Delaney, Colin et al. (2015) International Worm Meeting "The histone H4K20 methyltransferase SET-4 promotes dauer arrest in C. elegans insulin signaling mutants."

Moerman, Donald, Hu, Patrick, Graniel, Jacqueline, Delaney, Colin, Dumas, Kathleen, Flibotte, Stephan

[International Worm Meeting, 2015]

Insulin and insulin-like growth factor signaling (IIS) is highly conserved across metazoans and plays pivotal roles in development and aging. In C. elegans, the DAF-2/IIS pathway influences dauer diapause, a state of larval developmental arrest that occurs in response to stressful environmental conditions. Mutants that have reduced DAF-2/IIS pathway activity enter dauer constitutively in a manner requiring the FOXO transcription factor DAF-16/FOXO. DAF-16/FOXO function is antagonized by DAF-2/IIS signaling through AKT-mediated phosphorylation, leading to its nuclear export. When AKT activity is reduced, DAF-16/FOXO translocates to the nucleus, where it is inhibited by EAK-7/TLDC1, a conserved protein of unknown function. While eak-7 and akt-1 single mutants do not have strong dauer-constitutive phenotypes, eak-7;akt-1 double mutants always arrest as dauers.We designed a forward genetic screen to discover novel regulators of DAF-16/FOXO by mutagenizing eak-7;akt-1 worms and looking for suppressors of eak-7;akt-1 dauer arrest (seak mutants). Mapping and whole genome sequencing revealed that one seak mutant strain contained a mutation in set-4, which encodes a conserved histone H4 lysine 20 (H4K20) methyltransferase. Three independent set-4 mutants suppressed dauer arrest in an eak-7;akt-1 background, indicating that SET-4 functions to promote dauer arrest. SET-4 promotes dauer specifically through the IIS pathway, as set-4 mutation did not alter arrest in other conserved dauer pathways. Consistent with a role in DAF-16/FoxO regulation, set-4 mutants are short-lived. Furthermore, we found that expressing SET-4 under the control of the neuron-specific rab-3 promoter rescued dauer in set-4 mutants to a similar extent as the set-4 native promoter. We hypothesize that SET-4 acts in neurons to control dauer arrest by promoting DAF-16/FOXO activity. As both SET-4 and DAF-16/FoxO are conserved in mammals, our findings hint at a general role for histone H4K20 methylation in the regulation of FOXO transcription factor activity.

Delaney, Colin E et al. (2021) International Worm Meeting "Concentrates of histone methyltransferase MET-2 promotes gene silencing independent of its H3K9 methyltransferase catalytic activity"

Padeken, Jan, Delaney, Colin E, Kalck, Veronique, Gasser, Susan, Methot, Stephen P

[International Worm Meeting, 2021]

Segregation of genomic regions into accessible euchromatin and inaccessible heterochromatin is essential for temporal and tissue-specific gene transcription. In C. elegans, the SETDB1 homolog MET-2 promotes heterochromatic silencing of satellite repeats, transposable elements, and tissue-specific genes by di-methylating histone H3 lysine 9 (H3K9me2). Animals lacking met-2 are viable but show defects a loss of fertility, developmental delay, and are short-lived. We previously demonstrated that MET-2's ability to preserve heterochromatin repression requires concentration in nuclear foci through physical interaction with the intrinsically disordered protein LIN-65. Here we show that MET-2 foci have a second, non-catalytic function that contributes to gene repression. Genetic ablation of met-2 or dispersion of MET-2 foci with heat stress results in loss of silencing coincident with an increase in histone acetylation. Restoration of MET-2 foci deficient in methyltransferase activity prevents H3K9 and H3K27 hyperacetylation and mitigates gene derepression, infertility, and developmental delay. These results demonstrate that foci of a conserved heterochromatin histone methyltransferase are functionally relevant for gene silencing and can act in parallel with enzymatic activity.

Dumas, Kathleen et al. (2013) International Worm Meeting "Post-embryonic control of DAF-2 insulin-like signaling by the conserved dosage compensation protein DPY-21."

Hu, Patrick, Csankovszki, Gyorgyi, Flibotte, Stephane, Moerman, Donald, Delaney, Colin, Dumas, Kathleen

[International Worm Meeting, 2013]

During embryogenesis, an essential process known as dosage compensation is initiated to equalize gene expression from sex chromosomes. Although much is known about how dosage compensation is established, the consequences of modulating the stability of dosage compensation post-embryonically are not known. Here we define a role for the C. elegans dosage compensation complex (DCC) in the regulation of DAF-2 insulin-like signaling. In a screen for dauer regulatory genes that control the activity of the FoxO transcription factor DAF-16, we isolated three mutant alleles of dpy-21, which encodes a conserved DCC component. Mutations in dpy-21, dpy-28, and sdc-2, as well as RNAi depletion of other DCC components, suppressed dauer arrest, implicating the DCC in dauer regulation. In dpy-21 mutants, expression of several X-linked genes that promote dauer bypass is elevated, including four genes encoding components of the DAF-2 insulin-like pathway that antagonize DAF-16/FoxO activity. Accordingly, dpy-21 mutation reduced the expression of DAF-16/FoxO target genes by promoting the exclusion of DAF-16/FoxO from nuclei. Thus, dosage compensation enhances dauer arrest by repressing X-linked genes that promote reproductive development through the inhibition of DAF-16/FoxO nuclear translocation. This work is the first to establish a specific post-embryonic function for dosage compensation. The influence of dosage compensation on dauer arrest, a larval developmental fate governed by the integration of multiple environmental inputs and signaling outputs, suggests that the dosage compensation machinery may respond to external cues by modulating signaling pathways through chromosome-wide regulation of gene expression.

EK Round et al. (1999) International C. elegans Meeting "Cloning and characterization of aex-2 and aex-4, two genes required for defecation"

EK Round, JH Thomas

[International C. elegans Meeting, 1999]

C. elegans follows a stereotyped series of muscle contractions during defecation: first a posterior body-muscle contraction (pBoc), followed by an anterior body-muscle contraction (aBoc) and finally contraction of the enteric muscles which results in expulsion of the gut contents (Exp). In the presence of food, this defecation motor program occurs every 45 seconds. The neurotransmitter GABA specifically activates the expulsion since mutants lacking GABA have enteric muscle contractions in only 15% of defecation cycles and have no defect in aBoc or pBoc. In contrast, mutations in seven defined genes cause defects in both the aBoc and Exp steps of the defecation motor program (Aex phenotype)(1). Colin Thacker and Ann Rose have shown that aex-5 is a proprotein convertase in the kexin/subtilisin family(2), suggesting a role for peptide signaling in the process of defecation. With the hope of identifying molecules that act in the same pathway as aex-5 , we are mapping and cloning two other Aex genes. From a large number of recombinants, we obtained a precise map position for aex-2 between the anchor genes unc-110 and unc-115 on the X chromosome. These data were used to choose cosmids for transgenic rescue experiments. We have rescued the aex-2 phenotype with a pool of two cosmids and are currently working to identify the exact gene responsible for this rescue. We have mapped aex-4 between unc-78 and lin-18 on the X chromosome and are currently mapping aex-4 relative to polymorphisms in this region. 1. J.H. Thomas, 1990. Genetics 124 : 855-872. 2. C. Thacker and A.M. Rose, West Coast Worm Meeting Abstract 1998, #29.

Lynn Boyd (2001) International C. elegans Meeting "A simple lab using single worm PCR and dpy-5"

Lynn Boyd

[International C. elegans Meeting, 2001]

A simple lab has been designed for use in a sophomore genetics course. There are four primary objectives for this laboratory exercise: 1) students predict genotypes based on phenotype 2) students design primers for PCR 3) students use the PCR technique 4) students analyze results using gel electrophoresis The learning elements of the lab can be broken down into two main areas. First, it serves as a demonstration of the relationship between genotype and phenotype. Students observe the phenotype of worms and then make predictions about the genotype which they subsequently test. A second valuable learning element has been having the students design primers for PCR. Students must have a good understanding of both DNA structure and DNA polymerase activity to design primers successfully. This laboratory exercise makes use of a dpy-5 mutation that contains a deletion of 1009 bp (gracious thanks to Colin Thacker and Ann Rose for supplying the strain).. The mutation (e907) is semidominant. Therefore, students can identify all three genotypes in a population of worms containing the wild type and e907 alleles (+/+; +/e907; e907/e907). The investigation occurs over three lab periods. In the first lab, students become familiar with observing the Dpy phenotype and with picking worms. They also design primer pairs that will amplify both the wild type and e907 alleles. In the second lab, students use these primer pairs in single worm PCR reactions. They are asked to pick worms that represent each of the three genotypes and use these for PCR. In the third period, an agarose gel is run of the PCR reactions. Students determine the genotypes of the worms they chose and find out if their genotype predictions were correct. I have found that sophomore students are able to do this lab with a high percentage of success. If you would like more information or would like the lab handout, please contact Lynn Boyd at boydl@uah.edu.

Colin T. Dolphin et al. (2006) European Worm Meeting "C. elegans Reporter Fusion Genes Generated by Seamless Modification of Large Genomic DNA Clones"

Ian A. Hope, Colin T. Dolphin

[European Worm Meeting, 2006]

Colin T. Dolphin1 & Ian A. Hope2. By determining spatial-temporal expression patterns, reporter constructs can provide significant insight into the function of C. elegans genes. An improved reporter gene fusion would contain significant stretches of the 5 and 3 sequence flanking the protein-coding region to maximize the chances of including all associated regulatory elements such that the resulting expression pattern would be highly likely to recapitulate that of the endogenous gene. Furthermore, it would also be desirable, for various reasons, to be able to insert the reporter sequence, seamlessly and in-frame, at any specific position within the target gene. Lastly, the construction method should not be overly complicated and be potentially adaptable for high-throughput construct generation. We have developed such a method.. Reporter genes were inserted precisely, by homologous recombination in E.coli, into C. elegans genes cloned in fosmids. The homologous recombination, commonly referred to as recombineering, is mediated by the bacteriophage lambda Red system. This system is coupled with a simple and robust two-step counter-selection protocol; first a cassette with both positive and negative selectable marker genes is inserted at the target location and the cassette is then replaced with the reporter gene. This procedure allows straightforward, flexible and precise construction of translational reporter gene fusions directly from C. elegans fosmid clones with minimal alterations to the target gene.. We have used the approach to insert either gfp or cfp precisely at the C-termini of three C. elegans target genes, each located centrally within the approx. 35-40kb inserts of different fosmid clones and examined previously using conventional reporter approaches. Recombineered fosmids were used in transformation of C. elegans by microinjection. 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 reporter gene fusions highly likely to recapitulate endogenous gene expression and thus represents an important addition to the functional genomics toolbox. Its flexibility means the procedure will have many applications.

Ozdemir, Isa et al. (2021) International Worm Meeting "Regulation of transgenerational epigenetic H3K27me3 inheritance"

Steiner, Florian, Delaney, Kamila, Wenda, Joanna, Ozdemir, Isa

[International Worm Meeting, 2021]

A crucial process in life is the ability of cells to pass on information to their descendants. This transmission can occur at several levels from genetics to epigenetics. Epigenetic inheritance refers to the heritable phenotypes affected by gene expression without altering the DNA sequence and occurs through various factors including histone modifications. Interruption of this transmission process can lead to severe developmental defects and fertility diseases. Methylation of histone H3 at position 27 (H3K27me3) is an epigenetic mark that is associated with heterochromatin and repressed gene expression. Several studies have demonstrated that the genomic patterns of H3K27me3 can be inherited from one generation to the next by depositing H3K27me3 histones and PRC21. However, the limits of this inheritance are difficult to test because PRC2 mutants are maternal-effect sterile in C. elegans. We have recently shown that the expression of an H3.3K27M mutant acts in a dominant-negative manner and alters genomic H3K27me3 patterns and distribution of PRC2 and results in infertility phenotypes in C. elegans2. We used this strain to follow the trans-generational inheritance of the H3.3K27M-induced phenotypes. Strikingly, we observed that the altered patterning of H3K27me3 and the fertility defects are heritable for multiple generations after the H3.3K27M mutation is lost. These findings were also validated in a tetracycline-inducible system where we can abruptly switch on and off the H3.3K27M expression. We performed a targeted RNAi screen in our tetracycline-inducible system and detected several enhancer and suppressor modifiers of H3.3K27M defects such as chromatin remodeling complexes, the nuclear RNAi pathway, and histone writer and reader complexes. Overall, our results revealed that the transmission of H3K27me3 is an epigenetically programmed event that can last for generations and that several biological pathways seem to be involved in the regulation of transmission of H3K27me3 patterns across generations. 1. L. J. Gaydos, W. Wang, S. Strome. H3K27me and PRC2 transmit a memory of repression across generations and during development. Science 345, 1515-1518 American Association for the Advancement of Science (AAAS), 2014. 2. Kamila Delaney, Maude Strobino, Joanna M. Wenda, Andrzej Pankowski, Florian A. Steiner. H3.3K27M-induced chromatin changes drive ectopic replication through misregulation of the JNK pathway in C. elegans. Nature Communications 10 Springer Science and Business Media LLC, 2019.

Tsai, Hsin-Yue et al. (2009) International Worm Meeting "RDE-8 encodes a novel protein with a conserved domain required for RNAi."

Yates III, John R., Gu, Weifeng, Mello, Craig, Moresco, James J., Chen, Chun-Chieh G., Tsai, Hsin-Yue

[International Worm Meeting, 2009]

Several classes of small RNAs have been identified in mammals, zebrafish, Drosophila, and the nematode C. elegans. These include Dicer-dependent products (primary siRNAs and miRNAs) as well as secondary siRNAs produced by RNA-dependent RNA polymerase (RdRP). We have recently identified a new RNAi pathway component, RDE-8, which is essential for the accumulation of RdRP-derived small RNAs in both the exo- and endo- RNAi pathways. RDE-8 contains a novel protein motif that is highly conserved throughout all three kingdoms of life. Interestingly, in other organisms the majority of proteins containing the RDE-8 motif also contain RNA binding domains, most often of the CCCH zinc finger or KH domain families. Although RDE-8 itself is not highly conserved, one C. elegans RDE-8 family member (C30F12.1) has a human homolog in which both the amino acid composition and organization of its CCCH and RDE-8 motifs are well conserved. A third C. elegans RDE-8-motif protein also contains a Cytidine deaminase motif and is required for male fertility (See abstract by Colin Conine). These findings suggest that proteins containing the RDE-8 motif function in RNA binding and modification. To explore the role of RDE-8 in small-RNA pathways, we have utilized mass-spectrometry-based proteomics to identify protein interactors, and small-RNA deep sequencing to identify endogenous small RNAs whose biogenesis/stability depend on RDE-8. Our data suggests that RDE-8 is required for several distinct small-RNA pathways and resides in at least two protein complexes. RDE-8 interacts with the sap-domain exonuclease ERI-1b, and is required for the accumulation of ERI-pathway small RNAs. Interestingly, RDE-8 not only co-purifies with ERI-1b, but is required for a ssRNA trimming activity associated with the native ERI-1b/RDE-8 complex. We speculate that this trimming activity is necessary for the processing of substrates (or products) of the RdRP RRF-3. RDE-8 appears to form a second complex with components of the RNAi and transposon silencing small RNA pathway, including RDE-3, MUT-15, and MUT-16. We have not yet identified specific biochemical activities associated with this complex. However, it may also be involved in recruiting RdRPs: in this case, RRF-1 and EGO-1, which function to amplify silencing signals in the exo- and endo-RNAi pathways. We are currently investigating the biochemical properties of the RDE-8 complexes, and are exploring the activity of the RDE-8 motif.

Round EK et al. (2000) European Worm Meeting "aex-2 encodes a possible neuropeptide receptor required for defecation"

Thomas JH, Round EK

[European Worm Meeting, 2000]

The defecation motor program (DMP) in C. elegansconsists of three sets of muscle contractions that are initiated every 45 to 50 seconds when the animal is feeding. The cycle is initiated by a posterior body-wall muscle contraction (pBoc), followed by an anterior body-wall muscle contraction (aBoc) and finally gut contents are expelled by contraction of the enteric muscles (Exp). A signal from the intestine likely activates the pBoc (1), but how the aBoc and Exp steps are activated is unclear. GABA has been implicated as an important transmitter in signaling to the enteric muscles, but there remains at least one unknown signaling pathway that is important for completion of the DMP (2). Mutations in Aex genes cause defects in both the aBoc and Exp and may be the key to understanding which molecules are involved in the coordinate activation of the last two steps of the DMP (3). Colin Thacker and Ann Rose have shown that aex-5 is a proprotein convertase in the kexin/subtilisin family (4), suggesting a role for peptidergic signaling in the execution of the aBoc and Exp steps of the DMP. With the hope of identifying molecules that act in the same pathway as aex-5, we have mapped and cloned aex-2. aex-2 animals never have an active Exp contraction and occasionally have an aBoc. We found that aex-2 encodes a putative G-protein-coupled receptor that has weak homology to neuropeptide receptors. Two alleles correspond to mutations in predicted exons of aex-2: sa21 is a putative null and causes a stop codon in the fourth tramsmembrane domain, and sa3is a change from an Arg to a Gln in the sixth transmembrane domain. A 4.5 kb aex-2promoter and first exon fusion to GFP shows expression in many cells, including the enteric muscles, NSM, and several other neurons in the head. The weak homology of aex-2to neuropeptide receptors and the fact that aex-5 encodes a proprotein convertase indicate that AEX-2 may be a receptor for a neuropeptide processed by AEX-5. Expression of aex-2 in the enteric muscles suggests an important role for AEX-2 in their activation, since aex-2 mutants have a stronger Exp defect than GABA deficient mutants. AEX-2 may activate enteric muscle contractions directly or may be required for enteric muscle activation by some other signal. Additionally, aex-2 has an aBoc defect not seen in GABA-defective mutants suggesting that AEX-2-mediated signaling is important for the coordination of the aBoc and Exp steps of the DMP. 1. Dal Santo, et al., 1999. Cell 98: 757-67. 2. McIntire et al., 1993a. Nature 364: 334-37. 3. J.H. Thomas, 1990. Genetics 124: 855-872. 4. C. Thacker and A.M. Rose, West Coast Worm Meeting Abstract 1998, #29.