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.

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

Isiaka, Bolaji, Meister, Peter, Krebs, Arnaud, Semple, Jennifer

[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.

Wojtovich, Andrew et al. (2015) International Worm Meeting "Chromophore-assisted light inactivation of mitochondrial respiratory chain complex II in C. elegans using a miniSOG mev-1 fusion."

Wojtovich, Andrew, Nehrke, Keith, Brookes, Paul, Foster, Thomas, Sherman, Teresa

[International Worm Meeting, 2015]

Mitochondria play a critical role in meeting cellular energy demand and are implicated in cell death, stress responses and reactive oxygen species (ROS) signaling. Respiratory complex II occupies a unique position within the mitochondrial metabolism machinery, being both a component of the electron transport chain and the Krebs cycle. Moreover, all four subunits of complex II are nuclear-encoded. A point mutation in mev-1, the gene encoding the SDHC-1 subunit of Complex II, increases ROS production and decreases lifespan, while loss-of-function is lethal. These phenotypes have been recapitulated in an orthologous mouse model, suggesting an evolutionarily conserved role for complex II. Here, we developed an optogenetic tool to study mitochondrial respiratory chain function and determine how complex II contributes to systems physiology. We expressed the genetically-encoded 'singlet oxygen generator' miniSOG as a Pmev-1::MEV-1::miniSOG::mev-1 (3' UTR) fusion in trans to the endogenous mev-1 gene as single-copy MosSCI insertion. We then used this strain to conduct Chromophore-Assisted Light Inactivation (CALI) of complex II activity. In response to blue light, miniSOG produces ROS that are highly reactive and act locally, allowing for the selective inactivation of a target protein. The mev-1::miniSOG transgene complemented a lethal mev-1(tm1081) deletion, suggesting that it was active and targeted to the mitochondrial inner membrane. We isolated mitochondria from the transgenic strain and found a photo-titratable loss of complex II activity with no effect on mitochondrial citrate synthase (Krebs cycle) or complex IV (respiratory chain) activities. Using this transgenic model we determined the effects of in-vivo acute complex II inactivation on physiologic outputs including development, brood size, stress resistance, activation of stress resistance pathways, and lifespan. Collectively, our results show that complex II activity is important under conditions of high energy demand or stress. This optogenetic approach provides spatial and temporal control over mitochondrial function and represents a new model to interrogate the role of complex II activity in mitochondrial metabolism.

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

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

[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.

Siu Sylvia Lee et al. (2001) International C. elegans Meeting "A genetic screen to identify novel players in C. elegans lifespan regulation"

Gary Ruvkun, Siu Sylvia Lee, Raymond Lee

[International C. elegans Meeting, 2001]

Multiple genetic pathways play a role in worm lifespan control. Most notably, mutations leading to reduced daf-2 insulin-like signaling extend the lifespan of C. elegans up to 4-fold. The longevity phenotype associated with daf-2(lf) is completely suppressed by loss-of-function mutations in the forkhead transcription factor daf-16 , suggesting daf-16 likely regulates genes that control C. elegans lifespan. To identify daf-16 targets, as well as genes in other parallel pathways, which regulate lifespan, we performed a genetic screen to identify mutants that exhibit an extended lifespan phenotype in a daf-16 null background. From a pilot screen of 1600 haploid genomes, we recovered three independent mutants with lifespans significantly longer than the starting daf-16(mgDf47) strain. Among the three mutants, age-3(mg312) displayed the greatest lifespan extension, up to 3-fold that of control strain. age-3(mg312) is pleiotropic, including slight uncoordinated movement, arrested germ line development and sterility. These pleiotropies allowed rapid mapping of this mutation to a small region around +2 on chromosome I. Cosmid rescue and sequencing of genes in the region suggested that age-3(mg312) resulted from a nonsense mutation in the only C. elegans leucyl-tRNA synthetase gene. Single gene rescue experiments and further characterization of age-3 are underway. While it is not clear how such a fundamental player of gene expression specifically regulates lifespan, other basic cell machinery components have been previously implicated in longevity, including the WRN DNA helicase in human, the Indy Krebs cycle transporter in Drosophila , and the SIR2 histone deacetylase in worm and yeast. On the other hand, given the lifespan effects of germ line ablations, the sterility associated with age-3 could indirectly contribute to its lifespan phenotype. Phenotypic analysis and mapping of the other two longevity mutations is also in progress. In addition, a large-scale genetic screen to isolate more mutations with an extended lifespan phenotype is ongoing. Taking into account that mutations which affect global metabolism may modulate C. elegans lifespan, as well as other developmental processes, mutants with significant extension in lifespan, but no other pleiotropies, will be of the highest priority for mapping and cloning. Furthermore, we are also using the recently generated chromosome I RNAi library (A. Fraser, et. al ., Nature, 2000.) to survey for genes which, when inactivated, result in long lifespan.

Gallo, M. et al. (2009) International Worm Meeting "The longevity gene misc-1 modulates apoptosis in C. elegans and human cell lines."

Gallo, M., Riddle, D.

[International Worm Meeting, 2009]

We identified and characterized the C. elegans orthologue of human 2-oxoglutarate carrier (OGC) and called it misc-1 (MItochondrial Solute Carrier). The human orthologue is responsible for importing a-ketoglutarate, an intermediate of the Krebs cycle, and glutathione (GSH), a detoxifying molecule, into mitochondria. Because of its role in metabolism and detoxification, we thought misc-1 was a candidate longevity gene. misc-1 RNAi increased wild type and daf-2 mean adult lifespan by 20% and 42%, respectively, but it failed to induce significant longevity in eat-2 mutants, suggesting that misc-1 may be in the dietary restriction pathway. Unlike Mit mutants, misc-1 knock-down and knock-out allow a normal rate of pharyngeal pumping, normal body size and developmental timing. We hypothesized the absence of Mit phenotypes was due to the activation of compensatory detoxifying pathways in misc-1 mutants. We assessed gene expression levels for the sod genes. Only the Cu/ZnSOD sod-5 was upregulated (by ~40%) in misc-1. sod-5 is a known target of DAF-16 and strongly upregulated in daf-2 and dauer larvae. It is known that H2O2 - the end product of the dismutase reaction catalyzed by SOD-5 - inhibits most steps in the insulin signalling pathway. We believe misc-1 may modulate life-span by increasing sod-5 expression, reducing insulin signaling and causing a metabolic shift from glycolysis/mitochondrial respiration to other mitochondria-independent pathways, such as the glyoxylate cycle. misc-1 RNAi results in mitochondrial fragmentation. siRNA targeting OGC in HEK293 (Human Embryonic Kidney) cells also resulted in mitochondrial fragmentation. We showed that germline apoptosis in misc-1 knock-out animals is increased two-fold compared to wild-type N2, but that mitochondrial fragmentation alone is not sufficient to increase the apoptotic rate. It is known that OGC over-expression in human cells protects against chemically induced apoptosis. Germline apoptosis in C. elegans can be triggered by three different pathways, namely the DNA damage, physiological and stress-induced pathways. All pathways ultimately converge on the core apoptotic machinery composed of CED-9/Bcl-2, CED-4/Apaf1 and CED-3/Caspase-9. Epistasis experiments showed that misc-1 acts through the physiological apoptotic pathway and is dependent on LET-60/KRas. We propose a conserved, mitochondria-driven apoptosis mechanism dependent on control of mitochondrial Ca2+ stores by MISC-1 and CED-9/Bcl-2 and impinging on Ca2+ signalling mediated by LET-60/KRas.