Towbin, Benjamin D. et al. (2009) International Worm Meeting "Differentiation associated spatial separation of active and silent loci."

Towbin, Benjamin D., Meister, Peter, Gasser, Susan M., Pike, Brietta L.

[International Worm Meeting, 2009]

Chromatin is non-randomly organized in the interphase nucleus. In particular, silent heterochromatin is found adjacent to the nucleolus or at the nuclear periphery, where it is bound to the nuclear lamina. In yeast and flies, certain active genes are bound to nuclear pores. It is, however, still unclear whether clustering of silent genes at the nuclear envelope confers a biological function. In order to study the subnuclear distribution of genes in vivo and throughout development we have used recognition of lacO repeats by GFP-lacI for visualization of genetic loci in C. elegans. To this end, we applied microparticle bombardment to generate chromosomally integrated low-copy transgenes that contain arrays of LacO sites, and a developmentally regulated promoter driving mCherry (myo-3::mCherry or pha-4::mCherry-H2B). By expression of GFP-lacI from a ubiquitously expressed promoter, these transgenes are detected as fluorescent foci. In early embryonic cells, transgenes, like many endogenous loci, are randomly distributed throughout nuclear space. Over the course of differentiation, we observe spatial segregation of the transgenes, depending on their transcriptional status. Silent transgenes show high enrichment at the nuclear periphery while active ones become sequestered internally. In contrast to these low-copy transgenes created by bombardment, integrated repetitive transgene arrays derived form gonadal plasmid injection accumulate high levels of heterochromatic marks (H3K9me3 and H3K27me3). These silent domains are attached to the nuclear periphery throughout development. However, similar to low-copy transgenes, repetitive arrays can overcome peripheral anchoring upon activation of a developmentally regulated promoter located on the array. In summary, we observe spatial compartmentalization of active and silent genes in the nucleus, driven by differentiation. Our results suggest that the trigger for localization to the nuclear rim may be structural components of heterochromatin and not simply the binding of sequence specific factors. Preliminary experiments indicate that gene attachment to the nuclear envelope may stabilize heterochromatic silencing.

Towbin, Benjamin et al. (2021) International Worm Meeting "A folder mechanism ensures size uniformity among C. elegans individuals by coupling growth and development"

Towbin, Benjamin, Grosshans, Helge

[International Worm Meeting, 2021]

Animals increase by orders of magnitude in their volume during development. Hence, even small differences in the growth rates between individuals could generate large differences in their adult body size. Yet, such volume divergence among individuals is usually not observed in nature. We combined theory and experiment to understand the mechanisms of body size uniformity. Using live imaging, we measured the volume growth of hundreds of individuals of C. elegans over the entire span of their postembryonic development. We find that C. elegans grows exponentially in volume with a coefficient of variation of the growth rate of ~7%, but that individuals diverge much less in volume than expected from this heterogeneity. The mechanism counteracting size divergence does not involve size thresholds for developmental milestones. Instead, an inverse coupling of the growth rate and the duration of development produces a constant volume fold change per larval stage. The duration of larval stages of C. elegans is determined by the period of a developmental oscillator. Using mathematical modelling, we show that an anti-correlation between the growth rate and the oscillatory period emerges as an intrinsic property of a genetic oscillator. We propose that the robustness of body volume fold change is a hard-wired characteristic of the oscillatory circuit and does not require elaborate mechanisms of size control by cellular signalling. Indeed, the coupling of growth and development was unaltered by mutation of canonical pathways of growth control. This novel concept of size homeostasis may broadly apply to other multicellular systems controlled by genetic oscillators.

Towbin, Benjamin et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "High levels of the methyl donor S-adenosylmethionine (SAM) are required for perinuclear anchoring and silencing of repetitive DNA, histone methylation and germline integrity"

Towbin, Benjamin, Gasser, Susan M, Meister, Peter

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

Chromatin is non-randomly organized in the interphase nucleus. In particular, silent heterochromatin is often found at the nuclear periphery, close to the nuclear lamina. In C. elegans, this phenomenon is recapitulated by the peripheral localization of silent, repetitive transgene arrays (Meister et al., 2010). Here, we identified in a genome-wide RNAi screen that depletion of the cellular methyl donor S-adenosylmethionine (SAM) by knock-down of its synthetase sams-3 releases arrays from the nuclear periphery in early embryos. This correlates with a global reduction of histone methylation and hyperactivation of a GFP reporter encod ed by the array. However, transcriptional activation alone is neither sufficient nor required for array detachment: in embryos mutant for the histone methyltransferase (HMT) met-2, which is required for di-methylation of H3K9, arrays are greatly activated, but remain bound to the nuclear envelope; conversely, array detachment upon sams-3 RNAi does not depend on the presence of active promoters on the array. Remarkably, low levels of sams-3 do not impair embryonic development, but cause a strong brood size reduction. We propose that reduced cellular levels of the methyl-donor SAM result in improper histone methylation, which may be required for peripheral anchoring and silencing of heterochromatin. Our data also suggests that array silencing and anchoring are mediated by two independent methylation events. Analysis of array position and silencing in specific HMT mutants will allow us to test this model.

Pradhan, Sigma et al. (2021) International Worm Meeting "The role of parental diet on progeny's proteome and fitness"

Towbin, Benjamin, Pradhan, Sigma

[International Worm Meeting, 2021]

Animals adjust their phenotype to their environment, which is thought to provide a selective benefit under changing conditions. A famous example of such phenotypic plasticity is the slow down of growth and the delay of aging under dietary restriction (DR). Interestingly, parental DR also affects phenotypes of the F1 generation, for example, DR increases the starvation resistance of progeny (Jordan et al., 2019). Molecularly, DR is associated with a global change in proteome expression, including a reduction in ribosome levels and a proteome-wide slow-down of protein turnover (Dhondt et al., 2016; Visscher et al., 2016). Here, we ask if and how parental diet impacts the proteome of the F1 progeny. We will seek causal relationships between intergenerationally transmitted phenotypes and the inherited proteome. Finally, we aim to determine if the parental control of the proteome provides a selective benefit to their progeny. To address these questions, we have established a tool to precisely quantify ribosomal levels by measuring the luminescence from lysis of HiBit tagged to ribosomal proteins. We plan to globally analyze parental effects on the F1 proteome by proteomics and measure the dynamics of proteome changes, growth rates, and reproduction rates in different nutritional conditions by fluorescence live imaging in microchambers. The project will likely provide important insights about the molecular mechanisms and physiological significance of inter-generational inheritance induced by parental diet.

Tuomaala, Joel et al. (2021) International Worm Meeting "Decline of ribosomal proteins levels during L1 arrest"

Towbin, Benjamin, Dengjel, Jorn, Tuomaala, Joel

[International Worm Meeting, 2021]

Starvation of C. elegans upon hatching causes developmental arrest. Arrested animals can survive for more than to 2 weeks in the absence of food and restart growth when nutrition improves. However, upon re-feeding animals resume growth with a delay that increases proportionally with the duration of starvation. Survival during starvation requires autophagy, and the starvation response is under tight control by metabolic signaling. For example, we found that mutants with constitutively active mTOR signaling die within 1 week of starvation. The goal of this project is to characterize the dynamics of proteome turnover during starvation and during recovery, and to ask if specific proteins are preferentially degraded during starvation to maintain survival. We hypothesize that under extended periods of starvation proteins needed for growth and biosynthesis, such as ribosomes and mitochondria, are preferentially turned over, as compared to structural proteins that are essential for survival, such as collagens or histones. Resuming growth upon feeding would consequently require re-synthesis of the biosynthesis proteins, and thereby delay onset of growth in proportion to the duration of starvation. To test this hypothesis, we established an assay for protein quantification based on HiBit technology. HiBit relies on a split luciferase and allows for detection of specific proteins at high accuracy and sensitivity in a microplate-based readout. Using this assay, we find that indeed ribosomal proteins declined continuously during L1 arrest. We will now test if this decline relies on autophagy, and if the turnover rate of ribosomes can explain how survival is affected in mutants with perturbed autophagy and growth signalling. Finally, we will use proteomics to assess how the proteome composition globally changes during L1 arrest on a time scale of weeks.

Meister, Peter et al. (2011) International Worm Meeting "Towards molecular understanding of gene localization."

Towbin, Benjamin, Gasser, Susan, Rohner, Sabine, Meister, Peter

[International Worm Meeting, 2011]

To understand whether the spatial organization of the genome reflects the cell's differentiated state, we have examined whether genes assume specific subnuclear positions during C. elegans development. Monitoring the radial position of developmentally controlled promoters in embryos and larval tissues, we found that small integrated transgenes bearing three different tissue-specific promoters have no preferential position in nuclei of undifferentiated embryos. However, in differentiated cells they shifted stably towards the nuclear lumen when activated, or to the nuclear envelope when silent. We uncovered two additional parameters which influence subnuclear positioning. First, high copy number repeats targets arrays to the nuclear rim, even in the presence of active housekeeping promoters. Tissue-specific activation of promoters in the arrays overrides perinuclear anchoring. Second, some promoters contain sequences which target them to the nuclear periphery, both in their active and inactive form. Using genetic and biochemical methods, we are now using our system to uncover the molecular machinery involved in subnuclear positioning of genes.

Gonzalez Sandoval, Adriana V. et al. (2013) International Worm Meeting "CEC-4 is a novel chromodomain protein involved in perinuclear chromatin anchoring."

Gonzalez Sandoval, Adriana V., Kalck, Veronique, Yang, Teddy, Gasser, Susan M., Towbin, Benjamin D., Zhao, Kehao

[International Worm Meeting, 2013]

A genome-wide RNAi screen identified two histone H3K9 methyltransferases, MET-2 and SET-25, which deposit histone H3K9 me1, me2 and m3, respectively, as being necessary for both the anchoring and the repression of heterochromatic gene arrays (Towbin et al., 2012). H3K9m1 or me2 are sufficient for tethering, while trimethylated H3K9 was necessary for repression. To identify the proteins involved in heterochromatin anchoring at the nuclear envelope we screened "readers" of these histone marks in a targeted RNAi screen. Neither homologue of HP-1, HPL-1 nor HPL-2 were implicated in the anchoring, although loss of HLP-2 led to derepression. We screened 44 other Chromo-, MBT-, PHD- and Tudor- domain-containing genes in a strain deficient for HPL-1 and LIN-61, for loss of heterochromatin. We identified a previously uncharacterized chromo domain protein, CEC-4, as being essential for maintaining heterochromatin at the nuclear periphery. However, in contrast to the double mutant of met-2 and set-25, deletion of cec-4 does not cause array derepression. CEC-4 forms a ring around the nucleus, and colocalizes with the anchored array. We show that the chromodomain of CEC-4 binds all three methylated forms of H3K9, but no other methylated lysines in vitro. Using two points mutations within the chromodomain that lose affinity for methylated H3K9 in vitro, we confirm that CEC-4 requires a functional chromo domain to bind and tether heterochromatin, while interaction with the nuclear envelope requires the non-chromo domain portion of CEC-4. By LEM-2 ChIP-seq we determined that deletion of cec-4 globally reduces the interaction of chromosome arms to the nuclear periphery.

Meister, Peter et al. (2009) International Worm Meeting "Activation of heat-shock - inducible genes: interaction with nuclear envelope components."

Rohner, Sabine, Meister, Peter, Towbin, Benjamin D., Gasser, Susan M.

[International Worm Meeting, 2009]

Nuclear organisation has been implicated in gene expression in unicellular eukaryotes and some mammalian lineages. We have examined changes in nuclear organization during the differentiation of the multicellular organism C. elegans. Using fluorescence in situ hybridisation (FISH) in embryos and GFP-lacI/lacO systems, we showed that developmentally regulated promoters direct subnuclear positioning of genes and gene arrays. The conclusion of these studies is that silent genome segments and arrays are located at the nuclear periphery while active promoters are retained centrally. Surprisingly, heat-shock (HS) promoters behave in a different manner : an array containing a transcriptional fusion of hsp-16.2 fused to GFP is located at the nuclear periphery before activation and expands upon transcriptional activation but stays at the nuclear rim. This prompted us to ask whether HS promoters where anchored at the nuclear envelope. Using FISH, we show that the genomic hsp-16.2 is also preferentially located at the nuclear periphery and that enrichment for peripheral localisation is further increased after heat shock. Using microparticle bombardment, we generated chromosomally integrated low-copy transgenes that contain arrays of LacO sites and the hsp-16.2 promoter driving mCherry, as well as control transgenes containing only lacO sites. These were then observed in vivo by expression of a nucleus-targeted GFP-lacI fusion. We observed that the promoter alone is able to direct the transgene toward the nuclear periphery, while HS treatment of the embryos leads to an increase of the proportion of transgenes located at the nuclear rim. We are examining various components of the nuclear envelope as anchors for the HS promoter. These investigations will be presented.

Ferreira, Helder C. et al. (2013) International Worm Meeting "Developmental regulation of telomere anchoring in C. elegans."

Jegou, Thibaud, Ferreira, Helder C., Gasser, Susan M., Towbin, Benjamin

[International Worm Meeting, 2013]

Telomeres are specialized protein-DNA structures that protect chromosome ends. In lower eukaryotes, such as budding yeast, telomeres show a distinct nuclear organization, being bound to the nuclear periphery. By imaging telomere position in the metazoan C. elegans, we find that, here too, telomeres are preferentially located near the nuclear envelope. Peripheral telomere localization increases during embryogenesis and persists in later larval stages. We show that in early embryogenesis telomere position is independent of the Ku complex and of H3 K9 methylation but instead is regulated by the PIAS-like SUMO E3 ligase gei-17 and the nuclear envelope protein SUN-1. Moreover, we find that the shelterin component POT-1 but not POT-2 is required to maintain telomeres at the nuclear periphery. Interestingly, POT-1 inhibits recombination mediated telomere maintenance. To our knowledge, this is the first description of telomere position in C. elegans.

Meister, Peter et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Towards a molecular understanding of gene positioning in C. elegans"

Gasser, Susan M, Meister, Peter, Ponti, Aaron, Pike, Brietta, Towbin, Benjamin

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

To understand whether the spatial organization of the genome reflects the cells differentiated state, we have examined whether genes assume specific subnuclear positions during C. elegans development. Monitoring the radial position of developmentally controlled promoters in embryos and larval tissues, we found that small integrated transgenes bearing three different tissue-specific promoters have no preferential position in nuclei of undifferentiated embryos. However, in differentiated cells they shifted stably towards the nuclear lumen when activated, or to the nuclear envelope when silent. In contrast, large integrated arrays bearing the same promoters became heterochromatic and nuclea r envelopebound in embryos. Tissue-specific activation of promoters in these large arrays in larvae overrode the perinuclear anchorage. For transgenes that carry both active and inactive promoters, the inward shift of the active promoter was dominant. We are now using our system to specifically purify proteins bound to arrays in the active or the inactive form to uncover the molecular machinery involved in gene positioning.