Arnab Mukhopadhyay

Identification of direct DAF-16 targets controlling longevity, metabolism and diapause by chromatin immunoprecipitation

DAF-16, a forkhead transcription factor, is a key regulator of longevity, metabolism and dauer diapause in Caenorhabditis elegans. The precise mechanism by which DAF-16 regulates multiple functions, however, is poorly understood. Here, we used chromatin immunoprecipitation (ChIP) to identify direct targets of DAF-16. We cloned 103 target sequences containing consensus DAF-16 binding sites and selected 33 targets for further analysis. Expression of most of these genes is regulated in a DAF-16–dependent manner, and inactivation of more than half of these genes significantly altered DAF-16–dependent functions, including life span, fat storage and dauer formation. Our results show that the ChIP-based cloning strategy leads to greater enrichment for DAF-16 target genes than previous screening strategies. We also demonstrate that DAF-16 is recruited to multiple promoters to coordinate regulation of its downstream targets. The large number of target genes discovered provides insight into how DAF-16 controls diverse biological functions.

Chromatin immunoprecipitation (ChIP) coupled to detection by quantitative real-time PCR to study transcription factor binding to DNA in Caenorhabditis elegans

In order to determine how signaling pathways differentially regulate gene expression, it is necessary to identify the interactions between transcription factors (TFs) and their cognate cis-regulatory DNA elements. Here, we have outlined a chromatin immunoprecipitation (ChIP) protocol for use in whole Caenorhabditis elegans extracts. We discuss optimization of the procedure, including growth and harvesting of the worms, formaldehyde fixation, TF immunoprecipitation and analysis of bound sequences through real-time PCR. It takes ∼10–12 d to obtain the worm culture for ChIP; the ChIP procedure is spaced out over a period of 2.5 d with two overnight incubations.

A PP2A Regulatory Subunit Regulates C. elegans Insulin/IGF-1 Signaling by Modulating AKT-1 Phosphorylation

The C. elegans insulin/IGF-1 signaling (IIS) cascade plays a central role in regulating life span, dauer, metabolism, and stress. The major regulatory control of IIS is through phosphorylation of its components by serine/threonine-specific protein kinases. An RNAi screen for serine/threonine protein phosphatases that counterbalance the effect of the kinases in the IIS pathway identified pptr-1, a B56 regulatory subunit of the PP2A holoenzyme. Modulation of pptr-1 affects IIS pathway-associated phenotypes including life span, dauer, stress resistance, and fat storage. We show that PPTR-1 functions by regulating worm AKT-1 phosphorylation at Thr 350. With striking conservation, mammalian B56beta regulates Akt phosphorylation at Thr 308 in 3T3-L1 adipocytes. In C. elegans, this ultimately leads to changes in subcellular localization and transcriptional activity of the forkhead transcription factor DAF-16. This study reveals a conserved role for the B56 regulatory subunit in regulating insulin signaling through AKT dephosphorylation, thereby having widespread implications in cancer and diabetes research.

Worming pathways to and from DAF-16/FOXO

In Caenorhabditis elegans, the insulin/IGF-1 signaling pathway controls many biological processes such as life span, fat storage, dauer diapause, reproduction and stress response . This pathway is comprised of many genes including the insulin/IGF-1 receptor (DAF-2) that signals through a conserved PI 3-kinase/AKT pathway and ultimately down-regulates DAF-16, a forkhead transcription factor (FOXO). DAF-16 also receives input from several other pathways that regulate life span such as the germline and the JNK pathway [Hsin, H., Kenyon, C., 1999. Signals from the reproductive system regulate the lifespan of C. elegans. Nature 399, 362-366; Oh, S.W., Mukhopadhyay, A., Svrzikapa, N., Jiang, F., Davis, R.J., Tissenbaum, H.A., 2005. JNK regulates lifespan in Caenorhabditis elegans by modulating nuclear translocation of forkhead transcription factor/DAF-16. Proc. Natl. Acad. Sci. USA 102, 4494-4499]. Therefore, DAF-16 integrates signals from multiple pathways and regulates its downstream target genes to control diverse processes. Here, we discuss the signals to and from DAF-16, with a focus on life span regulation.

Complex expression dynamics and robustness in C. elegans insulin networks

Gene families expand by gene duplication, and resulting paralogs diverge through mutation. Functional diversification can include neofunctionalization as well as subfunctionalization of ancestral functions. In addition, redundancy in which multiple genes fulfill overlapping functions is often maintained. Here, we use the family of 40 Caenorhabditis elegans insulins to gain insight into the balance between specificity and redundancy. The insulin/insulin-like growth factor (IIS) pathway comprises a single receptor, DAF-2. To date, no single insulin-like peptide recapitulates all DAF-2-associated phenotypes, likely due to redundancy between insulin-like genes. To provide a first-level annotation of potential patterns of redundancy, we comprehensively delineate the spatiotemporal and conditional expression of all 40 insulins in living animals. We observe extensive dynamics in expression that can explain the lack of simple patterns of pairwise redundancy. We propose a model in which gene families evolve to attain differential alliances in different tissues and in response to a range of environmental stresses.

An endocytic pathway as a target of tubby for regulation of fat storage

The tubby loci provide a unique opportunity to study adult‐onset obesity. Mutation in either mammalian tubby or its homologue in Caenorhabditis elegans, tub‐1, results in increased fat storage. Previously, we have shown that TUB‐1 interacts with a new Rab GTPase‐activating protein, RBG‐3, for the regulation of fat storage. To understand further the molecular mechanism of TUB‐1, we identified the Rab GTPase downstream of RBG‐3. We found that RBG‐3 preferentially stimulates the intrinsic GTPase activity of RAB‐7 in both human and C. elegans. Importantly, either mutation or RNA interference knockdown in rab‐7 reduces stored fat in wild type and tub‐1 mutants. In addition, the small GTPase rab‐5 and genes that regulate Rab membrane localization and nucleotide recycling are required for the regulation of fat storage, thereby defining a role for endocytic recycling in this process. We propose that TUB‐1 controls receptor or sensory molecule degradation in neurons by regulating a RAB‐7‐mediated endocytic pathway.

A novel kinase regulates dietary restriction-mediated longevity in Caenorhabditis elegans.

Although dietary restriction (DR) is known to extend lifespan across species, from yeast to mammals, the signalling events downstream of food/nutrient perception are not well understood. In Caenorhabditis elegans, DR is typically attained either by using the eat‐2 mutants that have reduced pharyngeal pumping leading to lower food intake or by feeding diluted bacterial food to the worms. In this study, we show that knocking down a mammalian MEKK3‐like kinase gene, mekk‐3 in C. elegans, initiates a process similar to DR without compromising food intake. This DR‐like state results in upregulation of beta‐oxidation genes through the nuclear hormone receptor NHR‐49, a HNF‐4 homolog, resulting in depletion of stored fat. This metabolic shift leads to low levels of reactive oxygen species (ROS), potent oxidizing agents that damage macromolecules. Increased beta‐oxidation, in turn, induces the phase I and II xenobiotic detoxification genes, through PHA‐4/FOXA, NHR‐8 and aryl hydrocarbon receptor AHR‐1, possibly to purge lipophilic endotoxins generated during fatty acid catabolism. The coupling of a metabolic shift with endotoxin detoxification results in extreme longevity following mekk‐3 knock‐down. Thus, MEKK‐3 may function as an important nutrient sensor and signalling component within the organism that controls metabolism. Knocking down mekk‐3 may signal an imminent nutrient crisis that results in initiation of a DR‐like state, even when food is plentiful.

InAKTivation of insulin/IGF-1 signaling by dephosphorylation

Signal transduction pathways are tightly regulated by phosphorylation-dephosphorylation cycles and yet the mammalian genome contains far more genes that encode protein kinases than protein phosphatases. Therefore, to target specific substrates, many phosphatases associate with distinct regulatory subunits and thereby modulate multiple cellular processes. One such example is the C. elegans PP2A regulatory subunit PPTR-1 that negatively regulates the insulin/insulin-like growth factor signaling pathway to modulate longevity, dauer diapause, fat metabolism and stress resistance. PPTR-1, as well as its mammalian homolog B56β, specifically target the PP2A enzyme to AKT and mediate the dephosphorylation of this important kinase at a conserved threonine residue. In C. elegans, the major consequence of this modulation is activation of the FOXO transcription factor homolog DAF-16, which in turn regulates transcription of its many target genes involved in longevity and stress resistance. Understanding the function of B56 subunits may have important consequences in diseases such as Type 2 diabetes and cancer where the balance of Akt phosphorylation is deregulated.

Rifampicin reduces advanced glycation end products and activates DAF-16 to increase lifespan in Caenorhabditis elegans

Advanced glycation end products (AGEs) are formed when glucose reacts nonenzymatically with proteins; these modifications are implicated in aging and pathogenesis of many age‐related diseases including type II diabetes, atherosclerosis, and neurodegenerative disorders. Thus, pharmaceutical interventions that can reduce AGEs may delay age‐onset diseases and extend lifespan. Using LC‐MSE, we show that rifampicin (RIF) reduces glycation of important cellular proteins in vivo and consequently increases lifespan in Caenorhabditis elegans by up to 60%. RIF analog rifamycin SV (RSV) possesses similar properties, while rifaximin (RMN) lacks antiglycation activity and therefore fails to affect lifespan positively. The efficacy of RIF and RSV as potent antiglycating agents may be attributed to the presence of a p‐dihydroxyl moiety that can potentially undergo spontaneous oxidation to yield highly reactive p‐quinone structures, a feature absent in RMN. We also show that supplementing rifampicin late in adulthood is sufficient to increase lifespan. For its effect on longevity, rifampicin requires DAF‐18 (nematode PTEN) as well as JNK‐1 and activates DAF‐16, the FOXO homolog. Interestingly, the drug treatment modulates transcription of a different subset of DAF‐16 target genes, those not controlled by the conserved Insulin‐IGF‐1‐like signaling pathway. RIF failed to increase the lifespan of daf‐16 null mutant despite reducing glycation, showing thereby that DAF‐16 may not directly affect AGE formation. Together, our data suggest that the dual ability to reduce glycation in vivo and activate prolongevity processes through DAF‐16 makes RIF and RSV effective lifespan‐extending interventions.

A chromatin modifier integrates insulin/IGF‐1 signalling and dietary restriction to regulate longevity

Insulin/IGF‐1‐like signalling (IIS) and dietary restriction (DR) are the two major modulatory pathways controlling longevity across species. Here, we show that both pathways license a common chromatin modifier, ZFP‐1/AF10. The downstream transcription factors of the IIS and select DR pathways, DAF‐16/FOXO or PHA‐4/FOXA, respectively, both transcriptionally regulate the expression of zfp‐1. ZFP‐1, in turn, negatively regulates the expression of DAF‐16/FOXO and PHA‐4/FOXA target genes, apparently forming feed‐forward loops that control the amplitude as well as the duration of gene expression. We show that ZFP‐1 mediates this regulation by negatively influencing the recruitment of DAF‐16/FOXO and PHA‐4/FOXA to their target promoters. Consequently, zfp‐1 is required for the enhanced longevity observed during DR and on knockdown of IIS. Our data reveal how two distinct sensor pathways control an overlapping set of genes, using different downstream transcription factors, integrating potentially diverse and temporally distinct nutritional situations.