATA and CCAAT motifs occur about 90 and 120 bp upstream of the proposed initiator codon. The decanucleotide sequence TTTGCCTCTC is tandemly repeated about 200 bp upstream of this codon. [See Figure 1]

Toth, Marton L. et al. (2009) International Worm Meeting "HSF-1 is an Essential Downstream Mediator of Resveratrol-induced, SIR-2.1-dependent Longevity in C. elegans."

Soti, Csaba, Toth, Marton L., Csermely, Peter, Dancso, Balazs

[International Worm Meeting, 2009]

Major longevity assurance mechanisms include the heat-shock response governed by the heat-shock transcription factor (HSF-1) and the metabolic stress response, exemplified by the Sir2 deacetylase. Sir2, and recently HSF-1 have been implicated in the life-span extending effect of dietary restriction. Resveratrol, a polyphenol from red wine, is a dietary restriction mimetic that induces longevity and displays an impressive therapeutic potential against various degenerative diseases via Sir2. Resveratrol has also been shown to induce the heat-shock response in mammalian cells. In this study, we have asked how the heat-shock response is involved in resveratrol-induced/Sir2-related longevity in C. elegans. We find that resveratrol specifically induces chaperone expression, thermotolerance and longevity depending on both SIR-2.1 and HSF-1. Moreover, we show that SIR-2.1 is a potent activator of HSF-1-dependent thermotolerance. Finally, we identify HSF-1 as an essential downstream effector of resveratrol-induced, SIR-2.1-dependent longevity in C. elegans. Thus, our results demonstrate a direct interaction of metabolic and proteotoxic stress responses in life-span regulation and indicate that the robustness of the heat-shock response may determine the therapeutic response to resveratrol. Marton Toth''s current address: Rutgers, The State University of NJ, Molecular Biology & Biochemistry, Piscataway, NJ.

McDiarmid, Troy A et al. (2020) MicroPubl Biol

Kepler, Lexis D, McDiarmid, Troy A, Rankin, Catharine H

[MicroPubl Biol, 2020]

The Auxin-Inducible Degradation (AID) system is a powerful technique in the C. elegans toolkit that enables conditional and reversible protein depletion with high temporal and spatial specificity (Zhang et al. 2015; Martinez et al. 2020; Ashley et al. 2020; Martinez and Matus 2020). This system relies on tagging a gene of interest with a short AID degron sequence and transgenic expression of TIR1, an inducible E3 ubiquitin ligase normally found only in plants (Nishimura et al. 2009; Zhang et al. 2015). Upon exposure to the plant-derived hormone Auxin, TIR1 is activated and targets AID-tagged proteins for proteasomal degradation (Nishimura et al. 2009; Zhang et al. 2015) (Figure 1A). While there are qualitative reports that Auxin does not overtly affect the morphology or behavior of wild-type C. elegans (Zhang et al. 2015), this has not been quantitatively assessed. Determining whether Auxin significantly affects C. elegans morphology and behavior, even in subtle ways, is important given the C. elegans communitys rapid uptake of the AID system (Kasimatis et al. 2018; Nance and Frkjr-Jensen 2019; Ashley et al. 2020; McDiarmid et al. 2020). Here, we use our high-throughput machine vision tracking system, the Multi-Worm Tracker (MWT) (Swierczek et al. 2011), to investigate whether exposure to Auxin affects a suite of morphological, locomotor, mechanosensory, and short-term habituation learning phenotypes in our labs derivate of Bristol N2 wild-type worms and the CGC wild-type reference strain, PD1074 (Yoshimura et al. 2019) (Figure 1B).

Ow MC et al. (2008) Genes Dev "The FLYWCH transcription factors FLH-1, FLH-2, and FLH-3 repress embryonic expression of microRNA genes in C. elegans."

Martinez NJ, Silverman HS, Ow MC, Barrasa MI, Conradt B, Walhout AJ, Olsen PH, Ambros V

[Genes Dev, 2008]

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression post-transcriptionally via antisense base-pairing. Although miRNAs are involved in a variety of important biological functions, little is known about their transcriptional regulation. Using yeast one-hybrid assays, we identified transcription factors with a FLYWCH Zn-finger DNA-binding domain that bind to the promoters of several Caenorhabditis elegans miRNA genes. The products of the flh-1 and flh-2 genes function redundantly to repress embryonic expression of lin-4, mir-48, and mir-241, miRNA genes that are normally expressed only post-embryonically. Although single mutations in either flh-1 or flh-2 genes result in a viable phenotype, double mutation of flh-1 and flh-2 results in early larval lethality and an enhanced derepression of their target miRNAs in embryos. Double mutations in flh-2 and a third FLYWCH Zn-finger-containing transcription factor, flh-3, also result in enhanced precocious expression of target miRNAs. Mutations of lin-4 or mir-48&mir-241 do not rescue the lethal flh-1; flh-2 double-mutant phenotype, suggesting that the inviability is not solely the result of precocious expression of these miRNAs. Therefore, the FLH-1 and FLH-2 proteins likely play a more general role in regulating gene expression in embryos.

Chen, Jian et al. (2020) MicroPubl Biol

Chen, Jian, Mohammad, Ariz, Schedl, Tim

[MicroPubl Biol, 2020]

The auxin inducible degradation (AID) system was first introduced in C. elegans by Dernburg lab and has become a widely-used approach to study tissue-specific and/or temporal aspects of gene function (Zhang et al. 2015; Ashley et al. 2020; Martinez et al. 2020). The AID system utilized a plant derived E3 ubiquitin ligase, TIR1, to specifically degrade proteins fused with the degron tag following treatment with the plant growth hormone auxin. To increase the utility of the AID system, the Ward laboratory recently generated an expanded set of TIR1s transgenes, controlled by different tissue-specific promoters (Ashley et al. 2020). Here we aim to compare different germline-expressed TIR1 transgenes for their efficiency in degrading the transcription factor LAG-1, which is C-terminally tagged with degron (Chen et al., 2020). As indicated in Figure 1, these TIR1 transgenes are driven by the following promoters: gld-1p (Zhang et al. 2015), mex-5p (Ashley et al. 2020), sun-1p (Ashley et al. 2020), and pie-1p (Kasimatis et al. 2018), and contain the indicated C-terminal fluorescent proteins and 3 untranslated regions (Fig. 1A).