Amrit, Francis R.G. et al. (2010) Worm Breeder's Gazette "Differential expression of daf-16 splice variants"

May, Robin C., Amrit, Francis R.G.

[Worm Breeder's Gazette, 2010]

AMRIT, FRANCIS et al. (2017) International Worm Meeting "The Coordination of Reproductive Success and Stress Resilience by TCER-1/TCERG1."

Rana, Mainpal, Ghazi, Arjumand, AMRIT, FRANCIS, MASON, CARTER, Yanowitz, Judith

[International Worm Meeting, 2017]

Reproduction and stress-resistance capacity are life-history traits that govern the evolutionary success of all organisms. However, molecular mechanisms by which these processes are coordinated in metazoans are poorly described. Here, we report a role for TCER-1, the worm homolog of human transcription elongation and splicing factor, TCERG1, in enhancing reproductive capacity in the face of pathogen attack. In C. elegans, eliminating germline stem cells (GSCs) increases lifespan and elevates stress resistance. Previously, we demonstrated that TCER-1/TCERG1 specifically promotes the longevity of germline-less C. elegans but is critical for reproductive health in normal, fertile animals. Many genes that promote the longevity of GSC-less adults also enhance their stress tolerance. Surprisingly, we have discovered that TCER-1/TCERG1 inhibited stress tolerance in adult worms. tcer-1 mutants exhibited elevated resistance against multiple biotic and abiotic stressors, including thermal stress, oxidative stress, ER stress and infection by the opportunistic pathogen Pseudomonas aeruginosa. TCER-1/TCERG1 impairs stress resistance by inhibiting PMK-1, a conserved innate immunity-promoting kinase. PMK-1-target genes are up-regulated in tcer-1 mutants and the immunoresistance of tcer-1 mutants is dependent upon PMK-1. Overall, our data suggest that TCER-1/TCERG1, promotes reproductive fitness and represses stress resilience under normal conditions. Under stressful conditions, TCER-1/TCERG1 is repressed resulting enhanced stress resilience and reduced reproductive capacity. Unlike most pro-longevity genes, TCER-1/TCERG1 appears to have distinct regulatory effects on lifespan, stress resistance, and fertility, suggesting that the protein may function as a molecular rheostat for coordinating major life-history traits. The phenotypic uncoupling observed in tcer-1 mutants provides a unique platform to dissect the pathway that governs resource allocation between procreation, stress response and somatic maintenance in metazoans.

Amrit, Francis RG et al. (2021) International Worm Meeting "TCER-1-regulated alternative splicing promotes stress resilience"

Ghazi, Arjumand, Amrit, Francis RG, KUROYANAGI, Hidehito, Loose, Julia, Pfenning, Andreas

[International Worm Meeting, 2021]

Alternative RNA splicing augments proteomic biodiversity in eukaryotes, and has an emerging role in regulating longevity. This process has been shown to have various roles based on physiological demands including those in response to external stimuli. Here, we show that in the nematode Caenorhabiditis elegans, exposure to the Gram-negative pathogen, Pseudomonas aeruginosa, induces a shift in alternative splicing. In vivo analysis of transgenic reporters of splicing fidelity showed significant splicing changes in multiple tissues. Based on our recent discovery that TCER-1, worm homolog of the human transcription elongation and splicing factor, TCERG1, represses innate immunity in worms, we examined the role of TCER-1 in the modulation of splicing upon pathogen exposure. tcer-1 mutants exhibited aberrant splicing regulation in multiple tissues, and transcriptomic analysis identified 54 exon-skipping and 468 intron retention events upon tcer-1 inactivation. In vivo analyses of these events revealed a TCER-1 dependent splicing pattern that correlated with our stress resilience phenotypes. Further, CRISPR-mediated recreation of alternative splicing observed in tcer-1 mutants enhanced immunoresistance and motor function. Together our findings bring to the forefront the important role of alternative splicing in stress resilience and healthspan, and reveal TCER-1- to be a major regulator of this process.

RG Amrit, Francis et al. (2013) International Worm Meeting "The Dual Roles of TCER-1/TCERG1 in Balancing Reproductive Fitness and Longevity."

Ghazi, Arjumand, RG Amrit, Francis, Benos, Takis, Arora, Arshi

[International Worm Meeting, 2013]

The debilitating effects of advacing age on fertility are well-known, but recent studies have shown that reproductive status reciprocally alters aging. We describe a role for TCER-1, the worm homolog of human transcription elongation factor, TCERG1, in establishing the balance between reproductive efficiency and somatic maintenance based on the procreative status of the animal. In C. elegans, removal of germline-stem cells (GSCs) extends lifespan. TCER-1/TCERG1 promotes longevity specifically upon GSC loss, likely by influencing the activity of the conserved longevity determinant, DAF-16/FOXO. We used massively parallel RNA-Sequencing to map the contribution of TCER-1/TCERG1 and DAF-16/FOXO to the transcriptional profile of GSC(-) worms. We found that these proteins share about a third of their transcriptomes and jointly up-regulate a spectrum of genes that alter lipid metabolism and xenobiotic-stress resistance. These targets improve cellular homeostasis and are essential for the longevity of the sterile adult. Strikingly, TCER-1/TCERG1 represses more genes than it activates in GSC(-) worms, and these genes are highly enriched for reproductive roles. We found that tcer-1 mutants have reduced fecundity under normal conditions, including diminished brood size, poor egg viability and premature unfertilized-oocyte production. tcer-1 is expressed in the germline and its mutant phenotypes can be attributed to both defective sperm and eggs. Accordingly, many genes repressed by TCER-1/TCERG1 in GSC(-) worms are important for reproduction and reducing their functions impairs fertility. Our results reveal that TCER-1/TCERG1 plays a dynamic function depending on the reproductive status of the worm. In fertile adults, it promotes reproductive fitness. Upon GSC loss, it represses the expression of reproduction genes, and (in combination with DAF-16/FOXO) elevates the expression of pro-longevity genes. This molecular dexterity of TCER-1/TCERG1 facilitates communication between the germline and the soma, and orchestrates the balance between reproductive vigor and somatic preservation.

Amrit, Francis RG et al. (2015) International Worm Meeting "Molecular Jekyll and Hyde: The Opposing Roles of TCER-1/TCERG1 in Regulating Lifespan and Stress Resilience."

Amrit, Francis RG, Steenberge, Laura, Yanowitz, Judith, McClendon, Brooke, Ghazi, Arjumand

[International Worm Meeting, 2015]

Longevity and stress resistance are intimately linked. In worms, and in many other species, animals that exhibit increased lifespan almost inevitably also display enhanced resistance to various stressors. In most cases, genes that promote longevity also contribute to improved stress resilience. For instance, glp-1 mutants that are long-lived due to elimination of the proliferating germine-stem cells (GSCs) are also highly stress-resistant, and both the long life and stress-resilience of these animals is dependent on the longevity determinant, DAF-16/FOXO. Previously, we identified a conserved transcription elongation and splicing factor, TCER-1/TCERG1, that cooperates with DAF-16/FOXO to selectively augment the longevity of germline-depleted animals.Here, we demonstrate that, unlike DAF-16/FOXO, TCER-1 does not contribute to stress-resistance, instead it represses the animals' ability to combat adversity. tcer-1 reduction-of-function confers resistance to a multitude of biotic and abiotic stressors, both in fertile animals as well as sterile mutants. Using site of action studies and molecular genetic analysis, we have explored the basis of this phenotypic uncoupling. Our experiments reveal a unique role for TCER-1/TCERG1 in balancing length of life, reproductive health and stress resistance during adult life. These data will be discussed.

Amrit, Francis RG et al. (2015) International Worm Meeting "Transcription Factors that Coordinate Lipid Synthesis and Breakdown to Ensure Metabolic Homeostasis and Longevity."

Steenkiste, Elizabeth, Ratnappan, Ramesh, Amrit, Francis RG, Olsen, Carissa P, Yanowitz, Judith L, McClendon, T Brooke, Chen, Shaw-Wen

[International Worm Meeting, 2015]

The coordination of lipid production and degradation is essential for animals' health - lipid imbalances are characteristic of obesity and a feature of many reproductive pathologies and age-related diseases. But how these processes are balanced in multicellular organisms is poorly understood. In C. elegans, removing the germline, using mutations in genes such as glp-1, extends lifespan. Germline loss is a major metabolic challenge that compels the animal to stop fat deposition into eggs and remodel its lipid reserves. This phenomenon provides a unique platform to understand how complex metazoans retain metabolic homeostasis when challenged with alterations in fertility and age. Recent studies, including ours, have shown that germline loss activates conserved transcription factors such as DAF-16/FOXO and TCER-1/TCERG1 in the intestine, the worms' main fat depot. We recently demonstrated that the nuclear hormone receptor, NHR-49/PPARalpha, under partial regulation by DAF-16/FOXO and TCER-1/TCERG1, promotes glp-1 mutants' longevity by elevating fatty-acid beta-oxidation and desaturation- pathways involved in fat buildup and breakdown, respectively. We have now discovered that both lipid synthesis and degradation are widely and concurrently enhanced in response to germline loss. Using transcriptomics, molecular-genetic analyses and biochemical approaches, we demonstrate that these transcription factors activate multiple lipid anabolic (de novo lipid synthesis, fatty-acid desaturation and elongation and triglyceride production) and catabolic (triglyceride hydrolysis, fatty-acid oxidation) pathways in glp-1 mutants. Our data suggest that the coordinated enhancement of lipid synthesis and breakdown, by NHR-49, DAF-16 and TCER-1, facilitates the adaptation to germline loss by ensuring lipid homeostasis, and mediates longevity.

Francis Amrit et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "Evolution and Significance of DAF-16 signalling in the Caenorhabditis genus"

Francis Amrit, Robin May

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

In nematodes, lifespan and stress resistance have been shown to have both a gender specific and species specific variability and previous work has suggested that there is a common molecular mechanism or a molecular link which governs these processes. Using various Caenorhabditis species we are investigating whether the transcription factor DAF-16, a downstream component of the Insulin like/IGF-1 signalling pathway, acts as this molecular link. DAF-16 is orthologous to the FOXO family of transcription factors in mammals and is thought to play a key role in stress response, immune response and ageing by controlling expression of several hundred genes. We have shown that hermaphrodite species within the Caenorhabditis genus seem to age faster and possess a weaker immune response in comparison to the male/female (gonochoristic) species. This work focuses on establishing that it is the difference in expression of the DAF-16 transcription factor and its activity which is responsible for the difference in immune response and hence the difference in ageing among the various Caenorhabditid species. We propose that this could also explain the evolutionary conundrum of post-reproductive ageing since enhanced immunity would also increase lifespan even after the animal has reproduced.

Gandhi, Francis Amrit Raj et al. (2009) International Worm Meeting "Divergence in DAF-16 dependent phenotypic coupling within the Caenorhabditid nematodes."

Gandhi, Francis Amrit Raj, Boehnisch, Claudia, May, Robin

[International Worm Meeting, 2009]

In animals, ageing, immunity and stress tolerance are co-ordinately regulated by the forkhead transcription factors in response to upstream signals from Insulin-like growth factor-like signalling pathway. Previous work has emphasized that the transcription factor DAF-16, a key evolutionarily conserved gene regulator, functions as a "molecular link" that governs these processes. Although daf-16 is highly conserved in a broad range of organisms, it is unclear how the downstream co-regulation of immunity, lifespan and stress resistance changes during evolution. In nematodes, these phenotypes have been shown to have both a gender specific and species-specific variability. Here for the first time we investigate this phenotypic coupling by comparing ageing, stress-resistance and immunity in four species of Caenorhabditid nematode. We show that among these species there is a significant difference in expression levels between the daf-16 homologues. This expression level, correlates directly with the observed phenotypes of lifespan, stress resistance and immunity. Using constitutively active daf-16 mutants, backed by bioinformatic studies looking at daf-16 target regions, we propose that the divergence of DAF-16 responses, depending upon the niche and life history traits of the species concerned, could explain differences in stress response, immunity and aging among species. Finally, we suggest that the evolutionary conundrum of post-reproductive aging could be explained since enhanced immunity would also increase lifespan even after the animal has reproduced.

Naim, Nikki et al. (2021) International Worm Meeting "Modulation of small RNA pathways suppresses innate immunity"

Montgomery, Taiowa, Ghazi, Arjumand, Naim, Nikki, AMRIT, FRANCIS

[International Worm Meeting, 2021]

Procreation is widely known to cause immunosuppression yet the mechanisms by which immunity is suppressed during peak fertility remain unknown. tcer-1, which encodes the Caenorhabditis elegans homolog of the human transcription elongation and splicing factor, TCERG1, is once such gene involved in coordinating the immunity-fertility relationship. Our lab showed that TCER-1 is essential for healthy reproduction and prevention of age-related reproductive decline, and that it enhances lifespan in response to reproductive signals by ensuring lipid homeostasis. Our preliminary experiments have suggested that TCER-1 suppresses immunity, in part, through regulation of endogenous small RNA (smRNA) pathways. smRNA are short (~18-30nt) RNA molecules known for their ability to silence self and foreign genetic material to regulate host gene expression. Despite the fact endogenous smRNAs are reported to regulate immune-defense genes and microbes target host smRNA pathways during infection, little is known about the molecular mechanisms involved. We have discovered that loss-of-function mutations in biogenesis genes for endogenous small interfering RNAs (siRNAs) induce exceptional resistance against the human opportunistic pathogen Pseudomonas aeruginosa. Our epistasis analyses have demonstrated that these genes act in the same genetic pathway as TCER-1 to repress immunity. smRNA sequencing has also shown that tcer-1 mutation causes a ~10% reduction in total endogenous siRNAs. These observations, and other data, have led us to hypothesize that TCER-1 suppresses innate immunity through modulation of the endogenous siRNA pathway and their targeted immunoresistance genes.

Ratnappan, Ramesh et al. (2013) International Worm Meeting "Molecular Outsourcing: Reproductive Signals Deploy NHR-49/PPARa to Reorganize Lipid Homeostasis and Alter Lifespan."

Ratnappan, Ramesh, Ghazi, Arjumand, Gill, Hasreet, Ward, Jordan, Holden, Kyle, RG Amrit, Francis, Yamamoto, Keith

[International Worm Meeting, 2013]

Aging is an inherently entropic but remarkably plastic phenomenon. The rate of aging can be altered by modifications to an energy-intensive process such as reproduction. In worms, removal of the Germline-Stem Cells (GSCs) results in increased fat accumulation and the activation of a transcriptional network in intestinal cells that extends lifespan. Two key regulators in this network are DAF-16/FOXO and TCER-1/TCERG1. In a screen designed to identify nuclear hormone receptors (NHRs) that impact DAF-16/FOXO and TCER-1/TCERG1 function, one of the NHRs we isolated was NHR-49/PPARa, a conserved and crucial modulator of energy and fat metabolism. Independently, we also identified nhr-49 as a gene jointly upregulated by DAF-16/FOXO and TCER-1/TCERG1 through RNA-Seq transcriptomics. Predictably, nhr-49 mutation suppressed the longevity of GSC(-) worms, but did not appear to change the overall elevated fat content. A GFP-tagged translational reporter showed expression in the cytoplasm and nuclei of all somatic cells and rescued nhr-49(-) phenotypes. NHR-49/PPARa overexpression increased the lifespan of nhr-49 mutants substantially without a concomitant loss of fertility. NHR-49/PPARa regulated the expression of fatty-acid desaturases, specific mitochondrial b-oxidation pathway genes and acted in a feedback loop to increase DAF-16/FOXO and TCER-1/TCERG1 activity. At least part of these transcriptional changes may be brought about in co-operation with NHR-71, another NHR identified in our screen that interacted with both NHR-49/PPARa and TCER-1/TCERG1 in yeast two-hybrid experiments. Our results suggest that upon GSC loss, NHR-49/PPARa is recruited to fundamentally remodel the lipid metabolic profile of the worm. This reorganization not only allows the mobilization of fat that would otherwise have been transported to the oocytes, but also induces transcriptional and signaling events that direct the allotment of cellular resources towards somatic maintenance and longevity.