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.

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.

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.

Peter Meister et al. (2006) Development & Evolution Meeting "Functional implications of nuclear organization in C. elegans"

Peter Meister, Susan Gasser

[Development & Evolution Meeting, 2006]

Active and inactive genes are not randomly positioned in the eukaryotic nucleus, yet the impact of their localization is still not completely understood. Moreover, it is not yet established in the context of a whole organism with differentiated cells whether nuclear positioning of genes precedes or results from functional chromatin characteristics, such as timing of replication or transcriptional potential. We use the worm to study nuclear dynamics in the context of a multicellular organism with specialized differentiated cells. We created strains expressing GFP-lacI under transcriptional control of a ubiquitous promoter by injection and integration. Surprisingly, embryos and larvae expressing GFP-lacI show two bright nuclear spots per nucleus. By genetic and FISH analysis, we show that the array itself is recognized by the GFP-lacI protein. This is likely to be due to the presence of a single lacO site in the sequence of the plasmids used to create these arrays. Since most of the arrays created for the last 20 years involve plasmids containing lacO sites, this is a powerful technique to identify arrays localization when genes coded by the arrays are either active or inactive. Using this technique, we will now study array localization of a pharyngeal specific gene in vivo during differentiation of the pharynx.

Peter Meister et al. (2006) European Worm Meeting "Functional implications of nuclear organization in C. elegans"

Susan M. Gasser, Peter Meister, Brietta Pike

[European Worm Meeting, 2006]

Peter Meister, Brietta Pike and Susan M. Gasser. Active and inactive genes are not randomly positioned in the eukaryotic nucleus, yet the impact of their localization is still not completely understood. Moreover, it is not yet established in the context of a whole organism with differentiated cells whether nuclear positioning of genes precedes or results from functional chromatin characteristics, such as timing of replication or transcriptional potential. We use the worm to study nuclear dynamics in the context of a multicellular organism with specialized differentiated cells. We created strains expressing GFP-lacI under transcriptional control of a ubiquitous promoter by injection and integration. Surprisingly, embryos and larvae expressing GFP-lacI show two bright nuclear spots per nucleus. By genetic and FISH analysis, we show that the array itself is recognized by the GFP-lacI protein. This is likely to be due to the presence of a single lacO site in the sequence of the plasmids used to create these arrays. Since most of the arrays created for the last 20 years involve plasmids containing lacO sites, this is a powerful technique to identify arrays localization when genes coded by the arrays are either active or inactive.. Using this technique, we will now study array localization of a pharyngeal specific gene in vivo during differentiation of the pharynx. We are exploring the relationship between replication dynamics and active and inactive chromatin domains.

Peter Meister et al. (2007) International Worm Meeting "Nuclear position and gene array activation in C. elegans."

Brietta L. Pike, Peter Meister, Susan M. Gasser

[International Worm Meeting, 2007]

Active and inactive genes are not randomly positioned in the eukaryotic nucleus, yet the impact of their localization is not completely understood. Moreover, it has never been examined in the context of whole organismal differentiation whether changes of gene position precedes or results from functional chromatin characteristics such as timing of replication or transcriptional potential. We use the worm to study subnuclear organization and chromatin dynamics in the context of a multicellular organism with specialized differentiated cells. We created strains constitutively expressing nuclear GFP-lacI, with an RFP muscular marker. Surprisingly, embryos and larvae show two bright territories per nucleus. By genetic analysis, we show that this array itself is recognized by the GFP-lacI protein, and can be visualized thanks to the presence of a single lacO site in the sequence of the plasmids used to create the array. When the array is poorly expressed in embryos, its localization is completely perinuclear. Moreover, when peripheral, it bears the specific silent chromatin mark of histone H3 trimethyl K9. In contrast, when the RFP marker gets activated during muscle differentiation, we observe that the array is no longer peripheral, but gets specifically relocalized toward the nuclear lumen. Since most gene arrays created by worm labs are based on plasmids containing lacO sites, we are able to localize such gene arrays in the nuclei of worm embryos, under conditions that allow the promoters to be active or inactive. Arrays bearing heat-shock promoters were assayed to test whether induction by heat-shock is accompanied by relocalisation of the array into the nuclear interior. Interestingly, these arrays decondense without moving away from the nuclear envelope, remaining close to the periphery while active. These results suggest that gene activation can correlate with two different patterns of gene positioning: during differentiation a tissue-specific array moves from the periphery into the nuclear lumen, implying movement either before or after induction, while the heat-shock array simply decondenses close to the nuclear envelope. Dependence of this positioning on nuclear lamina or pores is under investigation.

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.

Rohner, Sabine et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Activation of heat-shock - inducible genes: interaction with nuclear envelope components"

Gasser, Susan M, Meister, Peter, Rohner, Sabine

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

Several stress-induced genes in different organisms have been shown to be sequestered to the nuclear periphery upon activation. These interact with the nuclear pore complexes (NPC), the gateway between the cytoplasm and the nuclear interior. Recent data in Caenorhabditis elegans (modENCODE, Jason Lieb) from ChIP on chip experiments on NPP-13 (Nuclear Pore Protein 13, homologous to the Nup93 subunit in vertebrates) indicates an interaction between the heat shock gene hsp-16.2 and the NPC. Using fluorescence in situ hybridisation in embryos, we show that the genomic hsp-16.2 locus is preferentially located at the nuclear periphery and that its peripheral localisation increases upon heat shock treatment . Moreover, the hsp-16.2 promoter on its own is able to direct peripheral localisation of small integrated bombarded transgenes. Upon HS treatment, the proportion of transgenes located at the nuclear rim increases. We are now examining the physiological importance of the peripheral localization of this gene. To do this we are identifying which components of the nuclear pore and pore-associated transcription and which sequences in the promoter are important for its peripheral localisation.

Isiaka, B.Nafisat. et al. (2019) International Worm Meeting "Examining transcription dynamics at the single molecule level in individual promoters."

Semple, Jennifer, Meister, Peter, Krebs, Arnaud, Isiaka, B.Nafisat.

[International Worm Meeting, 2019]

Transcription initiation is a dynamic, multi-step process, requiring the ordered recruitment of multiple factors to the core promoter. The highly dynamic nature of transcription means that at any given moment, a particular promoter might be in one of multiple states in different cells, resulting in heterogeneous signals from a population of cells. Most techniques that study transcription initiation, such as chromatin-IP or GRO-seq provide population average measurements of the transcriptional process. To really understand transcription regulation, it is necessary to examine changes in promoter state at the single molecule level. To achieve this, we applied to C. elegans a technique known as "dual enzyme Single Molecule Footprinting (dSMF)". The method involves in vitro methylation of chromatin in intact nuclei with bacterial CpG and GpC methyltransferases. Closed chromatin bound by proteins is protected from methylation, whereas accessible DNA is methylated. After bisulfite conversion, specific amplicons, or genome-wide DNA libraries are sequenced to identify footprints of accessible DNA. Correlation of dSMF data with publicly available genome wide datasets shows that dSMF footprints coincide with nucleosome free regions in promoters and other genomic regions such as HOT sites. To identify what the different footprints represent in terms of promoter states in the transcription cycle, we then inhibit individual steps of the transcription cycle either with chemicals or by blocking engineered cyclin-dependent kinases with ATP analogs. Upon mapping the promoter occupancy states in transcription inhibited conditions, we are able to correlate the different promoter footprints with specific protein complexes present during the different steps of transcription initiation. Our data strengthen our understanding of how chromatin and gene structure regulate gene expression at the level of transcription initiation.

Gomez Saldivar, Georgina et al. (2021) International Worm Meeting "Tissue-specific transcription footprinting in C. elegans using RNA PoI DamID (RAPID) and Nanopore sequencing"

Meister, Peter, Glauser, Dominique A., Gomez Saldivar, Georgina

[International Worm Meeting, 2021]

Differential gene expression across cell types is a major determinant of multicellular organism development and physiology. In C. elegans, it is notoriously difficult to characterize individual cell types due to the difficulty to obtain comprehensive tissue-specific gene transcription data. Available methods require tissue dissociation and cell sorting from large worm populations or the use of transgenic approaches to purify transcripts from the target cell type. This can be challenging when the considered cell type is hard to separate from surrounding cells or is a rare cell type. Here, we present the RNA Pol DamID (RAPID) approach, in which the Dam methyltransferase is fused to a ubiquitous RNA polymerase subunit in order to create transcriptional footprints via methyl marks on the DNA of transcribed genes. We implemented a Cre-lox recombination system to target specific tissue and 3rd generation sequencing (Oxford Nanopore Technologies) to eliminate the need for an external sequencing facility and streamline data acquisition to achieve analyses from DNA extraction to sequencing results in less than a week. To validate the method, we determined the polymerase footprints in whole animals and in different tissues from intact young adults. We obtained meaningful transcriptional footprints in line with RNA-seq studies in whole animals, muscle and intestine. To challenge the sensitivity of RAPID and demonstrate its utility to determine novel tissue-specific transcriptional profiles, we determined the transcriptional footprints of the pair of XXX cells, representing 0.2% of the somatic cell content of the animals. We identified 2362 genes potentially transcribed in XXX cells, among which the few known XXX markers, such as daf-9 and sdf-9. Using transcriptional reporters for a subset of new hits, we confirmed that the majority of them were indeed expressed in XXX. Interestingly, results of a gene ontology analysis on a refined list of 275 genes, whose expression is strongly enriched in XXX cells as compared to other profiled tissues, are in line with the endocrine function of these cells, with implications in signalling and dauer formation. A transcription factor predictive tool also revealed an association with specific transcription factors playing a role in dauer formation. Taken together, our work establishes RAPID as a valid method for the determination of polymerase footprints in specific tissues of C. elegans without the need for cell sorting or RNA tagging.