Bauer, Fanelie et al. (2013) International Worm Meeting "Functional analysis of H3K79 methylation."

Appert, Alex, Bauer, Fanelie, Ahringer, Julie

[International Worm Meeting, 2013]

H3K79 methylation has been linked to transcriptional activation however the mechanism of H3K79 targeting and function are not well understood. H3K79 methylation is carried out by the Dot1 family of histone methyltransferases, which are characterized by a class I SAM-dependent methyltransferase domain. In all studied organisms, DOT1 is solely responsible for all H3K79 methylation states as the knockout of this gene results in a total loss of mono- di- and tri- methylation of H3K79 methylation. In yeast, non-methylated H3K79 is found at silenced telomeres, whereas H3K79 methylation at telomeres disrupts transcriptional silencing. In humans, leukaemia causing MLL fusions to AF9 recruit Dot1, and inactivation of Dot1 reduces expression of MLL-AF9 targets and inhibits progression of the disease. Dot1 has also been implicated in DNA damage checkpoint control and repair, the cell cycle, and is required for early embryonic development in both mice and flies. We are carrying out a functional analysis of H3K79 methylation in C. elegans. The worm genome has six DOT1L homologs, of which only Y39G10AR.18 is highly expressed across all developmental stages. As in other organisms, H3K79me2 and H3K79me3 are present on the bodies of transcriptionally active genes in early embryos and L3 larvae (Liu et al 2011). However, the pattern of H3K79me1 differs in early embryos and L3 larvae. Regions of high H3K79me1 in early embryos often lack this modification in L3 larvae. We are assessing which genes carry out H3K79 methylation and studying the function and developmental regulation of H3K79 methylation.

Janes, Jurgen et al. (2017) International Worm Meeting "Genome-wide mapping and characterization of regulatory elements across development."

Appert, Alex, Dong, Yan, Ahringer, Julie, Janes, Jurgen

[International Worm Meeting, 2017]

The genome-wide identification of regulatory regions is necessary for understanding developmental gene regulation. Regulatory elements such as promoters and enhancers are usually nucleosome depleted, and so can be identified by their accessibility to digestion by nucleases such as DNAse I or to transposition by Tn5 (ATAC-seq). Using ATAC-seq, we have generated genome-wide maps of regulatory elements across the six C. elegans developmental stages and characterized their properties. We define more than 30,000 sites, with most regions showing developmental changes in accessibility. To annotate and characterise the regulatory regions, we further mapped transcription initiation, transcription elongation, and four histone modifications (H3K4me3, H3K4me1, H3K36me3 and H3K27me3) at the six developmental stages. Consistent with previous work showing that transcription initiation occurs at both promoters and enhancers, we find that more than 80% of the ATAC-seq sites have transcription initiation signal. In contrast, only a subset has clear transcription elongation signal linked to genes, indicative of promoter activity. Using these metrics, we annotated over 10,000 accessible regions as promoters; the remainder are annotated as putative enhancer elements. Examining histone modifications at promoters, we found that the classical promoter-associated signature of high H3K4me3 and low H3K4me1 is associated with broadly expressed genes but does not reliably identify promoters of developmentally regulated genes. Genes are associated with a range of patterns of regulatory elements, ranging from a single promoter to having multiple promoters and up to dozens of putative enhancers scattered upstream as well as within intronic regions. Characterizing accessibility of the 30,000 sites during development, we found that a quarter of sites show no changes. These are associated with genes broadly expressed across development and are enriched for H3K36me3. We also observed that a third of regions decrease and a third of regions increase in accessibility over development. Sites with increasing accessibility have lower GC content whereas sites with decreasing accessibility have higher GC content, and are enriched for H3K27me3. We also identified sequence features that are enriched at regulatory regions associated with transcription elongation. We identify and characterise features of C. elegans regulatory elements across development, providing an important new resource for studying transcription regulation.

Fabry, Martin et al. (2021) International Worm Meeting "A single cell multiomics approach to resolve genomic drivers of C. elegans development"

Dong, Yan, Appert, Alex, Fabry, Martin, van der Burght, Servaas, Marioni, John, Ahringer, Julie

[International Worm Meeting, 2021]

Animals develop from a single totipotent zygote that gives rise to all of the diverse cell types of the organism, which are genetically identical. During cell divisions in the embryo, most cells become specified to particular fates and initiate differentiation programs. Other cells maintain pluripotency and generate differentiated cells later in life. However, molecular events at decision making points when cells either maintain their cellular potency or commit to differentiation programs are poorly understood. To understand these processes, we need to determine the step-by-step changes in genomic remodelling that drive changes in gene expression from the birth of the pluripotent cell through its period of quiescence, activation, and production of different cell types. We are using 10X genomics single cell technology to jointly measure chromatin accessibility and RNA output in individual cells of different C. elegans lineages in order to follow molecular events that drive cell fate commitment and expression at various steps of development. Our data reveal differences in DNA accessibility before and after zygotic genome activation (ZGA). Following the activation of zygotic transcription, regulatory elements controlling key lineage-defining marker genes become accessible in subpopulations of cells and RNA output shows high temporal correlation with chromatin accessibility. Following the E lineage as an example revealed dynamic processes that result in the precise activation of specific master transcription factors at various steps of the E lineage trajectory. Using these data, we are developing new computational tools to recapitulate the real-time trajectory of individual cells throughout development by mapping cells onto the lineage tree and temporally ordering their age. With these methods we will be able to follow the precise molecular events resulting in the commitment of cells to specific developmental programs and finally understand and even predict the logic underlying cellular potency in C. elegans.

Pokhrel, Bharat et al. (2017) International Worm Meeting "Characterising C. elegans COMPASS complex in gene expression."

Chen, Ron, Appert, Alex, Chen, Yannic, Dong, Yan, Clifford, Rosamund, Pokhrel, Bharat, Ahringer, Julie, Stempor, Przemyslaw

[International Worm Meeting, 2017]

Trimethylation of histone lysine 4 (H3K4me3) is regarded as an active promoter mark across all eukaryotes. H3K4me3 is mainly deposited by an evolutionarily conserved COMPASS complex that contains two key subunits: SET-2, H3K4me3/2 methyltransferase, and CFP-1, a conserved CpG binding protein. Perturbation of this mark has been found to be associated with different diseases, development defects, and incorrect cell fate specification. However, it is unclear whether H3K4me3 play an instructive role in transcription or just a consequence of gene expression. To address this question, we phenotypically characterised and compared cfp-1(tm6369) mutants with a reported loss-of-function set-2(bn129) mutant allele. We observed dramatic loss of H3K4me3, poor fertility, slow growth and ectopic gene expression in both cfp-1 and set-2 mutants. We found that the role of H3K4me3 is to fine tune gene induction, likely by modulating other histone modifications that play an essential role in gene activation. Our results shed light on the role of H3K4me3 in chromatin regulation and function.

Gaarenstroom, Tessa et al. (2017) International Worm Meeting "Heterochromatin factors collaborate with small RNA pathways to combat repetitive elements and germline genotoxic stress."

Miska, Eric, Stempor, Przemyslaw, Wysolmerski, Brian, Dong, Yan, Sapetschnig, Alexandra, Gaarenstroom, Tessa, Appert, Alex, Ahringer, Julie, McMurchy, Alicia, Aussianikava, Darya, Kolasinska-Zwierz, Paulina, Huang, Ni

[International Worm Meeting, 2017]

Repetitive sequences derived from transposons make up a large fraction of the genome and must be silenced to protect genome integrity. Silencing of these elements is especially important in the germ line, where the piRNA pathway has been shown to be involved. Repeats are often found in heterochromatin, which in C. elegans are regions dispersed over the chromosome arms, however the roles and interactions of heterochromatin proteins are poorly understood. We have shown that a diverse set of heterochromatin factors act together with the piRNA and nuclear RNAi pathways to silence repetitive elements and prevent genotoxic stress in the germ line. HPL-2/HP1, LIN-13, LIN-61, LET-418/Mi-2 and the H2K9me2 histone methyltransferase MET-2/SETDB1 show genome-wide co-binding and enrichment at repetitive elements, and mutants show a derepression of a subset of transposons. Furthermore, heterochromatin mutants are characterized by functionally redundant and temperature sensitive sterility, and display increased germline apoptosis and activation of DNA damage signalling. Remarkably, fertility of heterochromatin mutants could be partially restored by inhibiting expression of MIRAGE1 DNA transposons or endogenous meiotic double strand breaks. Loss of CEP-1/p53 also ameliorates both their fertility and somatic defects, suggesting that DNA damage signalling contributes to the phenotypes observed. Through genetics and transcriptional profiling, we uncovered complex interactions between heterochromatin factors and the piRNA and nuclear RNAi pathways, including functional redundancy in repetitive element repression between let-418 and nrde-2. This redundancy underlies the importance of safeguarding the genome through multiple means. It is also becoming evident that heterochromatic silencing occurs through different mechanisms at different genomic elements. We are currently dissecting interactions between the various factors, as well as H3K9me2/3, to uncover their mechanisms of action at heterochromatic regions, as well as investigating their roles in development and adult homeostasis.

Sarfarazhussain Farooqui et al. (2006) European Worm Meeting "A GFP Reporter Based Screen for the Notch Target Genes during Vulval Development"

Sarfarazhussain Farooqui, Ivo Rimann, ALEX HAJNAL

[European Worm Meeting, 2006]

Sarfarazhussain Farooqui, Ivo Rimann and Alex Hajnal. During hermaphrodite vulval development, lateral signaling from the primary vulval precursor cell (VPC) P6.p activates the LIN-12 Notch pathway in the neighbouring secondary VPCs P5.p and P7.p. The LIN-12 Notch signal inhibits primary fate specification and promotes the secondary vulval fate by activating transcription factors of the CSL family, which turn on the expression of specific target genes.. LIN-12 Notch targets usually contain clusters of CSL binding sites (RTGGGAA) in their promoter/enhancer regions. To identify new target genes of the LIN-12 Notch pathway in a systematic way, we searched the C. elegans genome sequence for genes containing clusters of three or more CSL sites within 2 kbp upstream of the start codon. We further refined our list of candidate Notch targets by taking into consideration only those genes in which the putative CSL sites are conserved in the C. briggsae orthologs. Promoter-GFP fusion constructs for the 23 strongest candidate genes were made by fusion PCR and injected into wild-type worms to create transgenic lines. Details on the expression patterns of the reporter constructs generated so far will be presented at the meeting.

Tauffenberger, Arnaud et al. (2009) International Worm Meeting "Age-dependent modification of expanded CAG toxicity in simple C. elegans models."

Tauffenberger, Arnaud, Bel-Hadj, Samar, Parker, Alex

[International Worm Meeting, 2009]

Arnaud Tauffenberger1,2,3, Samar Bel-Hadj1, Alex Parker1,2, 3 1CRCHUM, 2Centre of Excellence in Neuromics, 3Department de pathologie et biologie cellulaire, Universite da Montreal, Montreal, Quebec, Canada The expansion of CAG trinucleotides coding for glutamine is the cause of at least nine late-onset neurodegenerative diseases. The expanded CAG (expCAG) diseases may have some pathogenic mechanisms in common as the disease threshold for all of these disorders is around 40 CAG repeats and they display misfolded intracellular protein aggregation phenotypes. We have turned to the model organism C. elegans to explore two areas of interest: 1. Age-dependent toxicity: Although the expCAG diseases show anticipation, the size of CAG repeat only partially correlates with the variation in age-of-onset. Evidence suggests that even for these single trait neurodegenerative diseases, disease onset and progression may have additional environmental and genetic components. Using C. elegans transgenic worms expressing expCAG in neurons we are exploring the links between aging and late-onset neurodegeneration. We hypothesize that genes regulating longevity, stress response, their associated signaling pathways and downstream effectors may encode new therapeutic targets for age-dependent proteotoxicity in humans. 2. Mechanisms of expCAG toxicity: Long homopolymeric CAG nucleotide stretches are unstable and may be prone to translation errors. Mistranslation of expCAG to alternate reading frames may produce hybrid proteins with additional toxicity. We have created transgenic strains to investigate this phenomenon. A summary of our data and recent findings will be presented.

Gal, C et al. (2015) International Worm Meeting "Regulation of gene expression by the DREAM complex."

Gal, C, Ahringer, J, Appert, A, Latorre, I, Chesney, M

[International Worm Meeting, 2015]

The conserved eight subunit DREAM (DP, Retinoblastoma [Rb]-like, E2F, and MuvB) complex is a regulator of cell cycle and quiescence genes and plays an important role in development and disease. The C. elegans DREAM members are LIN-35/Rb-like, EFL-1/E2F, DPL-1/DP1, LIN-8, LIN-37, LIN-52, LIN-53, and LIN-54. Recent work has demonstrated that C. elegans direct DREAM targets are de-repressed in the absence of the Rb-like protein LIN-35 and that high gene body levels of the histone variant H2A.Z are linked with target repression1. However, the mechanism that generates gene body H2A.Z enrichment, and the relationship between DREAM and H2A.Z are not currently understood. Furthermore, the functions and regulatory interactions of individual complex members are not clear. For example, LIN-35 has been demonstrated to be a transcriptional repressor by a number of groups, whilst the DP and E2F proteins have previously been reported to act as activators of transcription2. In addition, mutants of individual DREAM complex members do not all have the same phenotype, indicating different functions. Therefore, we are investigating the contribution of DPL-1 (DP) and EFL-1 (E2F) to DREAM target gene repression. We are also carrying out a GFP reporter screen to identify additional components required for DREAM-mediated repression, to elucidate the mechanisms behind DREAM mediated developmental control.1. Latorre, I., Chesney, M.A., Garrigues, J.M., Stempor, P., Appert, A., Francesconi, M., Strome, S., and Ahringer, J. (2015) 'The DREAM complex promotes gene body H2A.Z for target repression', Genes Dev., 29, 495-5002. Chi, W & Reinke, V. (2006) 'Promotion of oogenesis and embryogenesis in the C. elegans gonad by EFL-1/DPL-1 (E2F) does not require LIN-35 (pRB)', Development, 133, 3147-57.

Claudia Walser et al. (2006) European Worm Meeting "Distinct Roles of the Pumilio and FBF Translational Repressors during C. elegans Vulval Development"

Erika Frohli Hoier, Gopal Battu, Claudia Walser, ALEX HAJNAL

[European Worm Meeting, 2006]

Claudia Walser, Gopal Battu, Erika Frhli Hoier and Alex Hajnal. Members of the conserved PUF (Pumilio and FBF repeat) protein family regulate various aspects of germ line development by selectively binding to the 3 untranslated region of their target mRNAs and repressing translation. FBF-1 and FBF-2 regulate the sperm/oocyte switch and mitosis versus meiosis decision by repressing fem-3 and gld-1 translation, respectively (Zhang et al., 1997; Crittenden et al., 2002). We found that puf-8, fbf-1 and fbf-2 also act in the soma where they negatively regulate vulval development.. Loss-of-function mutations in puf-8 cause excess vulval differentiation when combined with mutations in negative regulators of the EGFR/RAS/MAPK pathway, and they suppress the Vulvaless phenotype caused by mutations that reduce EGFR/RAS/MAPK signaling. A PUF-8::GFP translational reporter is initially expressed in all six vulval precursor cells (the VPCs P3.p through P8.p) but gets restricted to the distal, uninduced (3) VPCs after vulval fate specification. Moreover, the fusion of the distal VPCs to the hypodermal syncytium is retarded in puf-8(lf) mutants. Thus, PUF-8 acts cell-autonomously to limit the temporal competence of the vulval precursor cells to respond to the extrinsic patterning signals. fbf-1 and fbf-2, on the other hand, function as redundant inhibitors of vulval cell fate specification in a distinct pathway that involves the repression of gld-1 translation. fbf-1 fbf-2 double mutants show ectopic 1 cell fate marker expression in adjacent VPCs. We conclude that the FBFs ensure that only one VPC (P6.p) is selected for the 1 cell fate.. Therefore, the PUF family translational repressors regulate various aspects of cell fate specification in the soma, and they may play a conserved role in modulating signal transduction during animal development.

Xiong, R. et al. (2019) International Worm Meeting "Cellular and molecular mechanisms of mok-1 function in left-right neuronal asymmetry."

Wang, X., Chuang, C.-F., Xiong, R.

[International Worm Meeting, 2019]

The developing nervous system generates a large diversity of cell types with distinct patterns of gene expression and functions. One way to establish neuronal diversity is to specify neuronal subtypes across the left-right axis. The C. elegansleft and right AWC olfactory neurons communicate to specify asymmetric subtypes, AWCOFF and AWCON. The default AWCOFF is specified by a Ca2+-regulated kinase cascade that is activated by influx of Ca2+ through the voltage-gated Ca2+ channel UNC-2/UNC-36. Intercellular communication between the two AWC neurons and other neurons through the NSY-5/innexin gap junction network antagonizes unc-2/unc-36 Ca2+ signaling in the induced AWCON cell. Our recent data suggest that voltage- and calcium-activated SLO BK potassium channels slo-1and slo-2acts redundantly downstream of nsy-5 to inhibit unc-2/unc-36 Ca2+ signaling in the specification of AWCON. To identify the genes required for slo-1function in inhibiting unc-2/unc-36 Ca2+ signaling for promoting AWCON, we performed a non-biased forward genetic screen to isolate mok (modifier of K+ channel) mutants that suppress the slo-1(gf) 2AWCON phenotype. mok-1(vy11) is one of the mutants identified from this screen. The 2AWCOFF phenotype of mok-1(vy11) mutants is suppressed by loss-of-function mutations in the Ca2+ channel gene unc-36, suggesting that mok-1 acts upstream of the unc-2/unc-36 Ca2+ signaling pathway. Together, our results suggest a model in which nsy-5 inhibits unc-2/unc-36 Ca2+ signaling through slo-1 and mok-1. We identified the mutation responsible for the vy11 phenotype using whole genome sequencing with the help of Alex Boyanov in the Oliver Hobert lab. Further genetic analysis and behavior analysis of the vy11 mutant, as well as molecular characterization of mok-1 will be presented in the meeting.