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[
International Worm Meeting,
2011]
We recently described the use of puromycin to select large populations of transgenic worms from lines carrying extrachromosomal arrays with the resistance gene (Nat Methods. 2010; 7(9):725-7.). Now, we have extended the technique to select for transgenic worms following bombardment. We have introduced some modifications in the standard bombardment protocol and created a three way Gateway compatible plasmid expressing both puromycin and neomycin resistance genes to make drug selection possible, despite the extremely high dilution ratio of transgenic to non-transgenic worms. The new protocol makes it possible to pick adult F1 generation worms only 8 days after bombardment, significantly faster than the most widely used selection protocol with
unc-119. Furthermore, this drug selection regime does not require any particular genetic background and we have successfully generated lines with integrated transgenes in worms from various different strains and species, namely C. elegans (N2, CB4856), C. briggsae (AF16, HK104) and C. remanei (PB4641).
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[
International Worm Meeting,
2009]
The commonly used transgenesis selection markers in worms involve the introduction, or rescue, of mutant phenotypes. Such mutations make the growth of the worms more labor intensive and can possibly interfere with the analysis of the transgene of interest. In addition, rescue of a mutant phenotype, such as the widely used
unc-119 system, is limited to species that contain an appropriate mutant worm strain. Here we report the development of a drug selection system in order to address these issues. Our experiments were carried out with mosaic animals expressing the gene from an extra-chromosomal array, suggesting that this system would be extremely useful for large scale genomic or proteomic studies where one could combine the ease of generating worms carrying extra-chromosomal transgenes with an easy protocol for enriching large populations of transgenic worms. We also anticipate that this selection system will be useful for other applications, such as stable worm transformation by bombardment, which requires selection of rare transformation events from among a large number of worms.
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[
European Worm Neurobiology Meeting,
2009]
Available methods for selecting transgenic C. elegans strains are based on the rescue of a detrimental mutation such as
unc-119 or the use of visible markers such as GFP. These approaches are often labour intensive and cannot be easily applied to other species. We have developed a selection system using the antibiotic puromycin. The selection is achieved with a plasmid that expresses the antibiotic resistance gene under the control of a ribosomal gene promoter. When different worm strains carrying an extrachromosomal array containing the puromycin resistance gene are treated with puromycin in liquid medium for 4 days, we routinely obtain populations in which 80-100% of the worms carry the transgene with a recovery rate of >50% of transgenic worms from the original population. We show that transgene selection with our vector can be carried out in at least two species of worms: C. elegans and C. briggsae. In summary, we describe a positive selection system based on puromycin which: - Allows for the easy and rapid selection of rare transformation events - Is independent of genetic background - Can be applied to other Caenorhabditis species - Constitutes a way to enrich large populations of worms carrying non-integrated transgenes.
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[
Evolutionary Biology of Caenorhabditis and Other Nematodes,
2010]
We have developed a drug selection system for transgene expression in nematodes. Worms carrying an extrachromosomal array containing the puromycin resistance gene are efficiently selected from a mixed population. This approach has a number of advantages over existing methods for transgene selection: 1) Large numbers of transgenic worms can be easily selected without the need for expensive, specialised equipment such as a worm-sorter. 2) Strains carrying extrachromosomal arrays with puromycin resistance can be maintained on puromycin plates without needing to pick individual worms. 3) Puromycin selection does not require a particular genetic background, eliminating the need to generate particular mutant strains for different species, which are often laborious to work with. We have successfully tested puromycin selection in both C. elegans and C. briggsae and anticipate that this system could be useful for transgenesis in a variety of Caenorhabditis species. 4) We have developed a protocol for rapid drug selection following bombardment of N2 worms.
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[
C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany,
2010]
We have developed a drug selection system for transgene expression in nematodes. Worms carrying an extrachromosomal array containing the puromycin resistance gene are efficiently selected from a mixed population. This approach has a number of advantages over existing methods for transgene selection: 1) Large numbers of transgenic worms can be easily selected without the need for expensive, specialised equipment such as a worm-sorter. 2) Strains carrying extrachromosomal arrays with puromycin resistance can be maintained on puromycin plates without needing to pick individual worms. 3) Puromycin selection does not require a particular genetic background, eliminating the need to generate particular mutant strains for different species, which are often laborious to work with. We have successfully test ed puromycin selection in both C. elegans and C. briggsae and anticipate that this system could be useful for transgenesis in a variety of Caenorhabditis species. 4) We have developed a protocol for rapid drug selection following bombardment of N2 worms.
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[
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.
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[
International Worm Meeting,
2021]
DNA is an antiparallel double helix; its transcription requires separating the two strands. This creates supercoils, underwound and overwound duplex DNA, which accumulate behind and in front of the transcription bubble, respectively. Overwound supercoils are detrimental to helix opening and transcription bubble formation, as they increase the energy necessary to separate strands. In vivo, these structures are resolved by the action of topoisomerases which release supercoils from DNA. Conversely, the action of condensins, molecular machines creating loops in chromatin, has been shown in vitro to increase supercoiling. The interplay between supercoiling, transcription and condensin action could be a powerful regulator of gene expression. To understand this interplay, we mapped supercoiling genome-wide in vivo in L3 larvae using biotinylated 4,5,8-trimethylpsoralen (bTMP). bTMP preferentially intercalates in negatively supercoiled DNA and is crosslinked to it using UV illumination. Using biotin pulldown, we can then enrich for negatively supercoiled DNA before high-throughput sequencing. Our results show highly reproducible supercoiling profiles. At chromosome scale, we did not observe a significant difference in supercoiling between chromosomes. In contrast, telomeric thirds of autosomes (perinuclear heterochromatic B domains) show a lower bTMP enrichment than at central domains (euchromatic A domains), in agreement with higher transcription levels in euchromatin. At the gene level, bTMP enrichment shows two peaks, one 5' of the transcription start site (TSS) and one at the transcription end site (TES). This suggests the accumulation of negative supercoils in the promoter region of genes and at the end of the transcription units. In agreement with a transcriptional cause, the bTMP enrichment profile along the genes correlates with gene expression levels. In hermaphrodite animals, a variant of condensin I called condensin IDC, is part of the dosage compensation complex which downregulates gene expression of X-linked genes. Condensins I/IDC purified from nematodes can induce the formation of supercoils in vitro. In vivo, we observe differential bTMP enrichment at X-linked TSS, coinciding with the location of the condensin IDC. This suggests that supercoiling may link the X-specific transcriptional repression and condensin loading.
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[
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 vitromethylation 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.
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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.
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Semple, Jennifer, Ridder, Jeroen de, Meister, Peter, Allahyar, Amin, Das, Moushumi, Straver, Roy
[
International Worm Meeting,
2019]
Understanding how our genes are regulated is a central question in biology. Studies have highlighted multiple levels of regulation for genomic expression, ranging from transcription factors binding to nuclear organization of the genome. Recent technical optimization of chromosome conformation capture (3C) analysis has revealed new levels of chromosome compartmentalization. Interphase chromosomes are arranged into specific kilo- to mega-base sized domains, known as Topologically Associated Domains (TADs). Functionally, most enhancer-promoter interactions occur within the same TAD. Moreover, during differentiation, genes clustered within the same TAD display a similar expression pattern, and alteration of these domains result in aberrant expression of the genes, suggesting their involvement in transcriptional regulation. TADs appear remarkably stable between cell types, but can differ in the level of compaction inside individual TADs. TADs encompassing inactive genes are more compact than TADs comprising active ones. This raises the question whether TAD compaction directly regulates or somehow limits gene expression. This project aims at answering this question using a model system, dosage compensation (DC) in the nematode, Caenorhabditis elegans. It has been previously shown that on the X chromosome, more compact TADs correlate with the down-regulation of X-linked genes in hermaphrodites compared to males. This provides an ideal system to understand the mechanism of transcriptional regulation by TAD formation. I have setup a chromatin conformation capture technique coupled with long molecule sequencing (Oxford Nanopore Technology) to capture gene structures at high resolution, in DC and non-DC environment. In contrast to standard HiC that captures pair-wise contacts, this approach can directly identify multi-way chromosomal contacts from within single cells. My aim is to understand how chromatin structure is modified at the gene level by TAD formation and whether and how this regulates gene expression, thereby deciphering the importance of genome folding in transcriptional regulation.