Das, Moushumi et al. (2021) International Worm Meeting "Condensin I organizes the C. elegans interphase genome"

Meister, Peter, Statzer, Cyril, Campos, Julie, Ewald, Collin, Semple, Jennifer, Das, Moushumi, Gitchev, Todor

[International Worm Meeting, 2021]

Recent data in mammalian cells have shown the role of cohesin, a Structural Maintenance of chromosomes (SMC) complex, in interphase genome organization into loops and domains. Since these complexes are involved in cell division, it is difficult to evaluate the functional consequences of their absence in animals. Here we use Caenorhabditis elegans, an animal, 90% of whose cells are post-mitotic at birth. Similarly to mammals, nematodes have three SMC complexes: cohesin, condensin I and condensin II. Additionally, condensin IDC, a variant of condensin I, is involved in dosage compensation (DC). To uncover which SMC complex(es) organize the C. elegans interphase genome and evaluate functional consequences of genome unfolding, we constructed strains in which individual SMC complexes - cohesin, condensin I/IDC and II - are acutely inactivated in vivo in fully differentiated animals. We then assessed their phenotype and carried out chromatin conformation capture (Hi-C) and RNA-seq. Our data shows that in contrast to mammalian cells, the major determinant of genome folding in C. elegans is condensin I/IDC and not cohesin. Its cleavage reduces short-range contact probabilities and causes genome-wide de-compaction on all chromosomes. In contrast, cohesin cleavage has marginal impact while condensin II cleavage has no consequence on genome folding. RNA-seq data show that about a third of expressed genes are significantly differently expressed upon cohesin and condensin II inactivation, however the effect sizes were very small, and a similar number of genes were up and down regulated. In contrast, cleavage of condensin I/IDC leads to up-regulation of 93% of all expressed X-linked genes versus only 1% down regulated, indicating the necessity of the constant presence of condensin IDC for maintenance of DC. Cleavage of cohesin and condensin II has little effect on animal post-embryonic survival, as their lifespan are similar to control animals. In contrast, inactivation of condensin I/IDC causes drastic reduction in life expectancy; yet additional experiments show that lifespan reduction is due to lack of DC rather than genome unfolding. Taken together, we discovered that condensin I is the main SMC complex folding the nematode interphase genome, yet genome unfolding has no major effect in C. elegans in standard laboratory conditions, apart from X-linked gene up-regulation and its consequences.

Yochem JJ et al. (1987) Worm Breeder's Gazette "sequence of the glutathione s-transferase P subunit of C elegans, or a step towards the complete sequence of the C. elegans genome."

Weston K, Yochem JJ, Greenwald IS

[Worm Breeder's Gazette, 1987]

In the process of completing the sequence of lin-12, we inadvertently obtained the sequence of the adjacent gene on the 3' side of lin-12. It is oriented in the opposite direction relative to lin-12. (We have data indicating that lin-12 runs 5' to 3' with respect to the genetic map.) The sequence presented below is a composite of genomic and cDNA sequence data; the cDNAs were obtained from a library kindly provided by Julie Ahringer and Judith Kimble. The predicted product is 40% identical to rat liver glutathione S- transferase P subunit (Suguoka et al., NAR 13: 6049, 1985); the degree of homology is higher if conservative substitutions are taken into account. Identical amino acids are boxed. The data indicate that there is a splice joining an exon (277-end) to a 5' exon(s). [See Figure 1]

Aurlie Blanc et al. (2005) International Worm Meeting "A French functional genomics platform."

Daniel Wong, Jerome Reboul, Jonathan Ewbank, Aurlie Blanc, Yohann Duverger

[International Worm Meeting, 2005]

For the last two years, we have offered a service of worm sorting based around the Union Biometrica COPAS platform. The COPAS machine is fully equipped with Zymark twister robot, allowing automated analysis of multiple 96 well plates, and the Profiler that generates 500 individual fluorescence measurements per worm. This equipment has been applied to a wide range of biological questions. They include quantifying the level of fluorescent reporter gene expression, large-scale RNAi and genetic screens and combinatorial library drug screening. Examples of each will be presented.This platform is now part of a fully integrated functional genomics facility open to the academic community (see http://www.ciml.univ-mrs.fr/EWBANK_jonathan/RIO.html). Other resources include the ORFeome (developed in Marc Vidals laboratory), and Julie Ahringers RNAi library, together with whole-genome microarrays. For the latter, through a collaboration with the Genome Sequencing Center at Washington, and the transcriptome platform at Nice, we have spotted the Illumina long oligo set onto glass slides and provide microarrays to the French C. elegans community.This French functional genomics platform has been made possible through funding from the National Genopole<sym02> network, Marseille-Nice genopole<sym02>, the CNRS and collaboration with Union Biometrica.

Yohann Duverger et al. (2007) International Worm Meeting "A French functional genomics platform."

Yohann Duverger, Jonathan Ewbank, Jerome Reboul, Sarah Scaglione, Daniel Wong

[International Worm Meeting, 2007]

For the last four years, we have offered a service of worm sorting based around the Union Biometrica COPAS platform. The COPAS machine is fully equipped with Zymark twister robot, allowing automated analysis of multiple 96 well plates, and the Profiler that generates up to 1000 individual fluorescence measurements per worm. This equipment has been applied to a wide range of biological questions. They include quantifying the level of fluorescent reporter gene expression, large-scale RNAi and genetic screens and combinatorial library drug screening. Examples of each will be presented. This platform is now part of a fully integrated functional genomics facility open to the academic community (see http://www.ciml.univ-mrs.fr/EWBANK_jonathan/RIO.html). Other resources include the ORFeome (developed in Marc Vidals laboratory), and Julie Ahringers RNAi library, together with whole-genome microarrays. For the latter, through a collaboration with the Genome Sequencing Center at Washington, and the transcriptome platform at Nice, we have spotted the Illumina long oligo set onto glass slides and provide microarrays to the French C. elegans community. This French functional genomics platform has been made possible through funding from the National Genopole&#174; network, Marseille-Nice genopole&#174;, INSERM, the CNRS and support from Union Biometrica.

Livingstone D (1989) Worm Breeder's Gazette "some more unc-31 sequence."

Livingstone D

[Worm Breeder's Gazette, 1989]

I am continuing the molecular work on the unc-31 gene started by Roger Hoskins. Roger isolated a 17.5kb genomic clone in lambda2001 which he showed was capable of transformation rescue of unc-31 mutants. He also partially sequenced a 4.5kb fragment contained within this clone which covers some of the polymorphisms associated with mutant alleles of the gene. [See Figure 1] I have made subclones of this phage in lambda2001 and have used them in an attempt to narrow down the position of the gene by transformation rescue. A phage containing the 13.5kb XhoI fragment seems to rescue although this result has still to be confirmed. No rescue has been obtained with phage containing the 13.5kb SacI fragment or the 10.5kb SacI-XhoI fragment. I am currently sequencing the 13.5kb rescuing fragment. Several open reading frames have been found which could be spliced together inframe. Some of these have been confirmed by sequencing partial cDNA clones isolated from Julie Ahringers mixed stage cDNA library. The gene covers more than 8kb of genomic DNA. The 5' end is near or beyond the SacI site and the 3' end is beyond the end of the 4.5kb HindIII fragment. No homology has been found between predicted amino acid sequences and protein sequences present in the PIR and Swissprot databases.

May Tran et al. (2005) International Worm Meeting "RNAi knockout of the mitochondrial carrier protein ucp-4: effects on metabolism"

May Tran, Craig LaMunyon, Aidyl Gonzalez-Serricchio

[International Worm Meeting, 2005]

We are investigating the phenotype of ucp-4 RNAi knockout worms. Mitochondrial uncoupling protein (UCP) function in humans has been linked to obesity. UCPs are mitochondrial carrier proteins, and human UCP-1, -2, and -3 are protonophores, transporting protons from the intermembrane space to the matrix, bypassing ATP synthase, and thereby raising resting metabolic rate in response to certain cues. The function of human UCP-4 is not clear, but it may be a mitochondrial fatty-acid transporter, and it is a homolog to the C. elegans ucp-4 (K07B1.3). Initial RNAi experiments by Dr. Julie Ahringers lab deemed knockout worms to be wild-type. However, we wondered if the RNAi phenotype might be a subtle alteration of metabolic activity. We obtained Dr. Ahringers RNAi feeding clone from the UK HGMP Resource Centre and measured egg laying and defecation rates. Compared to control worms fed HT115 bacteria containing the empty feeding vector, ucp-4 RNAi fed young adults that were exposed beginning as L1 larvae laid eggs at a significantly slower rate, although preliminary data on defecation rate suggests no difference between these treatments. If the trends in these data hold, it would suggest that ucp-4 RNAi knockout worms are processing food at the same rate, but converting that energy into offspring at a reduced rate.

Yohann Duverger et al. (2006) European Worm Meeting "A French Functional Genomics Platform"

Yohann Duverger, Jonathan Ewbank, Jerome Reboul, Daniel Wong

[European Worm Meeting, 2006]

Yohann Duverger1, Jrme Reboul2, Daniel Wong1 and Jonathan Ewbank1 For the last three years, we have offered a service of worm sorting based around the Union Biometrica COPAS platform. The COPAS machine is equipped with a Zymark twister robot, allowing automated analysis of multiple 96 well plates. We recently upgraded the machine to include the Profiler II that generates >1000 individual measurements per worm simultaneously for up to 4 channels (including 2 fluorescent). This equipment has been applied to a wide range of biological questions. They include quantifying the level of fluorescent reporter gene expression, large-scale RNAi and genetic screens and combinatorial library drug screening. Examples of each will be presented.. This platform is part of a fully integrated functional genomics facility open to the academic community (see http://www.ciml.univ-mrs.fr/EWBANK_jonathan /RIO.html). Other resources include the ORFeome (developed in Marc Vidals laboratory), and Julie Ahringers RNAi library, together with whole-genome microarrays. For the latter, through a collaboration with the Genome Sequencing Center at Washington, and the transcriptome platform at Nice, we have spotted the Illumina long oligo set onto glass slides and provide microarrays free of charge to the French C.elegans community and at cost price to academic researchers in Europe.. This French functional genomics platform has been made possible through funding from the National Genopole network, Marseille-Nice genopole, the CNRS and support from Union Biometrica.

Mingxue Cui et al. (2003) International Worm Meeting "Using RNAi to Identify New Genes Involved in C. elegans Vulval Differentiation"

Mingxue Cui, Min Han

[International Worm Meeting, 2003]

During vulval differentiation a signal from the gonad induces three of the six vulval precursor cells in the ventral epidermis to adopt vulval cell fates. A conserved RTK/Ras/MAPK signaling pathway acts in the precursor cells to transduce the signal. A number of genes that belong to class A or class B SynMuv genes negatively regulate vulval induction. A combination of mutations in both class A and class B genes, but not in either class, causes a Multivulva (Muv) phenotype. The lin-15 locus encodes two genes, lin-15a (a class A SynMuv gene) and lin-15b (a class B SynMuv gene). To identify new genes involved in regulating vulval differentiation, we have extended previous genetic screen for suppressors of the Muv phenotype of lin-15(lf) by carrying out a systematic RNAi screen. Using the chromosome I RNAi library described by Fraser et al. (2000)1, we searched for genes of which RNAi treatment can suppress the Muv phenotype of lin-15(n765ts). We have so far identified 15 genes through the screen. One of them is sur-2, a gene that is known for its function in vulval induction. 13 of these 15 genes have mammalian homolog. Most of them cannot effectively suppress the Muv phenotype of a stronger lin-15 allele, n309, neither can they suppress the Muv phenotype of let-60(n1046gf). It is possible that some of these genes represent a new class of information suppressor of the Muv phenotype of the lin-15(n765ts) allele. Currently, we are performing a number of biochemical and genetic experiments to understand the mechanism of the suppression. 1 Fraser, A.G., Kamath, R.S., Zipperlen, P., Martinez-Campos, M., Sohrmann, M., and Ahringer, J. Nature 408, 325-330 (2000).

Jolanta Polanowska et al. (2006) European Worm Meeting "Regulation of BRC-1 - Dependent Ubiquitylation at DNA Damage Sites"

Jolanta Polanowska, Simon Boulton, Tatiana Garcia-Muse, Julie Martin

[European Worm Meeting, 2006]

Jolanta Polanowska1,2, Julie Martin1, Tatiana Garcia-Muse1 and Simon Boulton1. The BRCA1 tumour suppressor and its heterodimeric partner BARD1 constitute an E3-ubiquitin (Ub) ligase and function in DNA repair by unknown mechanisms. We have previously described C. elegans BRCA1 and BARD1 orthologues (brc-1 and brd-1, respectively) that possess many of the functional domains present in their human counterparts, including RING, ankyrin, and BRCT domains (Boulton et al., 2004). Consistent with conserved roles in DNA repair, BRC-1 and BRD-1 interact to form a heterodimer via their respective RING domains. To explore the mechanistic role of BRC-1 and BRD-1 in DNA repair processes we have characterized a C. elegans BRC-1/BRD-1 complex (CeBCD) purified by tandem immunoaffinity before and at different time points after IR-treatment. This approach is a first for C. elegans and demonstrates that protein complexes purified in this manner are amenable to biochemical analysis and can be used in combination with genetics and cell biology to accelerate functional discoveries. We present evidence that the CeBCD complex possesses an E3-Ub ligase that is activated on chromatin in response to IR-treatment and further demonstrate that the DNA damage checkpoint promotes association of the CeBCD complex with E2-Ub conjugating enzyme, Ubc5(LET-70), to form an active E3-Ub ligase in response to DNA damage. We also show that ubiquitylation events at DNA damage sites require brc-1, brd-1, ubc5(let-70), mre-11 and atl-1, thus providing in vivo evidence to support our biochemical analysis.. Boulton, S.J., Martin, J.S., Polanowska, J., Hill, D.E., Gartner, A. and Vidal, M. (2004) Curr Biol, 14, 33-39.

P Askjaer et al. (2004) European Worm Meeting "IDENTIFICATION OF GENES REGULATING NUCLEAR ENVELOPE ASSEMBLY BY GENOME-WIDE RNAI"

V Galy, T Zimmermann, I Mattaj, M Gorjanacz, P Askjaer

[European Worm Meeting, 2004]

The nuclear envelope (NE) is essential for many cellular processes including regulation of gene expression, nuclear positioning and chromatin segregation during mitosis. In metazoans the NE disassembles when the cell enters mitosis and reassembles during anaphase or telophase to reestablish compartmentalization of the cell. Despite the importance of this process remarkably few genes have been identified, which are required for formation of a closed NE. One likely explanation is that overlapping and redundant pathways at least partially control NE formation: We and others (Liu et al., PNAS, 100:4598-4603) have found that while each of the two NE proteins emerin and MAN1 are dispensable in the worm co-depletion of the two proteins causes 100% embryonic lethality. To identify genes important for NE formation we have characterized an emerin deletion strain and use this strain in parallel with a control strain in a genome-wide RNAi screen to identify genes interacting with emerin. L1 larvae are incubated with RNAi-inducing bacteria from Julie Ahringer's RNAi library in 96-well plates enabling rapid screening of high numbers of samples. The individual wells are scored in three categories: wild type, adults without progeny (emb and ste), and larvae only (lva and lvl). In addition to visual analysis we have developed tools for automated scoring thereby standardizing data acquisition and providing documentation for each RNAi experiment. Data from our control strain allows us to compare our results with published data. Using a stringent threshold where only phenotypes with >80% penetrance are scored 13% of the RNAi constructs induce non-viable phenotypes, which is similar to other investigations. Furthermore, for previously identified essential genes we observe lethality or sterility in two thirds of the cases. We are currently retesting candidate genes in a secondary screen and will provide detailed information at the meeting.