Richard Roy
McGill University, Biology, Faculty Member
Developmental plasticity refers the ability of an organism to adapt to various environmental stressors, one of which is nutritional stress. Caenorhabditis elegans require various nutrients to successfully progress through all the larval... more
Developmental plasticity refers the ability of an organism to adapt to various environmental stressors, one of which is nutritional stress. Caenorhabditis elegans require various nutrients to successfully progress through all the larval stages to become a reproductive adult. If nutritional criteria are not satisfied, development can slow or completely arrest. In poor growth conditions, the animal can enter various diapause stages, depending on its developmental progress. In C. elegans, there are three well-characterized diapauses: the L1 arrest, the dauer diapause, and adult reproductive diapause, each associated with drastic changes in metabolism and germline development. At the centre of these changes is AMP-activated protein kinase (AMPK). AMPK is a metabolic regulator that maintains energy homeostasis, particularly during times of nutrient stress. Without AMPK, metabolism is disrupted during dauer, leading to the rapid consumption of lipid stores as well as misregulation of metabolic enzymes, leading to reduced survival. During the L1 arrest and dauer diapause, AMPK is responsible for ensuring germline quiescence by modifying the germline chromatin landscape to maintain germ cell integrity until conditions improve. Similar to classic hormonal signalling, small RNAs also play a critical role in regulating development and behaviour in a cell non-autonomous fashion. Thus, during the challenges associated with developmental plasticity, AMPK summons an army of signalling pathways to work collectively to preserve reproductive fitness during these periods of unprecedented uncertainty.
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During periods of energetic stress, Caenorhabditis elegans can undergo a global quiescent stage known as “dauer”. During this stage, all germline stem cells undergo G2 cell cycle arrest through an AMPK-dependent mechanism. In animals that... more
During periods of energetic stress, Caenorhabditis elegans can undergo a global quiescent stage known as “dauer”. During this stage, all germline stem cells undergo G2 cell cycle arrest through an AMPK-dependent mechanism. In animals that lack AMPK signalling, the germ cells fail to arrest, undergo uncontrolled proliferation and lose their reproductive capacity. These germline defects are accompanied by an altered chromatin landscape and gene expression program. We identified an allele of tbc-7, a RabGAP protein that functions in the neurons, which when compromised, suppresses the germline hyperplasia in the dauer larvae, as well as the post-dauer sterility and somatic defects characteristic of AMPK mutants. This mutation also corrects the abundance and aberrant distribution of transcriptionally activating and repressive chromatin marks in animals that otherwise lack all AMPK signalling. We identified RAB-7 as one of the potential RAB proteins that is regulated by tbc-7 and show tha...
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Environmental variation experienced early in life can result in long-term reproductive consequences. We have recently identified an important role for AMPK in the prevention of transgenerational defects following starvation of L1 stage... more
Environmental variation experienced early in life can result in long-term reproductive consequences. We have recently identified an important role for AMPK in the prevention of transgenerational defects following starvation of L1 stage larvae in C. elegans. Here we describe a means of analyzing these transgenerational defects following a single exposure to energy stress during early larval development. We also provide methods to quantify the histone modifications that are affected by this stress, along with the resulting reproductive defects that arise in later generations.
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5'-AMP-activated protein kinase (AMPK) has been called "the metabolic master switch" because of its central role in regulating fuel homeostasis. AMPK, a heterotrimeric serine/threonine... more
5'-AMP-activated protein kinase (AMPK) has been called "the metabolic master switch" because of its central role in regulating fuel homeostasis. AMPK, a heterotrimeric serine/threonine protein kinase composed of alpha, beta, and gamma subunits, is activated by upstream kinases and by 5'-AMP in response to various nutritional and stress signals. Downstream effects include regulation of metabolism, protein synthesis, cell growth, and mediation of the actions of a number of hormones, including leptin. However, AMPK research represents a young and growing field; hence, there are many unanswered questions regarding the control and action of AMPK. This review presents evidence for the existence of AMPK signaling pathways in Caenorhabditis elegans, a genetically tractable model organism that has yet to be fully exploited to elucidate AMPK signaling mechanisms.
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C. elegans cki-1 encodes a member of the CIP/KIP family of cyclin-dependent kinase inhibitors, and functions to link postembryonic developmental programs to cell cycle progression. The expression pattern of cki-1::GFP suggests that cki-1... more
C. elegans cki-1 encodes a member of the CIP/KIP family of cyclin-dependent kinase inhibitors, and functions to link postembryonic developmental programs to cell cycle progression. The expression pattern of cki-1::GFP suggests that cki-1 is developmentally regulated in blast cells coincident with G1, and in differentiating cells. Ectopic expression of CKI-1 can prematurely arrest cells in G1, while reducing cki-1 activity by RNA-mediated interference (RNAi) causes extra larval cell divisions, suggesting a role for cki-1 in the developmental control of G1/S. cki-1 activity is required for the suspension of cell cycling that occurs in dauer larvae and starved L1 larvae in response to environmental signals. In vulva precursor cells (VPCs), a pathway of heterochronic genes acts via cki-1 to maintain VPCs in G1 during the L2 stage.
Research Interests: Caenorhabditis elegans, Biology, Life Sciences, Cell Cycle, Enzyme Inhibitors, and 14 moreCell Biology, Development, Medicine, Cell Division, Starvation, Biological Sciences, RNA interference, Cell Differentiation, Animals, Cell Molecular Biology and Developmental Biology, Cell Cycle Proteins, GFP, Gonads, and Medical and Health Sciences
During starvation, organisms modify both gene expression and metabolism to adjust to the energy stress. We previously reported that Caenorhabditis elegans lacing AMP-activated protein kinase (AMPK) exhibit transgenerational reproductive... more
During starvation, organisms modify both gene expression and metabolism to adjust to the energy stress. We previously reported that Caenorhabditis elegans lacing AMP-activated protein kinase (AMPK) exhibit transgenerational reproductive defects associated with abnormally elevated trimethylated histone H3 at lysine 4 (H3K4me3) levels in the germ line following recovery from acute starvation. Here, we show that these H3K4me3 marks are significantly increased at promoters, driving aberrant transcription elongation resulting in the accumulation of R-loops in starved AMPK mutants. DNA-RNA immunoprecipitation followed by high-throughput sequencing (DRIP-seq) analysis demonstrated that a significant proportion of the genome was affected by R-loop formation. This was most pronounced in the promoter–transcription start site regions of genes, in which the chromatin was modified by H3K4me3. Like H3K4me3, the R-loops were also found to be heritable, likely contributing to the transgenerational ...
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<p><b>(A)-(B)</b> Signal overlap between ATGL-1::GFP (Green) and C<sub>1</sub>-BODIPY-C<sub>12</sub>-stained lipid droplets (Red) was compared in control <i>daf-2</i> and... more
<p><b>(A)-(B)</b> Signal overlap between ATGL-1::GFP (Green) and C<sub>1</sub>-BODIPY-C<sub>12</sub>-stained lipid droplets (Red) was compared in control <i>daf-2</i> and <i>daf-2; aak(0)</i> mutant animals at 32 <b>(A)</b> and 48 hours <b>(B)</b> after shifting to restricted temperature. ATGL-1::GFP signal was closely associated with the labeled lipid droplets in <i>daf-2; aak(0)</i> mutant animals (white arrowheads in the insets) while the signals are clearly distinguishable from each other in control <i>daf-2</i> animals. Scale bar = 10μm. Insets were generated by selecting the same size of frame on each image and amplified by the same magnification. <b>(C)</b> Western blot analysis of the endogenous ATGL-1 levels in isolated lipid droplets (L) and cytoplasm (C) obtained from total day 0 (48 hours after shifting to restricted temperature) dauer extracts of control <i>daf-2</i> and <i>daf-2; aak(0)</i> mutant animals. Protein concentration was measured and 30μg of total protein was loaded in each sample lane. Actin was used as a loading control for the total protein level according to the recent proteomic study on <i>C</i>. <i>elegans</i> lipid droplets [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130480#pone.0130480.ref039" target="_blank">39</a>]. <b>(D)</b> Lipid droplet isolation method verified by significant C<sub>1</sub>-BODIPY-C<sub>12</sub> staining and triglyceride enrichment in the isolated lipid droplets portion comparing to the cytoplasm (remaining portion of the total lysate) from <i>daf-2</i> day 0 dauer larvae.</p
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<p><b>(A)</b> CGI-58::GFP was expressed in the hypodermis and the intestine at comparable levels in both control <i>daf-2</i> and <i>daf-2; aak(0)</i> mutant dauer larvae. Both strains carry the... more
<p><b>(A)</b> CGI-58::GFP was expressed in the hypodermis and the intestine at comparable levels in both control <i>daf-2</i> and <i>daf-2; aak(0)</i> mutant dauer larvae. Both strains carry the same <i>ex[Pcgi-58</i>::<i>cgi-58</i>::<i>GFP; rol-6(gf)]</i> transgene (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005284#sec010" target="_blank">Materials and Methods</a>). Scale bar = 20 μm. <b>(B)</b> CGI-58::GFP localizes to the surface of the lipid droplets at dauer day 0 (48 hours after being shifted to 25°C at the L1 stage) in both <i>daf-2</i> and <i>daf-2; aak(0)</i> mutant dauers. Both strains carry the same <i>ex[Pcgi-58</i>::<i>cgi-58</i>::<i>GFP; rol-6(gf)]</i> transgene. Scale bar = 10μm. Insets (B' and B") were generated by selecting the same size of frame for each image followed by proportionate amplification to the same magnification. <b>(C)</b> Optimal ATGL-1 lipase activity requires <i>cgi-58</i>. ATGL-1-dependent lipase activity was determined in <i>daf-2; aak(0)</i> mutant dauer larvae with wild type or compromised <i>cgi-58/atgl-1</i> function. ** indicates statistical significance (P<0.01) compared to all three of the other genotypes. Error bars indicate SD of three independent experiments. <b>(D)</b> Elimination of <i>cgi-58</i> resulted in the dissociation of ATGL-1 from the lipid droplets. Both strains carry the same <i>hjIs67[Patgl-1</i>::<i>atgl-1</i>::<i>GFP]</i> transgene. Scale bar = 10μm. Insets (D' and D") were generated by selecting the same size of frame for each image followed by proportionate amplification to the same final magnification. <b>(E)</b> ATGL-1 association with the lipid droplets is dependent on appropriate CGI-58 levels. Immunoblot analysis was used to determine the levels of ATGL-1 in isolated lipid droplets (L) and cytoplasm (C) obtained from total day 0 dauer extracts of each genotype. Protein concentration was measured and 30μg of total protein was loaded in each sample lane. Actin was used as a loading control for the total protein level. <b>(F)</b> CGI-58 does not contribute to ATGL-1 stability in AMPK mutant dauers. ATGL-1 levels were determined by immunobloting using anti-ATGL-1 antisera in lysates obtained from AMPK mutants with or without <i>cgi-58</i>. <b>(G)</b> CGI-58 physically interacts with ATGL-1 in vivo in both control and AMPK mutant dauers. Co-immunoprecipitations were performed with <i>daf-2</i> and <i>daf-2; aak(0)</i> day 0 dauer larvae carrying the same <i>ex[Pcgi-58</i>::<i>cgi-58</i>::<i>GFP;rol-6(gf)]</i> transgene using anti-ATGL-1 serum for pull down and blotted with anti-GFP serum.</p
Research Interests: Biochemistry, Genetics, Physiology, Biophysics, Developmental Biology, and 4 moreCell Biology, CGI, PUFA, and Function
<p><b>(A)</b> Comparison of ATGL-1::GFP levels between control <i>daf-2</i> and <i>daf-2; aak(0)</i> animals during the early dauer stage. Dauer day 1 is defined as 72 hours after shifting to... more
<p><b>(A)</b> Comparison of ATGL-1::GFP levels between control <i>daf-2</i> and <i>daf-2; aak(0)</i> animals during the early dauer stage. Dauer day 1 is defined as 72 hours after shifting to restrictive temperature (25°C) at the L1 stage (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130480#sec002" target="_blank">materials and methods</a>). Scale bar = 10μm. <b>(B)</b> Western blot analysis of GFP levels in control <i>daf-2</i> and <i>daf-2; aak(0)</i> mutant animals during early dauer stage. <b>(C)</b> Western blot analysis of endogenous ATGL-1 levels in control <i>daf-2</i> and <i>daf-2; aak(0)</i> mutant animals during early dauer stage. <b>(D)</b> Quantification of ATGL-1::GFP mRNA levels in control <i>daf-2</i> and <i>daf-2; aak(0)</i> mutant dauer day 4 animals using semi-quantitative RT-PCR. <i>act-1</i> was used as loading control.</p
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During periods of starvation organisms must modify both gene expression and metabolic pathways to adjust to the energy stress. We previously reported that C. elegans that lack AMPK have transgenerational reproductive defects that result... more
During periods of starvation organisms must modify both gene expression and metabolic pathways to adjust to the energy stress. We previously reported that C. elegans that lack AMPK have transgenerational reproductive defects that result from abnormally elevated H3K4me3 levels in the germ line following recovery from acute starvation1. Here we show that H3K4me3 is dramatically increased at promoters, driving aberrant transcription elongation that results in the accumulation of R-loops in the starved AMPK mutants. DRIP-seq analysis demonstrated that a significant proportion of the genome was affected by R-loop formation with a dramatic expansion in the number of R-loops at numerous loci, most pronounced at the promoter-TSS regions of genes in the starved AMPK mutants. The R-loops are transmissible into subsequent generations, likely contributing to the transgenerational reproductive defects typical of these mutants following starvation. Strikingly, AMPK null germ lines show considerab...
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Regulation of germline stem cell proliferation downstream of nutrient sensing
When faced with suboptimal growth conditions, Caenorhabditis elegans larvae can enter a diapause-like stage called "dauer" that is specialized for dispersal and survival. The decision to form a dauer larva is controlled by three... more
When faced with suboptimal growth conditions, Caenorhabditis elegans larvae can enter a diapause-like stage called "dauer" that is specialized for dispersal and survival. The decision to form a dauer larva is controlled by three parallel signaling pathways, whereby a compromise of TGFβ, cyclic guanosine monophosphate, or insulin/IGF-like signaling (ILS) results in dauer formation. Signals from these pathways converge on DAF-12, a nuclear hormone receptor that triggers the changes required to initiate dauer formation. DAF-12 is related to the vitamin D, liver-X, and androstane receptors, and like these human receptors, it responds to lipophilic hormone ligands. When bound to its ligand, DAF-12 acquires transcriptional activity that directs reproductive development, while unliganded DAF-12 forms a dauer-specifying complex with its interacting protein DIN-1S to regulate the transcription of genes required for dauer development. We report here that din-1S is required in parall...
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Stem cells have recently attracted significant attention largely due to their potential therapeutic properties, but also because of their role in tumorigenesis and their resemblance, in many aspects, to cancerous cells. Understanding how... more
Stem cells have recently attracted significant attention largely due to their potential therapeutic properties, but also because of their role in tumorigenesis and their resemblance, in many aspects, to cancerous cells. Understanding how stem cells are regulated, namely with respect to the control of their proliferation and differentiation within a functional organism, is thus primordial to safely profit from their therapeutic benefits. Here, we review recent advances in the understanding of germline stem cell proliferation control by factors that respond to the nutritional status and/or insulin signaling, through studies performed in C. elegans and Drosophila. Together, these data uncover some shared fundamental features that underlie the central control of cellular proliferation within a target stem cell population in an organism. These features may indeed be conserved in higher organisms and may apply to various other stem cell populations.
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Genetic analysis has demonstrated that the decision to execute dauer development is controlled by three parallel pathways. Little is known however concerning the downstream effectors common to these three pathways that mediate the... more
Genetic analysis has demonstrated that the decision to execute dauer development is controlled by three parallel pathways. Little is known however concerning the downstream effectors common to these three pathways that mediate the important metabolic and ...
The genetic basis underlying C. elegans adult lifespan regulation is emerging from studies carried out in several laboratories. It appears that, among others, genes regulating dauer formation such as those involved in insulin signaling,... more
The genetic basis underlying C. elegans adult lifespan regulation is emerging from studies carried out in several laboratories. It appears that, among others, genes regulating dauer formation such as those involved in insulin signaling, have a dramatic effect on ...
During adverse environmental conditions, C. elegans larvae can enter a diapause-like stage called" dauer", and three parallel signalling pathways are implicated in this decision (insulin-like, TGF-, or cGMP). The dauer larva is... more
During adverse environmental conditions, C. elegans larvae can enter a diapause-like stage called" dauer", and three parallel signalling pathways are implicated in this decision (insulin-like, TGF-, or cGMP). The dauer larva is specialized for long-term survival ...
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In C. elegans, reduced insulin-like signalling induces developmental quiescence, reproductive delay and lifespan extension. We show here that the C. elegans orthologues of LKB1 and AMPK cooperate during conditions of reduced insulin-like... more
In C. elegans, reduced insulin-like signalling induces developmental quiescence, reproductive delay and lifespan extension. We show here that the C. elegans orthologues of LKB1 and AMPK cooperate during conditions of reduced insulin-like signalling to establish cell cycle quiescence in the germline stem cell population, in addition to prolonging lifespan. The inactivation of either protein causes aberrant germline proliferation during diapause-like `dauer' development, whereas the loss of AMPK uncouples developmental arrest from lifespan extension. Reduced TGF-β activity also triggers developmental quiescence independent of the insulin-like pathway. Our data suggest that these two signalling pathways converge on the C. elegans PTEN orthologue to coordinate germline proliferation with somatic development during dauer formation, via the regulation of AMPK and its upstream activator LKB1, rather than through the canonical insulin-like signalling cascade. In humans, germline mutatio...
Research Interests: Caenorhabditis elegans, Biology, Cell Cycle, Cell Biology, Transcription Factors, and 14 moreDevelopment, Medicine, Signal Transduction, Biological Sciences, Longevity, Germ Cells, Protein Kinases, Mutation, Animals, Larva, Transforming Growth Factor Beta, Cell Proliferation, Receptor Insulin, and Medical and Health Sciences
The protein kinase LKB1 is a crucial regulator of cell growth/proliferation and cell polarity and is the causative gene in the cancer-predisposing disease Peutz-Jeghers syndrome (PJS). The activity of LKB1 is greatly enhanced following... more
The protein kinase LKB1 is a crucial regulator of cell growth/proliferation and cell polarity and is the causative gene in the cancer-predisposing disease Peutz-Jeghers syndrome (PJS). The activity of LKB1 is greatly enhanced following its association with the Ste20-like adapter protein STRAD. Unlike LKB1 however, mutations in STRAD have not been identified in PJS patients and thus, the key tumour suppressive role(s) of LKB1 might be STRAD independent. Here, we report that Caenorhabditis elegans strd-1/STRAD mutants recapitulate many phenotypes typical of par-4/LKB1 loss of function, showing defects during early embryonic and dauer development. Interestingly, although the growth/proliferation defects in severe par-4 and strd-1 mutant dauers are comparable, strd-1 mutant embryos do not share the polarity defects of par-4 embryos. We demonstrate that most of par-4-dependent regulation of germline stem cell (GSC) quiescence occurs through AMPK, whereby PAR-4 requires STRD-1 to phosphor...
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Caenorhabditis elegans larvae can undergo developmental arrest upon entry into the dauer stage in response to suboptimal growth conditions. Dauer larvae can exit this stage in replete conditions with no reproductive consequence. During... more
Caenorhabditis elegans larvae can undergo developmental arrest upon entry into the dauer stage in response to suboptimal growth conditions. Dauer larvae can exit this stage in replete conditions with no reproductive consequence. During this diapause stage, the metabolic regulator AMP-activated protein kinase (AMPK) ensures that the germ line becomes quiescent to maintain germ cell integrity. Animals that lack all AMPK signalling undergo germline hyperplasia upon entering dauer, while those that recover from this stage become sterile. Neuronal AMPK expression in otherwise AMPK-deficient animals is sufficient for germline quiescence and germ cell integrity and its effects are likely mediated through an endogenous small RNA pathway. Upon impairing small RNA biosynthesis, the post-dauer fertility is restored in AMPK mutants. These data suggest that AMPK may function in neurons to relay a message through small RNAs to the germ cells to alter their quiescence in the dauer stage, thus chal...
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Caenorhabditis elegans can endure long periods of environmental stress by altering their development to execute a quiescent state called "dauer". Previous work has implicated LKB1 - the causative gene in the autosomal dominant,... more
Caenorhabditis elegans can endure long periods of environmental stress by altering their development to execute a quiescent state called "dauer". Previous work has implicated LKB1 - the causative gene in the autosomal dominant, cancer pre-disposing disease called Peutz-Jeghers Syndrome (PJS), and its downstream target AMPK, in the establishment of germline stem cell (GSC) quiescence during the dauer stage. Loss of function mutations in both LKB1/par-4 and AMPK/aak(0) result in untimely GSC proliferation during the onset of the dauer stage, although the molecular mechanism through which these factors regulate quiescence remains unclear. Curiously, the hyperplasia observed in par-4 mutants is more severe than AMPK-compromised dauer larvae, suggesting that par-4 has alternative downstream targets in addition to AMPK to regulate germline quiescence. We conducted three genome-wide RNAi screens to identify potential downstream targets of the protein kinases PAR-4 and AMPK that m...
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Acute starvation can have long-term consequences that are mediated through epigenetic change. Some of these changes are affected by the activity of AMP-activated protein kinase (AMPK), a master regulator of cellular energy homeostasis. In... more
Acute starvation can have long-term consequences that are mediated through epigenetic change. Some of these changes are affected by the activity of AMP-activated protein kinase (AMPK), a master regulator of cellular energy homeostasis. In Caenorhabditis elegans, the absence of AMPK during a period of starvation in an early larval stage results in developmental defects following their recovery on food, while many of them become sterile. Moreover, the loss of AMPK during this quiescent period results in transgenerational phenotypes that can become progressively worse with each successive generation. Our recent data describe a chromatin-based mechanism of how AMPK mediates adjustment to acute starvation in the germ cells, however, the heritable aspect of this AMPK mutant phenotype remains unresolved. Here, we explore how AMPK might affect this process and speculate how the initial transcription that occurs in the germ cells may adversely affect subsequent germline gene expression and/o...
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Life cycle delays are beneficial for opportunistic species encountering suboptimal environments. Many animals display a programmed arrest of development (diapause) at some stage(s) of their development, and the diapause state may or may... more
Life cycle delays are beneficial for opportunistic species encountering suboptimal environments. Many animals display a programmed arrest of development (diapause) at some stage(s) of their development, and the diapause state may or may not be associated with some degree of metabolic depression. In this review, we will evaluate current advancements in our understanding of the mechanisms responsible for the remarkable phenotype, as well as environmental cues that signal entry and termination of the state. The developmental stage at which diapause occurs dictates and constrains the mechanisms governing diapause. Considerable progress has been made in clarifying proximal mechanisms of metabolic arrest and the signaling pathways like insulin/Foxo that control gene expression patterns. Overlapping themes are also seen in mechanisms that control cell cycle arrest. Evidence is emerging for epigenetic contributions to diapause regulation via small RNAs in nematodes, crustaceans, insects, an...
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The stress associated with starvation is accompanied by compensatory behaviours that enhance foraging efficiency and increase the probability of encountering food. However, the molecular details of how hunger triggers changes in the... more
The stress associated with starvation is accompanied by compensatory behaviours that enhance foraging efficiency and increase the probability of encountering food. However, the molecular details of how hunger triggers changes in the activity of neural circuits to elicit these adaptive behavioural outcomes remains to be resolved. We show here that AMP-activated protein kinase (AMPK) regulates neuronal activity to elicit appropriate behavioural outcomes in response to acute starvation, and this effect is mediated by the coordinated modulation of glutamatergic inputs. AMPK targets both the AMPA-type glutamate receptor GLR-1 and the metabotropic glutamate receptor MGL-1 in one of the primary circuits that governs behavioural response to food availability in C. elegans. Overall, our study suggests that AMPK acts as a molecular trigger in the specific starvation-sensitive neurons to modulate glutamatergic inputs and to elicit adaptive behavioural outputs in response to acute starvation.
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Life history events, such as traumatic stress, illness, or starvation, can influence us through molecular changes that are recorded in a pattern of characteristic chromatin modifications. These modifications are often associated with... more
Life history events, such as traumatic stress, illness, or starvation, can influence us through molecular changes that are recorded in a pattern of characteristic chromatin modifications. These modifications are often associated with adaptive adjustments in gene expression that can persist throughout the lifetime of the organism, or even span multiple generations. Although these adaptations may confer some selective advantage, if they are not appropriately regulated they can also be maladaptive in a context-dependent manner. We show here that during periods of acute starvation in Caenorhabditis elegans larvae, the master metabolic regulator AMP-activated protein kinase (AMPK) plays a critical role in blocking modifications to the chromatin landscape. This ensures that gene expression remains inactive in the germ-line precursors during adverse conditions. In its absence, critical chromatin modifications occur in the primordial germ cells (PGCs) of emergent starved L1 larvae that corr...
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The human BTF2 basic transcription factor (also called TFIIH), which is similar to the delta factor in rat and factor b in yeast, is required for class II gene transcription. A strand displacement assay was used to show that highly... more
The human BTF2 basic transcription factor (also called TFIIH), which is similar to the delta factor in rat and factor b in yeast, is required for class II gene transcription. A strand displacement assay was used to show that highly purified preparation of BTF2 had an adenosine triphosphate-dependent DNA helicase activity, in addition to the previously characterized carboxyl-terminal domain kinase activity. Amino acid sequence analysis of the tryptic digest generated from the 89-kilodalton subunit of BTF2 indicated that this polypeptide corresponded to the ERCC-3 gene product, a presumed helicase implicated in the human DNA excision repair disorders xeroderma pigmentosum and Cockayne&#39;s syndrome. These findings suggest that transcription and nucleotide excision repair may share common factors and hence may be considered to be functionally related.
Research Interests: Genetics, Science, DNA repair, Transcription Factors, Multidisciplinary, and 15 moreSequence Analysis, DNA, Humans, Protein Kinases, Cockayne syndrome, Trypsin, Nucleotide Excision Repair, Transcription Factor, Amino Acid Sequence, Recombinant Protein, Recombinant Proteins, Binding Site, Adenosine Triphosphate, Immunoblotting, and binding sites
The brain tissues of the rat and mouse express two types of corticosteroid binding proteins, the glucocorticoid (GR) and aldosterone (MR) receptors. Unlike the type II (GR) receptor, type I receptor has a high affinity for aldosterone... more
The brain tissues of the rat and mouse express two types of corticosteroid binding proteins, the glucocorticoid (GR) and aldosterone (MR) receptors. Unlike the type II (GR) receptor, type I receptor has a high affinity for aldosterone (ALDO) and corticosterone and is structurally similar to the kidney mineralocorticoid receptor (MR). The results reported in this study provide direct evidence for the interaction of dexamethasone (DEX), triamcinolone acetonide (TA), dexamethasone-21-mesylate (DXM) and 11-deoxycorticosterone (DOC) with human MR expressed in cells by transient co-transfection of a hMR expression vector. The interactions of hMR with DEX, TA, DXM, DOC, promegestone (R5020) and methyltrienelone (R1881) were measured by trans-activation of mouse mammary tumor virus long terminal repeat fused to bacterial chloramphenicol acetyltransferase (MMTV-tk-CAT) in gene co-transfection experiments and by cell free hormone binding assay. The incubation of various steroid hormones in the presence of [3H]ALDO in a competition assay with extracts prepared from HeLa cells co-transfected with hMR expression vector, showed that hMR expressed under these conditions has a high relative affinity for DEX which is similar to ALDO, TA and DOC. Incubation with DXM under these conditions showed very little competition, as was observed with R1881 and R5020. Incubation of the co-transfected cells with DEX, ALDO, DOC, R5020, TA, R1881 and DXM demonstrated that the level of trans-activation did not reflect the previously observed order of binding affinity for the hMR. The level of transactivation was always higher with DEX and TA compared to ALDO and DOC. Analysis of the binding of labeled glucocorticoid regulatory element (GRE) and hMR incubated with DEX, ALDO and DXM by gel shift analysis demonstrated that the trans-activation of MMTV-tk-CAT by hMR is a result of the interaction of hMR with GRE in the MMTV-LTR.
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C. elegans cki-1 encodes a member of the CIP/KIP family of cyclin-dependent kinase inhibitors, and functions to link postembryonic developmental programs to cell cycle progression. The expression pattern of cki-1::GFP suggests that cki-1... more
C. elegans cki-1 encodes a member of the CIP/KIP family of cyclin-dependent kinase inhibitors, and functions to link postembryonic developmental programs to cell cycle progression. The expression pattern of cki-1::GFP suggests that cki-1 is developmentally regulated in blast cells coincident with G1, and in differentiating cells. Ectopic expression of CKI-1 can prematurely arrest cells in G1, while reducing cki-1 activity by RNA-mediated interference (RNAi) causes extra larval cell divisions, suggesting a role for cki-1 in the developmental control of G1/S. cki-1 activity is required for the suspension of cell cycling that occurs in dauer larvae and starved L1 larvae in response to environmental signals. In vulva precursor cells (VPCs), a pathway of heterochronic genes acts via cki-1 to maintain VPCs in G1 during the L2 stage.
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Animals have developed diverse mechanisms to adapt to their changing environment. Like many organisms the free-living nematode C. elegans can alternate between a reproductive mode or a diapause-like "dauer" stage during larval... more
Animals have developed diverse mechanisms to adapt to their changing environment. Like many organisms the free-living nematode C. elegans can alternate between a reproductive mode or a diapause-like "dauer" stage during larval development to circumvent harsh environmental conditions. The master metabolic regulator AMP-activated protein kinase (AMPK) is critical for survival during the dauer stage, where it phosphorylates adipose triglyceride lipase (ATGL-1) at multiple sites to block lipid hydrolysis and ultimately protect the cellular triglyceride-based energy depot from rapid depletion. However, how the AMPK-mediated phosphorylation affects the function of ATGL-1 has not been characterised at the molecular level. Here we show that AMPK phosphorylation leads to the generation of 14-3-3 binding sites on ATGL-1, which are recognized by the C. elegans 14-3-3 protein orthologue PAR-5. Physical interaction of ATGL-1 with PAR-5 results in sequestration of ATGL-1 away from the l...
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TFIIH is a multiprotein factor involved in transcription and DNA repair and is implicated in DNA repair/transcription deficiency disorders such as xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. Eight out of the nine... more
TFIIH is a multiprotein factor involved in transcription and DNA repair and is implicated in DNA repair/transcription deficiency disorders such as xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. Eight out of the nine genes encoding the subunits forming TFIIH have already been cloned. We report here the identification, cDNA cloning and gene structure of the 52 kDa polypeptide and its homology with the yeast counterpart TFB2. This protein, along with p89/XPB, p62, p44 and p34, forms the core of TFIIH. Moreover, using in vitro reconstituted transcription and nucleotide excision repair (NER) assays and microinjection experiments, we demonstrate that p52 is directly involved in both transcription and DNA repair mechanisms in vitro and in vivo.
Research Interests: Genetics, Fluorescence, DNA repair, Transcription Factors, Sequence Analysis, and 27 moreWestern blotting, In Situ Hybridization, Transcription, Biological Sciences, Saccharomyces cerevisiae, Molecular, Antibodies, Humans, Fluorescence in situ hybridization, Sequence alignment, Cockayne syndrome, Monoclonal Antibodies, Nucleotide Excision Repair, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Cloning, Transcription Factor, Gene Structure, Support, Molecular cloning, Electrophoresis, Monoclonal Antibody, Amino Acid Sequence, Polyacrylamide Gel, Chromosomes, Xeroderma Pigmentosum, Monoclonal, and Molecular Sequence Data
ERCC2 is involved in the DNA repair syndrome xeroderma pigmentosum (XP) group D and was found to copurify with the RNA polymerase II (B) transcription factor BTF2/TFIIH that possesses a bidirectional helicase activity. Antibodies directed... more
ERCC2 is involved in the DNA repair syndrome xeroderma pigmentosum (XP) group D and was found to copurify with the RNA polymerase II (B) transcription factor BTF2/TFIIH that possesses a bidirectional helicase activity. Antibodies directed towards the 89 kDa (ERCC3) or the p62 subunit of BTF2 are able to either immunoprecipitate ERCC2 or shift the polypeptide in a glycerol gradient. Conversely, an antibody directed towards ERCC2 also retains or shifts BTF2. ERCC2 could be resolved from the other characterized components of BTF2 upon salt treatment, while its readdition enhanced BTF2 transcription activity. ERCC2, ERCC3 and p44 are three repair proteins found in association with BTF2. Two of them, ERCC2 and ERCC3, are responsible for atypical forms of XP disorders which confer a high predisposition to skin cancer. This includes clinical features that lack an adequate rationalization on the basis of nucleotide excision repair (NER) deficiency but which may now be explained better in te...
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BTF2/TFIIH from human, delta from rat, and factor b from yeast are multisubunit basal transcription factors that have been shown to be closely associated with a protein kinase capable of phosphorylating the carboxyl-terminal domain of the... more
BTF2/TFIIH from human, delta from rat, and factor b from yeast are multisubunit basal transcription factors that have been shown to be closely associated with a protein kinase capable of phosphorylating the carboxyl-terminal domain of the large subunit of RNA polymerase II (Lu, H., Zawel, L., Fischer, L., Egly, J. M., and Reinberg, D. (1992) Nature 358, 641-645; Serizawa, H., Conaway, R. C., and Conaway, J. W. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 7476-7480; Feaver, W. J., Gileadi, O., and Kornberg, R. D. (1991) Cell 67, 1223-1230). We report here that a DNA-dependent ATPase and the previously characterized helicase (Schaeffer, L., Roy, R., Humbert, S., Moncollin, V., Vermeulen, W., Hoeijmakers, J., Chambon, P., and Egly, J. M. (1993) Science 260, 58-63) are both associated with BTF2 and reside with the p89 polypeptide subunit. The DNA requirement, the effect of Sarkosyl and staurosporine inhibitors, as well as nucleotide competition experiments, clearly distinguished ATPase/heli...
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The association between lipoproteins, cholesterol and cholesteryl esters is very well known to facilitate both the transport in plasma and the entry of these non-polar compounds into the cellular compartment. However, recent observations... more
The association between lipoproteins, cholesterol and cholesteryl esters is very well known to facilitate both the transport in plasma and the entry of these non-polar compounds into the cellular compartment. However, recent observations suggest that in addition to cholesterol, lipoproteins contain several other steroids in their lipoidal metabolite forms which may be transported in the very low, low and high density lipoproteins in human serum. Using the important androgen and oestrogen precursor, dehydroepiandrosterone (DHEA), the biosynthetic formation of lipoidal DHEA was demonstrated in human serum. Serum was also fractionated into its lipoprotein components during the course of its incubation with tritiated DHEA. A progressive movement of the label from the fraction containing the conventional steroid binding-proteins to the lipoproteins was observed with the fraction containing the low density lipoproteins demonstrating the greatest incorporation of the label. This displaceme...
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The precise control of cell division during development is pivotal for morphogenesis and the correct formation of tissues and organs. One important gene family involved in such control is the p21/p27/p57 class of negative cell cycle... more
The precise control of cell division during development is pivotal for morphogenesis and the correct formation of tissues and organs. One important gene family involved in such control is the p21/p27/p57 class of negative cell cycle regulators. Loss of function of the C. elegans p27 homolog, cki-1, causes extra cell divisions in numerous tissues including the hypodermis, the vulva, and the intestine. We have sought to better understand how cell divisions are controlled upstream or in parallel to cki-1 in specific organs during C. elegans development. By taking advantage of the invariant cell lineage of C. elegans, we used an intestinal-specific GFP reporter in a screen to identify mutants that undergo cell division abnormalities in the intestinal lineage. We have isolated a mutant with twice the wild-type complement of intestinal cells, all of which arise during mid-embryogenesis. This mutant, called rr31, is a fully dominant, maternal-effect, gain-of-function mutation in the cdc-25...
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Regulation of chain elongation by RNA polymerase II can have an important effect on gene expression (Bentley, D. (1995) Curr. Opin. Genet. Dev. 5, 210-216; Yankulov, K., Blau, J., Purton, T., Roberts, S., and Bentley, D. (1994) Cell 77,... more
Regulation of chain elongation by RNA polymerase II can have an important effect on gene expression (Bentley, D. (1995) Curr. Opin. Genet. Dev. 5, 210-216; Yankulov, K., Blau, J., Purton, T., Roberts, S., and Bentley, D. (1994) Cell 77, 749-759); however the mechanisms that control this step in transcription are not well understood. The adenosine analogue 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) has long been used as an inhibitor of RNA polymerase II elongation, but its target is not known. We show that DRB is a potent inhibitor of Cdk-activating kinase, associated with the general transcription factor TFIIH. Two other inhibitors of this kinase, H-7 and H-8, also inhibited transcriptional elongation. Furthermore, TFIIH kinase bound specifically to the herpes simplex virus VP16 activation domain which stimulates polymerase II elongation in addition to initiation (Yankulov, K., Blau, J., Purton, T., Roberts, S., and Bentley, D. (1994) Cell 77, 749-759). Our results suggest that DRB affects transcription by inhibiting the TFIIH-associated kinase and that this kinase functions in the control of elongation by RNA polymerase II.